Abstract

Synthesis of cynuric chloride based various chalcones A1-A19 by condensation reaction of Cyanuric chloride with 1-(4-aminophenyl)ethanone to yields product 1-(4-(4,6-dichloro-1,3,5-triazin-2-ylamino)phenyl)ethanone which upon condensation with various aromatic aldehyde. All prepared chalcones were further reflux with urea to give pyrimidines B1-B19. Characterization of all synthesized pyrimidones were done using various spectroscopic techniques such as 1HNMR, 13CNMR, IR, MASS.  Biological evaluation of all prepared pyrimidones   were done using against two gram positive bacteria such as Staphylococcus aureus, Bacillus megaterium and two gram negative bacteria Escherichia coli, Proteus vulgaris. Most of the synthesized products exhibited moderate to good potency against bacteria as compared to standard drugs.

Keywords

Pyrimidone, Cyanuric Chloride, Chalcone, Aldehydes, Antimicrobial Activity, Urea, Spectroscopy

Cite This Article

Solanki, J. A., Rakeshkumar, Vyas, K. B. (2021). Synthesis of Various Cynuric Chloride Based Pyrimidones And Their Antimicrobial Activity. International Journal for Pharmaceutical Research Scholars, 10(1);53 -60.

Abstract

The Various chalcones A1-A19 were obtained by one pot condensation of 1-chloro-4-methyl benzene with 1-(4-aminophenyl) ethanone followed by condensation with various aromatic aldehydes. All prepared chalcones were further reflux with urea to give pyrimidines B1-B19. All the synthesized pyrimidines were characterized in by 1HNMR, 13CNMR, IR, MASS spectroscopic techniques. Biological evaluation of all synthesized pyrimidines were done using f gram positive bacteria such as Staphylococcus aureus, Bacillus megaterium and gram negative bacteria Escherichia coli, Proteus vulgaris. Most of the prepared compounds shows moderate to good activity against bacteria as compared to standard drugs.

Keywords

Pyrimidone, Chalcone, Aldehydes, Antimicrobial activity, Urea, Spectroscopy

Cite This Article

Bhatt., C. A., Rakeshkumar, Vyas, K. B. (2021). Study of Synthesis And Antimicrobial Activity of Novel Pyrimidones From Chalcones and Urea. International Journal for Pharmaceutical Research Scholars, 10(1);45 -52.

Department of Pharmacology and Pharmacy Practice, K. B. Institute of Pharmaceutical Education and Research, Gandhinagar, Gujarat, India

Abstract

Gentamycin-induced nephrotoxicity mainly involves the generation of reactive oxygen species. Phytic acid is an abundantly found compound in various grains with antioxidant properties. We studied the protective effect of phytic acid against gentamicin-induced nephrotoxicity in rats. Male Sprague Dawley rats were divided into 4 groups each of 6 animals. All the animals except normal control received gentamycin (100 mg/kg, i.p, oid) from day 1 to 8. Group I and II served as normal and disease control respectively. Test groups III and IV received phytic acid 150 mg/kg and 300 mg/kg respectively (p.o, oid from day 1 to 8). Serum creatinine, serum uric acid (UA), blood urea nitrogen (BUN), urine volume (24 hr), urine creatinine, urine UA and total protein were measured. Creatinine clearance (CLCr), fractional excretion of UA (FEUA), and fractional excretion of sodium (FENa %) were calculated. Superoxide dismutase (SOD) and catalase (CAT) activity; malondialdehyde (MDA) and reduced glutathione (GSH) levels were measured in kidney homogenate. Phytic acid treatment significantly lowered the levels of serum creatinine, serum UA, BUN, urine creatinine, urine UA; and total protein in the urine as compared to disease animals. Phytic acid significantly increased GSH content, SOD and CAT activity; and lowered MDA level.  Creatinine clearance and FEUA were significantly higher in treatment groups. Histopathological studies of the kidney showed less tubular, and glomerular damage and fewer signs of inflammation in treated groups. It can be concluded that phytic acid shows a protective effect in gentamycin-induced nephrotoxicity in rats due to its antioxidant property.

Keywords

Nephrotoxicity, gentamycin, phytic acid, reactive oxygen species, antioxidant

Cite This Article

Shah P.A., Gupta, A.D., Deshpande, S.S. (2022). Protective Effect of Phytic Acid in Gentamycin Induced Nephrotoxicity in Rats. International Journal for Pharmaceutical Research Scholars, 11(3);01 - 10.

INTRODUCTION

Nephrotoxicity is characterized by reduced glomerular filtration rate (GFR), elevated serum creatinine and blood urea nitrogen (BUN); and proteinuria. It is the major adverse effect of aminoglycoside antibiotics, especially gentamycin, account for 10 % to 15 % of all cases of acute renal failure. Gentamycin toxicity is due to its marked accumulation and retention in the proximal convoluted tubules1.  Gentamycin mainly causes acute tubular necrosis. It is postulated that gentamycin may enhance the generation of reactive oxygen species (ROS) by damaging renal mitochondria. Gentamycin may alter mitochondrial respiration; generates superoxide anion and hydroxyl radical in renal mitochondria2, 3, 4. Gentamycin-iron complex causes lipid peroxidation In vitro and is a potent catalyst for free-radical formation5. The role of ROS is well-established in gentamycin-induced nephrotoxicity. Studies showed that hydroxyl radical scavengers are protective in gentamicin-induced acute renal failure in rats 6,7. Co-administration of antioxidants, vitamin E, and selenium is protective against gentamycin-induced nephrotoxicity 8. Phytic acid (myo-inositol hexaphosphoric acid) is an abundant plant constituent, chiefly found in edible legumes, pollens, nuts, cereals, and seeds about almost 1-5% by weight. Phytic acid can chelate iron and it is a potent inhibitor of the iron-driven formation of reactive oxygen species 9. It has anticarcinogenic activity partly attributable to its antioxidant property 10. In this study, we evaluated the protective effect of phytic acid in gentamycin-induced nephrotoxicity in rats.

MATERIALS AND METHODS

Drugs and Chemicals

Gentamicin Sulphate Powder (Morvel Laboratory, Mehsana, Gujarat), 40 % phytic acid solution (purity 98 %) (Sigma Aldrich, St. Louis, USA); uric acid kit (Beacon Diagnostic Pvt. Ltd, Navsari, India), creatinine kit, BUN and total protein kit from (Span Diagnostic Pvt. Ltd, Surat, India) were purchased.

Ethics Approval

The project was approved by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) and the Institutional Animal Ethics Committee K. B. Institute of Pharmaceutical Education & Research, Gandhinagar; Project no. KBIPER/12/303.

Animals and study groups

Healthy male Sprague-Dawley rats (body weight 200 to 250 g) were selected and divided into 4 groups of 6 animals in each. The rats were acclimatized to standard condition of temperature (22 ± 1°C), relative humidity (40 to 60 %), and 12:12 hr alternate light & dark cycle. They were fed with the standard pellet diet (Pranav Agro Ltd, Gujarat, India) and water ad libitum. Nephrotoxicity was induced by administering gentamycin (100 mg/kg/day, i. p, once a day, days 1 to 8) in all animals except the normal control group. The normal control group (I) received a normal saline solution (0.5 mL, i. p). Group II served as the disease group and received gentamycin only. Test groups III and IV received phytic acid 150 mg/kg and 300 mg/kg respectively (p. o, once a day, from day 1 to 8).

Table 1: Groups and Treatments

Parameters:

Body weight, food, and water intake (24 hr) were measured (day1 & 8). The blood was collected from retro orbital plexus of each animal using isoflurane inhalation anaesthesia (day 1 & 8). The sample was centrifuged at 4000-5000 rpm for 15 minutes and the serum was separated. Serum uric acid (UA), creatinine & blood urea nitrogen (BUN) were estimated. Urine was collected from each animal on day 1 & 8 (at 24 hr). Urine volume & pH of urine (using pH strip) were measured. Uric acid, creatinine & total protein were estimated in urine. Creatinine clearance (CLCr), fractional excretion of UA (FEUA), and fractional excretion of sodium (FENa %) were calculated. On day 8, all the animals were sacrificed using a carbon dioxide chamber. The kidney was isolated, washed with saline, and weighed. It was further processed for histopathology. Levels of reduced glutathione (GSH)¹¹ and malondialdehyde (MDA)¹², superoxide dismutase (SOD)^13, and catalase (CAT) activity¹⁴ were measured in kidney homogenate.

Statistical Analysis

Data were expressed as mean ± SEM (n=6). P<0.05 was considered statistically significant. One-way Analysis of Variance (ANOVA) was carried out followed by the post hoc Tukey test. Statistical analysis was performed using Sigma Plot for Windows version 12 software developed in 2011.

RESULTS AND DISCUSSION

Percentage change in body weight (%)

Animals in disease group (-44.02±4.32) showed significant % change (reduction) in body weight (BW) as compared to control group (17.29±1.42) on day 8. Percentage change in BW was significantly different in animals treated with phytic acid 150 mg and 300 mg (6.48±1.42 and 15.57±3.17 respectively) as compared to the gentamycin group (Fig. 1).

Food intake (gm)

Animals in the disease group (16.16±0.30) showed significantly lower food intake as compared to the control group (22.83±0.60) on day 8. Animals treated with phytic acid 150 mg and 300 mg showed significantly higher food intake (23.16±0.60 and 24±0.60 respectively) as compared to the disease group on day 8 (Fig. 2).

Water intake, urine volume, and urine pH

No significant differences were found amongst normal, disease, and treatment groups.

Kidney weight (gm)

In the gentamycin group, kidney weight (0.56±0.03) was significantly lower as compared to the control group (0.78±0.02). Concurrent administration of phytic acid (150 and 300 mg/kg) with gentamycin showed significantly higher kidney weight (0.75±0.05 and 0.83±0.02 respectively) as compared to the gentamycin group (Fig. 3).

Blood urea nitrogen (BUN) (mg/dL)

BUN level was significantly higher in the gentamycin group (8.35±0.26) as compared to the control group (0.69±0.06) on day 8. Concurrent administration of phytic acid (150 and 300 mg/kg) with gentamycin significantly decreased BUN level (5.40±0.15 and 5.37±0.17 respectively) as compared to the gentamycin group respectively (Fig. 4).

^# indicates the significant difference from normal control, * indicates the significant difference from the gentamycin (disease) group, (p<0.05, One way ANOVA followed by Tukey test)

Serum and urine creatinine (mg/dL)

Serum and urine creatinine were significantly higher in the gentamycin group as compared to the control group. Concurrent administration of phytic acid (150 and 300 mg/kg) with gentamycin showed significantly decreased levels of serum and urine creatinine as compared to the gentamycin group. Phytic acid 300 mg/kg treatment significantly lowered serum creatinine levels as compared to 150 mg/kg dose (Table 2).

Table 2: Effect of Phytic Acid on Serum and Urine Creatinine (mg/dL)

^# indicates the significant difference from normal control, * indicates the significant difference from the gentamycin (disease) group, ^$ indicates the significant difference from test group I. (p<0.05, One way ANOVA followed by Tukey test).

Serum and urine uric acid (mg/dL)

Serum and urine uric acid were significantly higher in the gentamycin group as compared to the control group on day 8. Concurrent administration of phytic acid (150 and 300 mg/kg) with gentamycin significantly decreased serum and urine uric acid levels as compared to the gentamycin group (Table 3).

Table 3: Effect of Phytic Acid on Serum and Urine Uric Acid (mg/dL)

# indicates the significant difference from normal control, * indicates the significant difference from the gentamycin (disease) group, (p<0.05, One way ANOVA followed by Tukey test).

Urine total protein (g/dL)(24hr)

Urine total protein was significantly higher in the gentamycin group (38.26±2.62) as compared to the control group (19.12±2.25) on day 8. Concurrent administration of phytic acid (150 and 300 mg/kg) with gentamycin significantly decreased total protein level in urine (22.07±1.19 and 19.52±1.15 respectively) as compared to the gentamycin group (Fig. 5).

Creatinine clearance (mL/min)

Creatinine clearance (CLcr) was significantly lower in the gentamycin group (0.20±0.03) as compared to the control group (0.59±0.06) on day 8. Concurrent administration of phytic acid (150 and 300 mg/kg) with gentamycin significantly increased CLcr (0.70±0.02 and 0.71±0.02 respectively) as compared to the gentamycin group (Fig. 6).

^# indicates the significant difference from normal control, * indicates the significant difference from the gentamycin (disease) group. (p<0.05, One way ANOVA followed by Tukey test).

Fractional excretion of uric acid (FEUA) and sodium (FENa) (%)

FEUA was significantly lower in the gentamycin group as compared to the control group on day 8. Concurrent administration of phytic acid (150 and 300 mg/kg) with gentamycin significantly increased FEUA as compared to the gentamycin group respectively (Table 4). Fractional excretion of sodium (FENa) was significantly higher in the gentamycin group as compared to the control group on day 8. Concurrent administration of phytic acid (150 & 300 mg/kg) with gentamycin significantly decreased FENa as compared to the gentamycin group (Table 4).

Table 4: Effect of Phytic Acid on Fractional Excretion of Uric Acid (FEUA) and Sodium (FENa) (%)

# indicates the significant difference from normal control, * indicates the significant difference from gentamycin (disease) group, (p<0.05, One way ANOVA followed by Tukey test).

Effect of phytic acid on total protein in kidney, GSH level, MDA level, SOD and CAT activity (day 8) (Table 5)

The protein level in the gentamycin group was significantly lower than in the control group. Administration of phytic acid (150 and 300 mg/kg) significantly increased protein levels in the kidney as compared to the gentamycin group. Total protein in the kidney was significantly higher in phytic acid 300 mg dose as compared to 150 mg dose. The level of GSH was significantly decreased and MDA was increased the in gentamycin group. Treatment with phytic acid 150 and 300 mg increased the levels of GSH and decreased MDA. Similarly, SOD and Catalase activity were significantly decreased in the gentamycin group. The activity of both enzymes was increased in phytic acid-treated groups.

Table 5: Effect of Phytic Acid on Total Protein in Kidney, GSH level, MDA level, SOD and CAT Activity (day 8)

^# indicates the significant difference from normal control, * indicates the significant difference from the gentamycin (disease) group, ^$ indicates the significant difference from test group I. (p<0.05, One way ANOVA followed by Tukey test).

Histopathology of kidney (Magnification 100x × 40X )

Normal control  (a) shows normal architecture in the tubular and glomerular parts in the cortex and medulla and shows normal papilla. There is no sign of inflammation or tissue damage. Gentamycin group (b) shows the presence of only renal glomeruli with loss of renal epithelial cells and increased vascularity. Phytic acid 150 (c) and phytic acid 300 (d) show almost intact glomeruli as well as normal tubular epithelial cells (Fig. 7).

DISCUSSION

The results showed that administration of Gentamycin induced nephrotoxicity in rats. This was demonstrated by decreased body weight, kidney weight, food intake and CLcr; increased serum creatinine, BUN and uric acid levels; decreased protein content in kidney and increased urine protein levels. Oxidative stress was increased shown by decreased GSH level, increased SOD and Catalase activity; and increased lipid peroxidation. Phytic acid improved the markers of nephrotoxicity and improved antioxidant parameters. It has been proven that phytic acid promotes the oxidation of Fe2+ to Fe3+ which results in less Fe2+ available for hydroxyl radical formation 15. It is found to be affecting pathological targets in Alzheimer’s disease ¹⁶. Phytate can chelate iron, zinc, copper, and magnesium which are involved in ROS ¹⁷. In our study, results show that phytic acid treatment improved antioxidants in the kidney and showed a nephroprotective effect.

CONCLUSION

Phytic acid decreased creatinine, BUN, uric acid, and urine protein excretion. It improved total protein in the kidney, creatinine clearance, FEUA, and FENa. It improved antioxidant parameters along with histopathological changes induced due to gentamycin. It can be concluded that phytic acid has a nephroprotective effect due to its antioxidant activity.

REFERENCE

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Abstract

Microemulsions are thermodynamically stable, fluid, optically clear dispersions of two immiscible systems. Recent interest in micro emulsion system has resulted from utility in a broad range of applications including enhanced oil recovery, consumer and pharmaceutical formulations, nanoparticles synthesis and chemical reaction media. In last few decades ionic liquids (ILs) have been widely considered as a green solvent and they are used in various fields. ILs can be used in the formation of micro emulsion as a dispersed medium, polar domain and recently as a surfactant. ILs with a certain surface activity having long alcohol chain substitutes can self-aggregate and form Ionic Liquid Micro Emulsion with high-temperature insensitivity.

Keywords

Microemulsion, ionic liquids, surfactant, self-aggregates

Cite This Article

Gallani, N. S., Choudhary, R. Y., Dighade, S. J. (2022). Novel Ionic Liquid Based Microemulsion Formulation: A Potential Tool for Drug Delivery. International Journal for Pharmaceutical Research Scholars, 11(1);34 - 43.

INTRODUCTION

The existence of micro emulsions was first described by Schulman in (1943)and Winsor (1954) and the definition of micro emulsion described by Lindman is as “micro emulsion is a system of water oil an empty file which has a single optically isotropic and thermodynamically stable liquid solution”.^1-3 However, the history of discovery of Micro emulsion was well before the study of Schulman and the first commercial micro emulsion, liquid boxes was discovered in 1928 and later it gained significant attention in the oil industry.⁴ Nowadays it has potential application in separation science, environmental science ,materials science and reaction engineering with certain unique advantages.⁵

can be used as a Nano reactor to form a mono dispersed nanoparticles of drug delivery vehicle due to the fast exchange between the droplets in micro emulsions. ^6-7

showing serious adverse effects. This is a significant step forward in the delivery of poorly soluble drugs. Micro emulsion systems are also now being increasingly investigated for transdermal, ocular, nasal, pulmonary, vaginal, rectal and intravenous drug delivery. ⁸ Micro emulsions are generally classified as water in oil, oil in water or bi-continuous system depending on surfactant type, sample environment etc and they are characterized by ultra-low interfacial tension between oil and water phases. In water in oil in micro emulsion, the surfactant rich oil phase coexist with the surfactant pour acquiesce face and in oil in water micro emulsion, the surfactant rich water phase coexist with the oil phase where surfactant is only present as monomer at small concentration micro emulsion, which contain water or highly polar solvents like methanol(CHOH) acetone nitrile(ACN), dimethyl form amide (DMF) in the core, have been studied extensively.^9-16 The size of the micro emulsion is varied in the order of nanometer and it is generally characterized by the R/W value (R/W=[water or polar solvents]/[surfactants]). Recently, several attempts have been made for the preparation of micro emulsion where various polar solvents with high dielectric constant and which are miscible in nonpolar solvents are used instead of water in polar core.^17-19 These micro emulsions are important in their article as well as experimental point of view and recently they are widely applied to semiconductors, solar energy conversion, micro colloids etc.^20-21 Moreover these waterless micro emulsions have significant advantages over water-based micro emulsions in different organic reactions such as Diels -Alder reaction, esterification, polymerization etc.²² Recently room temperature ionic liquids received an increasing number of attention is because of their unique physio-chemical properties such as low volatility, high thermal stability and high ionic conductivity and the drawbacks associated with the non-aqueous solvent or organic solvents related to environment, health or safety can be conquered by using the room temperature ionic liquids.^23-28 More importantly, the cationic and anionic constituents of the room temperature ionic liquids can be modified to obtain the desired property of the solvent and therefore they are termed as designer solvent. For this they are frequently used in organic synthesis, catalyses, electrochemical studies and other chemical and technological applications. ^29-33 IL exist as liquid in room temperature because of their chemical structure. The cations and anions of the ILs are chosen in such a way that they destabilize the solid face Crystal and they are stabilized by the strong electrostatic interaction and other weak non-specific interaction between them.

At first, researchers were attempting to solubilize different ionic liquids replacing water from the core of the micro emulsion.^34-35 Later it was observed that IL can also play the role of organic solvent and from the green chemistry point of you it is very much promising for applications. Besides this water and oil can also solubilize in oil-based surfactant.

DEFINITION: Micro emulsion is defined “as system of water, oil, and amphiphile which is optically isotropic and thermodynamically stable liquid solution”.

IUPAC DEFINITION: Micro-emulsion- Dispersion made of water, oil, and surfactant(s) that is an isotropic and thermodynamically stable system with dispersed domain diameter varying approximately from 1 to 100 nm, usually 10 to 50 nm.

STRUCTURE OF MICROEMULSION:

Micro emulsions are dynamic systems in which the interface is continuously and spontaneously fluctuating.38 Structurally, they are divided into oil-in- water (o/w), water in-oil (w/o) and bi continuous micro emulsions. In w/o micro emulsion, water droplets are dispersed in the continuous oil phase while o/w micro emulsion is formed when oil droplets are dispersed in the continuous aqueous phase. In systems where the amounts of water and oil are similar, a bi continuous micro emulsion may result. In all three types of micro emulsions, the interface is stabilized by an appropriate combination of surfactants and/or co-surfactants. The mixture of oil, water and surfactants is able to form a wide variety of structures and phases depending upon the proportions of the components. The flexibility of the surfactant film is an important factor in this regard. A flexible surfactant film will enable the existence of several different structures like droplet like shapes, aggregates and bi continuous structures, and therefore broaden the range of micro emulsion existence. The micro emulsion structure is shown in Fig. 1.The schematic representation given in Fig. 2 gives an indication of a few of the wide variety of possible self-association structures that surfactants can form in the presence of water, oil or combinations of all three. The possible structural representations of the three different types of micro emulsions which are most probably likely to be formed depending on their individual composition. It can be seen that while the oil-in-water (o/w) and water-in-oil composition (w/o) droplets are represented as spheres, they may be also be asymmetric in shape, frequently looking like the shape of a prolate ellipsoid.

The likely presence of o/w micro emulsion droplets is a feature in micro emulsions where the volume fraction of oil is low. Conversely, w/o droplets are likely when the volume fraction of water is low and in systems where the amount of water and oil are similar, a bi continuous micro emulsion may result.

COMPONENTS OF MICROEMULSION

• Oil Phase
• Surfactant
• Co-surfactant

OIL PHASE: The oil represents one of the most important excipients in the formulation not only because it can solubilized the required dose of the lipophilic drug, it can increase the fraction of lipophilic drug transported via the intestinal lymphatic system, thereby increasing absorption from the GI tract depending on the molecular nature of the triglyceride.³⁹ The oil component influences curvature by its ability to penetrate and hence swell the tail group region of the surfactant monolayer. Short chain oils penetrate the tail group region to a greater extent than long chain alkanes, and hence swell this region to a greater extent, resulting in increased negative curvature. Saturated (for example, lauric, myristic and capric acid) and unsaturated fatty acids (for example, oleic acid, linoleic acid and linolenic acid) have penetration enhancing property of their own and they have been studied since a long time.

SURFACTANTS: The surfactant chosen must be able to lower the interfacial tension to a very small value which facilitates dispersion process during the preparation of the microemulsion and provide a flexible film that can readily deform around the droplets and be of the appropriate lipophilic character to provide the correct curvature at the interfacial region.⁴⁰ Surfactants used to stabilize microemulsion system may be: i.Non-ionic, ii. Zwitterionic, iii. Cationic, or iv. Anionic surfactants. It is generally accepted that low HLB (3-6) surfactants are favored for the formulation of w/o microemulsion, whereas surfactants with high HLB (8-18) are preferred for the formation of o/w microemulsion. Surfactants having HLB >20 often require the presence of co-surfactants to reduce their effective HLB to a value within the range required for microemulsion formation.

CO-SURFACTANT: In most cases, single-chain surfactants alone are unable to reduce the o/w interfacial tension sufficiently to enable a microemulsion to form. The presence of co-surfactants allows the interfacial film sufficient flexibility to take up different curvatures required to form microemulsion over a wide range of composition. If a single surfactant film is desired, the lipophilic chains of the surfactant should be sufficiently short, or contain fluidizing groups (e.g. unsaturated bonds). Short to medium chain length alcohols (C3-C8) are commonly added as co surfactants which further reduce the interfacial tension and increase the fluidity of the interface. Typical co-surfactants are short chain alcohols (ethanol to butanol), glycols such as propylene glycol (PG), medium chain alcohols, amines or acids.

FACTORS AFFECTING THE FORMATION OF MICROEMULSION: The formation of oil or water swollen micro emulsion depends on the packing ratio, property of surfactant, oil phase, temperature, the chain length, type and nature of co-surfactant.^41-42

METHOD OF PREPARATION OF MICROEMULSION:

 4.1.      PHASE TITRATION METHOD: Microemulsions are prepared by spontaneous emulsification method (phase titration method) and can be depicted with the help of phase diagrams. Construction of phase diagram is a useful approach to study the complex series of interactions that can occur when different components are mixed. Microemulsions are formed along with various association structures (including emulsion, micelles, lamellar, hexagonal, cubic, and various gels and oily dispersion) depending on the chemical composition and concentration of each component. The understanding of their phase equilibria and demarcation of the phase boundaries are essential aspects of the study. As quaternary phase diagram (four component system) is time consuming and difficult to interpret, pseudo ternary phase diagram is often constructed to find the different zones including microemulsion zone, in which each corner of the diagram represents 100% of the particular component.

PHASE INVERSION METHOD: Phase inversion of microemulsions occurs upon addition of excess of the dispersed phase or in response to temperature. During phase inversion drastic physical changes occur including changes in particle size that can affect drug release both in vivo and in vitro. These methods make use of changing the spontaneous curvature of the surfactant. For non-ionic surfactants, this can be achieved by changing the temperature of the system, forcing a transition from an o/w microemulsion at low temperatures to a w/o microemulsion at higher temperatures (transitional phase inversion).

5.         CONSTRUCTION OF PSEUDO TERNARY PHASE DIAGRAM:

When water, oil and surfactants are mixed, microemulsion is only one of the association structures. Preparation of a stable, isotropic homogeneous, transparent, non-toxic microemulsion requires consideration of a number of variables. Construction of phase diagrams reduces a number of trials and labor. Phase diagrams help to find the microemulsion region in ternary or quaternary system and also help to determine the minimum amount of surfactant for microemulsion formation. Pseudo-ternary phase diagrams of oil, water, and co- surfactant/ surfactants mixtures are constructed at fixed cosurfactant/surfactant weight ratios. Phase diagrams are obtained by mixing of the ingredients, which shall be pre-weighed into glass vials and titrated with water and stirred well at room temperature. Formation of monophasic/biphasic system is confirmed by visual inspection. In case turbidity appears followed by a phase separation, the samples shall be considered as biphasic. In case monophasic, clear and transparent mixtures are visualized after stirring the samples shall be marked as points in the phase diagram. The area covered by these points is considered as the microemulsion region of existence. Fig 3. shows hypothetical pseudo- ternary diagram at constant surfactant to co-surfactant ratio. It also shows that single phase or multiphase regions of microemulsion domains are near the center of diagram in areas containing large amounts of surfactant that is toxic. The phase behavior of surfactants, which form microemulsion in absence of co-surfactant, can be completely represented by ternary diagram.⁴³

TYPES OF MICROEMULSION SYSTEMS: According to Winsor there are 4 types:

WINSOR I: The microemulsion composition corresponding to Winsor I is characterized by two phase, the lower oil/water (O/W) microemulsion phase in equilibrium with excess oil.

WINSOR II: The microemulsion composition corresponding to Winsor II is characterized by very low interfacial tension and maximal solubilization of oil and water for a given quantity of surfactant. Since, in this phase, microemulsion coexists with both excess phases, no one can distinguish the dispersed phase from the continuous phase.

WINSOR III: This phase comprises of three phases, middle microemulsion phase (O/W plus W/ O, called bicontinuous) in equilibrium with upper excess oil and lower water.

WINSOR IV: Microemulsions can be distinguished from the micelles by its inner core swollen with oil. The microemulsion structure depends on the chemical composition, temperature and concentration of the constituents.

APPLICATIONS OF MICROEMULSIONS:

It can be used as a delivery system by the various routes as below:

1. Oral Delivery: Eg. – Piroxicam
2. Parenteral Delivery :Eg. -Flurbiprofen
3. Topical Delivery: Eg. -Dexamethasone
4. Ophthalmic Delivery: Eg. -Chloramphenicol
5. Nasal Delivery: Eg. – Sumatriptan
6. Drug Targeting
7. Brain Targeting
8. Periodontal Delivery
9. Cellular Delivery

ADVANTAGES OF MICROEMULSION:

1. Microemulsion acts as super solvent for drug.
2. Ability to carry both hydrophilic and lipophilic drug.
3. Due to small droplet size it has large interfacial area of globule so drug is rapidly released in external phase when absorption takes place.

DISADVANTAGES OF MICROEMULSIONS:

 Require large amount of surfactant and co-surfactant.

1. Limited solubility for high melting substances.
2. Stability influenced by environmental parameters such as temperature and pH

DIFFERENT ROLE OF IL IN THE

FORMULATION OF MICROEMULSION

Based on the role of ILs, microemulsions can be classified into three categories:

• Non aqueous IL microemulsions: The polar domain of the microemulsion constitutes the IL.
• Aqueous IL microemulsion: The nonpolar part constituents the IL and water is used in polar domain.
• Microemulsions with IL as surfactants: Different Surface Active Ionic Liquids (SAIL) are used as surfactant and in the polar domain aqueous as well as non aqueous solvents including IL are used.

EVALUATION OF MICROEMULSION:

CONCLUSION

The use of microemulsions as drug delivery vehicle has been an exciting and attractive area of research because of its any potential and extraordinary benefits. In microemulsions, one can design the interface of such nanometer sized droplets so that droplet stability and lifespan in humans can be made to last from a few milliseconds to minutes, or even to hours. The interfacial rigidity of the microemulsion droplets plays a key role in the flux of the drugs from such droplets to the cells and Tailoring of microemulsion systems to control the flux of the drugs can be done so as to customize drug delivery according to individual patient requirements or to specific pharmaceutical needs. Microemulsions offer an interesting and potentially quite powerful alternative carrier system for drug delivery because of their high solubilization capacity, transparency, thermodynamic stability, ease of preparation, and high diffusion and absorption rates when compared to solvent without the surfactant system. In last few decades ionic liquids (ILs) have been widely considered as a green solvent and they are used in various fields. ILs can be used in the formation of micro emulsion as a dispersed medium, polar domain and recently as a surfactant. ILs with a certain surface activity having long alcohol chain substitutes can self-aggregate and form Ionic Liquid Micro Emulsion with high-temperature insensitivity.

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Abstract

Immune system is the organization of different oragans and cells in human body. It's function is to protect and to fight against the host from any non-self particles like viruses, microbes, fungi, parasites, etc. Our body requires vast variety of micro (i.e. vitamins and minerals) as well macro(carbohydrates, proteins, fats) nutrients  synthesize different  kinds of immune cells. Viral disease like the  Covid-19 can be prevented by a strong immune system. In terms of Covid-19 and it's origin, transmission, clinical aspects and diagnosis. However here, we have formulated the novel concept hitherto ancient means of traditional medicines or herbal plants to beat this pandemic. In this paper we studied the literature on the immune supportive properties, finding revealed that a variety of natural herbs like Spirulina, Tulsi, Neem, Triphala, Garlic, Clove, Turmeric, Ginger, Black pepper, etc. are some of proven ancient herbs that enhance the immunity.

Keywords

Potential, immunity, mechanism, natural herbs and immunity enhancer, COVID-19  

Cite This Article

Digambar, S. S., Prabhakar, N. Y., Bhandari, A. S., Shantling, S. A., Shinde, R. R. (2021). Potential Natural Immunity Enhancers Against Covid-19 Pandemic. International Journal for Pharmaceutical Research Scholars, 11(1);01 -33.

INTRODUCTION

The world experienced coronavirus for the first time in 2002–2003 through severe acute respiratory syndrome (SARS), and in 2011, Middle East Respiratory Syndrome (MERS) for the first time. The causative agents for both cases (SARS-CoV and MERS-CoV) were newly identified coronaviruses of zoonotic origin in the genus Beta-coronavirus [1]. The present coronavirus (SARS-CoV-2) Covid-19 appeared for the first time in Wuhan, China, at

the end of 2019. People are being affected by human-to-human transmission due to close contact [2,3], and people affected by Covid-19 suffer from severe respiratory illness [4].

Nearly, 213 countries of all continents have been affected in less than three months by this pernicious virus. After studying its clinical characteristics, experts affirmed that it is quite similar to pneumonia and therefore, named as Novel Coronavirus. However, in the second week of March, 2020, Covid-19 was stated as the  pandemic by World Health Organization(WHO) [5]. In essence, it has been known to transmit through droplets such assaliva or nose or even through air-borne transmission [6]. SARS-CoV-2 infection is often categorized into three stages: first, asymptomatic phase; second, non-severe symptomatic phase; and third, severe respiratory symptomatic phase. Usually, a small number of patient’s progress to the severe stage and develop ARDS and/or multiorgan failure. SAR-COV-2 include fever, cough, fatigue and sputum production. Symptoms shortness of breath, myalgia, headache this are common it includes gastrointestinal symptoms such as vomiting, diarrhoea, anosmia.

As no specific therapy is available for SARS-CoV-2, the proposed therapy is based on the previous experience from SARS or Middle East Respiratory Syndrome (MERS) coronavirus. These therapeutic molecules, for example viral methyltransferase inhibitors, nitazoxanide, protease inhibitors (such as lopinavir/ritonavir), interferon, therapeutic peptides, RNA synthesis inhibitors (such as ribavirin, flavirapivir, and remdesivir), anti-inflammatory drugs, [7] but the remdisivir had many side effects such as hepatotoxicity, respiratory toxicity, cardiovascular toxicity, nephrotoxicity, reproductive toxicity, [8,9] so it’s risky to treat.

At this time, prevention is more necessary than cure, in this pandemic immunity plays a key role by[10]

• Washing hands regularly for 20 seconds with soap and water or alcohol-based hand rub. • Cover nose and mouth when sneeze or cough with a disposable tissue.
• Avoiding close contact with people who are sick. • Stay at home and self-isolate from others if feel unwell.

People in certain previous sicknesses like diabetes, hypertension, cardio vascular disease and respiratory issues are at a higher danger of having Covid-19 entanglements, it likewise with age as the immunity decreases as you get older. In the younger age with no prolonged severe disease, Covid-19 can bring about minor symptoms on the individuals who have stronger immunity [10].

Mechanism of immunity against Covid-19 :-

The immune system is the best defense because it supports the body’s natural ability to defend against pathogens (eg. Viruses, bacteria, fungi, protozoan and worms) [11,12] and resists infection. As long as the immune system is functioning normally, infections such as Covid-19 go unnoticed. There are three type of immune system innate immunity (rapid response), adaptive immunity (slow response) and passive immunity. There are two types of passive immunity natural immunity, this recieve from maternal side, and artificial immunity recieve from medicine. The innate, passive and adaptive immune response could be triggered in response to the SARS-COV-2 infection. Blood sample of sympathomimetic hospitalised patients with mild to moderate SARS-COV-2 infection, immunological changes such as increase the number of activated CD4+ helper T cell and CD4+ killer cells, follicular helper T cells, IgE, IgM were detected. No registered the medicine or vaccine against Covid-19 immune system is the best defense because it support the body’s natural immunity to defence against the pathogens [11,12], begins the inflammatory response of skin when body is affected [13,14]. Our immune system is essential for our survival. Without immune system, our body would be open to attack from bacteria, viruses, parasites and other microbes. It is immune system that keep us healthy as we drift through a sea of pathogens. The immune system is spread throughout the body and involves by many type of cells, organs, protien and tissues. It can distinguish our tissue from foreign tissue i.e., form non self. Dead and faulty cells are also recognised and cleared away by the immune system. If the immune system is encounter a pathogens, for instance, a bacterium, virus or parasite, it means so called immune response. An antigen is any substance that can spark an immune response. Once B lymphocytes spot the antigens. Antibodies are special protein that lock on specific antigen. Antibodies are part of a large family of chemical called immunoglobulins, which play many roles in the immune response: IgG, IgM, IgD, IgA, IgE [13,14]. But when the immune system response is low, weak open the invitation for infection, overall gut microbiom health which makes up to 85% of the body’s immune system. Patients of coronavirus must have plenty of water that will keep the mucous membrane moist which can lower the chances of cold and flu [15]. Although drinking water does not ensure that you not contract the coronavirus, remaining hydrated can improve your health and make sure the immune system can defect the virus. The drinking water is work to help your cells to oxygenated. All cells of our body compete at their best if they get enough oxygen that helps them protect the body from any infections agent that enter, if they do fight against them(16).

Medicinal herbs are ‘Gifted Gods’ for supporting, healing and rehabitating the patients. No any type of substantiation is present, but different studies on herbal plant are that have the ability enhance, boost or strengthen the immune system. Some phytocompounds are being recognised the characterised the herbs in mitigating the incidence of various type of infections. There are various type of traditional medicinal plants- Ayurveda, Unani, Siddhi, Homeopathy Romanian, Persian, Chinese. Examples of traditional medicinal plant currently check the effectiveness this virus [17,18]. Various type of herbal plant utilized conventual medicine and in aboriginal health services to combat disease. Herbal medicine enhance immune system increasing potential against Covid. More than 25,000 herbal formulations used In folk remedies in Ayurveda alone. About plant sources and their usage is chiefly indispensable employe it under right condition [19, 20]. Medicinal herbs are life saving drugs. These days are research is being conducted on them and promote usage in treatment of Covid-19 patients due to their potential possessing anti-inflammatory, antioxidant and antiviral property. It is necessary to maintain the hygiene sorrounding [21]. There are various type of traditional herb medicine Tulsi, Amla, Neem, Bhringraj, Triphala, Turmeric, Ginger, Aloe, Ashwagandha, Peppermint, Eucalyptus, Green tea, Night flowering Jasmin, Ginsang, Gulvel, Clove. They help for the increase or enhance the immunity and our body potential. Various active constituents help to enhance the potential. example garlic, ginsang they participate in cytokinin secretion modulation. Above traditional herb drive have their own immunomodulatory action. All herbal medicines have less side effects. Various type of traditional herb and food play important role in boost the potential and immunity [22].

Various herbal plants which enhance the immunity

1. Spirulina

Introduction-

Botanical name is Spirulina platensis or Spirulina maxima belongs to family oscillatoriaceae. Spirulina is a high quality source of pigments, minerals and vitamins. The beneficial effect of Spirulina as a nutritional and dietary supplement. Several research studies recommended that Spirulina can be a potential alternate therapy against virus diseases due to the possible synergistic effect of many bioactive compounds present in the whole cell. It has major beneficial activity i.e.,immunomodulatory and antioxidants. Spirulina has the potential to enhance immune components and reduce physiobiochemical stress, and therefore could be used as a supplement along with treatments or prevent Covid-19 infection and related symptoms[23,24].

History

The term Spirulina remains in use is because of historical reasons (Vonshak, 1997). In sixteenth century, S.platensis was first isolated from Lake Texcoco by the Aztecs and they devised the term “tecuitlatl” for Spirulina (Habib M. et.al., 2011). It has a long history of use as food and it has been reported that it has been used during the Aztec civilization[25].In America, spirulina is sold in health food stores as a powder or tablet. In Russia, it has been approved to treat symptoms of radiation sickness, because the carotenoids it contains absorb radiation[26].The first documented report on spirulina dates back to the 16th century and spirulina is believed to have been a nutritional source for the Aztecs and Mesoamericans[27].

Chemical Constitute-

Spirulina is a protein-rich food product(approximately 55-70% dry weight), with a relatively low carbohydrate content of around 15% dry weight. It also contains several trace of  minerals, vitamins and pro- and pseudo- vitamins. It contains phycocyanin- containing phycobiloproteins which are active ingredient[28,30].

How to administer-

One tablespoon, which is around gram of dried spirulina powder contains protein (4 grams), Vitamin B1 (11% RDA), Vitamin B2 (15% RDA), Vitamin B3 (4% RDA), Copper (21% RDA) and Iron (11% RDA). 7 grams of powdered spirulina has around 20 calories and 1.7 grams of digestible carbs.

Mechanism of action-

The aqueous extract of spirulina was found to have a major impact on the immune system by increasing the phagocytic activity of macrophages, stimulating the NK cells[28,29]. For decades, users have anecdotally reported a decrease in colds and flu from spirulina use. Several pre-clinical animal studies have shown good immunostimulatory effects in a variety of species. Extracts from spirulina biomass have also been found active against herpes virus, cytomegalovirus, influenza virus, etc. Spirulina extracts have also been shown capable of inhibiting carcinogenesis[30,31].

Role against Covid 19-

Spirulina (LED Spirulina), at a concentration of 0.1 μg/mL, decreases macrophage and monocyte-induced TNF-a secretion levels by over 70% and 40% respectively. The administration of spirulina could enhance nonspecific preventive measures, such as the activation of CD4+ cells, which further enhance the production of IFN-y in humans, for the prevention of viral infections[32,33].

Pharmacological action of phytocostituent-

Therapeutic uses-

In diabetes mellitus, anticancer properties, in radioprotective, antiviral properties, immunomodulatory properties, antioxidants, also as cardiovascular benefits[32,35,36].

Antiviral property-

The researchers concluded that aqueous spirulina platensis extracts contain antiretroviral activity that may be of potential clinical interest. Calcium Spirulana inhibited the replication of enveloped viruses such as Herpes simple type 1, human cytomegalovirus, measles, mumps, influenza A and HIV-1[12]. Calcium was seen to play an essential role in a dose-dependent manner for inhibiting the cytopathic role of such viruses.[23] In addition, in undernourished children spirulina has been found to improve weight gain and correct anemia in both HIV-infected and HIV-negative cases[35,36].

Side effects-

Although few adverse effects are associated with the use of spirulina, consuming spirulina may cause headaches, allergic reactions, muscle pain, sweating, and insomnia in some cases. People with allergies to seafood, seaweed, and other sea vegetables should avoid spirulina.

2. Tulsi

Introduction-

Botanical name Ocimum sanctum belongs to family lamiaceae. Tulsi commonly known as holy basil. It has been used for treatment of wide range variety of ailments in many parts of world. Tulsi tea or kadha is commonly used for relieving bronchitis and asthma. It is an essential ingredient in preparation of ayurvedic cough syrup[37]. Leaves of Ocimum sanctum contains water soluble phenolic compounds and various constitute such as eugenol, methyl eugenol, caryophyllene that may acts as immunostimulant. Ayurveda consider tulsi as one of most enriching herbs and ‘queen of herbs’ and reverse as an ‘elixir of life’ that is equal for both medicinal and spiritual properties[38].

History-

Tulsi has been used for thousands of years in Ayurveda, a Hindu form of medicinal science for its diverse healing properties. It is mentioned in Charaka, Samhita and ancient Ayurvedic text. If one make a paste of tulsi leaves and smears it over his body and worship Vishnu, it is worth several ordinary Pujas and Lakhs of Godan (Donation of cows)[39].

Chemical constituents-

It contains volatile oils. The oils contain about 70% eugenol, 20% methyl eugenol, beta caryophyllene, carvacol, cineole, linalol. Active ingredients are eugenol, thymol, beta caryophyllene, rosamarinic acid and carvacol[40].

Extract use-

-Tulsi is rich in vitamin C and zinc. It act as natural immunity booster and keeps infection at away. It is abundant in antioxidants and micronutrients that provides powerful immune protection from free radical damage and increase body capacity to fight against disease and infection[41,42].

How to administer-

It can be administered in different way. To prepare kadha there is a method of preparation is given:- 10-15 tulsi leaves, 1 inch ginger, 1 inch raw turmeric, 4 stick mulethi, 10 black peppercorn, 10 cloves, 3-4 cinnamon sticks, 8 cups of water. Pour water in deep pan and add above ingredients. Boil water for a hour in low medium flame. After a hour, switch off stove, allow it for cooling and take it for boosting immune system[41,46].

Mechanism of action-

Tulsi leaves are natures best antibiotics. Chewing tulsi leaves purifies blood and help several coomon elements. Kadha of tulsi useful to boost immunity. It is best home remedies to boost immunity by Ministry of health GOI [43].

Role against Covid-19

Tulsi extremely useful for treating bacterial and fungal infection as well as immunological disorders like allergies and asthma. Tulsi has natural essential oils like camphene, cinolene and eugenol which will reduce cold congestion in chest. It contain bioflavonoids and antioxidants compound such as Rosamarinic acid which is good for antimicrobial agents for treating infection in the respiratory tract. Tulsi leaves extract increase the T-helper cells and natural killer cells activity, boosting immune system[44].

Pharmacological action of phytocostituent-

Therapeutic uses-

In bronchitis asthma, anticancer activity, anti-oxidant, antidiabetic, antimicrobial, immunomodulatory, anti-inflammatory, antistress activity, hepatoprotective, analgesic, antiarthritic, radioprotective, anti-aging effect[37,43].

Antiviral property-

All extract of Ocimum sanctum (crude extract, terpenoid and polyphenols) shows significant virucidal activity. Depending upon type of extract, the antiviral activity of Ocimum sanctum has been assessed against many important viral agent as fish pathogenic viruses viz., Infectious hematopoietic necrosis viruse (IHNV), Herpes virus (HSV), Adenoviruses, etc[44,45,46,47].

Side effects-

Not suitable in pregnant women, may not good for diabetics patients, Interfere with blood thinning medicine, may stain your teeth[48].

3. Neem

Introduction-

Botanical name Azadirachta indica belong to family meliaceae. Neem is one of the most useful traditional medicinal plant in India. Azadirachta indica is fast growing, evergreen tree and it is native in India, America and Africa. As Covid-19 is responsible for severe Cytokine Storm induced complications and coagulopathies[49]. The neem can be useful as a Single Silver Bullet in Covid-19 in both prophylactic and curative aspects and also useful in post Covid complications. Neem has called the wish fulfilling tree and pinchumada or destroyer of leprosy.

History-

The Vedas called Neem as “SARVA ROGA NIVARINI” which means, one that cures all ailments and ills. This tree considered to be divine origin. The ancient Indian found many therapeutic uses of tree and also observed that tree could survive and grow almost anywhere as long as it warm and dry. Ayurvedic Text described neem tree by associating its remarkable healing properties from as for back as 5000 BC[50].

Chemical constituents-

Leaf- Quercetin, nimbosterol, nimbin

Flower- Nimbosterol, kaempferol, melicitrin

Bark- Nimbin, nimbidin, nimbosterol, margosine

Seed- Azadirachitin, azadiradione, nimbin, vepinin, vilasinin, fraxinellone[51,52].

Extract use-

According to Exam research, an aqueous plant extract from Azadirachta indica and it’s chromatographic fraction F1(neem extract) exerted immunomodulating of antimetastatic activities in BALB. Neem extract can be regarded immunomodulating and antimetastatic substances which holds promise for further experimental and clinical condition[53].

How to administer-

Take Some neem leaves and soaked in water for 5 minutes. Put these soaked neem leaves in grinder. Then add some water, lemon and sugar. Churn it well. This drink will freshen and cleanse your body.

Mechanism of action-

It helps to boost your immune system by cooling your body internally. It also purifies blood. It has proinflammatory, cytokine inhibitor and immunomodulator effects[54].

Role against Covid-19

It was shown that 20+ compounds in neem leaves show high inhibition against Covid-19. The main protease (6 LU7) with value ranging- 14.3Kcal/mol to a minimum of -9.1Kcal/mol and in addition to compound there are other components from neem leaves which exhibit minimum binding affinity with Covid-19 protease(6LU7). Research suggest ethanol extract of neem leaves show in vitro antibacterial activity against Staphylococcus aureus and MRSA[54].

Pharmacological action of phytocostituent-

Antiviral properties-

The evidence suggested that presence of a battery of compound beside flavonoids, triterpenoids and their glycoside in NCL-11 have antiviral action for Coxsackie B group of in vitro. Neem leaves extract powder/crude leaves contents might inhibit Covid-19 virus by prevent from replicating[55,56].

Side effects-

Vommiting, diarrhoea, drowsiness, blood disorders and contraindicated during pregnancy [54].

4. Triphala

Introduction-

Emblica officinalis (Family euphorbiaceae), Terminalia bellerica (Family combretaceae), and Terminalia chebula (Family combretaceae). It is one of important rasayanas in Ayurveda. It is used in traditional Indian system of medicine. It is three fruits together so called Triphala. Its synonyms are Vara, Phalatrikam and Sresthatmam. The three ingredients are Amalaki (Phyllanthus emblica), Bibhitaki (Terminalia bellirica), and Haritaki (Terminalia chebula). It contains Vitamin C[57]. The immunomodulatory activity of Amalaki, Haritaki and Bibhitaki was proved by experimental study so that it would be used in various ayurvedic formulation. It is widely used in many disorders due to its various pharmacological activities. It is natural remedy for a variety of health condition. The relationship between the pre- and post-symptoms of COVID-19 and the therapeutic activity of ‘Triphala’ gives us a ray of hope to use Triphala as an anti-corona therapeutic supplement during the pandemic as well as in near future[58].

History-

Triphala is used in Ayurveda over 2000 years. Reference to the use of Triphala can be found in the Sushruta Samhita, which is dated to 1500BC. As both Ayurveda and western medicine agree that health and disease begin in the gut, Triphala represents an essential foundational formula as it promotes efficient digestion, absorption, elimination and rejuvenation. According to Charak,  taking Triphala Rasayana (Triphala with honey and ghee) daily has potential to make a person live for one hundred years devoid of old age and diseases. The ‘Triphala’ have been acting as ‘one formula therapy’ since the time of the Ayurveda, and the Covid-19 is not an exception[59].

Chemical constituents-

It contains major four chemical constitutes such as galic acid, tannic acid, syringic acid and epicatechin along with ascorbic acid. The composition of triphala is rich in various antioxidants such as ascorbic acid, ellagic, gallic as well as chebulinic acid and several classes of flavonoids like (querecetin, luteolin), saponin, anthraquinone, amino acid, fatty acids and various carbohydrates[57,59].

Extract use-

Administration of the fruits/methanol extract of the leaves/compounds isolated from the fruits showed protective effects against cognitive deficits, biochemical abnormalities, apoptosis induced by aluminum chloride, and tau hyperphosphorylation cadmium-induced neurotoxicity in mice CCl 4-induced oxidative injuries and tissue damage of lungs of rats, peroxide-induced injury in PC12 cells and chemical-induced liver injuries in several animal models. The fruit extract reversed the immunosuppressive effect of Cr (VI), enhanced white blood cell count an  % lymphocyte distribution in mice, and also activated macrophages and the isolated compounds, geraniin and isocorilagin, stimulated splenocyte proliferation[60,61].

How to administer-

Triphala churna is more effective than using the individual herb’s to boost immunity and improve overall health.

Triphala is available in the form of churna (powder), rasa (juice), tablet and capsule.

Triphala churna: Take 1/2–1 teaspoon of triphala churna/powder with honey twice a day after meals. Use it at least for 1-2 months for effective results.

Triphala tablet: You can take 1-2 tablets with water after meal to reap its benefits.

Triphala capsule: To boost immunity take one capsule each after lunch and dinner with water.

Triphala juice: Take 15–20 ml of triphala juice in a glass and add equal amount of water to dilute it and have it on an empty stomach[60].

Role against Covid-19

The role of Triphala and its extract has been emphasized in stimulating neutrophil function. Under stress condition such as noise, Triphala significantly prevents elevation of IL-4 levels as well as corrects decreased IL-2 and IFN-γ levels. Under the condition of inflammatory stress its immunosuppressive activity is attributed to its inhibitory action on complement system, humoral immunity, cell mediated immunity and mitogen-induced T-lymphocyte proliferation. The aqueous and alcoholic extracts of the individual constituents reportedly enhance especially the macrophage activation due to their free radical scavenging activity and the ability to neutralize reactive oxygen species. This study thus concludes the use of Triphala and its three individual constituents as potential immunostimulants and/or immunosuppressants further suggests them to be a better alternative for allopathic immunomodulators[61,62].

Pharmacological action of phytocostituent-

Therapeutic uses-

Hypercholesteramic effect, Antiinfammatory effects, Gastrointestinal effect, Strees reducing effects, Antiobesogenic effect, Antidiabetic effects, Antineoplastic effect, Immunomodulating effect, Analgesic effect, Bronchodilator effect[59,60].

Antiviral property-

THL can play an antiviral role by regulating immunity. As a potential immune stimulant and/or immunosuppressant, it can significantly prevent the increase in interleukin-4 (IL-4), increase the decrease in interleukin-2 (IL-2) and interferon γ (IFN-γ) levels, and inhibit cellular immunity, mitogeen-induced T lymphocyte proliferation and humoral immunity under inflammatory stress[62].

Side effects- Mild laxative, depending on preparation used, side effects like these may occur with even smaller doses, triphala might interact with others medication, it cause gastrointestinal side effects.

5. Clove:-

Introduction-

Botanical name Syzygium gromaticum belongs to family myrtaceae [63]. That grows in tropical climates and has been widely used in Ayurveda and Chinese traditional medicines for over 2000 years. Cloves are currently used in three different forms, as whole dried buds (commonly referred to as “cloves”), ground spice, and essential oil. Though all forms share similar biomedically relevant properties, they differ in the degree of potency, with the oil showing the highest potency and thus, often being dilute CBC, the spice generally losses most of the essential oil [64].

History-

Clove is an ancient spice, which is believed to be originated in the first century, before Christ. The first clue Hussain et al., 2017A.D. The origin and source of clove was a mystery, until the discovery of Indonesia or Moluccas Island, by Portuguese, in 16th century. In 17th and 18th century in India East India Company introduced clove in 1800A.D.

Chemical constituent-

Clove buds contain 15–20% essential oil, which is dominated by eugenol (70–85%), eugenyl acetate (15%) and β-caryophyllene (5–12%). Other essential oil ingredients of clove oil are vanillin, crategolic acid, tannins, gallotannic acid, methyl salicylate, flavonoids eugenin, kaempferol, rhamnetin, eugenitin and triterpenoids like oleanolic acid. The constituents of the oil also include methyl amyl ketone, methyl salicylate, α and β-humulene, benzaldehyde, β-ylangene and chavicol. The minor constituents like methyl amylketone, methyl salicylate etc., are responsible for the characteristic pleasant odour of cloves. Gopalakrishnan et al. (1984) characterized six sesquiterpenes, namely: α-cubebene (1.3%), α-copaene (0.4%), β-humulene (9.1%), β-caryophyllene (64.5%), γ-cadinene (2.6%) and δ-cadinene (2.6%) in the hydrocarbonfraction of the freshly distilled Indian clove bud oil [65].

Extract-

Clove extract and clove oil may increase the production of gastric mucous and help protect against stomach ulcers.

How to administer-

Hot clove tea is common way to use cloves for respiratory disorders like coughs, colds, asthma, bronchitis, and sinusitis [64]. To chew cloves for treating soreness of throat and inflammation of the pharynx [64]. In mixtures with honey, it helps in the case of chronic coughs and is mentioned to be specifically useful in the case of shortness of breath [65].

Mechanism of action-

Clove extract and clove oil may increase the production of gastric mucous and help protect against stomach ulcers. Clove extract could suppress the T- cell cellular immunity and enhance humoral immune response. In clove affection cytokine pattern shifted toward modulatory and Th2 response and accelerator for humoral immunity cytokine.

Role against Covid-19

The traditional therapeutic use of clove in respiratory disorders and its activity against different types of viruses, alongside its anti-inflammatory, immunostimulatory, and antithrombotic properties, are all attractive features highlighting its potential in the fight against the Covid-19 disease. To prevent and control the SARS-CoV-2 associated disease, together with Eucalyptus globulus, Cymbopogon citratus, Zingiber officinale, and other plants endowed with the advantage of being inexpensive and abundantly available around the globe [66]. More than 93% of the interviewed Indian people believed that spices are helpful in curing Covid-19 or other viral infections and can help in boosting the immunity[67].

Pharmacological action of phytoconstituent-

Therapeutic use

Anesthetic, antimicrobial, antiviral, antifungal, antioxidant, antimutagenic, antithrombotic, anti-inflammatory, antiseptic, gum infections and burns, respiratory and digestive disorder, anticancer, antiparasite [68-73].

Antiviral property

Eugenol being the major constituent of cloves, was  investigated for its antiviral activity by several research groups. The above-mentioned Tragoolpua and Jatisatienr [74] used pure eugenol as the reference compound in their anti-HSV studies and found that it exerted a higher antiviral activity than the ethanol extracts of whole clove buds. Similar finding where obtained by benencia and courregescourreges[75]. Same study, eugenol was virucidal, whilst no compound-associated cytotoxicity was revealed at the concentrations tested [76]. Eugenol also showed antiviral activity against the influenza A virus (IAV), being able to inhibit IAV replication [77]. Finally, it was also found active as an inhibitor of the Ebola Virus in vitro [78].

Side effects-

Lactic acidosis, muscle pain, nausea, upper stomach pain, dizziness, fever sore throat, jaundice, erection problems, itching, rash, mild skin irretation.

6. Garlic:-

Introduction-

Garlic has the botanical name Allium sativum belongs to family lillaceae. Garlic contains numerous compounds that have the potential to influence immunity [79,80]. In recent reports, garlic and its complex constituents have been investigated as promising candidates for improving immune system. Allium sativum seems to counteract most of the negativities caused by Covid-19 infection. The administration of this plant will contribute to the immune system elements during the fight against this pathogen. This functional food may contribute to the prevention and treatment of pathologies such as obesity, metabolic syndrome, cardiovascular disorders, gastric ulcer, and even cancer [81,82].

History-

Historically, it is believed that Louis Pasteur described the antibacterial effect of garlic in 1858 for the first time, although no reference is available. More recently, garlic has been proven to be effective against a plethora of gram-positive, gram-negative, and acid-fast bacteria. garlic extract showed in vitro activity against influenza A and B [83], cytomegalovirus [84], rhinovirus, HIV, herpes simplex virus 1 [85], herpes simplex virus 2 [86], viral pneumonia and rotavirus.

Chemical constituents-

Allicin- active against malaria parasites, cytomegalovirus, protozoan parasite [79,87].

Alitridin- fight against cytomegalovirus [87]

Allin (s-allyl cystein sulfoxide) [79]

Diallyl sulfide (DAS) , E/Z ajoene, S- allyl cysteine (SAC) [79].

Extract-

Age garlic extract might be used as a herbal medicine against Covid-19. Aged garlic extract suppresses the production of proinflammatory cytokines such as TNF-α and CRP in the liver [88]. In the hypothalamus, aged black garlic (ABG) treatment induced a decrease in leptin receptor (LepR) mRNA levels.

How to administration-

According to megha krishna, clinical Ntritionalist- the main ingredient of garlic which fight against germ cells is allicin best way to use as an immune booster eat it raw. Chewing garlic releasing the alicin in mouth which is absorbed by body, but when it taken with food or in the form of pills it’s effectiveness is very less.

Mechanism of action

Garlic participates in cytokine secretion modulation, which may provide a mechanism of action for many of its therapeutic effects. Alliin is the main organosulfur compound in garlic and has been shown to induce a decrease in the expression of proinflammatory cytokines[81,82]. The increasing hemoglobin production and increasing hemoglobin availability for oxygen binding [89]. It is also hypothesized that patients with severe Covid-19 infection. It stimulates macrophages, lymphocytes, NK cells, DC and eosinophils, by mechanisms including modulation of cytokine secretion [90].

Role against Covid-19

1. Prevention and treatment of obesity.
2. To reserve some sign and symptoms observed in Covid patients.
3. Reincraese or regain the decrease or lost gustatory sence.
4. Increase the number of T- cells [89,90].
5. To increase cytotoxic and helper T- cells [89,90].
6. Decrease the level of leptin and increase appetite [89,90].
7. To decrease interleukin-6 concentration [89,90].
8. Stimulate NK cells [89,90].
9. Prevent this viral agent from spreading all over the body.
10. Suppress TNT-alpha and c- reactive protein [89,90].

Pharmacological action of phytoconstituent-

Antiviral property-

Garlic and it’s sulphur constituents verified antiviral activity against coxsakie virus species, herpes, simplex type 1 and 2, influenza B, parainfluenza virus type-2 vaccinia virus, rhinovirus type 2, immunodeficiency type.

Side effects-

Have few side effects as compared to chemotherapy in treating cancers caused by substances like aflatoxin B1 [91].

7. Turmeric:

Introduction:

Turmeric (Curcuma longa) belonging to the family zingiberaceae. Medicinal plants have provided a reliable source of preparation of new drug as well as combating diseases, from the dawn of civilization. The extensive survey of the literature revealed that curcuma longa L. or turmeric is highly regarded as a universal panacea in the herbal medicine with a wide spectrum of pharmacological actvity. The coloring principle of turmeric is called curcumin which has yellow color and essential components of this plant [92]. Some experts warn that turmeric may interfere with the body’s response against Covid-19. There is also good data to supporting using turmeric for Covid-19. Follow healthy lifestyle choices and proven prevention methods instead[93].

History :

Turmeric has been used in Asia for countries and is a major part of ayurveda, siddha medicine, traditional Chinese medicine, unani and the animistic ritual of austronesian peoples. It was first used as a dye and then later for it’s supposed properties in folk medicine. From Indian it spread to Southeast Asia along with Hinduism and Buddhism, as the yellow dye used to color the robes of monks and priests turmeric has also been found in Tahiti, Hawaii and eastern islands before European contact. Turmeric was found in farmana, dating to between 2600 and 2200 BCE and in a merchant’s tomb in megiddol Israel dating from the second millennium BCE.In medieval Europe turmeric was called “Indian saffron “[94].

Chemical constitutes:

Curcumin, demethoxycurcumin(DMC), bisdemethoxycurcumin(BDMC), Eugenol, dihydrocurcumin, azulene, Borneo, d- champagne, acrylic acid, turmerone[95].

Extract use:

Turmeric, the bright yellow spices extracted from the tuberous rhizomes of the plant curcuma longa, has been used in traditional Indian and Chinese systems of medicine for centuries to treat a variety of ailments, including jaundice and hepatic disorders, rheumatic, anorexia, diabetes wound, and menstrual difficulties. Immunomodulators effects of curcumin on various facets of the immune response and cytokine production[96].

How to Administer:

Turmeric paste is 1/4 to 1/2 Tsp of the water 1 cup, ginger of pinch grated, lemon juice is 5ml and honey as per your taste. Place a pan on medium heat and add grated ginger and water.  Then add the turmeric paste and allow to boil. Finally add lemon juice and honey and mix well. Strain it in a glass and serve.

Mechanism of action:

Turmeric is one of the most widely used spices ingredients, derived from Curcuma longa of the zingiberaceae plant family. Curcumin, known for its therapeutic effects especially in cancer, also recognized as a potent modulators of the immune system curcumin has been shown to exert immunomodulators effects on several cells and organs of the immune system. The immune system has evolved to various specialized cells and soluble molecules that are organized into a number of organs tissue including bone marrow and thymus as the central lymphoid organ and lymph spleen nodes spleen as well as mucosal lymphoid tissue as peripheral ones [97,98].

Role against Covid-19:

Turmeric has been used for centuries with a good safety profile. It is shown promising efficacy against influence A viral infection by regulating immune response to prevent injury to pulmonary tissue well defined randomized studies should be performed to evaluate the efficacy of turmeric derivative against SARS-COV-2 and assess its value as a possible treatment for this deadly virus [99].

Pharmacological action of phytoconstituent:

Therapeutic uses:

Improve skin health, boost immune system, improve digestion, help control diabetes.

Antiviral properties:

Turmeric may be alternative antimicrobial agents against bacterial infections. The utilization of essential oil of turmeric leaves significantly inhibit fungal growth, as well as aflatoxin B1 and G1 production [102].

Side effects:

Turmeric usually doesn’t cause serious side effects, some people can experience mild side effect such as stomach upset, nausea, dizziness or diarrhea [103].

8. Ginger:

Introduction:

Ginger (Zingiber officinale) is the herbaceous plant native to south Asia belonging family of zingiberaceae. The characteristic pungent flavor of ginger rhizome is used extensively in food and beverages[104]. Ginger is a common Indian spice and traditional medicinal plants have important pharmacologic activities such as antioxidant, analgesic and antipyretic properties. Fresh ginger possesses anti-viral activity against human respiratory syncytial virus due to presence of bioactive phenolic phytocompound 6-gingerol[105]. Hence, the present study aims to examine phytocompound 6-gingerol from the ginger plant (Zingiber officinale) that could act as a promising drug against Covid-19 protein and screened through in to silico approach.

History:

Ginger an herbaceous perennial plant of the family zingiberaceae probably native to southeastern Asia, or its aromatic pungent, rhizome is used as a spice flavouring food and medicine. It generic name zingiber is derived from the greek zingiberies, which comes from the Sanskrit name of the spice singabera. Its use in India and China has been known from ancient times, and by the first century, traders had taken ginger into the mediterranean region by the eleventh century it was well known in England. The Spaniards brought it to west indies and mexico soon after the conquest, and by 1547 ginger was being exported from Santiago to spain[106].

Chemical Constituents:

The ginger rhizome contain 0.6 to 3.3% essential oil, comprising more than 150 secondary metabolites. Around one quarter is 6-gingerol. Ginger rhizome further contains organic acids, fats around 50% sugar and slimes[107].

Extract use:

Ginger extract may be more efficient and convenient because of its small usage in diet compared with ginger root powder. This trial was designed to investigated the effect of ginger extract on production performance, antioxidant capability, immunity and also inflammation of laying hens, trying a find a natural and effective feed additive in poultry production[108].

How to Adiminister:

1.Firstly, consuming 4 tsp ginger juice with 4 tsp honey and 2 tsp lemon juice with water reduce cold. 2. Sun dry the peeled and cut ginger pieces in a covered bottle for 12 days. Consuming 2-4 pieces everyday solves digestive issues. 3. Also dried ginger mixed with little jiggery and 1 glass of milk. Consumed every morning cures stomach ache and increases digestion[109].

Mechanism of action:

Ginger has been used for medicinal purposes, due to its rich nutritional properties. Even in several Ayurvedic medicines ginger has been used as an active ingredient and this is due to the presence of Gingerol, an active compontent that makes ginger a perfect immunity booster. Apart from that, ginger has antibacterial and anti-inflammatory properties, which help keeping several ailments at and help fighting infection[110].

Role against Covid-19:

Since the onset of the Covid-19, people have shifted to healthier, nutritional option to fight the virus and boost immunity. Health has becomes a top priority and many of us are trying to find home remedies to fight the deadly virus[111].

Ginger a modulates genetic pathway, acts on tumor suppression of genes, good anti-platelet and cyclooxygenase-I inhibitory propertie, anti-inflammatory action on prostaglandin synthesis also help in relieving menstrual cramps antimicrobial effect.

Pharmacological action of phytocostituent-

Therapeutic uses:

Treat  hair loss, boost digestion, control nausea , fight infection.

Antiviral properties:

Due to the presence of some phenolic compound in it, ginger has shown great antimicrobial activities and effectiveness in controlling certain viral, bacterial and fungal diseases. Ginger is used in many countries for the preservation of food [113].

Side effect:

Ginger can cause mild side effect including heartburn, diarrhea, burping, and general stomach discomfort(114).

9. Tinospora cordiofolia ( Guduchi ,Gilly )

Introduction-

It consists of biological source Tinospora cordiofolia and family menispermaceae. Tinospora cardifolia (Willd.) Miers. (Menispermaceae) is one of the most glabrous, succulent, woody found throughout India. It is known as Guduchi in Sanskrit and Giloe or Amrita in Hindi[115,116]. It is designated as Rasayana in  traditional system Ayurveda. It is recommended that it enhances general body resistance[117]. Different type of active constitute form from the plant such as alkaloids, glycosides, steroids and diterpenoid lactones has been isolated from the different parts of the plant, such as root, stem and whole plant[118].

History –

Research from center for advanced studies Pune published a paper titled “immunomodulatory effect” of tinospora cordiofolia on macrophages activation. This reaserch prove that guduchi can sharpen and hasten ones immunity response to invading bacteria and virus and help combat such threats from pathogens faster and better. A paper by Cornell university submitted on may 29, 2020, titled “in silico investigation of phytoconstituent from Indian medicinal herb “tinospora cordiofolia”as potential inhibitor against SARS-CoV-2 tried to throw light on this[119].

Chemical constituents

It contains effective chemical constituents in stem and root contain berberine, tinosporin, palmatine, tetrahydropalmatine it’s alkaloid type. Tinosporon, colymbin, tinocordiofolin, heptacosanol  contain in whole plant is diterpenoid type. It containt various elements Cl, k, Ca, Cr, Mn, Fe, Ni, Cu, Zn, Br, etc [120].

Extract use_

It’s extract is very effective, it contains methanol, antimicrobial effectiveness against virus strains- which are staphylococcus aureus, Klebsiella pneumoniae, Echericha coli, Shigella flexneri, Salmonella typhi, Enterobacter aerogene, Psedomonas aeruginosa, Seratia marcesenses, Proteus vulgaris, etc. Mix of extract giloy+Tulsi(6leaves), ginger1/2tsp+Kali Mirch(4-6seeds) all crush and grind them together and use as herbal tea or mix it with honey and consume it[121].

How to administer

Extract(juice) of guduchi be taken orally.

Mechanism of action-

It’s have effective mechanism of action dry stem crude extracts of Tinospora cordifolia with a polyclonal B cell mitogen, G1-4A on binding to macrophages have been reported to enhance immune response in mice by inducing secretion of IL-1, together with activation of macrophages. Tinospora cordifolia in prevention of oxidative damage[122].

Role against Covid 19-

Active compounds in aqueous extracts of Tinospora cordiofolia like alkaloids, di-terpenoid lactones, glycosides, steroids, sesquiterpenoid, phenolics, aliphatic compounds or polysaccharides[123]in experimental rat model have been reported for their cytotoxic action. Dry stem crude extracts of Tinospora cordifolia with a polyclonal B cell mitogen, G1-4A on binding to macrophages increase immune response in mice by inducing secretion of IL-1, together with activation of macrophages[124]. The (1,4)-alpha-d-glucan (alpha-d-glucan) derived from the Tinospora cordifolia activate human lymphocytes and downstream synthesis of the pro- and anti-inflammatory cytokines, in vitro[125]. Tinospora cordiofolia it’s contain large active constituent responsible for the boost  the immunity.

Pharmacological action of phytocostituent-

Action Anti-microbial activity.

The methanol extracts of Tinospora cordifolia have potential against microbial infections[126]. The anti-bacterial activity of Tinospora cordifolia extracts has been effective  against Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Proteus vulgaris, Salmonella typhi, Shigella flexneri, Salmonella paratyphi, Salmonella typhimurium, Pseudomonas aeruginosa, Enterobacter aerogene, and Serratia marcesenses (Gram-positive bacteria)(127). In models of mice, TCE has been bacterial clearance and improved phagocytic and intracellular bactericidal capacities of neutrophils(128). TCE has been proved it’s immunostimulant properties on macrophages.

Immunomodulatory action

Active compounds 11-hydroxymustakone, N-methyl-2-pyrrolidone, N-formylannonain, cordifolioside A, magnoflorine, tinocordiside and syringin[129]. Have potential immunomodulatory and cytotoxic effects[130]. There function is effective by boosting the phagocytic activity of macrophages, production of reactive oxygen species (ROS) in human neutrophil cells[131], increase nitric oxide (NO) production by stimulation of splenocytes and macrophages indicative of anti-tumor effects[132]. Aqueous Tinospora extracts is effective and influence the cytokine production, mitogenicity, stimulation and activation of immune effector cells it helps to boost the immunity. Tinospora cordifolia extracts has been shown effective  result in up-regulation of IL-6 cytokine, resulting in acute reactions to injury, inflammation, activation of cytotoxic T cells, and B cell differentiation[133].

Uses

Antimicrobial, antidaibetic, antioxidant, anti-inflammatory, antiperiodic, antipasmodic, anti-arhythmatic, antitoxic, antistress, wound healing, cardiotonic, bittertonic, blood purifier, improve digestion, boost immunity, reduces stress and anxiety, detoxification of blood, treat type DM2, fight against respiratory issue, improve eye vision, treat asthma and arthritis, help in chemotherapy, etc[134].

Side effects

Pregnant women shall avoid regular intake of giloy.

10. Panax quinquefolius L. (Ginseng):

Introduction

It consists biological source obtained from the dried roots of panax ginseng and family araliaceae. Ginseng (the root of Panax ginseng Meyer, Family araliaceae), well-known oriental medicinal herbs. It’s used as an herbal remedy for various disorders [135]. Natural-dried ginseng is known as white ginseng and red ginseng is prepared by steaming fresh ginseng root priority to drying on the purpose of enhancing it’s efficacy, safety, and preservation[136]. Different types of ginseng panax ginseng, panax quinquefolius, panax trifollius, panax notoginseng, panax japonicas, etc.

History

A 2018 report examined accuracy of calm improve thinking process and cognition. A 2016 study effect of Korean and ginseng on cognitive function in patient with Alzheimer’s disease. Ginseng reduces inflammation according to 2020 study[137].

Chemical constituents

It contains large active constituent tetracyclin triterpenoid saponins (ginsenoids), polyacetylenes, polyphenolic compound, phytosterols, sesquiterpenes, Alkaloids, flavonoids. Active constitute effective against the boost the immunity[138].

Extract use

In ginseng ginsenoids Rg3 enriched this exhibited immunity mediated antitumor effect invitro and invivo. Anticancer effect of ginseng extract due to immunity boosting action against colon cancer cell[139].

Mechanism of action

It’s mechanism of action is very effective stimulation of T cells via IL-2, IL-12 by dendritic cells, production of antibody, activation of macrophages and NK cells activation it show immunomodulator effect. Cytokine that regulates cells of innate immune system. Dendritic cells (DC) play important role in innate immune response to infection and linking innate and adaptive immune response[140].

Role against Covid-19

Immune cells differentially responds to ginseng treatment. It contains gensenoside Rg3, antitumor effect invitro and invivo. It shows stronger antigrowth and propptoic effect in human gastric cell. Immune response is mediated by T-cell and NK cells is most effective against different virus infected cells and intracellular bacteria[140]. It protect against infectious bacteria and virus. Increaaes natural killer cells, increase macrophages, act as radiation protecting Cytokine that regulate the cells of innate immune system. Production of antibody, activation of macrophages, NK cell activation, shows immunomodulator action, dentitric cells(DC) play important role in innate immune response to infection and linking innate and adaptive immune responses[140,141].

Pharmacological action of phytocostituent-

Action

Antiviral

Intranasal administration of ginseng extract within influenza virus A/PR8 significant increase IgE as well as total IgG observed in blood, lungs, vaginal lavage and fecal extract in mice[142].

Antibacterial

Ginseng polysaccharide interact with microbes, interrupt microbial adhesion to host cell and block initiation of infectious disease[143].

Antimicrobial_

Plant continuously contact with different microorganisms such as virus, bacteria, fungi. Interation between plant and microbes beneficial for plant[144].

Respiratory_

Ginseng produces numerous action on respiratory system, especially on asthma related with antiallergic properties[145].

Uses

Active constitute effective against various disorder Ginseng used as in treatment of erectile disfunction, anti inflammatory effect, sharper cognitive function, increase energy, enhance immunity, anticancer property, combating various cardiovascular disease, neurological disorder, diabetes, antimicrobial[143], antiviral[142], antibacterial[143] etc.

Ginseng contains various pharmacological components include tetracyclic triterpenoid saponins (ginsenosides), polyacetylenes, polyphenolic compounds, and acidic polysaccharides. Ginsengs Roots (mostly), stems, leaves and their extracts have been used for maintaining immune homeostasis[146].

Side effects

Tissue injury (in inflammatory disease).

Other herbal immunity enhancers-

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Abstract

Oxalate is via way of means of product that is obviously present in the body for metabolism, and in everyday people it's far excreted harmlessly. Oxalate may be poisonous due to its tendency to crystallize at physiological pH and shape calcium oxalate crystal. There changed into a minute correlation oxidative strain and extrude in urine evaluation with the pomegranate extract supplement. Stone formers have extensively extended tiers of oxidative strain, renal tubular harm, and DNA oxidative damage. After administration of ethylene glycol, produce excessive tiers of urinary calcium oxalate, results in speedy crystal deposition and nephrolithiasis. Pomegranate extract supplementation with 1,000 mg polyphenol extract each day can also additionally present a few modest advantages in reducing supersaturation of calcium oxalate. The iNOS-mediated NO production is extensively excessive while there may be accelerated tiers of oxidative strain. Renal tubular molecular harm ends in the formation of cystic fibrosis stem cells. There are numerous promoters of stone formation just like the Tamm-Horsfall proteins and osteopontin. The THP make a contribution closer to stone formation while the ionic electricity is excessive and the pH is low. But if the THP gene is inactivated in mouse embryonic stem cells, it effects in impromptu formation of calcium crystals. The formation of stones relies upon the stability among the promoters and inhibitors of urolithiasis.

Keywords

lipid peroxidation, oxalate, oxidative stress, Renal tubular injury, crystal deposition, pomegranate extract supplementation, singular serum constituents, hyperoxaluria

Cite This Article

Begum, N., Khan, E. F., Sadiq, M., Raniya, K. (2021). Kidney Stone Treatment By Pomegranate (Punica Granatum). International Journal for Pharmaceutical Research Scholars, 10(4);09 -19.

INTRODUCTION

Hyperoxaluria (unrestricted urinary excretion of oxalate) is the major consequence for formation of stone disease[1].

Oxalate is by product which is naturally present in the body for metabolism, and in normal individuals it is excreted harmlessly. However, Oxalate can be toxic because of its tendency to crystallize at physiological pH and form calcium oxalate(CaOx) crystal which are deposited in the kidney[3,4].Acute and chronic production of CaOx crystals leads to lipid peroxidation; therefore, this process plays an important role in CaOx stone formation[5].

Oxalate and calcium oxalate crystals cause damage to Renal epithelial cells[6,7] and induces lipid peroxidation which leads to functional impairment of cellular components by reactive oxygen species (ROS) such as superoxide[8,9] . ROS act as mediators to signaling molecules such as p38-MAPK (mitogen-activated protein kinase) and transcription factors such as nuclear factor kappa-B (NF-kB) [10,11].

(NF-kB) is ubiquitous tanscriptional factors of the many genes including iNOS, host defences process[12] Mitogen-activated protein kinases (MAPK) are major mediators involved in the intracellular network of interaction proteins that transduce extracellular stimuli to intracellular responses[13].Three distinct MAPK pathways have been described: extracellular signal regulated and terminal kinase, and p38 MAPK[14].Both Ox and CaOx crystals selectively activated p38-MAPK signal transduction pathways in the proximal tubular epithelial cells[15,16]. The activation of p38-MAPK was found to be essential for the reinitiation of Ox- induced DNA synthesis.

Nitric oxide(NO) regulates inflammation and vasorelaxation and have a major role in eradication of tumor cells and pathogens[17]. Although in elevated production of NO is oxidized to ROS, which results in disruption of cell signaling and uncontrolled systemic inflammation[18].

Malondialdehyde (MDA) is a phenomenal marker of lipid peroxidation. In excessive amount it cause tissue injury and DNA damage. When combine with proteins, forms MDA- modified protein adducts[19].

Glutathione(GSH) is important intracellular antioxidant[20].

COMPONENTS OF POMEGRANTE

• Pomegranate is a rich source of potent polyphenolic, glucose, ellagic acid, garlic acid , flavonoids (anthocyanin effective in inhibition of lipid peroxidation[22,23]). Fresh juice is enriched with vitamin C and polyphenolic compounds[32].
• Edible parts of pomegranate fruit has 78% juice and 22% seeds[25].
• Fresh fruit contains 85% water, 10% total sugars and 1.5% pectin, ascorbic acid and polyphenolic flavoids[1].

Pomegrante seed is rich source of crude pectin, fibres and sugars[28]. It shows that pomegranate can act as a NF-kB inhibitory effect on kidney stone formation[29].

Pomegranate has become more popular because of major effective physiological properties, such as anticancer,[42,43] cholesterol lowering, cardioprotective,[44]etc. Many experiments have reported that pomegranate and its derivatives have free radical scavenger and potent antioxidant activity.[45,46,47] It has also been shown that pomegranate can suppress NF-kB activation through a novel mechanism in vascular endothelial cells.[48]

CHARACTERISTICS OF POMEGRANATE

Pomegranate is used as a traditional medicine from ancient time. It exhibit both anti- hypercalciuria and anti- urolithiasis effect . Its phytochemicals are responsible for muscle relaxation in the urinary tract which can easily remove stones from the kidney. The extract and juice of pomegranate inhibits the hyperoxaluria – induced oxidation renal tubular damages by reducing the levels of ROS, NO and NF-kB and regulates the level  of creatinine , urea, and uric acid[1,8,9,18].

MATERIAL AND METHOD 

Patients following, repetitive stone formers (RSFs; >2 earlier scenes), 18–70 years of age, were selected from our stone facility. Potential subjects needed to have been recently advised on broad dietary rules to diminish the “stone facility impact.” Only patients with calcium containing (non-irresistible, non-uric corrosive, noncystine) stones who had gone through something like a 24-hrs urine study were included. There were chosen from an information base of volunteers and coordinated with three to one as to age, sex, and weight record (BMI). Subjects as of now on clinical treatment for stone anticipation or with any clinical inclination to stone arrangement were prohibited (i.e., essential gout, LeschNyhan condition, Von Gierke infection, persistent loose bowels, insulin opposition, neoplastic issues, renal hyperuricosuria, hyperparathyroidism, renal cylindrical acidosis, innate hyperuricemia)[2].

SUPPLEMENT

Members got a solitary 1,000 mg pomegranate separate case day by day for 90 days administered by our exploration drug store. Pill checks were performed at the finish of the examination to evaluate adherence[2].

OXIDATIVE PRESSURE MARKERS  

Urinary 8-hydroxy-deoxyguanosine (8-OHdG) is a marker of oxidative DNA get damage by receptive oxygen species (ROS)[33]. Lipid peroxidation (LPO) has been evidence to be associated with the pathogenesis of an assortment of infections, for example, end stage renal illness, coronary vein sickness, atherosclerosis, and stroke. As basal degrees of oxidation are regularly low and might be impacted by singular serum constituents, we utilized the free extreme generator 2.2′-azobis (2amidinopropane) hydrochloride to incite oxidation to quantify serum peroxides and assist with anticipating a person’s capacity to react to oxidative pressure[34] . Basal and inducible oxidative states were estimated for every persistent during each stage[34]. AAPH prompted serum lipid peroxidation was controlled by incubating serum tests (weakens x 4 with Phosphate-cushioned saline) with AAPH (100 mol/L) for 2 hrs at 37 °C [21]. The degree of lipid peroxidation was then estimated by the

TBARS[Thio barbituric corrosive responsive substances] examine and by the lipid peroxides test[24]. For correlation among gatherings, we used AAPH-prompted values, as it is the best address a person’s capacity to react to oxidative pressure.

Exceptionally sensitive C-receptive protein (hsCRP) has been linked in elevating the level of oxidative pressure in diabetes and coronary artery disease[2].

STATISTICAL ANALYSIS

Values for demographic profile of patients, urine samples serum samples and oxidative stress markers were calculation for each individual patient earlier enlisted and following supplement intervention to pre and post examine. Linear mixed model analysis  for repeated measures was used to test for differences in urine values and oxidative stress values between non stone formers and stone formers at pre and post changes between the groups. For correlation analysis between urine values and oxidative stress among stone formers could be detected with 0.80 power [2].

RESULT

17 recurrent stone formers, 5 non stone formers. There were no adverse effects reported from supplements during the study period. The standard age of participants was 40.4 years and 57% were female with standard BMI of 29. Baseline and post intervention values for markers of oxidative stress in RSFs and NSFs. RSFs patient had significantly increase oxidative state at baseline when compared to NSFs patient[2].

Urinary 8-OHdG was significantly increased in RSFs by 350%. Additionally, there was higher susceptibility of the patients’ serum to AAPH-induced lipid peroxidation as measured by lipid peroxides and TBARS. However, there was no baseline difference between RSFs and NSFs in serum PON1 activity, or in the inflammatory marker hsCRP. Following supplementation serum PON1 activity was seen increased significantly in RSFs patients. Supplementation decreased the serum hsCRP levels insignificant by 29 % in the RSFs patients. 24 hrs  urine analysis showed that RSFs had lower urinary pH and saturation of calcium phosphate and higher saturation of uric acid than NSFs at baseline. Following to pomegranate extract supplementation, the only difference in urinary analysis of RSFs patients was an increase in calcium, sodium, chloride, and magnesium for RSFs patient. Otherwise, no urinary risk factor Changes were seen significantly.  In general the was a minute correlation oxidative stress and change in urine analysis with the pomegrante extract supplement. Although there was a negative correlation between lipid peroxides and uric acid i.e, a patient with higher baseline lipid peroxides levels had a greater reduce in urinary uric acid with the following supplementation. Significant changes were seen in the levels of AAPH-induced serum TBARS, lipid peroxides, hsCRP, and urine 8-OHgG did not correlate with changes in urine analysis values following supplementation in RSFs. i.e, patients with high levels of PON1 pomegranate extract supplementation were more likely to have a decline in supersaturation of CaOx[2].

DISCUSSION

Stone disease has been linked to obesity, hypertension[35], diabetes[36], metabolic syndrome[37], and chronic kidney disease[38]. As these diseases have been associates to oxidative stress[39].

Oxidative stress also plays a pathophysiologic role in nephrolithiasis. Earlier studies in RSFs suggest that reactive oxygen species-induced renal cellular injury and inflammation are likely involved in idiopathic nephrolithiasis, as demonstrated by increased in level than normal levels of urinary gamma-glutamyl transpeptidase, angiotensin 1 converting enzyme, beta-galactosidase, Nacetyl-betaglucosaminidase (NAG) activity, TBARS, and 8-OHdG[33,40].

Stone formers have significantly increased levels of oxidative stress (in serum and urine), renal tubular injury, and DNA oxidative damage, than NSFs as showed by significant differences in urine 8-OHdG, serum TBARS, and lipid peroxide levels. The association between oxidative stress and calcium-containing crystals is not fully understood[2].

Tentatively studies suggested that renal epithelial exposure to high calcium oxalate results in significant increase in markers of oxidative stress[27], implicit that the stone constituents themselves may be an causing factor in renal tubular damage and ROS radical formation. Atypical, deposition of calcium apatite in the renal papillary interstitium may lead to production of ROS and recruitment of monocytes and macrophages, which phagocytize or coat crystals in macromolecules which causes further cellular damage[41]. If crystal formation continual unregulated, localized injury and inflammation cause collagen deposition, mineralization, and renal tubular damage, providing sites for crystal attachment. As crystals continue to enlarge, they ulcerate through the papillary urothelium, where exposure to pelvic urine leads to heterogeneous nucleation of calcium salts[50]. After administration of ethylene glycol produce high levels of urinary calcium oxalate, leads to rapid crystal deposition and nephrolithiasis [31].

Pomegranate plants, which are rich  source of antioxidants, including polyphenols hydrolyzable tannins, anthocyanins, and ellagic acid derivatives[52], have been shown to suppress NF-kappaB activation in vivo and inhibit lipid peroxidation. In a intended diet-controlled study,[51,53] demonstrated that prophylactic pomegranate juice administration in rats receiving ethylene glycol helpfully no crystal formation compared to the development of severe crystallization and epithelial degradation seen in animals without any supplementation. Eventually[8, 9], in a similar type of animal nephrolithiasis. Found that crystal accumulation, inducible oxide synthase (iNOS), p38-MAPK and p65NFκB activity, and oxidative stress markers were decreased by pomegranate supplementation[30].It is concluded that p38-MAPK and NF-kB pathways are activated in a variety of models of renal inflammatory disease, including nephritis[49].The iNOS-mediated NO production is significantly high when there is elevated levels oxidative stress[26].

CONCLUSION 

Recurrent stone formers have markedly excessive levels of oxidative stress than NSFs. Pomegranate extract supplementation with 1,000 mg polyphenol extract daily may present some modest benefit in lowering supersaturation of calcium oxalate.

The correlation between elevated serum PON1 activity with lower saturation of calcium oxalate may help  explain the reduced risk of calcium oxalate stone formation with pomegranate shown in earlier animal studies[2].

FORMATION OF STONES

Factors causing “nidus” formation: – Lithogenic drugs, Altered pH, Microorganisms, Gout, Genetic Disorder, Solute precipitation

Nidus/Nucleus formation

Crystallization of nucleus

Crystal growth

Crystal aggregation on renal tubule

Crystal cell interaction

Renal tubular cell injury & these injured cells act as site of binding

Crystal retention

STONE FORMATION

There are several promoters of stone formation like the Tamm-Horsfall proteins and osteopontin. Some of the proofs indicate that a primary interstitial apatite crystal formation leads to CaOx stone formation (54). The lipids in human cellular membranes are chiefly involved in the crystal nucleation (55). The renal cells which get injured due the crystal sell interaction produce PT-1 or other anionic proteins lead to COM crystal aggregation (56). The THP contribute towards stone formation when the ionic strength is high and the pH is low (57). The THP is also assumed to provide protection from the stone agglomeration when it has low ionic strength and high pH, as reported by Hess (58). The mucopolysaccharides also act as the binders by increasing nucleation and aggregation (59). But according to a study, if the THP gene is inactivated in the mouse embryonic stem cells, it results in impromptu formation of calcium crystals. This is a conclusive confirmation theta the THP is an important inhibitor of stone formation (60). The presence of ROS due to oxalate can damage the mitochondrial membrane leading to apoptosis (61).  According to a study, when oxalic acid is added to the HK-2 cells, it causes activation of IL-2R beta mRNA and IL-2R beta proteins that lead to inflammation. This whole process may activate the p38 MAPK signalling, however the mechanism is still not known (62). The promoters are the substances which promote urolithiasis by different mechanisms (63). The idiopathic stone formers are those in whom stone are formed due to various drugs. In these patients, the stones are attached to the interstitial site of the Randall’s plaque (64). Some molecules like phosphatidylserine, CD44 and hyaluronan also act as binders for the stones (65, 66). The stone formation can be prevented by blocking the binding molecules like the monocyte chemoattractant protein-1, hyaluronic acid (67). There are also some promoters of urolithiasis which are the calcitriol hormones (69), phospholipids, cholesterol, glycolipids (68), oxalic acid, cystine, sodium, low urine volume, calcium (70). The formation of stone primarily depends upon the balance between the promoters and the inhibitors.

There are certain food colours which may have adverse effect on the kidneys and liver. According to a study Tartrazine and Carmoisine were administered in two male albino rats by the oral route in two divided doses amongst which one was low and the other was high. This was continued for 30 days. After the completion of the pre-determined duration of drug administration the tissue and serum samples were collected and LFT, RFT, blood glucose, lipid profile was done.

The research data estimated an increase in the levels of urea, creatinine, albumin, ALT, AST, ALP, Total protein. The study concludes that both tartrazine and carmosine can unfavourably effect the biomarkers of organs like the liver and kidney not only at higher but also at lower doses (71).

Histopathology in livers of rats fed tartrazine (Tz) and ameliorative effects several doses of curcumin (CUR). A) Normal structure of liver tissue of control showing the central vein, normal arrangement of hepatic cords, normal blood sinusoids(s) and hepatocytes, HE, X 400; B) Liver tissue of rats exposed to Tz showing dilation of blood sinusoids, and central vein with hemorrhage and necrosis (*), HE, X 400; C) Liver tissue of rats fed Tz in combination with 1.0 g/ kg dry mass (dm) diet of CUR, showing less necrosis and moderate degenerative changes compared to the control and rats fed Tz alone (N), HE, X 400; D) Liver tissue of rats fed a diet containing Tz in combination with 2 g/ kg, dm diet of CUR, showing little necrosis (N) compared to the controls or rats exposed to lesser amounts of CUR. HE, X 400; E) Liver tissue of rats fed a diet containing Tz supplemented with 4.0 g/ kg, dm diet of CUR, showing little necrosis (N), H&E, X 400.

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Abstract

Benzimidazole is an example of aromatic heterocyclic organic compound. It is a bicyclic compound. It contains the fusion of benzene with imidazole which ultimately gives a privileged structure. Benzimidazole and its derivatives play an important role in the medicinal chemistry and drug discovery with many pharmacological activities. Substitution of various chemicals on benzimidazole nucleus gives important synthetic product and strategy in the drug discovery process. Benzimidazole derivatives contain versatile nitrogen containing heterocyclic compounds. The methods for the synthesis of benzimidazole and its derivatives have become a focus of synthetic organic scientist, Benzimidazole and its derivatives were used as building blocks for the important therapeutic compounds in medicine. Benzimidazole nucleus plays a very important role as a therapeutic agent. Benzimidazole and its derivatives exhibit pharmacological activities such as antimicrobial, antiviral, anticancer, anti-inflammatory, analgesic activity, anti-ulcer, anti-diabetic activity etc. Benzimidazole nucleus gives active sites for the reaction like 2 and 5 position which gives potent therapeutic agents. The present review covers the chemistry and pharmacological activities of substituted benzimidazole. In the present review, benzimidazole derivatives with different synthetic derivatives and their pharmacological activities are given. The main aim of review is to help medicinal chemists for the development of SAR on benzimidazole for each activity and to review the work reported, chemistry and pharmacological activities of benzimidazole derivatives during past years.

Keywords

OPD, Antimicrobial, Antiviral, Anti-inflammatory, Analgesic Activity, Anti-ulcer, Anti-diabetic Activity

Cite This Article

Pawar, S., Bhor, R. J., Magar, S. (2021). Novel New Research Strategies of Benzimidazole Derivatives: A Review. International Journal for Pharmaceutical Research Scholars, 10(4);01 - 08.

INTRODUCTION

Benzimidazole is an example of aromatic heterocyclic organic compound. The new method of benzimidazole based on poly heterocycles draw the attention of pharmacists from last few decades^[1]. It has important pharmocophore in medicinal chemistry and pharmacology. Benzimidazole and its derivatives are an example of bicyclic compound consisting of the fusion of benzene with imidazole^[2]. The structure of benzimidazole and its derivatives are given below; The worsening of asthma at night, is commonly referred to as nocturnal asthma (NA).

It has magical properties with many pharmacological properties. Benzimidazole possess many biological activities such as anti- microbial, anti-fungal, anti-histaminic, anti-inflammatory, anti- viral, anti-oxidant, anti-cancer, anti-ulcerative^[4-6] etc so that it having important moiety for the development of molecules of pharmaceutical interest. Many benzimidazole derivatives having heterocyclic building blocks is due to the structural similarity to purine nucleobase. It selectively inhibits the endothelial cell growth and then suppresses the process of angiogenesis in vitro as well as in vivo biological activity ^[7]. Benzimidazole is also known as 1H-benzimidazole or 1,3-benzodiazole. Five-membered nitrogen-containing heterocyclic ring was present in the structures of various biologically active synthetic compounds. The magical properties of the benzimidazole related drugs have encouraged the medicinal chemists to synthesize a large number of novel derivatives with chemotherapeutic agents. Benzimidazole and its derivatives are commonly used for the prevention and treatment of parasitic infections ^[8]. Some examples of benzimidazole containing drug are given below;

• Omeprazole
• Rabeprazole
• Lansoprazole
• Pantoprazole
• Esomeprazole

They are well known discovered benzimidazole drugs. There are some other drugs with different hetero atoms like Thiabendazole (TBZ); Parbendazole (PAR) Cambendazole (CAM) Mebendazole (MBZ) Oxibendazole.  Thiabendazole (TBZ)was the first benzimidazole to be marked over 40 years ago^[9].

Benzimidazoles having the well known heterocyclic compounds which have common and characteristic features of a variety of medicinal agents. Benzimidazole is soluble in water and other polar solvents. Benzimidazoles exist in two equivalent tautomeric forms because the hydrogen atom can be located on either of the two nitrogen atoms. It having calculated dipole of 3.61D, and it was soluble in water [10]. Benzimidazole and its derivatives are classified as aromatic due to the presence of a sextet of π-electrons, consisting of a pair of electrons from the protonated nitrogen atom and one from each of the remaining four atoms of the ring. When it having acidic pka is 14.5, then it gives less acidic than carboxylic acids, phenols, and imides, but slightly more acidic than alcohols. When it having basic pka is approximately 7 then it gives benzimidazole approximately sixty times more basic than pyridine [11].

SYNTHETIC APPROACHES FOR BENZIMIDAZOLE DERIVATIVES:

Synthesis of benzimidazole and its derivatives through the direct condensation reaction by OPD i.e. o-Phenylene diamine and formic acid was reported but sometimes in many synthetic reaction; a few shortcomings like less yield, extreme reaction conditions, longer reaction time and difficult workup process were noticed. Some synthetic reaction of benzimidazole and its derivatives are given below;

Scheme 1:

Synthesizing Benzimidazoles through the coupling of benzne-1,2-diamine or OPD with different Aldehydes in ethanol at room temperature. These chemical easily available cheap catalysts are more efficient than various reported expensive heterogeneous catalysts. Yields of Pt, TiO2 catalyst are higher (72-88%) in shorter reaction time (3-7h) (Scheme 1).

Scheme 2:

Literature survey has revealed that o-phenylenediamine or benzne-1,2-diamine react readily with most carboxylic acids to give 2-substituted benzimidazole, usually in very good yields. (Scheme 2).

Scheme 3:

The most commonly used (Phillip’s method[12], involves the condensation of o-diaminobenzenes or Benzne-1,2-diamine with carboxylic acids or its derivatives, including heating the reagents together in the presence of concentrated hydrochloric acid (Scheme 3),

Scheme 4:

In the same context, Ghosh[13] and coworkers employed a new, potent, less toxic catalyst: CH3CN for the synthesis of substituted benzimidazole from 1, 2-phenylenediamine with aryl, heteroaryl aldehydes (Scheme 4). High yield, clean reaction profile, cheaper and green in nature are the significant features of this methodology.

Scheme 5:

In the same context, phylum[14] and coworkers employed a new, potent, less toxic catalyst such as DBSA; for the synthesis of substituted benzimidazole from 4 methylbenzne-1,2-diamine and aliphatic aldehyde; it gives different benzimidazole derivatives (Scheme 5).

Scheme 6:

Hollan[15] et al. who have reported the reaction of the appropriate Trichloro acetimidate with o- phenylenediamine or benzene-1,2-diamine; it gives the 2-trichloromethyl benzimidazole (Scheme 6) only at room temperature.

Scheme 7:

o-phenylenediamine (0.01 mol) and different aromatic acid (0.01 mol) in the presence of ammonium chloride as catalyst at 80–90 °C (Scheme 7). The reaction is green and economic.

Scheme 8:

Recently Saberi[16] has reported synthesis of 2-benzimidazoles under microwave irradiation and solvent-free conditions which is catalyzed by zeolite HY. As shown in scheme 8, o-phenylenediamine (2 mmol) with aromatic, aliphatic and heterocyclic carboxylic (2 mmol) and 50 mg of Zeolite was mixed thoroughly in a mortar. The reaction mixture was then irradiated in a domestic microwave oven for 5–9 min at 160–560 W (Scheme 8).

Scheme 9:

Recently Saberi has reported synthesis of 2-benzimidazoles under microwave irradiation and solvent-free conditions which is catalyzed by Silica Gel. As shown in scheme 9, o-phenylenediamine (2 mmol) with aromatic, aliphatic and heterocyclic carboxylic (2 mmol) and 50 mg of Silica gel was mixed thoroughly in a mortar. The reaction mixture was then irradiated in a domestic microwave oven for 5–9 min at 160–560 W(Scheme 9).

Scheme 10:

Recently Saberi has reported synthesis of 2-benzimidazoles under microwave irradiation and solvent-free conditions which is catalyzed by Alumina. As shown in scheme 10, o-phenylenediamine (2 mmol) with aromatic, aliphatic and heterocyclic carboxylic (2 mmol) and 50 mg of Alumina was mixed thoroughly in a mortar. The reaction mixture was then irradiated in a domestic microwave oven for 5–9 min at 160–560 W. (Scheme 10).

Scheme 11:

Under  the  correct  conditions  aldehydes  may  react  with  o- phenylenediamines   to   yield   2-substituted   benzimidazoles(Scheme 11).

Scheme 12:

Heating in the presence of nitro benzene Mann[17] et al used a mixture of unsubstituted or substituted phenylenediamine  and  appropriate  aldehyde  in  nitrobenzene  heated  at 140 °C., the mixture was cooled and filtered after adding water which gives benzimidazole (Scheme 12).

Scheme 13:

The other way was synthesized by Lin[17] et al. involving a direct  one  step  synthesis  of  various  benzimidazoles  from phenylenediamines and aldehydes that includes air as oxidant(Scheme 13).

Conclusion

The review has concluded with the key synthetic approaches and pharmacological activities of the Benzimidazole. It gives one of the most useful biological activities. Benzimidazoles and its derivatives are utilized in much therapeutic or biological activity such as anti-inflammatory, anti anxiety and antimicrobial activity. The efficient and economical methods of synthesizing benzimidazole and its derivatives by condensation reaction between OPD i.e. orthophenylenediamine and various compounds in the presence of various conditions presented in this review. This review helps to other chemist chemists to get the first hand information for the synthesis of benzimidazole and become very useful for chemists and workers in this field. This can be developed from year to year to produce new economical and environmental clean protocols for the large scale production of important heterocyclic compounds.

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Lakshmi Narain Collage of Pharmacy (RCP), Indore, India

Abstract

The endeavor of the present study was to formulate and evaluated an oral pulsatile drug delivery system of Terbutaline sulphate intended for treatment of asthma attack. Mostly asthma attack occurs at mid night (nocturnal asthma), hence it is very inconvenient for patients to take medication at mid night. To overcome this problem pulsatile form of immediate and burst release of a drug is suitable, which provides drug release at the time of attack and at a target site. With the help of pulsatile delivery system, drug releases with a lag time of few hours and a burst release of drug at the peak duration (early morning hours) of asthmatic attack. Solution layering technique assisted spherical shaped Terbutaline sulphate loaded non pareil seeds of immediate release and enteric release polymer. Five batches were formulated using varying concentrations of cellulose acetate phthalate and constant concentration of ethyl cellulose (2%) in acetone till 2-6% weight gain. These layers restrict the release of the drug from pellets in stomach and provided sufficient lag time to the formulation. All the batches were characterized for drug content and in- vitro release. The results indicated efficient pulsatile drug release i.e. drug release after lag time of 8 hours post administration (first 2hrs in acidic medium and 6hrs in basic medium).

Keywords

Nocturnal Asthma, Circadian rhythm, Pulsatile delivery, Terbutaline sulphate, Cellulose acetate phthalate, Solution layering technique, Lag time, Burst release

Cite This Article

Reddy, R., Saini, D., Mathew, S. E., Ghodke, A., Borasi, M. (2021). Formulation and Characterization of Pulsatile Drug Delivery System of Terbutaline Sulphate. International Journal for Pharmaceutical Research Scholars, 10(3);24 - 33

INTRODUCTION

Asthma is a chronic inflammatory disease of the airways, characterized by hyper responsiveness to a variety of stimuli. The role of circadian rhythms in the pathogenesis and treatment strategies of asthma indicates that airway resistance increases progressively at night in asthmatic patients. In normal lung function, circadian changes are seen, which reaches a low point in the early morning hours. The worsening of asthma at night, is commonly referred to as nocturnal asthma (NA). The dose is administered at bedtime but should release drug during morning hours. The bronchi divide into smaller bronchi, and then into bronchioles which ultimately terminate in the alveoli – the folded membranes where gas exchange takes place. The bronchi and bronchioles have a muscular layer in the wall which allows them to contract. In an acute asthma attack, this muscular layer contracts and leads to narrowing of the airways. The symptoms of asthma are mostly found at night than during the day. Many circadian dependent factors appear to contribute to the worsening of nocturnal asthmatic symptoms. For example, cortisol levels reached maximum at the time of awakening and were lowest in the middle of night and histamine concentration were found maximum at the time of 4:00 am. Approach to achieve Chronotherapeutic Drug Release Various methods have been developed and applied to design chronotropic system to achieve pulsatile drug release. These method are mainly classified into three major categories.

• Time controlled chronotropic systems.
• Stimuli induced pulsatile drug delivery systems.
• Externally regulated pulsatile drug delivery system.

Pulsatile drug delivery system

• Temperature sensitive pulsed- release delivery systems.
• Inflammation induced systems.
• Enzyme dependent pulsatile-release systems.
• Glucose concentration dependent insulin release systems.
• Intelligent gels responding to antibody concentration.
• pH sensitive pulsatile drug delivery systems.

Disease requiring pulsatile drug delivery

Circadian rhythm regulates many body functions in human, viz metabolism, behaviour, physiology, sleep pattern, hormone production etc. Asthma is one such disease where pulsatile drug delivery system can be useful. Circadian changes are seen in normal lung functions, which reaches a low point in the early morning hours. In case of cardiovascular disease, BP is at its lowest during the sleep cycle and rises steeply during the early morning period. Platelets agreeability is increased and fibrinolytic activity is decreased in the morning, leading to a state of relative hypercoagulability of the blood. Circadian increases in the blood sugar level after meal has been observed in diabetes mellitus. Circadian variations seen in DOPA level in afternoon in case of attention deficit syndrome.

Material and Method

Terbutaline sulphate (Yarrow chem labs), Isopropyl alcohol (Lobachem Pvt. Ltd), Polyvinyl Pyrrollidone k30 (Lobachem Pvt. Ltd), cellulose acetate phthalate, Ethyl cellulose, Acetone, Propylene glycol, Span 80, Castor oil, Titanium di oxide, Methylene chloride. (Lobachem Pvt. Ltd).

Formulation and Development

A pulsatile drug delivery of Terbutaline sulphate was prepared by coating Non pareil seeds approximately 14/16#. It was prepared in three stages.

Drug loading

Drug loading was carried out by solution layering technique, using conventional coating pan. The weighed non-Pareil seeds of approximately 14/16# were charged into pan and Terbutaline sulphate solution (1%w/v) in water: IPA (1:1) containing PVP-k30 (2%), as a binder, was sprayed over the non pareil seed till mass put on 1% hot air (60-70ºc) was blown to evaporate the solvent.

Coating of drug loaded pellets

Dried pellets were sprayed with the solution of cellulose acetate phthalate (2%w/v, 4%w/v, 6%w/v, 8%w/v, and 10%w/v) and ethyl cellulose (2% constant) in Acetone till 2-6% weight gain. These layers restrict release of drug from pellets upto 6 hours until it reaches to intestine.

Final drug loading

At last an initial dose was encrusted, fourth layer, as similar as the first layer, for immediate action. For that Terbutaline sulphate solution (1%w/v) in water: IPA (1:1) containing PVP-K30 (2%), as a binder, was sprayed over the non pareil seed till mass put on 1%, Hot air (60-70ºc) was blown to evaporate the solvent. The solution was applied at pressure 20 psi. The speed of revolution of coating pan was 20-30 rpm. Hot air was supplied by hair dryer which, was placed at a distance of 15 cm from pan.

Table 1:  Composition of coating solution

Table 2: Processing conditions for coating

Result and discussion

Preformulation

Melting point determination

The melting point of Terbutaline sulphate was found to be 118-120ºC which is same as reported in literature.

 Determination of wavelength using UV spectrophotometric analysis

The maximum wavelength of Terbutaline sulphate was found to be 275nm. The reported wavelength is 276-280nm.

Preparation of calibration curves

The calibration curves of Terbutaline sulphate in various solvents e.g. Distilled water, Phosphate buffer 6.8 and 0.1 N HCl was prepared.

Table 3: Absorbance data of Terbutaline sulphate in distilled water at 275 nm

Determination of solubility of Terbutaline sulphate in various medium

The solubility of Terbutaline sulphate in various medium were studied and results of study shown in table 6.

Fig 2:  Calibration curve of Terbutaline sulphate in distilled water

Drug-excipient interaction study

Table 4:  Absorbance data of Terbutaline sulphate in phosphate buffer 6.8 at 278 nm

The drug Terbutaline sulphate was found to be compatible with various excipients which were selected for formulation of pulsatile drug delivery. The compatibility was assessed by TLC and retention factors of all ratio found similar. The Rf factor of Terbutaline is 0.34 and mobile phase is Chloroform: Methanol (4:1).

Table 5:  Absorbance data of Terbutaline sulphate in 0.1 N HCl at 273nm

Table 6:   Solubility data of Terbutaline sulphate in different medium at 37°C

Evaluations of capsule filled coated pellets

% Drug content of each formulations

Table 7: % Drug content

Practical yield

Table 8: % practical yield in each formulation

In – vitro drug release study          

Table 9:  In 0.1 N HCl   Immediate release layer

Table 9:   In vitro % drug release in pH 6.8 Phosphate buffer       

In Phosphate buffer pH 6.8:  Burst release layer

  F1 contained 2 % CAP and EC 2% the drug release was found to be 80.9 % in 2 hrs, F2 contained 4 % CAP and 2% EC the drug release was found to be 82.7 % in 3.5 hrs., F3 contained 6% CAP and 2% EC drug release was found to be 82.9 % in 4.5 hrs., F4 contained 8% CAP and 2% EC drug release was found to be 91.6 % in 5.5 hrs. and F5 contained 10 % CAP and 2% EC drug release was found to be 91.7 % in 6 hrs.

 Disintegration test for capsule

The disintegration time for hard gelatin capsule was found to be in the range of 2.5±0.816 to   10.33±0.471 min. in HCl.

 Stability study

 There were no changes in appearance and percentage drug content of pellets loaded capsules stored at different temperature 40ºC ±2% RH and 75 ºC ±5% RH.

Table 10: Stability studies              

 Conclusion

Over the past decades, it has been observed that asthmatic attack occur at mid night it is very difficult to take medication at mid night for patient or it is inconvenient for patient to take medicines repeatedly in an interval. To overcome such problems, an effort was made to formulate and evaluate time controlled pulsatile dosage form of Terbutaline sulphate. The drug loading of Terbutaline was processed with suitable carrier (non pareil seed) by employing solution layering technique. Solution layering technique assisted spherical shaped drug loaded non pareil seed of immediate release and enteric release polymers. With the use of non pareil seed the delivery of a drug at the target site may be achieved by avoiding dose dumping.

Initial drug release was observed in first 2 hours in acidic medium which can offer immediate relief, on the other hand enteric coated polymers releases the drug with a lag time of 5-6 hours and a burst release in a single pulse. Burst release of the drug after a lag time (requirement for chronotherapeutics) was achieved with the developed formulation.

To study the better release of the drug, five batches were prepared on the basis of cellulose acetate phthalate and ethyl cellulose, it was found that the batch F5 (10% CAP and 2% EC) was the best as it showed 95.4% drug content and 91.7% drug release after lag time of 8 hours (first two hrs in acidic medium and 6 hrs in basic medium) This batch was accurate for treatment of nocturnal asthma according to pulsatile drug delivery system.

REFERENCE

1. Anonymous-www.pharmatutor.org-Art-2657
2. Ricciotti, E., & FitzGerald, G. A. (2011). Prostaglandins and inflammation. Arteriosclerosis, thrombosis, and vascular biology, 31(5), 986-1000.
3. A review of Ayurvedic medicated oils,Ayur times Dr.Jagdev Singh.
4. Ghasemian, M., Owlia, S., & Owlia, M. B. (2016). Review of anti-inflammatory herbal medicines. Advances in pharmacological sciences, 2016.
5. Mukherjee, P. K. (2002). Quality control of herbal drugs: an approach to evaluation of botanicals. Business Horizons.
6. Khandelwal, K. (2008). Practical pharmacognosy. Pragati Books Pvt. Ltd.
7. Pharmacopoeia, I. (2014). Ghaziabad: Indian Pharmacopoeial Commission. Govt. of India-Ministry of Health and Family Welfare, 1948.

Abstract

With the growing interest in Herbal Therapies among persons associated with pain, inflammation, Arthritis, Surgery or other medical conditions. There exists a need for formulation & evaluation of Herbal Analgesic oil. The present work intends to formulate & evaluate Herbal Analgesic oil.

Keywords

Anti-inflammatory, Analgesic, Evaluation

Cite This Article

Ramakrishna, S., Kulkarni, S., Harshith, K., Ravikumar, S.R., Milan, K.T., Harshitha, M., Rekhashree, N. (2021). Formulation And Evaluation of Herbal Analgesic Oil. International Journal for Pharmaceutical Research Scholars, 10(3);18 - 23

INTRODUCTION

Analgesics are derived from Greek word ‘An’ means without & ‘Algos’ means Pain. Analgesic agents act by inhibiting cyclooxygenase Enzymes¹. NSAIDS ( Non steroidal Anti inflammatory drugs) are the drug of choice for inflammation & its associated symptoms , but these drugs shows severe Gastro-intestinal irritations , ulcers Kidney failure & liver toxicity. Hence for this Ayurvedic formulations are the best alternate², Herbal analgesic oils one of the Ayurvedic formulation in which herbs and base oils are used for the formulation. The different Tailas have been prepared using different herbs accordingly to the purpose and of its use.

The herbal Ayurvedic formulations are popular worldwide because they don’t show any side effects. Herbal analgesic oil shows anti-inflammatory action, analgesic action, and antimicrobial activities.

Herbs used for formulation provides essential nutrients for treatment of the diseases. Herbal formulations are comparatively causing fewer side effects than chemical formulations³.

The herbal products have dual importance due to the presence of variety of phytochemical which helps in body care and improves the biological functions of the human body⁴.

Hence an attempt is made to formulate & Evaluate Analgesic oil from Bilva, Guduchi, sweet flag, pipal (long pepper), Moringa gum, Gingelly oil & Mustard oil.

Table: 1 Ingredients

SL NO	NAME OF THE DRUG	FIGURE	BOTANICAL NAME	USES
1.	BILVA		Aegle marmelos	Anti-inflammatory , antianalgesic
2.	GUDUCHI		Tinospora cardifoila	Anti-inflammatory,  antimicrobial
3.	SESAME OIL		Sesamum indicum L	Helps in joint strengthening and bone rejuvenation.
4.	MUSTARD OIL		Brassica campestris	Reduces inflammation, blocks microbial growth.
5.	SWEET FLAG		Acorus calamus	Anti-inflammatory, flavouring agent
6.	PIPAL (LONG PEPPER)		Pipar longum	Antibacterial , antidiabetic
7.	MORINGA GUM		Moringa olefera	Antioxidant

Table: 2 Formulation of Herbal Analgesic Oil

Materials And Methods

Preparation of Kalka (Paste)

The Kalka were prepared by mixing Sweet flag,  Pipal, ,Guduchi,Moringa gum, Bilva with 3 to 4 volumes of water & heat it for 2 hours until Decoction is obtained.  Filter the Decoction & concentrate until Kalka ( Paste )is obtained

Preparation of Analgesic Oil.

  Take 1 litre of Sneha dravya ( Sesame oil) & add 50 ml of mustard oil and heat it until it forms homogeneous mixture. Add Kalka to the preparation and stir it until a strong base is formed. Then add 9 litres of water and constantly heat it until the oil is fully extracted. Filter & store in food grade glass container⁵.

Evaluation

The prepared herbal analgesic oil was subjected to under mentioned evaluations.

1. Organoleptic evaluation:

Different parameters were studied such as:Colour,Physical state, Odour and solubility and it is tabulated in Table No-3.

Table No-3

2. Phytochemical evaluation:

The formulated herbal analgesic oil were subjected to qualitative chemical analysis for identification of various plant constituents like alkaloids, saponins, glycosides, protein, steroids, carbohydrates and flavonoids.The results are tabulated in Table No-4.

Table No-4

3. Physical evaluation of herbal analgesic oil :

The physicochemical evaluation was carried out with respect to Acid value, Saponification value,  Iodine value, Specific gravity, Density, pH & Skin irritation test were carried out  as per standard methods^6,7.

The observations for the physical evaluations were reported in Table-5 where it was found that the formulation has all the value within the standard limits

Table No-5

CONCLUSION

This research provides guidelines on the use of herbal ingredients in the preparation of herbal analgesic oil having minimal (or) no side effects. The good quality and purity of the analgesic oil has met with almost all the parameters and comes under the specified limits. The ball can be rolled on by further clinical trials which will fetch further benefits of analgesic oils.

REFERENCE

1. Anonymous-www.pharmatutor.org-Art-2657
2. Ricciotti, E., & FitzGerald, G. A. (2011). Prostaglandins and inflammation. Arteriosclerosis, thrombosis, and vascular biology, 31(5), 986-1000.
3. A review of Ayurvedic medicated oils,Ayur times Dr.Jagdev Singh.
4. Ghasemian, M., Owlia, S., & Owlia, M. B. (2016). Review of anti-inflammatory herbal medicines. Advances in pharmacological sciences, 2016.
5. Mukherjee, P. K. (2002). Quality control of herbal drugs: an approach to evaluation of botanicals. Business Horizons.
6. Khandelwal, K. (2008). Practical pharmacognosy. Pragati Books Pvt. Ltd..
7. Pharmacopoeia, I. (2014). Ghaziabad: Indian Pharmacopoeial Commission. Govt. of India-Ministry of Health and Family Welfare, 1948.

Abstract

Heterocyclic moieties are abundant in environment & are of great importance to life because their constitutional subunits exist in a lot of natural compounds such as hormones, vitamins and antibiotics; therefore, they have involved significant awareness in the design of biologically potency molecules & advanced organic chemistry. Organic chemistry involving number of synthesis in which heterocyclic compound may synthesized using several catalysts may include both homogeneous and heterogeneous type. In this context we have developed environment benign novel protocol for synthesis of various substituted naphthaoxazole was prepared using aromatic aldehydes and 3-amino-2-naphthol by Microwave heating method. This protocol of synthesis of PEG-SO3H catalyzed naphthaoxazole using condensation reaction between 3-amino-2-naphthol and various aldehydes under ethanol as solvent using microwave heating method. A series of compounds were prepared and characterized by 1HNMR, 13CNMR, IR and Mass spectroscopy. Prepared products were checked for their antimicrobial activity against gram +ve & gram –ve bacteria.

Keywords

3-amino-2-naphthol, PEG-SO3H, Antimicrobial study, Microwave, naphthaoxazole, Spectral Characterization

Cite This Article

Pandya, K. A., Rakeshkumar, Vyas, K. B. (2021). Microwave Assisted Synthesis of Naphthaoxazole Using Peg-So3h As Heterogeneous Catalyst and Its Antimicrobial Activity. International Journal for Pharmaceutical Research Scholars, 10(1); 36 - 44

INTRODUCTION

Heterocyclic compounds consist of cyclic structures in which one or more of the ring atoms are of elements other than carbon. The common hetero atoms are nitrogen, oxygen and sulphur. About a third of known organic compounds are heterocycles.

They can be divided into alicyclic and aromatic compounds, which possess five or six member rings. The chemistry of heterocyclic compounds is one of the most complex branches of organic chemistry. It has seen unparalleled progress owing to their wide natural occurrence, specific chemical reactivity and widespread utility in the field of therapeutics.

Benzo fused azoles are an important class of compounds. They provide a common heterocyclic scaffold in biologically active and medicinally significant compounds [1-6]. Benzoxazoles are found in a variety of natural products [7] and are important targets in drug discovery [8]. They also find applications in material science as photochromic agents and laser dyes [9].

There has been a recent surge in the development of new benzoxazole syntheses because of their potential uses as cytotoxic agents [10-12], cathepsin S inhibitors [13], HIV reverse transcriptase inhibitors [14], estrogen receptor agonists [15], selective peroxisome proliferator activated receptor antagonists, anticancer agents [16] and orexin-1 receptor antagonists [17]. They have also found application as herbicides and as fluorescent whitening agent dyes [18]. 2-Arylbenzoxazoles are an important group of target molecules by virtue of their special photo physical properties [19-21] and biological activities, including antitumor, antimicrobial, and antiviral properties [22]. It has also been reported that arylbenzoxazole-containing amino acids have high fluorescence quantum yields and can be engineered into convenient fluorescent probes [23-25]. Recently, it has been reported that 2-arylbenzoxazoles are novel cholesteryl ester transfer protein inhibitors [26], and some 2-arylbenzoxazoles are highly selective amyloidogenesis inhibitors [27].

Our aim to developed novel protocol for synthesis of Naphthaoxazole using green heterogeneous catalyst PEG-SO₃H under Microwave Irradiation in shorter reaction time with good yield and all prepared compounds were checked for biological activities.

1.2 Materials and Methods

1.2.1 Chemicals and Reagents

Laboratory grade chemicals used are 3-amino-2-naphthol, Aldehydes, PEG-6000, Sulfonicacid, methylenedichloride, ether and alcohol were purchased from Samir Tech Chem. Pvt. Ltd., Vadodara, India.

1.2.2 Instruments

Bruker Avance-400 instrument was used for Proton NMR study and 100MHZ frequency instrument was used for ¹³C NMR. Parts per million unit was used to expressed chemical shift value. ABB Bomem Inc. FT-IR 3000 Spectrophotometer was used for Infrared Spectral study. Data obtained was expressed in cm^-1 unit. Shimadzu LCMS-2010 was used for MASS spectral analysis.  Perkin Elmer-2400 Series II CHNS/O Elemental Analyzer was used for Composition measurement.

1.2.3 Synthesis of PEG-SO₃H catalyst

A catalyst was prepared by report method (Hasaninejad et al., 2011) at 0^ᵒC, 10 mmol Chlorosulfonic acid (1.16 g) was mixed up with PEG-6000 solution (6 g, 1 mmol) in 10ml CH₂Cl₂. Shake the mass at room temperature for few minutes followed by allow to stand overnight and concentrated under vaccum. Followed by addition of 60ml ether & precipitates was obtained are filter and washed with 30 ml of ether three times to afford the PEG-SO₃H.

1.2.4 Methods for preparation of compounds K15-K28

 Take 1 mmol benzaldehyde in 250ml round bottom flask, add 1 mmol amount of 3-amino-2-naphthol, 5 mmol% of PEG-SO₃H with respect to 3-amino-2-naphthol and 5 ml ethanol as the solvent, reflux the mixture under microwave with optimize power level 240Watt for 3 minutes. TLC (Thin layer Chromatography) is used for monitoring reaction. Once reaction is over, remove solvent by evaporation with proper care & separated the finest product obtained. This has been confirmed using spectroscopic techniques and Melting point determination as well as elemental analysis.

Table 1 The characteristic data showing the synthesis of Naphthaoxazole

^aTLC method used for completion of reaction; ^bIsolated yields.

 1.3 Result and Discussion

1.3.1 Reaction Scheme

3-amino-2-naphthol (0.01 mole) and benzadehyde (0.01 mole) react by using catalytically amount of 5 mmol % PEG-SO₃H with respected to weight of 3-amino-2-naphthol under 10ml ethanol as solvent using Microwave to affords Naphthaoxazole (Scheme 1).

1.3.2 Optimization of reaction condition

Model reaction was carried out for optimization by taking 0.01mol benzaldehyde 1a and 0.0105mol 3-amino-2-naphthol 2 by convectional energy source to produced compound 2-aryl substituted naphthaoxazoles K-15 (Scheme 1). PEG-SO3H a heterogeneous catalyst was used in the ratio of mmole % with respect to 3-amino-2-naphthol. The reaction optimization was done in terms of reaction duration and amount of catalyst required to produced maximum yield. Reaction was monitored using thin layer chromatography. The resultant data obtained are shown in Table 2.

Table 2 Effect of different catalyst on the condensation of 3-amino-2-naphthol and benzaldehydes in ethanol under microwave

^a Reaction was monitored by TLC, ^b Isolated yields.

The same model reaction was optimized under microwave irradiation by taking different amount of catalyst, it was found that best result again obtained by taking 5mmol% amount of catalyst. The microwave power level was also optimized by taking 5mmol% amount of PEG-SO₃H with respect to diamine. It was found that best result was obtained at power level 240 Watt. At this power level reaction was completed in 3minutes with 83% yield of product as shown in Table 3.

Table 3 Data representing the optimization for synthesis of Naphthaoxazole by the assistance of MWI technique. ^{a, b}

^aReaction was monitored by TLC;  ^bIsolated yields.

1.4 Antimicrobial Activity of Compounds K15-K28

From the above Figure-1 it shown that following are the results with maximum and minimum zone of restriction vale.

(a) Against Staphylococcus aureus:

Compounds K-19 and K-23 shows maximum potency with zone of restriction-11.0 m.m. whereas minimum potency was shown by compounds K-15 and K-26 with zone of restriction 4.0 m.m.

(b) Against Bacillus megaterium:

Compounds K-17, K-19, K-23 and K-27 shows maximum potency with zone of restriction-12.0 m.m. whereas minimum potency was shown by compounds K-18 and K-25 with zone of restriction 4.0 m.m.

(c) Against Escherichia coli:

Compounds K-17, K-23 and K-26 shows maximum potency with zone of restriction-13.0 m.m. whereas minimum potency was shown by compounds K-18, K-24 and K-28 with zone of restriction 5.0 m.m.

(d) Against Proteus vulgaris:

Compounds K-22, K-23 and K-26 shows maximum potency with zone of restriction-12.0 m.m. whereas minimum potency was shown by compounds K-15, K-18 and K-21 with zone of restriction 4.0 m.m.

1.5 Characterization

K15 as a sample compound was taken for characterization. Following Spectroscopic results were obtained and its well agreement with proposed structure of 2-phenylnaphtho[2,3-d] oxazole (K-15).

Compound K-15         2-phenylnaphtho[2,3-d]oxazole
Molecular Formula	C17H11NO
Molecular Weight (g·mol-1)	245.10
Melting Point (°C)	165
1H NMR (DMSO, 400 MHz): δ ppm 7.3 (2H, m), 7.6-7.4 (3H, m), 7.7 (1H, m), 7.8 (1H, m), 8.3 (4H, m).
13C NMR (DMSO, 100 MHz): δ ppm 110.6 119.9, 124.5, 125.1, 127.3, 127.5, 128.9, 131.5, 142.2, 150.8, 156.1, 158.0, 163.0.
DEPT-135: Up peaks: 163.0, 158.0, 156.1, 150.8, 142.2, 131.5                     Down peaks: 128.9, 127.5, 127.3, 125.1, 124.5, 119.9, 110.6
IR (KBr) cm-1: 3047 (w), 1454, 1404, 1260, 968, 750 cm−1
LC-MS: 245.10
% C, H, N Analysis:  Calculated: C, 83.25; H, 4.52; N, 5.71 Observed:  C, 83.34; H, 4.60; N, 5.73

Conclusion

We have reported environment benign novel protocol for synthesis of various naphthaoxazole using 3-amino-2-naphthol and different aromatic aldehyde under microwave heating method. Reaction product was obtained in shorter reaction time with good yield of product. Prepared compounds were also tested for antimicrobial activities and it was found that K-15, K-17 to K-22 shows good activity.

Reference

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2. Kumar, D.; Jacob, M. R.; Reynolds, M. B.; Kerwin, S. M. Bioorg. Chem. 2002, 10, 3997-4004
3. DeLuca, M. R., & Kerwin, S. M. (1997). The para-Toluenesulfonic Acid-Promoted Synthesis of 2-Substituted Benzoxazoles and Benzimidazoles from Diacylated Precursors. Tetrahedron, 53(2), 457-464.
4. Gong, B., Hong, F., Kohm, C., Bonham, L., & Klein, P. (2004). Synthesis and SAR of 2-arylbenzoxazoles, benzothiazoles and benzimidazoles as inhibitors of lysophosphatidic acid acyltransferase-β. Bioorganic & medicinal chemistry letters, 14(6), 1455-1459.
5. Paramashivappa, R., Kumar, P. P., Rao, P. S., & Rao, A. S. (2003). Design, synthesis and biological evaluation of benzimidazole/benzothiazole and benzoxazole derivatives as cyclooxygenase inhibitors. Bioorganic & medicinal chemistry letters, 13(4), 657-660.
6. Edwards, P. D., Meyer Jr, E. F., Vijayalakshmi, J., Tuthill, P. A., Andisik, D. A., Gomes, B., & Strimpler, A. (1992). Design, synthesis, and kinetic evaluation of a unique class of elastase inhibitors, the peptidyl. alpha.-ketobenzoxazoles, and the x-ray crystal structure of the covalent complex between porcine pancreatic elastase and Ac-Ala-Pro-Val-2-benzoxazole. Journal of the American Chemical Society, 114(5), 1854-1863.
7. Rodríguez, A. D., Ramírez, C., Rodríguez, I. I., & González, E. (1999). Novel Antimycobacterial Benzoxazole Alkaloids, from the West Indian Sea Whip Pseudopterogorgia e lisabethae. Organic Letters, 1(3), 527-530.
8. Brown, R. N., Cameron, R., Chalmers, D. K., Hamilton, S., Luttick, A., Krippner, G. Y., … & Watson, K. G. (2005). 2-Ethoxybenzoxazole as a bioisosteric replacement of an ethyl benzoate group in a human rhinovirus (HRV) capsid binder. Bioorganic & medicinal chemistry letters, 15(8), 2051-2055.
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10. Davidson, J. P., & Corey, E. J. (2003). First enantiospecific total synthesis of the antitubercular marine natural product pseudopteroxazole. Revision of assigned stereochemistry. Journal of the American Chemical Society, 125(44), 13486-13489.
11. Sato, S., Kajiura, T., Noguchi, M., Takehana, K., Kobayashi, T., & Tsuji, T. (2001). AJI9561, a new cytotoxic benzoxazole derivative produced by Streptomyces sp. The Journal of antibiotics, 54(1), 102-104.
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Abstract

7-chloro-1-methyl-5-phenyl-3H-1,4-Benzodiazepin-2-one is the well-known psychoactive drug of class benzodiazepam. A simple, precise, accurate, reproducible RP-HPLC method was developed and validated for the simultaneous estimation of 7-chloro-1-methyl-5-phenyl-3H-1,4-Benzodiazepin-2-one in bulk and in pharmaceutical dosage forms. Chromatographic separation was carried out on a Thermo-hypersil C8 column (250mm×4.6mm i.d,5μm) utilizing a mobile phase consisting of acetonitrile and 0.01M ammonium phosphate buffer (pH adjusted to 3.0 with ortho phosphoric acid) in the ratio of 55:45 v/v at a flow rate of 1ml/min with UV detection at 242nm. The retention times of 7-chloro-1-methyl-5-phenyl-3H-1,4-Benzodiazepin-2-one were 2.23 min. The developed method was validated in terms of selectivity, sensitivity, accuracy, precision, linearity, specificity, limit of detection and limit of quantification. The linear range was found to be 20-100 μg/ml.

Keywords

7-chloro-1-methyl-5-phenyl-3H-1,4-Benzodiazepin-2-one, Selectivity, Sensitivity, Reverse Phase HPLC, Validation, Psychoactive Drug

Cite This Article

Makwana, K., Vyas, K. B. (2021). Development and Validation of 7-Chloro-1-Methyl-5-Phenyl-3h-1,4 Benzodiazepin-2-One by RP HPLC. International Journal for Pharmaceutical Research Scholars, 10(3);10 - 17.

INTRODUCTION

Benzodiazepines (BZDs) represents one of the important and highly explored class of seven-membered aromatic heterocycles containing two ring nitrogen that are critical for numerous applications in the pharmaceutical industry and organic synthesis of complex molecules [1]. Due to their diverse spectrum of biological activities, they were considered as “privileged structures” in medicinal chemistry.

Further, they are key synthons for the synthesis of various fused ring compounds [2,3]. As such, further development of BZDs has gained significant attention of organic and medicinal chemists in a quest to discover new and efficacious benzodiazepine based therapeutic agents [4].

7-chloro-1-methyl-5-phenyl-3H-1,4-Benzodiazepin-2-one have been majorly prescribed in many parts of the world; first as anxiolytics and then as hypnotics [5]. They are extensively indicated for various CNS disorders such as anxiolytics (chlordiazepoxide and diazepam), anticonvulsants (clonazepam, and clobazam), muscle relaxants, anesthesia (midazolam) and insomnia, for some motor disorders and in psychoses (olanzapine and clozapine) [6]. The benzodiazepines were classified as short-acting, intermediate acting and long-acting depending upon their duration of action [7]. The BDZs exert their effect by binding to the central benzodiazepine receptors which are located at the post and presynaptic membranes. However, certain side effects are associated with the short- and long-term use of benzodiazepines which includes confusion, drowsiness, amnesia, and ataxia [8].

Following the guidance provided by structure-activity relationship studies, compounds with high potency and expanded spectrum of activity, improved absorption, and distribution properties were synthesized and biologically evaluated in various disease areas. Benzodiazepine derivatives were reported to possess various pharmacological activities such as antimicrobial, anticancer, anti-anxiolytic, antidepressant, anticonvulsant, antitubercular, anti-inflammatory, analgesic, antihistaminic and anti-anxiety activities [9-11]. Various benzodiazepine-based compounds have different groups or substituents attached to their core structural motif at positions 1, 2, 5, or 7 respectively [12]. These different side groups affect the binding properties of molecules with the relevant target proteins or receptors (such as BET) and hence modulate their pharmacological properties, the potency of biological response and the pharmacokinetic profile [13].

In presence study, we have reported method for development and validation of 7-Chloro-1-Methyl-5-Phenyl-3h-1,4-Benzodiazepin-2-One by well-known RP-HPLC method.

1.2       Experimental Part

1.2.1    Apparatus and Software

Chromatography was performed on Shimadzu (Shimadzu Corporation, Kyoto, Japan) chromatographic system equipped with Shimadzu LC-20AT pump and Shimadzu SPD20AV absorbance detector. Samples were injected through a Rheodyne 7725 injector valve with fixed loop at 20 μl. Data acquisition and integration was performed using Spinchrome software (Spincho biotech, Vadodara). The chromatographic elution of analyte was obtained by using Thermo-hypersil C8 column (250mm×4.6mm i.d,5μm).

1.2.2    Reagents and Chemicals

7-chloro-1-methyl-5-phenyl-3H-1,4-Benzodiazepin-2-one was provided as gift sample from Samir Tech Pvt. Ltd. Vadodara, India. HPLC grade Acetonitrile and Ortho Phosphoric Acid, Dipotassium monohydrogen phosphate AR grade was purchased from Samir Tech Pvt. Ltd. Vadodara, India. The pharmaceutical samples used in the present study include 7-chloro-1-methyl-5-phenyl-3H-1,4-Benzodiazepin-2-one 5% ointment.

1.2.3    Chromatographic Conditions

The mobile phase comprised of Acetonitrile: Dipotassium monohydrogen phosphate buffer pH 3.2 in the proportion of 55:45. Resulting solution was degassed by ultrasonication for 10minutes.

1.2.4    Preparation of Standard Solution Of 7-Chloro-1-Methyl-5-Phenyl-3h-1,4-Benzodiazepin-2-One[14]

Stock solution of (1000 µg/ml) was prepared by accurately weighing 10 mg of 7-Chloro-1-Methyl-5-Phenyl-3h-1,4-Benzodiazepin-2-Onein 10 ml volumetric flask. The drug was dissolved in Acetonitrile and the solution was diluted to volume. Further dilutions were made from this stock solution and the injection volume was kept 20 μL. A calibration curve was plotted between concentrations against their respective area for 7-Chloro-1-Methyl-5-Phenyl-3h-1,4-Benzodiazepin-2-One. From the calibration curve, it was found that linearity range is between 20- 100ug/ml.

1.3       Analysis of Marketed Formulation

Extraction Procedure:

An amount equivalent to 10mg [0.2g for Ointment [15](5%), 0.5g for Gel (2%), and 0.46ml for Injection (2%), 0.1g equivalent to four sprays for Aerosol (10%)] was taken and dissolved in 10 ml of ACN to get 1000ug/ml of stock concentration. The stock solution was sonicated for 10 minutes and was filtered through Whatman filter paper. From the stock solution 0.6ml was taken in 10 ml volumetric flask. The volume was made up to the mark with ACN to get solution of 60ug/ml. The solution was finally filtered through 0.2um syringe filter was injected into HPLC Transdermal patch (5%). An amount equivalent to 10 mg(0.2g) was taken in 10 ml of Dipotassium monohydrogen phosphate buffer 10 mM of pH3.0 and was magnetically stirred for 2 hours. The solution was then sonicated for 15 minutes and was then filtered through Whatman filter paper. From this stock solution 0.6 ml was taken in 10 ml volumetric flask. The volume was made up to the mark with ACN to get solution of 60ug/ml. The solution was finally filtered through 0.2um syringe filter was injected into HPLC

1.4       Result and Discussion

1.4.1    Optimization of Chromatographic Conditions

To optimize the chromatographic conditions, the effect of chromatographic variables such as composition of mobile phase, ratio of mobile phase and flow rate were studied. The resulting chromatograms were recorded and the chromatographic parameters such as capacity factor, asymmetric factor, and theoretical plates were calculated. Finally, a simple and inexpensive method was developed by using a combination of Acetonitrile and Dipotassium monohydrogen phosphate buffer in ratio 55:45. Optimized chromatographic conditions are listed in Table 1.

1.4.2    Method Validation [16].

1.4.2.1 Linearity

The calibration curve was constructed by plotting concentrations of 7-Chloro-1-Methyl-5-Phenyl-3h-1,4-Benzodiazepin-2-Oneversus peak areas, and the regression equations were calculated. The linearity of the method was investigated by using concentrations in the range20-100μg/ml.Retention time for 7-Chloro-1-Methyl-5-Phenyl-3H-1,4-Benzodiazepin-2-One was found to be2.23 min.. The linear regression equation is Y=  0.831x+1.583(r2=0.999). The plot obtained from linear regressions given in Figure 1.

1.4.2.2 Limit of Detection and Limit of Quantification

The limit of detection (LOD) and limit of quantification (LOQ) were calculated according to the 3.3 σ/s and 10 σ/s criteria, respectively, where σ is the standard deviation of the peak area and s is the slope of the corresponding calibration curve[16].The LOD and the LOQ for HPLC were found to be 1.54ug/ml and 4.68ug/ml.

1.4.2.3 Precision

The precision of the proposed method was assessed as intraday and inter day precision. Three replicate injections of specific standard at various time intervals on the same day were injected into system for intraday precision and were repeated on three different days for Inter day precision. The % RSD (Relative Standard Deviation) of the results was calculated

 Table 2: Intraday precision of7-Chloro-1-Methyl-5-Phenyl-3H-1,4-Benzodiazepin-2-One

Table 3: Inter day precision of7-Chloro-1-Methyl-5-Phenyl-3H-1,4-Benzodiazepin-2-One

1.4.2.4 Accuracy

Accuracy of the method was studied using standard addition method at three different levels (80, 100, and 120%) by recovery experiments. Known amounts of standard solutions containing 7-Chloro-1-Methyl-5-Phenyl-3H-1,4-Benzodiazepin-2-One (48, 60,72ug/ml) were added to one of the marketed formulations of concentration 60 ug/ml to reach 80%, 100% and 120% levels. Percentage Recovery was the mean of three determinations at each standard addition level

Table 4: Accuracy data of 7-Chloro-1-Methyl-5-Phenyl-3H-1,4-Benzodiazepin-2-One

1.4.2.5 Analysis of Marketed Formulation [6]

When the 7-Chloro-1-Methyl-5-Phenyl-3H-1,4-Benzodiazepin-2-Onemarketed formulation was analyzed by these proposed HPLC method, sharp peaks were obtained at tR 5.43 minutes, when scanned at 263nm. The amount of the label claim measured is given in table 6, all the formulations are within the limits (95%-105%), for patch the limits are (90%-110%)

Table 6: Assay results of marketed formulation

CONCLUSION

The proposed reverse phase high performance liquid chromatography method has been developed for the analysis of 7-Chloro-1-Methyl-5-Phenyl-3H-1,4-Benzodiazepin-2-Onein their marketed formulation. The method was validated as per ICH guideline. % Assay values of marketed formulation were found to be in the prescribed range. Thus, the proposed HPLC method can be used for routine quality control analysis of 7-Chloro-1-Methyl-5-Phenyl-3H-1,4-Benzodiazepin-2-Onefrom its various Pharmaceutical dosage forms.

REFERENCE

1. Sternbach, L. H. (1979). The benzodiazepine story. Journal of medicinal chemistry, 22(1), 1-7.
2. Walser, A., & Fryer, R. I. (1991). Dihydro‐1, 4‐Benzodiazepinones and Thiones. Chemistry of Heterocyclic Compounds: Bicyclic Diazepines: Diazepines with an Additional Ring, 50, 631-848.
3. Qadir, M. I. (2015). Recent structure activity relationship studies of 1, 4-benzodiazepines. Open Journal of Chemistry, 1(1), 008-012.
4. Bernardy, N. C., & Friedman, M. J. (2015). Psychopharmacological strategies in the management of posttraumatic stress disorder (PTSD): what have we learned?. Current Psychiatry Reports, 17(4), 20.
5. Kaufmann, C. N., Spira, A. P., Depp, C. A., & Mojtabai, R. (2018). Long-term use of benzodiazepines and nonbenzodiazepine hypnotics, 1999–2014. Psychiatric Services, 69(2), 235-238.
6. Paton, C. (2002). Benzodiazepines and disinhibition: a review. Psychiatric Bulletin, 26(12), 460-462.
7. Batlle, E., Lizano, E., Viñas, M., & Pujol, M. D. (2019). 1, 4-Benzodiazepines and New Derivatives: Description, Analysis and organic synthesis. Medicinal chemistry, 63-90.
8. Goodman, L. S. (1996). Goodman and Gilman’s the pharmacological basis of therapeutics(Vol. 1549). New York: McGraw-Hill.
9. Barbui, C., Cipriani, A., Patel, V., Ayuso-Mateos, J. L., & van Ommeren, M. (2011). Efficacy of antidepressants and benzodiazepines in minor depression: systematic review and meta-analysis. The British Journal of Psychiatry, 198(1), 11-16.
10. File, S. E. (1990). The history of benzodiazepine dependence: a review of animal studies. Neuroscience & Biobehavioral Reviews, 14(2), 135-146.
11. Sawada, N., Uchida, H., Suzuki, T., Watanabe, K., Kikuchi, T., Handa, T., & Kashima, H. (2009). Persistence and compliance to antidepressant treatment in patients with depression: a chart review. BMC psychiatry, 9(1), 1-10.
12. Wick, J. (2013). The history of benzodiazepines. The Consultant Pharmacist®, 28(9), 538-548.
13. López-Muñoz, F., Álamo, C., & García-García, P. (2011). The discovery of chlordiazepoxide and the clinical introduction of benzodiazepines: half a century of anxiolytic drugs. Journal of anxiety disorders, 25(4), 554-562.
14. Gandhi, M., & Mashru, R. Development and Validation of RP-HPLC Method for Estimation of Lidocaine in Various Pharmaceutical Dosage Forms.
15. Kumar, B. K., Rajan, V. T., & Begum, N. T. (2012). Analytical method development and validation of Lidocaine in ointment formulation by UV spectrophotometric method. International Journal of Pharmacy and Pharmaceutical Sciences, 4(2), 610-4.
16. Gandhi, M., & Mashru, R. Development and Validation of RP-HPLC Method for Estimation of Lidocaine in Various Pharmaceutical Dosage Forms.

Abstract

Moyamoya disease (MMD) is a rare and unique cerebrovascular disease. The term “moyamoya” is Japanese and refers to a hazy puff of smoke or cloud. In people with moyamoya disease, this is how the blood vessels appear in the angiogram. MMD is characterized by the progressive stenosis of the distal internal carotid artery (ICA) resulting in a hazy network of basal collaterals called moyamoya vessels. This may be a consequence of Mutations in a few genes. In addition, MMD is also associated with many genetically transmitted disorders, including neurofibromatosis, Down syndrome, Sickle cell anemia, and Collagen vascular disease. It follows bimodal age distribution. Younger populations present with ischaemic symptoms, whereas adults show hemorrhagic symptoms The exact cause remains unknown. Immune, genetic and other factors contribute to this disease. It follows complex pathophysiology resulting in neovascularization as a compensatory mechanism. Diagnosis is based on cerebral angiography using the DSA scale. Treatment involves managing symptoms with medicine or surgery, improving blood flow to the brain, and controlling seizures. Revascularization helps to rebuild the blood supply to the underside of the brain.

Keywords

Moyamoya disease, intracarotid artery, cerebrovascular, ischemia, haemorrhage, seizures, revascularization

Cite This Article

Ahamadi, A., Fatima, A., Siddiqua, A., Ali, A. A., Nayak, S. P. S. (2021). A Clinical Review on Moyamoya Disease Management. International Journal for Pharmaceutical Research Scholars, 10(3);01 - 09

INTRODUCTION

Moyamoya disease (MMD) is a unique cerebrovascular disease characterized by the progressive stenosis of the distal internal carotid artery (ICA) that results in a hazy network of basal collaterals called moyamoya vessels. It is of unknown etiology. The diagnosis is based on angiographic findings. The angiographic findings differ according to the stages of MMD.

But sometimes these angiographic findings may not be sensitive. Therefore the current diagnosis is based on the presence of basal collaterals. [1,2]

Higher incidents are seen in the Asian population compared to the European and North American populations. There are 2 clinical signs of moya moya disease -1. Cerebral ischemia, 2. Cerebral hemorrhage. These differ in the pediatric and adult populations. In most, pediatric transient cerebral ischemic attacks and cerebral infarctions are seen along with mental decline and seizures. Whereas in adults 50 percent population experience intracranial hemorrhage and the remaining 50 may experience an ischaemic stroke.[3]

Studies have shown that approximately 10% of the population has genetically inherited this disease. Mutations in few genes may cause MMD. In addition, MMD is also associated with many genetically transmitted disorders, including neurofibromatosis, Down syndrome, sickle cell anemia, and collagen vascular disease.[1,4,5,6,7]

HISTORY

MMD was first described in Japanese literature by Takeuchi and Shimizu in 1957 as a case of “hypoplasia of the bilateral internal carotid arteries.”[8] It was first published in English literature by Kudo, who described it as a “spontaneous occlusion of the circle of Willis” in 1968.[9] The popular terminology “MM” was suggested soon after in 1969 by Suzuki and Takaku. “MM” is a Japanese expression that refers to “something hazy, like a puff of cigarette smoke drifting in the air.” Suzuki and Takaku coined the term MM to describe the characteristic angiographic appearance of the dilated collateral arteries that develop at the base of the brain.[8]

NEUROANATOMY

The brain receives blood supply from anterior and posterior circulation.

1. Anterior circulation receives blood from the internal carotid artery and supplies it to the cerebral hemisphere [frontal, parietal, temporal lobes]
2. Posterior receives from vertebral arteries and supplies to the hindbrain [brain stem, cerebellum, occipital lobe, and posterior part of cerebral hemisphere]

The posterior and anterior blood vessels connect and form an anatomical structure called the “circle of Willis”

1. Structure: The anterior communicating artery connects with the bilateral anterior cerebral artery these further connect to inter carotid artery, which runs into the brain through the neck. The inter carotid artery intersects with the anterior cerebral artery and the remaining inter carotid artery forms the middle cerebral artery. Posteriorly this middle carotid artery is connected to the posterior cerebral artery via the posterior communicating artery. The basilar artery is formed by the fusion of the posterior cerebral artery. These then divide into the vertebral artery and further gives rise to the single anterior spinal artery.[10]
2. Function: The function of the cow is to provide blood flow between the anterior and posterior regions of the brain and to protect against ischemia and other blood vessel diseases.[11]

EPIDEMIOLOGY  

Most common in Asian countries like china, japan, and Korea. in japan prevalence was 3.16 per 100,000 in 1994, which raised to 10.5 per 100,000 in 2006. The US and Europe have one-tenth of prevalence compared to that of Japan. Females are twice as prone compared to males. Age-wise it is seen in either pediatrics (5-10 yrs) or adults (25-49yrs) this is called bimodal age distribution. The younger population shows ischaemic symptoms, whereas adults show hemorrhagic symptoms.[12]The presence of genetic susceptibility has been shown to play role in existing data.[13]

GENETIC ASSOSCIATION  

According to a Japanese study, 5 different chromosomal regions are associated: 3p24-p26, 6q25, 8q23, 12p12, and 17q25.

1. RNF213 as a susceptible gene for MMD :

Also known as ring finger 213 is identified as the strongest gene for MMD. The studies show that there are two variants of the RNF213 gene. They are, PR4810KRNF213 variants are related to the ischaemic type of MMD while non-PR4810KRNF213 is related to haemorrhagic type. [1,4,5,6,7]

2. Pathophysiology according to gene involvement:

Data suggest that it is not an inflammatory disease but still inflammation plays an important role.(fig.1)

RNF213 is also associated with vascular risk like HTN resulting in hemodynamic stress. [1]

ETIOLOGY

The exact cause is not known. It is genetically inherited. Immune, genetic and other factors contribute to this disease. Inflammation plays a role.[14]

Inherited conditions and/or association:

• Sickle Cell Disease or trait
• Down Syndrome (Association)
• Neurofibromatosis type 1 (Association)

Acquired conditions:

• Head and/or neck irradiation
• Chronic meningitis
• Skull base tumor
• Atherosclerosis of skull base arteries
• Arteriosclerosis
• Cerebral vasculitis [15]

PATHOPHYSIOLOGY 

This is a complex process. The pathophysiological features of the stenotic segment are the presence of microcellular thickening of the intima, irregularities in an elastic lamina, medial thinness, and a decreased outer diameter. There is a proliferation of endothelial cells and stenosis as a result of fibrocellular thickening of intima. Neovascularization is seen as a result of an increase in stenotic segment. Neovascularization is a compensatory mechanism for decreased cerebral blood flow. The new vessels formed are known as moyamoya vessels. It is an active process. They have dilated perforating arteries characterized by pathological findings like fibrin deposits in walls, fragment elastic lamina, attenuated media, and formation of aneurysms. [1] Recent studies have also shown the involvement of progenitor cells and circulating stem cells in the pathogenesis of MMD. [13]

CLINICAL PRESENTATION

Symptomatology in MMD is related to the compromised blood flow to the cerebral hemisphere and its consequences. The most common symptom thus is an ischemic stroke, and the signs vary according to the vascular territory affected. Anterior circulation is more often affected than posterior circulation.

The MM vessels which develop in response to stenosis of ICA are small in caliber and under hemodynamic stress. They are vulnerable and prone to bleed. Microaneurysms tend to develop in these vessels, and these aneurysms can rupture under stress. The incidence of aneurysms in MMD varies from 1% in children to 6.2% in adults.[9] Thus, the second common presentation in MMD is a cerebral bleed. Dilation of meningeal and leptomeningeal collateral vessels can result in headache.

Chronic infarcts with resultant gliosis can also result in seizures, focal deficits, and involuntary movements [Figure 2]. Chronic global hypoperfusion can result in a progressive decline in neurocognitive function. Involvement of the posterior circulation territory may result in visual field defects, diplopia, scintillating scotomas, ataxia, and vertigo.[8]

bleeding is more common in adults [Figure 3].[16-17]^] One characteristic feature in children is that ischemic events get precipitated by strenuous events, such as coughing, crying, hyperventilating, or blowing. Hypocapnia-induced vasoconstriction, accompanied by a transient reduction in cerebral blood flow (CBF) in an already compromised cerebral circulation, is responsible for these events.[18] Cerebral hemorrhage is more common in adults. Hemorrhage is the presentation in 40%–65% of adult patients, and the common locations are the basal ganglia (40%), thalamus (15%), or ventricular system (30%).[19-20] Metastatic carcinoma is also seen.[21] Younger population shows ischemic symptoms, where as adults shows hemorrhagic symptoms.[12] Growth hormone deficiency, Headache, Hemorrhagic stroke, Transient ischemic attack are also few symptoms. [21]

DIAGNOSIS

Diagnosis is based on cerebral angiography. Japan has made guidelines for MMD in the year 1996, based on angiographic findings, and is as follows

1. Anterior cerebral artery and medial cerebral artery occlusion.
2. Abnormal vascular network.
3. Bilateral manifestation.[3,22]

The recent guidelines were received in 2012 and remained the gold standard. The below figures show the staging of MMD (table.1) according to digital subtraction angiography [DSA].[23]

Table. 1 staging of MMD

The MRA results are evaluated by adding the scores and then staging. A score of 0-1 represents stage 1 (DSA stage 1 and 2), A Score of 2-4 represents stage 2 (DSA stage 3), A Score of 5-7 represents stage 3 (DSA stage 4), A Score of 8-10 represent stage 4 (DSA stage 5 and 6).[3,22,23]

MANAGEMENT

1. Pharmacotherapy

Calcium channel blockers (nimodipine, verapamil, nicardipine, etc) were given for symptomatic treatment. Studies showed no further episodes of transient ischemic attacks, seizures, or headaches in patients. Studies show nicardipine may have a beneficial effect on cerebral hemodynamics and may prevent ischemic sequelae from optimizing existing collateral circulation.[24] Additionally, antiplatelets and antiepileptics may also be used for MMD associated seizures disease. Drug therapy is usually ineffective and has only a symptomatic effect, revascularization surgery is the only option.[25]

2. Non-pharmacological management

Maintain spco2 levels between 35-40 mmHg. During direct arterial bypass measures like mild hypothermia (33-35degree centigrade) are maintained. Barbiturates are used to maintain mild HTN.[26-27] They are 3 types of surgeries currently available.

3. Direct vascularization :

In this technique, STA is connected to MCA. It has the benefit of immediately increased blood flow compared to indirect vascularization. This is preferred usually in adults.[28]

STA-MCA bypass: parietal branch of STA is taken as a donor and the posterior branch of MCA as the recipient’s vessel. Sometimes anterior branch can be taken as donor and M2 as a recipient. Papevarin is used preoperatively for dilating arteries. And is irrigated post-operatively. Aspirin 325mg/dl is administered post-operatively.[25] Few cases present with hemorrhagic stroke with dangerous choroidal anastomosis on cerebral angiography.[29]

4. Indirect revascularization :

Usually done in children(presence of small arteries). As the name specifies this procedure is indirect and relies on the subsequent formation of collateral vessels. To enhance blood delivery, They don’t provide immediate revascularization and hence delayed blood flow is a drawback.[25] This further includes :

1. ENCEPHALOMYSYNANGIOSIS: In this technique, temporal muscle is implanted on the lateral surface of the brain, and suturing is done in place to the dural edge.
2. PIAL SYNOPSIS: It involves opening of arachnoid along with dural and suturing with STA.

• ENCEPHALODUROARTERIOMYOSYNANGIOSIS: It allows the formation of collateral from STA and deep temporal artery.

1. OTHER TECHNIQUES: Includes omental transplantation, multiple burr holes, cervical carotid sympathectomy.[30,31,32,33]
   5. Combining direct and indirect revascularization :

It has the advantage of immediate revascularization and also revascularization of brain regions outside the distribution of a single major arterial division. Usually, STA-MCA direct bypass is combined with encephaloduralarterysynangiosis. The parietal branch of STA is selected for encephaloduralarterysynangiosis, while frontal for direct bypass. [25]

CONCLUSION

Moyamoya disease (MMD) refers to Cerebrovascular abnormalities with a hazy network of angiographic findings who may have genetic susceptibilities but no associated conditions. The angiographic findings differ according to the stages of MMD. The current diagnosis is based on the presence of basal collaterals.

Cerebral ischemia and Cerebral hemorrhage are the distinctive signs. These signs differ in pediatric and adult populations. Studies have shown that approximately 10% of the population has genetically inherited this disease. Pathophysiology is complex following neovascularization and involvement of progenitor cells and circulating stem cells.

MMD has varying degrees of severity, so not every patient needs surgery immediately. For patients with little to no symptoms, it is often managed with medication to control the contributing risk factors. In case of stroke or hemorrhage, surgical procedures to improve blood flow to the brain are often recommended. The management differs in the pediatric and adult populations.

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4. Ikeda, H., Sasaki, T., Yoshimoto, T., Fukui, M., & Arinami, T. (1999). Mapping of a familial moyamoya disease gene to chromosome 3p24. 2-p26. The American Journal of Human Genetics, 64(2), 533-537.
5. Inoue, T. K., Ikezaki, K., Sasazuki, T., Matsushima, T., & Fukui, M. (2000). Linkage analysis of moyamoya disease on chromosome 6. Journal of child neurology, 15(3), 179-182.
6. Sakurai, K., Horiuchi, Y., Ikeda, H., Ikezaki, K., Yoshimoto, T., Fukui, M., & Arinami, T. (2004). A novel susceptibility locus for moyamoya disease on chromosome 8q23. Journal of human genetics, 49(5), 278-281.
7. Sakurai, K., Horiuchi, Y., Ikeda, H., Ikezaki, K., Yoshimoto, T., Fukui, M., & Arinami, T. (2004). A novel susceptibility locus for moyamoya disease on chromosome 8q23. Journal of human genetics, 49(5), 278-281.
8. Kudo, T. (1968). Spontaneous occlusion of the circle of Willis: a disease apparently confined to Japanese. Neurology, 18(5), 485-485.
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21. Chiu, D., Shedden, P., Bratina, P., & Grotta, J. C. (1998). Clinical features of moyamoya disease in the United States. Stroke, 29(7), 1347-1351.
22. Fukui, M. (1997). Guidelines for the diagnosis and treatment of spontaneous occlusion of the circle of Willis (‘moyamoya’disease). Research Committee on Spontaneous Occlusion of the Circle of Willis (Moyamoya Disease) of the Ministry of Health and Welfare, Japan. Clinical neurology and neurosurgery, 99, S238-40.
23. on the Pathology, R. C. (2012). Guidelines for diagnosis and treatment of moyamoya disease (spontaneous occlusion of the circle of Willis). Neurologia medico-chirurgica, 52(5), 245-266.
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¹Department of Pharmacy Practice, Shri Sarvajanik Pharmacy College, Mehsana-384001, Gujarat, India ²Department of Pharmaceutics, Shree Dhanvantary Pharmacy College, Kim, Surat-394110, Gujarat, India ³School of Pharmaceutical Sciences, Jaipur National University, Jaipur-302017, India

Abstract

To check the prevalence of Antimicrobial Resistance Pattern in Tertiary Care Hospital. It was a single centric prospective observational study. This study collected the data of 250 samples, in patients who were admitted to in-patient department and in whom at least one antibiotic was prescribed.  The data such as patient demographics, diagnosis, the laboratory data such as count of White Blood Cells (WBC), C- Reactive Protein (CRP) and Procalcitonin (PCT). The culture sensitivity reports were collected from the Microbiology Department of the hospital. The positive samples were further evaluated for the study. Statistical analysis is done by using Microsoft Excel 2007. In this present study, out of 250 samples, 87 (35%) samples were positive. The most common specimen which had shown highest growth was Tracheal (38%), followed by Blood sample (21%) and third was Urine (15%). The most common organisms which were found to be resistant included Klebsiella Pneumoniae (24%), Acienobacter Baumanii (21%), Pseudomonas Aeruginosa (18.39%) and E Coli (11.50%). The maximum resistance was shown by five groups viz, carbapenem (54%), fluoroquinolones (46%), penicillins (45.51%), cephalosporin (37.12%) and aminoglysocides (33.51%). According to antibiogram, all of these four bacteria had shown highest resistance against fluoroquinolones. The results obtained from this study can guide rationale use of antibiotics which can contribute to effective application of Antimicrobial Stewardship Program. Antimicrobial resistance, Tertiary care hospital, sample, bacteria, Antimicrobial Stewardship Program.

Keywords

Antimicrobial resistance, Tertiary care hospital, sample, bacteria, Antimicrobial Stewardship Program

Cite This Article

Shethia, F. K., Suryawanshi, M. V., Anand, I. S. (2021). Prevalence of Antimicrobial Resistance Pattern in Tertiary Care Hospital: A Prospective Observational Study. International Journal for Pharmaceutical Research Scholars, 10(2);01 - 17

INTRODUCTION

When micro-organism such as bacteria, viruses, fungi and parasites changes over the period of time and also they no longer respond to the treatment leading to infections difficult to treat, which results in the increasing the spread of infection, the severe illness and death, it is when Antimicrobial Resistance (AMR) occurs.

This leads to the drug resistance, this decreases the effectiveness of the antibiotics and other antimicrobial medicines, therefore leading to difficulty in treating the infection and either resistance is increased that much that makes impossible to treat the infection. ^[1]

The first commercialized antibiotic – Penicillin, was come into existence in 1928 discovered by Alexander Fleming. Since then, the new discovery of antibiotics and the resistance were all together. The microorganisms always try to survive and resist the new drugs. In this way, it’s difficult to manage the resistance developed by it. ^[2]

AMR naturally occurs over the period of time, the most common cause is through genetic changes. These antimicrobial resistant organisms are found almost everywhere in people, plants, animals, food and also in the surrounding environment (in soil, air and water).

The organisms are easily transmitted from one individual to another or between the individuals or they are also transmitted from people and animals, also including from food of animal origin.

The main causes of the antimicrobial resistance includes the its misuse and/or its overuse; poor infection and disease prevention and control in health-care facilities and farms; lack of access to clean water, sanitation and hygiene (WASH) for both humans and animals; poor access to quality, vaccines and diagnostics; affordable medicines, lack of enforcement of legislation; and also the lack of awareness and knowledge. ^[1].

The antimicrobial resistance affects the high, middle as well as low income countries. There are specific type of diseases such as tuberculosis (TB) and gonorrhoea which have high rates of the antimicrobial resistance.

A reliable data estimation of the global scenario of the prevalence of antimicrobial resistance is difficult to know as the data are not collected consistently and systematically.

Yet, the data collected from Europe indicates the excess mortality rate because of the resistant bacteria in the hospital infections which exceeds 25,000 annually, with the annual cost of at least worth €1.5 billion.

Also the data of outpatient from the US indicates that more than 63,000 people die in a year because of resistant bacterial infections.

A combination having the higher rates of infections, less developed health systems and also low quality and improper use of antibiotics, as among the other factors, in turn, it increases the burden of the antimicrobial resistance in middle and low income countries.^[3]

It is not easy to get the accurate data of the drug resistance, but though it is predicted that the burden of antimicrobial resistant infection will increase to nearly 10 million deaths per year by 2050 and also it will increase the total GDP loss of $100.2 trillion by 2050 if proper steps are not taken to prevent. ^[4]

Although antimicrobial resistance occurs usually naturally, it is also occurs by inexistent programmes for infection prevention and control (IPC), misuse of antimicrobial medicines, weak laboratory capacity, inadequate surveillance, insufficient regulation of the use of antimicrobial medicine and poor-quality medicines.

Several intrinsic factors such as gene amplification, point mutation as well as extrinsic factors like horizontal transfer of resistant gene between bacteria within and across species by transposons, integrins or plasmids has contributed for the development of resistance, and this cannot be reduced once developed even after the stopping its usage. Also social factors like deficient hygienic practices, overcrowding and demographic changes contributes to the development of AMR. ^[5]

In India, the major causes of AMR are the unregulated access to the antibiotics, which also includes the over the-counter sale and sale without prescription or with invalid prescription; rising incomes, and it also includes the preservation of the financial incentives to the providers for prescribing antibiotics, this often can be by the patient demand and their expectations.

The global attention to the AMR issue in India was drawn by the controversy of the nomenclature of the New Delhi Metallo-beta-lactamase-1 (NDM-1), and since then the AMR issue has gained a lot of attention in context to public health concern.

In India, AMR is of special concern as it as it includes the factors like high rates of the bacterial infections, increasing of intensive animal farming, especially in small holder settings with minimal oversight and quality control and also the poor sanitary and hygiene conditions.

This issue related to AMR is now not only limited to the clinical or hospital setting but is also spread to the animal/food/livestock sector, and also the environmental contamination as a source for the spread of resistance genes as well as the antibiotic residues which in turn promotes the selection pressure.^[6]

India is one of the nation in which there is highest burden of the infections which are related to bacteria. In India today, the crude mortality from infectious diseases is 417 per 100,000 persons. As a result of which, AMR is likely to be high in India. The resistance not only occur from the older and more frequently used classes of drugs but also is emerging from the newer and more expensive drugs viz, carbapenems.^[7]

The higher rates of AMR were observed from toxin-producing E. Coli which were isolated from the calves with diarrhoea in Kashmir Valley and the Gujarat.

All of the strains from the Gujarat were observed to be resistant to at least 3 antibiotics, and also almost half were resistant to 8 or more of the 11 antibiotics which were tested.

Resistance was found everywhere for kanamycin and cephalexin and it was above 50% for 7 of the antibiotics which were tested. ^[8]

Also antimicrobial resistance for H. pylori treatment is a growing problem in Gujarat. ^[9]

There is wide availability of the effective antibiotics which is under the threat, and it is jeopardizing the today’s modern healthcare. The forecasts of economic costs, which is similar to those 2°C rise in global average surface temperature above the preindustrial levels.

AMR is becoming an urgent need of attention for policy makers, and this in turn raising the pressure to secure international commitments to solve the problem of the AMR. ^[10]

During this pandemic situation of coronavirus disease 2019 (COVID-19), there are threats which are potentially affecting the antimicrobial stewardship activities leading to antimicrobial resistance. For example, the many of the individuals are presenting with mild disease without pneumonia or moderate disease with pneumonia is receiving antibiotics.

Data published by World Health Organisation (WHO) shows that shows that azithromycin is being widely used with Hydroxychloroquine although it is not yet recommended outside of COVID-19 clinical trials. ^[11]

Both economically and also for human health and its lives, the cost of the antimicrobial resistance is extremely large.

The new report published (Stemming the superbug tide, 7 Nov 2018) by Organization for Economic Co-operation and Development (OECD) predicts that 2.4 million people in Australia, Europe and North America will die from the infections with resistant microorganisms in the next 30 years as well as it could cost up to US$3.5 billion per year. ^[12]

AMR leads to global health issues and also contributes to the economic rise. To achieve the issues related to costs, the medical strategy is to reduce the prescribing unnecessary antibiotics, to protect the effectiveness of current antimicrobials in the long term of time. To gain future benefits, economic evaluations of health care interventions can be done for short term costs. ^[13]

Optimized use and appropriate prescription of antimicrobials gives direction to the principles of antimicrobial stewardship activities, together with the quality diagnosis and treatment, and the reduction and the prevention of infections. ^[11]

AMR occurs when microorganisms like bacteria and fungi contribute to develop the resistance which is used for treatment. In this way, microorganisms are continually grow and not killed. AMR can affect any stage of their life, it also affects the veterinary, healthcare as well as it affects the agricultural industries, resulting a urgent threat to global health problems. In the United States (US) in each year at least 2.8 million people are infected with the resistant microorganisms and as a result of which more than 35,000 people die. ^[2]

Material and Method

Material

This was a Single Centric Prospective Observational Study which had studied the prevalence of antimicrobial resistance pattern.

This study was conducted in Tertiary Care Hospital at Gandhinagar. The data of patients which were admitted to In Patient Department from February 2021 to April 2021 were collected for the study. The sample size of the study was calculated by online sample size calculator and that was 250 samples.

Inclusion criteria

1. Data of all patients who were admitted to in Patient Department were included in the study irrespective of the age.
2. Data of both genders were included.
3. Data of only those patients for whom at least one antibiotic was prescribed and at least one culture report had been sent were included in the study.
4. Culture data of all the sample types (blood, tracheal, urine swab, pus, sputum, fungus, others and tissue) were included in the study.

Exclusion criteria

1. Data of Outpatient Department (OPD) were excluded.
2. The data of patient with incomplete information were excluded.

Data of each patient enrolled in the study were recorded in the paper Case Report Form (CRF) which was prepared for the study.

Prior approval was obtained from Sarvajanik Clinical Research Ethics Committee before the study was initiated. Data collected were kept confidential and it won’t be disclosed to anyone.

Data collection procedure

 The study data of each patient was recorded in paper CRF.

• The files of patient were checked for the collection of demographic details as well as the laboratory reports along with WBC count, CRP and PCT level.
• The culture reports were collected from Microbiology Department of the hospital to check the status of the antibiotics.
• The culture reports of patients were collected for the analysis for the AMR resistance pattern.
• The name of isolated organism was collected to identify the common microorganism causing the resistance.
• Type of culture was taken into context to identify which type of culture showed the maximum resistance pattern.
• The name of antibiotics was collected to identify common resistant antibiotics.
• The statistical analysis was done by using Microsoft Excel 2007.

RESULT

1. Analysis for Total number of Samples

In this present study, the data of 250 samples were collected, out of which 87 (35%) samples were found positive and the remaining were negative (65%).

Further analysis was done from the positive samples which were collected for the study.

Table: 1 Total number of Samples

2. Analysis for Type of Sample

• Out of the positive samples, the most common samples that were found to be resistant were Tracheal (38%), then Blood (21%), followed by Urine sample (15%).
• The remaining ones were swab, pus, sputum, fungus, others and tissue.

Table 2 Type of sample

3. Analysis of resistant organisms

• The most common organisms which were found to be resistant included Klebsiella Pneumoniae (24%), Acienobacter Baumanii (21%), Pseudomonas Aeruginosa (18.39%) and E Coli (11.50%).

The complete list of organisms that were found to be resistant is enlisted in Table 3.

Table 3 Name of organism

4. Analysis for Resistance among antibiotics

–           The maximum resistance was shown by five groups viz, carbapenem (54%), fluoroquinolones (46%), penicillins (45.51%), cephalosporin (37.12%) and aminoglysocides (33.51%).

Table 4 Resistance among antibiotics

• From the abovementioned classes of antibiotics, the top three drugs from each class that showed highest resistance are enlisted in Table 5:

Table 5 Top Resistance

3. Antibiogram of Organism to Antibiotic

Table 6 Antibiogram of Klebsiella Pneumoniae

i). Klebsiella Pneumoniae

– According to the data obtained, Levofloxacin (90%), followed by Ofloxacin and Ciprofloxacin (86%) had shown maximum resistance against Klebsiella Pneumoniae.

– These results indicate that Klebsiella Pneumoniae show the maximum resistance to fluoroquinolones.

– The complete list of antibiotics that were found to be resistant against Klebsiella Pneumoniae is mentioned in Table 6.

According to the data obtained, Tracheal specimen (33%), followed by urine specimen (29%) had shown maximum growth of Klebsiella Pneumoniae.

Table 7 Sample Growth of Klebsiella Pneumoniae

ii). Acienobacter baumanii

 –           It was observed that Acienobacter Baumanii was resistant to all the antibiotics as shown in Table 8 and figure 7.0.

Table 8 Antibiogram of Acienobacter baumanii

• According to the data obtained, Tracheal specimen (81%), followed by blood specimen (9.5%) had shown maximum growth of Acienobacter Baumanii.

Table 9 Acienobacter baumanii Growth in Sample

iii) Pseudomonas Aeruginosa

 –           Pseudomonas Aeruginosa showed highest resistance to Ofloxacin (50%), followed by Cefoperazone + Sulbactam (37.5%). The complete list of antibiotics along with their level of resistance is listed in Table 10.

Table 10 Antibiogram of Pseudomonas Aeruginosa

–           According to the data obtained, Tracheal specimen (33%), followed by urine specimen (24%) had shown maximum growth of Pseudomonas Aeruginosa.

Table 11 Pseudomonas Aeruginosa Growth in Sample

1. iv) Escherichia Coli

Escherichia Coli showed maximum resistance to Levofloxacin (80%). The complete list of antibiotics along with their level of resistance is listed in Table 12.

Table 12 Antibiogram of Escherichia Coli

According to the data obtained, Tracheal specimen, blood and urine specimen had shown maximum growth of Escherichia Coli.

Highlighted = Maximum Percentage of antibiotic that is resistant to particular organism. This antibiogram shows that fluoroquinolons group of antibiotics are resistant to the entire organisms. Apart from the fact that maximum resistance is shown by carbapenem group.

Table 14 Antibiogram (n = 250)

DISCUSSION

As per present study results, the maximum resistance was shown against antimicrobials such as imipenem, doripenem, meropenem, then second most resistant group was flouroquinolones, then penicillin (ticarcillin, piperacillin, ampicillin), then cephalosporin and least resistance was shown by aminoglycoside group of antibiotics. According to the study conducted by Saravanan R [14], et al, showed the maximum resistance against commonly used first line antimicrobials such as co-trimoxazole, ampicillin, amoxicillin, amoxyclav, fluoroquinolones, third generation cephalosporins and nalidixic acid. These results were not in line with the study results with the present study. According to Mohammed MA et al ^[15], they found that Cephalothin (80.6%) and Ampicillin (90.0%) were highest resistance. These results were also not in line with the present study.

 As per present study results, the most common isolates were Klebsiells pneumoniae (24%), then Acienobactor baumanii (21%), Pseudomonas aeruginosa (18.39%) and E.coli (11.5%). A study performed by Kaur N et. al, in patients with urinary tract infection (UTI), they found that the most common bacterial isolates were E.coli (45.4%) followed by Klebsiella (16.7%) and Enterococcus spp (13.2%). Isolation of candida (21.1%) was found in four of the patients, and that were maximum from ICU of 63.1%. [16] These results were not in with the present study. A study carried out by Veena Kumari HB, et. al, their study showed that most common isolates were non fermentative gram negative bacilli which was followed by P. Aeruginosa and Klebsiella spp.  The results obtained from this study are as similar as present study. [17] The most frequently bacteria which were isolated were Staphylococcus aureus (n = 100; 22.8%), Klebsiella pneumoniae (n = 65; 14.8%) and Escherichia coli (n = 41; 9.3%). The results of this study are found similar to the present study. [18]

A study performed by Saxena [19], the Klebsiella species and Acinetobacter species were resistant to beta lactam group of antibiotics such as cephalosporins and piperacillin-tazobactam, while as per the present study, the the Klebsiella pneumoniae and Acinetobacter baumanii were resistant to fluoroquinolones of antibiotics such as olfoxacin, levofloxacin and ciprofloxacin. According to another study carried out by Moremi N, et. al, in their study, third generation cephalosporins had shown most resistance against high proportion of gram-negative isolates. [18] As per this study conducted, the flouroquinolones had shown most resistance against the resistant isolates.

In a retrospective study conducted by Warren C, et. al [20], in which they evaluate the antibiotic susceptibility patterns of pathogens in laboratory of Ndola teaching hospital. They collected the 693 total specimens. They found that the common specimens were urine (58.6%), blood (12.7%) and wound swabs (8.5%), but according to the present study the common specimens were found to be tracheal (38%), then followed by blood (21%) and then urine (15%). This study was done for 5 years. They evaluated the types of bacterial isolates and their AMR profile. The study was performed at Debre Markos Referral Hospital, Northwest Ethiopia. They found that the common samples in which growth was shown in stool culture 68 (28.3%) followed by urine (23.3%), ear discharge (22.5%) and wound swabs at (10.8%) whereas the results of present study wasn’t in the line with the above study results. [21]

In a study carried out by Yaw Effah C, et. al [22], the K. pneumoniae was found to be resistant to amikacin (40.8%), aztreonam (73.3%), ceftazidime (75.7%), ciprofloxacin (59.8%), colistin (2.9%), cefotaxime (79.2%), cefepime (72.6) and imipenem (65.6%). But according to the present study, the K. pneumoniae was found to be resistant to imipenem (71%), doripenem (76%), meropenem (71%), ofloxacin (86%), levofloxacin (90%), ciprofloxacin (86%), cefipime (71%), ceftazidime (71%), cefoperazone + sulbactam (76%), gentamicin (67%), tobramycin (71%) and neitilimycin (48%). The results from both the studies are differing. Yet, it can be said that K. Pneumoniae had shown the resistance to imipenem.

In a prospective study carried out by Gill MK, et. al [23], K. Pneumoniae was studied from January 2019 to July 2019 in North India. They studied 194 non-repeat isolates. Most of them, they were multi drug resistant, ESBL and Carbapenemase producers. They show 100% showed resistance to Ampicillin. These results are somewhat are in line with the present study, as in the present study, the K. Pneumoniae had been shown highest resistance to imipenem (carbapenems), followed by fluoroquinolones.

A cross sectional study performed by Motbanior H, et. al, in 238 patients [24] showed following results. They collected blood, urine and swab specimens. They only performed study on Acinetobacter baumannii and Pseudomonas aeruginosa isolates. They were 100% resistant to ampicillin and piperacillin. A. baumannii isolates were 33.3% against meropenem and 44.5% resistance against ciprofloxacin while as per present study, Acinetobacter baumannii had shown 100% resistance to group of antibiotics such as carbapenem (mostly to imipenem, doripenem and meropenem), also it had shown the resistance to fluoroquinolones, penicillin, cephalosporins and also to aminoglycoside. As per their study, P.aeruginosa isolates showed against ciprofloxacin of 36.4 % and 45.5% resistance against meropenem. But as per present study, Pseudomonas aeruginosa had shown highest resistance to ofloxacin group of about 50%.

The results of the study carried out by Al-Samaree Y. M, et. al, had shown that Acinetobacter baumannii was highest resistant to Piperacillin (94%), this result is not in line with the present study. [25]

In a study by Thapa DB, et. al [26], E. coli isolates were resistant to Ampicillin (100%) Co-trimoxazole (86.40%), Doxycycline (46.60%), Levofloxacin (45.63%), Nitrofurantoin (26.21%) and Amikacin (10.68%). And others were resistant to multidrug resistance to two or more antimicrobials. But as per present study, the E. coli isolates were highly resistant to Levofloxacin (80%), followed by ofloxacin, ciprofloxacin and cefipime (each of about 70%).

In this retrospective data collection by Kibret M, et. al [27], they review the culture of specimen of urine, ear discharge, eye discharge and pus swab from wounds. They analyzed total of 3149 specimen culture reports. Among them, 446 (14.2%) had shown E. coli growth. They found that, urine samples had shown maximum growth of E. coli of 45.5%, but as per present study, the tracheal, blood and urine samples shown the same growth of E. coli. E. coli was highly resistant to erythromycin (89.4%), amoxicillin (86.0%) and tetracycline (72.6%), these results are not in with the present study as per present study, the E. coli showed highest resistance to levofloxacin (80%).

In a study by Javiya VA, et. al [28], the highest growth of Pseudomonas aeruginosa was found in urine, pus and then sputum. But as per present study conducted the highest growth was observed in tracheal specimen, then urine followed by blood.

CONCLUSION

The antibiotic groups which showed the maximum antibiotic resistance pattern were carbapenem, fluoroquinolones, penicillins, cephalosporin and aminoglysocides. The antibiogram shows that the fluoroquinolons group of antibiotics are resistant to the all the tested organisms. By using this data, the particular centre or hospital can use this data to guide the consultant/doctor to select proper antibiotics with help of microbiologist. This data can be useful for further future analyzes related to rational use of antibiotics which can contribute to effective application of Antimicrobial Stewardship Program. Apart from the fact that maximum resistance is shown by carbapenem group. After The antibiogram shows that the fluoroquinolons group of antibiotics are resistant to the all the tested organisms. And before the sentence By using this data, the particular centre or hospital can use this data to guide the consultant/doctor to select proper antibiotics with help of microbiologist.

ACKNOWLEDGEMENT

I want to express my kind gratitude to Mr. Nipul Kapadia and Mr. Sanket Shah, as they had been my mentors at study site (Apollo Hospital). Without their guidance, this won’t be accomplished.

ETHICS DECLARATIONS

Shethia Foram, Suryawanshi Meghraj declares no conflict of interest.

HUMAN AND ANIMAL RIGHTS AND INFORMED CONSENT

This article does not include studies conducted by any of the authors on human or animal subjects.

AUTHOR’S CONTRIBUTION

Shethia Foram – Conceptualization, literature search and writing of the manuscript

Suryawanshi Meghraj- Conceptualization, literature search, writing, and  reviewing of the manuscript

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25. Al-Samaree, M. Y., & Al-Khafaji, Z. M. (2016). Antibiogram of Acinetobacter baumannii isolated from Baghdad Hospitals.  J. Adv. Res. Biol. Sci, 3(4), 238-242.
26. Thapa, D. B., & Chapagain, A. (2020). Antibiogram of Escherichia coli Isolated from Avian Colibacillosis in Chitwan District of Nepal. International Journal of Applied Sciences and Biotechnology, 8(1), 52-60.
27. Kibret, M., & Abera, B. (2011). Antimicrobial susceptibility patterns of E. coli from clinical sources in northeast Ethiopia. African health sciences, 11, 40-45.
28. Javiya, V. A., Ghatak, S. B., Patel, K. R., & Patel, J. A. (2008). Antibiotic susceptibility patterns of Pseudomonas aeruginosa at a tertiary care hospital in Gujarat, India. Indian journal of pharmacology, 40(5), 230.

Abstract

To development of transethosome loaded with the poorly water soluble irbesartan which could be utilized for increasing solubility. Transethosome loaded irbesartan are prepared by cold method. Irbesartan were successfully loaded into transethosome and confirmed through vesicle shape, vesicle size, polydispersivity index, zeta potential, calibration curve, entrapment efficiency, FTIR, in-vitro drug release, SEM. Transethosome are useful carrier for poorly soluble drug and provide a novel vehicle for delivery of drug.

Keywords

Transethosome, Irbesartan, Solubility, Carrier, Novel vehicle

Cite This Article

Verma, A., Mishra, M. (2021). Formulation and Characterization of Transethosomal-Loaded Nanoparticles of Irbesartan. International Journal for Pharmaceutical Research Scholars, 10(1); 28 - 35

INTRODUCTION

Irbesartan is an angiotensin receptor blocker used mainly for the treatment of hypertension. Angiotensin 2 the principal pressor agent of rennin angiotensin system is responsible for effects such as vasoconstriction stimulation of synthesis and release of aldosterone. Cardiac stimulation and renal reabsorption of sodium. Irbesartan is a specific competitive antagonist of AT1 receptor with much greater affinity for the AT2 receptor than for the AT2 receptor and no agonist activity. Irbesartan’s inhibition of angiotensin 2 binding to the AT1 receptor leads to multiple effect including vasodilation, reduction in the secretion of vasopressin, and reduction in the production and secretion of aldosterone. The resulting effect is a decrease in blood pressure. Irbesartan effectively lowers BP in patients with hypertension without effecting heart rate¹.

The transethosomal system contains the basic components of classical ethosomes and the additional compound, such as penetration enhancers or surfactants in their formula. The research showed that different types of surfactants and penetration enhancers made ethosomal systems with better physico-chemical characteristics. In view of this, nano-carrier drug delivery systems possess high biocompatibility and excellent drug delivery potential².

Material and Method

Material

Irbesartan was obtained as a gift sample. Soya lecithin was purchased by urban platter. Cholesterol, Tween80, Ethanol was supplied by Thomas baker Mumbai. All the chemicals and solvent were used as analytical grade. Distilled water was used in all the experimental studies.

Method

In the preparation of transethosome loaded irbesartan was prepared by cold method. In a beaker taken the irbesartan, soyalecithin, and cholesterol was dissolved in specified amount of ethanol at 30℃ or room temperature. Tween80 was dissolved in distilled water in a separate beaker. Aqueous phase is added into the organic phase through syringe (22gauge) drop by drop. Stirring are continuing through the magnetic stirrer at 700-1500rpm at 30min. after the stirring process sonication are done by the bath sonicator at 30min. finally transethosome was prepared³.

Characterization of transethosome

Vesicle shape, Vesicle size and Polydispersity index – Vesicle shape and vesicle size are measured by microtrac nanotrac wave. Dilute the sample (2 drop) with distilled water and calculate them. Polydispersivity index was measured by mastersizer 2000⁴.

Zeta potential

Zeta potential measurement of the samples was carried out by using Malvern zeta sizer. It indicates charge present on the surface of treansethosome which is responsible for stability of the formulation and interact with membrane⁵.

Entrapment efficiency

The percentage entrapment of the drug added is called entrapment efficiency. Free unentrapped drug from irbesartan transethosomes was separated by centrifugation at 20,000 rpm for 1 h at 4°C using a cooling centrifuge. The pellets that are formed after centrifugation were washed twice with 5 ml of phosphate buffer (pH 7.4) and re-centrifuged again for 1 h. The encapsulation efficiency of the drug was determined after lysis of the pellets with 5 ml of methanol and sonication for 10 min. The concentration of irbesartan in methanol was determined using UV-Visible spectrophotometer at 224nm. Entrapment was determined using the following equation –

% Entrapment Efficency = (Total Drug Added-Free Unentraped Drug)/(Total Drug Added) X 100

Calibration Curve  

The UV spectrophotometer was utilized for the detection and quantification of Irbesartan. For the preparation of the stock solution, 10 mg of the pure drug was accurately weighed and dissolved in 10 ml phosphate buffer solution 7.4pH and then the volume was made up to 100 ml with phosphate buffer solution 7.4pH   to give standard stock solution 100 μg/mL. From the above stock solution, different concentration (10 μg/mL) was prepared by appropriate dilution to prepare 2, 4, 6, 8 and 10μg/mL concentration solution. The sample was filtered and scanned in the range 200-400 nm using a UV spectrophotometer to determine λmax. The absorbance of the above dilution was determined at 224nm λmax on the UV spectrophotometer. The absorbance values corresponding to each concentration were then statistically evaluated and plotted as a standard graph between absorbance and concentration⁶.

In-vitro Drug Release

In-vitro drug release was measured by using franz diffusion cell. A cellophane dialysis membrane with molecular weight cut-off of 8000 daltons was hydrated with phosphate buffer saline 7.4pH. Vesicular formulation of 1ml of irbesartan was placed in the donor compartment. The receptor compartment was filled with 7.4pH and stirred with a magnetic bead at 300-400rpm and the temperature of the system was maintained at 32± 1℃ to mimic human skin. 1ml aliquot was withdrawn at predetermined time intervals and was immediately replaced with an equal volume of fresh buffer. All samples were analyzed for irbesartan content by U.V spectrophotometry at 224nm⁷.

FTIR studies

FTIR spectra of Irbesartan, lecithin, formulation and physical mixture were recorded by using Thermoscintific FTIR spectroscopy in the range of 4000-500 cm-1. A 10 mg sample was mixed with potassium bromide (200-400 mg) and compressed. The compressed disc was placed in the light path and spectra were obtained. After conducting the exhibition, important themes were identified related to the major working groups^{8, 9}.

SEM studies

A scanning electron microscope (SEM) is a type of electron microscope that produces images of a sample by scanning the surface with focused electrons. Different magnification of transethosome can be measured for surface morphology^{10, 11}.

Result

 Vesicle shape, Vesicle size and Polydispersity index

 The result of the vesicle shape, vesicle size and polydispersity index are given in Fig No. 2 and Table No. 1 respectively. Vesicle shape of the transethosome is irregular. Vesicle size of F4 formulation are optimized range is 104.63nm±0.90 and polydispersivity is 20.73.

 Zeta potential

The result of the zeta potential is given in Fig No. 1 and Table No. 1 respectively. The zeta potential of the optimized formulation F4 was found to be -23.9mV which indicate stable formulation.

Table No. 1: Vesicular Size, PDI, Zeta Potential, % Entrapment Efficiency

ENTRAPMENT EFFICIENCY

The result of the entrapment efficiency is given in Fig No.3 and Table No.1 respectively. The formulation F4 shows the highest entrapment efficiency is 76%.

Calibration Curve

Table No. 2: Calibration Curve of Irbesartan

The result of the calibration curve is given in Table No.2 and Fig No.4 respectively. Calibration curve of irbesartan in the phosphate buffer 7.4pH was prepared and take the absorbance at 224nm.

In-Vitro Drug Release

The results of the in-vitro drug release are given in Table No. 3 and Fig No. 5 respectively. The formulation (F4) was optimized in which after 8 hours %cumulative drug release is 45%.

Table No. 3: % Cumulative Drug Release

FTIR studies

The result of the FTIR studies is given in Fig No.6. FTIR spectra of Irbesartan, lecithin, formulation and physical mixture were recorded by using Thermoscintific FTIR spectroscopy in the range of 4000-500 cm-¹.

SEM studies

The results of the SEM studies are given in Fig No. 7. A scanning electron microscope (SEM) is a range is about 200nm.

Conclusion

Irbesartan loaded with transethosome formulations were successively prepared using cold method. Characterization of transethosome as vesicle shape, vesicle size, PDI, zeta potential, entrapment efficiency, calibration curve of UV, % drug release, FTIR, SEM as responses.

Reference

1. Lake, Y., & Pinnock, S. (2000). Improved patient acceptability with a transdermal drug‐in‐adhesive oestradiol patch. Australian and New Zealand Journal of Obstetrics and Gynaecology, 40(3), 313-316.
2. Li, G., Fan, Y., Fan, C., Li, X., Wang, X., Li, M., & Liu, Y. (2012). Tacrolimus-loaded ethosomes: physicochemical characterization and in vivo evaluation. European journal of pharmaceutics and biopharmaceutics, 82(1), 49-57.
3. Ricci, M., Giovagnoli, S., Blasi, P., Schoubben, A., Perioli, L., & Rossi, C. (2006). Development of liposomal capreomycin sulfate formulations: effects of formulation variables on peptide encapsulation. International journal of pharmaceutics, 311(1-2), 172-181.
4. Sonjoy, M., Thimmasetty, J., Ratan, G. N., & Kilarimath, B. H. (2011). Formulation and evaluation of carvedilol transdermal patches. International Research Journal of Pharmacy, 2(1), 237-248.
5. Charoo, N. A., Anwer, A., Kohli, K., Pillai, K. K., & Rahman, Z. (2005). Transdermal delivery of flurbiprofen: permeation enhancement, design, pharmacokinetic, and pharmacodynamic studies in albino rats. Pharmaceutical development and technology, 10(3), 343-351.
6. Albash, R., Abdelbary, A. A., Refai, H., & El-Nabarawi, M. A. (2019). Use of transethosomes for enhancing the transdermal delivery of olmesartan medoxomil: in vitro, ex vivo, and in vivo evaluation. International journal of nanomedicine, 14, 1953.
7. Abdulbaqi, I. M., Darwis, Y., Abou Assi, R., & Khan, N. A. K. (2018). Transethosomal gels as carriers for the transdermal delivery of colchicine: statistical optimization, characterization, and ex vivo evaluation. Drug design, development and therapy, 12, 795.
8. Maurya, S. D., Prajapati, S., Gupta, A., Saxena, G., & Dhakar, R. C. (2010). Formulation development and evaluation of ethosome of stavudine. Int J Pharm Edu Res, 13, 16.
9. Negi, P., Singh, B., Sharma, G., Beg, S., & Katare, O. P. (2015). Biocompatible lidocaine and prilocaine loaded-nanoemulsion system for enhanced percutaneous absorption: QbD-based optimisation, dermatokinetics and in vivo evaluation. Journal of microencapsulation, 32(5), 419-431.
10. Dubey, V., Mishra, D., Dutta, T., Nahar, M., Saraf, D. K., & Jain, N. K. (2007). Dermal and transdermal delivery of an anti-psoriatic agent via ethanolic liposomes. Journal of controlled release, 123(2), 148-154.
11. El Zaafarany GM, Awad GA, Holayel SM, et al. Role of edge activators and surface charge in developing ultradeformable vesicles with enhanced skin delivery. Int J Pharm. 2010;397: 164–172.

Abstract

The present study is an attempt to formulate microspheres of Glimepiride, an orally administered ant-diabetic drug with a view of improving its oral bioavailability and giving a prolonged release of drug, where here the microspheres with polymers such as Ethyl cellulose and Eudrajit RS 100 and guar gum were successfully prepared by emulsification solvent evaporation method and the particle size analysis revealed that the size of microspheres was increased with increase in the concentration of polymer. The study comprises that floating microsphere of Glimepiride, model drug, may increase the gastric residence time. The floating microspheres are prepared by the emulsification solvent diffusion technique using polymers Ethyl cellulose, Eudragit RS100 and Guar gum, PVA, Dichloromethane in different ratio. The formulated microsphere was evaluated for the percentage yield, percentage encapsulation efficiency, percentage buoyancy and in-vitro drug release. Floating microspheres prolong the release of the drug and gastric residence time, release almost 82.35% drug within 24hrs.

Keywords

Glimepiride, Eudragit RS 100, Floating Microsphere, Solvent evaporation techniques

Cite This Article

Kumar, M., Mishra, M.K., Srivastava, R., Patel, A.K. (2021). Formulation and characterization of a floating microsphere of glimepiride by using solvent evaporation technique. International Journal for Pharmaceutical Research Scholars, 10(1); 12-27.

NTRODUCTION

Glimepiride is a drug which is absorbed from the gastrointestinal tract (GIT) and has a short half-life and eliminated quickly from the blood circulation, so it required frequent dosing. To avoid this drawback, the oral sustained-controlled release formulations have been developed in an attempt to release the drug slowly into the GIT and maintain an effective drug concentration in the serum for longer period of time.

The microspheres are characteristically free flowing powders consisting of proteins or synthetic polymers, which are biodegradable in nature. Solid biodegradable microspheres incorporating a drug dispersed or dissolved throughout particle matrix have the potential for controlled release of drugs where their spherical particles ranging from 1 to 1000 micrometers. Where here the microspheres are prepared from the solvent evaporation method, microspheres are prepared by two particles core material and coating material, where the core material is made up of drug and particulate is of polymers. Microencapsulation for oral use has been employed to sustain the drug release, and to reduce or eliminate gastrointestinal tract irritation.

Multiparticulate delivery systems spread out more uniformly in the gastrointestinal tract. This results in more reproducible drug absorption and reduces local irritation when compared to single-unit dosage forms such as no disintegrating, polymeric matrix tablets.

Microsphere is used to modify and retard drug release; due to its small particle size they are widely distributed throughout the gastrointestinal tract which improves drug absorption and reduces side effects due to localized build-up of irritating drugs against the gastrointestinal mucosa. [1-4]

Floating drug delivery systems (FDDS) or hydro dynamically balanced systems (HBS) are among the several approaches that have been developed to increase the gastric residence time of dosage forms. This Gastro retentive floating drug delivery system (GRFDDS) have a bulk density lower than that of gastric fluids and thus remains buoyant in the stomach without affecting gastric emptying rate for a prolonged period of time. While the system is floating on gastric contents, the drug is released slowly at a desired rate from the system. [5]

The use of oral antidiabetic drugs for the treatment of Type 2 diabetes is increasing rapidly. Glimepiride is an antidiabetic drug belongs to second generation sulphonylurea drug. It lowers the blood glucose level in patients with Type 2 diabetes (non-insulin dependent diabetes mellitus) by stimulating the release of insulin from the pancreatic β-cells. In this way it exerts a long-term effect of reducing the blood glucose levels. In addition, extra pancreatic effects may also play a role in the activity of glimepiride. It has low risk of hypoglycemia because of preservation of physiological suppression of insulin secretion in response to low blood glucose levels1. It is completely (100%) absorbed following oral administration. It has rapid onset of action, 24 h duration of effect with a half-life of 5 h and once a day dosing. [6-7]

Materials and Methods

 Glimepiride was obtained as a gift sample from Medley Pharmaceuticals Ltd., Daman Unit, Andheri East, Mumbai, India., Eudragit RS100 were obtained from Natco Pharma; Hyderabad, India and Ethyl cellulose from obtained were from Merck, all other reagents and solvents used were of analytical grade. obtained were from Merck.

PREFORMULATION STUDIES

Preformulation studies mainly focus on those physiochemical properties of the drug that could affect performance and development of an efficacious dosage form such as determining the purity of Active Pharmaceutical Ingredient (API) before formulation any dosage form. Preformulation studies are useful in determining the formulation components and physiochemical properties of new drug substance that will affect the final dosage form and its stability.

Description of Drug

The sample of drug was observed for colour, state and odour.

Drug Excipients Compatibility Study

Before formulating a dosage form it is essential to confirm that drug is not interacting with the polymer under certain experimental studies. Interacting among drug and polymer may affect the efficacy of final dosage form.

Fourier transform infra-red spectrum of pure drug, Eudragit RS 100, Ethyl cellulose, Guar gum and their Physical mixture were recorded. Drug and different excipients were taken in 1:1 ratio. The samples of pure drug and physical mixture of polymer and drug were taken and subjected to FTIR study.

Standard Calibration Curve

A stock solution of glimepiride (10μg/mL) was prepared by dissolving 10 mg of glimepiride with different buffer solution such as 0.1N HCl and phosphate buffer pH 7.4. Further various dilutions were made with these different pH buffer solutions containing concentration 2, 4, 6 & 8 μg/mL of glimepiride and absorbance was measured against blank at λ_max 262 nm and a standard calibration curve between concentration and absorbance was plotted as shown in figure. All spectral absorbance measurements were made on Shimadzu-1700 UV-visible spectrophotometer.

Preparation of Formulations

Formulation of drug-loaded microspheres was carried out by the emulsion solvent diffusion-evaporation method. The polymers ethyl cellulose, Eudragit RS100, Guargum, PVA, Dichloromethane was used in different ratios. Initially a solvent mixture of 10mL of dichloromethane was prepared in the considering their volumes.

An accurately weighed quantity of drug and polymer was co-dissolved at room temperature in a solvent mixture. This solution was introduced into 100 mL of 1% PVA aqueous solution at room temperature and dispersed to form emulsion at stirring rates of 800 rpm using a mechanical stirrer equipped with 4-blade propeller. Agitation provided by stirrer breaks the poured polymer solution to form an oil-in-water (O/W) type emulsion. This emulsion was then stirred for about 45 min at room temperature. After stirring, the solidified microspheres were recovered by filtration and dried.

Table 1: Formulation of batches of floating microsphere of Glimepiride

Evaluation of the Formulated Microsphere

Percent Yield of Microspheres

Microspheres dried at room temperature were then weighed and the yield of microspheres. Preparation was calculated using the following formula.

% Yield = (Actual weight of dried microsphere)/(Total weight of drug and excipient)×100

Drug Entrapment Efficiency

Encapsulation efficiency of the microspheres was evaluated by deriving percent drug encapsulation. The drug content of drug-loaded microspheres was determined by dispersing 100 mg of microspheres in 50 mL ethanol or the solvent choose according to its solubility followed by agitation with a magnetic stirrer for about 30 min to dissolve the polymer and to extract the drug. After filtration through a 5μm membrane filter, the drug concentration in the ethanol phase was determined by taking the absorbance of this solution spectrophotometrically at 298nm. Eudragit RS100 and ethyl cellulose did not interfere under these conditions. Drug concentration was then calculated. Thus, the total drug encapsulated in total yielded microspheres from the procedure was calculated. It was expressed in percentage called as “Percent drug entrapment” calculated as.

% Drug Efficiency = (Practical amount of drug present in mg)/(Theoretical amount of drug taken in mg) × 100

Percentage Buoyancy

The floatation studies were carried out to ascertain the floating behavior of various polymers   Combinations. Beaker method was initially used to have an idea of the floatation behavior of the proposed dosage form. 50 mg of floating microsphere were placed in each of four 50 mL beakers containing 20 mL of 0.1N HCl containing 0.02% tween 80. The beakers were shaken in a biological shaker at 37◦C ± 0.5◦C at 40 rpm. Floating microspheres were collected at 4,8 and 12 h and dried till constant weight was obtained. The percentage of floating microspheres was calculated by the following equation.

% Buoyancy = Wf/(Wf+Ws) × 100

where Wf and Ws = weights of the floating and settled microspheres, respectively.

Differential scanning colorimetry (DSC)

Differential scanning colorimetry (DSC) a type of thermo analytical process. In this heat require to rise temperature of sample and reference.

Temperature of sample and reference maintain nearly same all over experiment. Sample holds temperature increase linearly as function of time.

These technique used mainly physical change of sample and also determine process is exothermic and endothermic.

When solid sample melt into a liquid require more energy and temperature increase so all over process is endothermic but when liquid change into a solid the temperature decrease to all over process is exothermic.

SEM (Scanning Electron Microscope) Studies

Surface morphology of drug loaded microsphere was inspected using SEM (CIF, IIT BHU, Varanasi). Little quantity of drug loaded microsphere was spread manually on a carbon tape, which is attached to an aluminum stub. Samples were analyzed by SEM with direct data capture of the image on to a computer screen.

Micromeritic properties

Microsphere were characterized for micromeritic backdrop like bend of repose, aggregate density, broke density, carr’s basis and hausners ratio.

Bulk density

An exact quantity “M” of microsphere was taken and was placed into a measuring cylinder. Volume “V” occupied by the microspheres was noted without disturbing the cylinder and bulk density was calculated using the following equation;

Bulk density (Pb) = M/V

Tapped Density

The tapping method was used to determine the tapped density in which the cylinder containing known amount (M) of microspheres was subjected to a fixed number of taps (approximately 100) until the bed of microspheres had reached the minimum. The final volume after tapping “Vo” was recorded and the tap density was calculated by the following equation.

Tapped Density (Pp) = M/Vo

Angle of Repose

This property was determined to predict flowability Angle of repose of the microspheres is the maximum angle possible between the surface of the pile of microspheres and the horizontal plane, was obtained by fixed funnel method using the formula

Angle of repose (ф) = tan-1[2h/d]

Where, h is height and d is the diameter of the microsphere pile that is on a paper after making the microspheres flow from the glass funnel.

Carr’s Index or % Compressibility

A high Carr’s index is indicative of the tendency to form bridges can be calculated by using following formula:

Carr index or %compressibility Index or C = [1- V0/V] × 100

Hausner Ratio

Hausner’s ratio is measures of the propensity of a powder to be compressed and the flow ability of granule. A higher Hausner ratio indicates greater cohesion between particles.

Hausner Ratio = [100/100+C]

Where C is Carr’s Index.

In Vitro Drug Release Studies

The drug release rate from microspheres was determined using USP basket-type dissolution apparatus. A weighed amount of microspheres equivalent to 5 mg drug was filled into a capsule (size 0) and placed in the basket. Dissolution medium used was phosphate buffer 7.4 for first hour and maintained at 37 ± 0.5°C at a rotation speed of 100 rpm. Prefect sink conditions prevailed during the drug release studies.

5 mL of sample was withdrawn at each 1 h interval; later this interval was extended to 2 h. Sample was then passed through a 5 μm membrane filter, and analyzed spectrophotometrically at 300 nm to determine the concentration of drug present in the dissolution medium. The initial volume of dissolution medium was maintained by adding 5 mL of fresh dissolution media after each withdrawal. The dissolution study was continued for next 24 h.

Release kinetics

Release kinetic models, which described the overall release of drug from the dosage forms. Because qualitative and quantitative changes in a formulation may alter drug release and in vivo performance, developing tools that facilitate product development by reducing the necessity of bio-studies is always desirable. In this regard, the use of in vitro drug dissolution data to predict in vivo bio-performance can be considered as the rational development of controlled release formulations. Data obtained from the in vitro release studies were fitted to various model dependent kinetic equations such as zero order, first order, Higuchi model and Korsmeyer- Peppas model.

Higuchi Release Model

To study the Higuchi release kinetics, the release rate data was fitted to the following equation

F = KH.t 1/2

Where, F is the amount of drug release

KH is the release rate constant

t is the release time

When the data was plotted as a cumulative percentage drug release versus square root of time, yields a straight line, indicating that the drug released by diffusion mechanism the slope is equal to k.

Korsmeyer and Peppas Model

The release rate data were fitted to the following equation,

Mt/M8 =KM.tn

Where, Mt/M8 is the fraction of drug release

KM is the release constant

t is the release time

N is the diffusional exponent for the drug release that dependent on the shape of the matrix dosage form. When the data is plotted as log percentage release versus log time, yields as straight line with a slope equal to n and the k can be obtained from y- intercept. For non-fickian release the n values falls between 0.5 and 1.0 while for fickian (case I) diffusion n= 0.5 and zero order release (case II transport) n=1.0.

Zero order release rate kinetics

To study the zero order release kinetics the release rate data are fitted to the following equation

F= K0t

Here, F is the fraction of drug release

K0 is the rate constant

T is the release time

Hen data is plotted a cumulative percentage drug release versus time, if the plot is linear then the data obey zero order release kinetic, with a slope equal to K0.

First order model

This model has also been used to describe absorption and /or elimination of some drug, the release of the drug which followed first order kinetic can be expressed by the equation

LogC = logC0-Kt/2.303

Where, C0 is the initial concentration of drug

K is the first order rate constant

t is the time

The data obtained are plotted as log cumulative percentage of drug m remaining Vs time. This yields a straight line with a slope of –K/2.303.

RESULT AND DISCUSSION

Table 2: Organoleptic properties

Drug Identification

The accurately weighed quantity of drug was dissolved in sufficient volume of acetone and scan was obtained on UV-VIS spectrophotometer. The wavelength at which maximum absorbance obtained was considered as maximum wavelength (λmax) i.e. 262 nm for the drug.

Drug- Excipients Compatibility Study

Drug and polymers identified by infra-red spectrum which are compared with its standard IR. The IR spectrum given below shown that the peaks obtained in the test spectrum is similar to that given in standard.

Fig 5: FTIR Spectrum of Glimepiride and guar gum

Fig 6: FTIR Spectrum of best formulation

Table 3: Major peak observe in FTIR Spectrum

The IR spectrum of Glimepiride showed characteristic N-H stretch peaks of urea at 3370 and 3289 cm-¹, peaks at 1707 and 1673 cm-¹corresponding to the carbonyl group, and peaks at 1348 and 1144 cm-¹ related to the sulfonamide group, peaks at 2850 and 2960.42 cm-¹ corresponding to the CH aromatic group, and peaks at 3000 and 2915 cm-¹ corresponding to the CH aliphatic groups.

Preparation of standard calibration curve: Obtained absorbances are shown in the tables 4 and standard calibration curves of glimepiride in a methanol solvent of a phosphate buffer pH 7.4 are shown in figures

Table 4: Standard curve of Repaglinide

Fig 7: Standard curve of Glimepiride

Table 5: Percentage yield of floating microsphere of Glimepiride

The entrapment efficiency was found to be abruptly increasing when both polymers were used together. Entrapment efficiencies of batches GMF1-GMF9 ranged from 36.24% to 87.24%. Maximum encapsulation efficiency was observed of the batch GMF5, where ratio of 2:2:1 of the ethyl cellulose and Eudragit RS100 and Guargum was used. Table no.6 and Fig. 8 represent the entrapment efficiency.

Table 6: % Entrapment Efficiency of floating microsphere of Glimepiride

Percentage Buoyancy

The percentage Buoyancy was found to be abruptly increasing when both polymers were used together. Percentage Buoyancy of batches GMF1-GMF9 ranged from 58.13% to 85.42%. Maximum percentage Buoyancy was observed of the batch F5, where ratio of 2:2:1 of the ethyl cellulose and Eudragit RS100 and Guargum was used. Table no.7 and Fig. 13 represent the floating behavior.

Table 7: Percentage buoyancy of floating microsphere of Glimepiride

The Differential Scanning Calorimetry (DSC)

The differential scanning calorimetry (DSC) thermograms of pure Glimepiride, Eudragit, Guar gum was found to be 221, 210, 80 °C respectively in figure A,B,C. It was found that the DSC thermogram of GM showed a single endothermic peak at 221°C.

Scanning Electron Microscopy

Surface morphology of drug loaded microsphere was inspected using SEM (CIF, IIT BHU, Varanasi). Little quantity of drug loaded microsphere was spread manually on a carbon tape, which is attached to an aluminum stub. Samples were analyzed by SEM with direct data capture of the image on to a computer screen.

Micromeritic properties

Results of micromeritic properties such as bulk density, tapped density, hausner’s ratio, angle of repose and carr’s index were within standard limits.

Value of bulk density, tapped density, carr’s index, Hausner’s ratio and angle of repose of optimized were found to be 0.38±0.017 gm/cm³, 0.45±0.022 gm/cm³, 19.33±1.47, 1.16±0.02 and 25.11±1.80 respectively indicating good flow property.

Table 8: Micrometric properties of floating Microspheres of Glimepiride

Table 9: Percentage drug release

Table 10: Percentage drug release of optimized Batch (GMF 5)

In Vitro Drug Release Studies

In-vitro drug release study were done for determining %drug release of drug from formulation and gives an idea about the effective formulation development. In-vitro drug release was done by open end tube at one end it was tied by dialysis membrane.

Drug Release Kinetics

The required amount of sample was taken in open end tube and the dialysis membrane was activated by soaking it for 24 hours in phosphate buffer 7.4. The aliquots was withdrawn and change with fresh phosphate buffer.  absorbance of the prepared dilution were taken at 240nm.  The % drug release of optimized batch is 82.35.

CONCLUSION

The present study has been a satisfactory attempt to formulate microspheres of Glimepiride, an orally administered antidiabetic drug with a view of improving its oral bioavailability and giving a prolonged release of drug. FT-IR spectra of physical mixture showed no significant shifting of the peaks therefore it reveals that the drug is compatible with the polymer used. Microspheres with polymers such as Ethyl cellulose and Eudragit RS 100 and guar gum were successfully prepared by emulsification solvent evaporation method. The percentage yield obtained in all the formulations was good and in the range of 54-84.32%. The particle size analysis revealed that the size of microspheres was increased with increase in the concentration of polymer.

 It may be concluded that this sustained release formulation would be a promising drug delivery system to sustain the drug release for about 24 h enhancing the patient compliance. In the formulation, the combination of cost-effective and biocompatible polymers Eudragit RS100, Ethyl cellulose and Guargum had been successfully used and there is scope of scale up of the batches to the commercial level. The best formulation from the 9 batches, found to be efficient with good recovery yield, percent drug entrapment and drug release was F5, prepared using 1:2:2:1:1:1 ratio of polymer.

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5. Sheth, P. R., & Tossounian, J. (1984). The hydrodynamically balanced system (HBS™): a novel drug delivery system for oral use. Drug Development and Industrial Pharmacy, 10(2), 313-339.
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7. Bala, S., Kataria, M. K., Bilandi, A. Studies on Solid Dispersion Techniques Implemented for Dissolution Enhancement of Glimepiride Am.J.PharmTech,2014,(4),1-8.

Abstract

A simple, rapid and precise RP-UHPLC method is developed for the simultaneous estimation of Olmesartan Medoxomil and Metoprolol Succinate in bulk drug and pharmaceutical dosage form. The quantification is carried out using Shimadzu Shim-pack XR ODS II (50mm X 1.9mm, 2.0 µm) column with mobile phase-A consist of 50mM phosphate buffer pH-6.8±0.05: Acetonitrile (95:5 %v/v) & mobile phase-B consist of 50mM phosphate buffer pH-6.8±0.05: Acetonitrile (34:66 %v/v) with gradient elution. The flow rate is 0.8 mL/min using 40°C column oven temperature. The eluent is measured at 225 nm. The retention times of Olmesartan Medoxomil and Metoprolol Succinate are about 1.7 min and 0.7 min respectively. The method is validated in terms of linearity, precision, accuracy, specificity, limit of detection and limit of quantitation. Linearity of Olmesartan Medoxomil and Metoprolol Succinate are in the range of 25-75µg/ml for Olmesartan and 62.5-182.5µg/ml for Metoprolol with regeression co-efficient more than 0.999. The percentage recoveries of both the drugs are between 98 to 102%. The stress testing of dosage form is carried out under acidic, alkaline, oxidation, photo-stability and thermal degradation (dry heat and wet heat) conditions and the drugs are well resolved from its degradation products with good resolution. Hence the method is accurate and precise and can be employed for routine analysis of Olmesartan and Metoprolol in different dosage forms.

Keywords

Olmesartan Medoximil, Metoprolol Succinate, Stability Indicating Assay method, Validation, UHPLC

Cite This Article

Thakker, N., Shinde, G/. Dharamsinh, A., Patel, R. (2021). Development and Validation of Stability Indicating Assay Method for Estimation of Olmesartan Medoximil And Metoprolol Succinate in Combined Dosage Form By UHPLC. International Journal for Pharmaceutical Research Scholars, 10(1); 01-11.

INTRODUCTION

Worldwide, raised blood pressure is estimated to cause 7.5 million deaths, about 12.8% of the total of all deaths. This accounts for 57 million disability adjusted life years (DALYS) or 3.7% of

total DALYS. Raised blood pressure is a major risk factor for coronary heart disease and ischemic as well as hemorrhagic stroke. Treating high blood pressure can take a multi-pronged approach including diet changes, medication, and exercise.

Fixed dose combination (FDC) drugs are standard practice in the treatment of infectious diseases, but their role in chronic non-communicable diseases is unclear. FDC anti-hypertensive drugs are an attractive option to improve compliance by reducing the number of pills taken daily, particularly in elderly patients who generally require more than one drug to control blood pressure and often are on multiple other medications.

Olmesartan is an angiotensin II receptor blocker (ARB) used in the treatment of Hypertension and it works by relaxing blood vessels so that blood can flow more easily (Fig.1a). Metoprolol succinate is a selective beta-1 blocker and is to treat angina (chest pain) and hypertension (high blood pressure). It is also used to lower your risk of death or needing to be hospitalized for heart failure (Fig.1b).

The combination of Olmesrtan Medoximil (20 mg) and Metoprolol succinate (25 mg) was tentatively approved by US Food and Drug Administration (USFDA) in Oct 2016 for the treatment of hypertension in adults. Pharmaceutical companies are focusing on achieving ever shorter times of drug to market, so it is vital that a tailored, pragmatic approach is adopted when conducting method development for active pharmaceutical ingredients (API) or drug products (DP). Although methods require a high degree of robustness, the overall strategy should encompass full evaluation of the regulatory requirements applicable to the particular phase of the drug lifecycle; this is pivotal to ensure a successful regulatory submission, whereby the applicant must demonstrate suitable validation of all methods used to support the filing. Successfully developed (and validated) analytical methods can reduce overall turnaround times from preclinical to commercial release. Methods should have the desired flexibility built in during early stages to allow easy translation from API to DP, thus potentially reducing costs throughout the product lifecycle.

Analytical method development, validation, and transfer are key elements of any pharmaceutical development program. The need to develop new analytical methods with extremely high sensitivity along with precision and accuracy thus became mandatory. These methods are needed for assurance of quality, safety and efficacy of medicine and pharmaceuticals. There are five analytical tests that are considered universal by the FDA for formulated products: description, identification (ID), assay, dissolution and impurities. Description is a physical characteristic of the finished product but for identification, assay, dissolution and impurities analytical methods required. Fixed dose combination of Olmesartan medoximil and Metoprolol succinate is not official in Indian pharmacopeia.

Assay by UHPLC method for the estimation of Olmesartan Medoximil and Metoprolol Succinate is not available in any pharmacopeia and not reported in any scientific journal. This present work describes the development of simple, selective, accurate and precise RP-HPLC method for the determination of Assay by UHPLC of Olmesartan Medoximil and Metoprolol Succinate in Tablet formulation.

Materials and Methods

Chemicals and Reagents:

Potassium bromide, Sodium dihydrogen phosphate, hydrochloric acid (35%), sodium hydroxide, Hydrogen peroxide and HPLC grade acetonitrile were purchased from Merck.  Analytical standards were provided as gift samples by Piramal Enterprises Ltd, Pharmaceutical Development Service Ltd. Tablet Olmetor-M tablet formulation purchased from market.

Instrument used:

The UHPLC used for method development and validation was Shimadzu N-Series and Nexera X2 UHPLC.

API Evaluation

Identification by UV test was performed for evaluation of Olmesartan Medoxilmil and Metoprolol Succinate. Solution of 10ppm of Olmesartan Medoxilmil and Metoprolol Succinate individually was prepared in a mixture of 50 volumes of water and 50 volumes of acetonitrile and scanned from 200-400 nm in UV spectrophotometer. Identification by IR was carried out for all APIs. Accurately weighed 2 mg of individual API was mixed with 200 mg of previously dried potassium bromide at 105°C for 1 hr. and triturated to get homogenous mixture. This sample was scanned in the range of 400-4000cm-1 in FTIR. Melting point determination was carried out for all APIs. Melting point was determined by capillary method using Lab India melting point apparatus.

Method Development

The step wise logical scientific method development has been described along with the reasoning.  Method development was initiated by converting a HPLC method from literature to UHPLC method. Initial trial were taken using 0.1% OPA in water and Acetonitrile on a C18 column. But it was observed that the peak shape was not proper for Olmesartan. Later the ratio was changed, but it always led to more retention of Olmesartan peak and run time was increasing. Then we shifted from 0.1% OPA in water to phosphate buffer and found improvement in peak shapes. Later trials with various gradient composition were taken during forced degradation development to assure specificity. Final optimized method parameters are as follows: 50mM phosphate buffer pH-6.8±0.05: Acetonitrile (95:5 %v/v) as a mobile phase-A and 50mM phosphate buffer pH-6.8±0.05: Acetonitrile (34:66 %v/v) as a mobile phase-B. Samples were injected in C18 column Shimadzu Shimpack XR ODS II (50mm X 1.9mm, 2.0 µm) which was eluted at 0.8mL/min. Injection volume kept 10µL. UHPLC column temperature was set to 40°C and auto sampler temperature kept ambient. Selected gradient was as follows: 0.0-1.5 min, linear gradient 40-70% B 1.5-2.5 min, isocratic 70% B; 2.5-3.5 min, linear gradient 70-40% B; 3.5-40 min,  isocratic 40% B. Forced degradation study was performed on tablet formulation to check stability indicating nature of method. In forced degradation study acid-base hydrolysis, oxidation using hydrogen peroxide, thermal stress and photo stability stress were carried out. Acid, base and peroxide hydrolysis was performed at 100°C. For thermal stress tablet formulation was kept at 105°C for 24 hours and photo stress carried out at 1 ICH Cycle.

Method Validation

The performance characteristics considered for validation of the optimized method were: system suitability, specificity, filter study, linearity, accuracy, precision and robustness.

System suitability

System suitability of analytical method was checked throughout whole analysis by measuring the %RSD for known standard, Tailing factor, resolution and plate count.

Specificity

Specificity was performed by checking interference from blank, placebo (excipients of formulation) at the retention time of both these active peaks. Peak purity of Olmesartan and Metoprolol were checked for specificity Forced degradation study was also performed.

Filter study

Filter study was performed to select suitable filter to get clear solution. Filters were evaluated against centrifuged sample solution and absolute difference between filter and centrifuged sample was calculated.

Linearity

Linearity was assessed visually and by means of a lack-of-fit test. The working range was defined as the interval between the upper and the lower levels of the analytes within the calibration curve. Linearity was evaluated from 50% level to 150% level.

Accuracy

Accuracy of analytical method was evaluated by recovery study. Known amount of API spiked in placebo mixture preparation at 50%, 100% and 150% level.

System precision

The five replicate injections of standard preparation were injected to determine the reproducibility of the instrument.

 Method precision

The six different sample sets were prepared and injected to determine the repeatability of method.

Standard solution preparation

Accurately weighed and transferred about 20 mg OLM and 25mg of MET into 100 mL of clean, dry volumetric flask. 50 mL of diluent (Water: Acetonitrile (1:1)) added and sonicated to dissolve and volume made up to the mark with diluent. 5mL of standard stock transferred into 50mL of volumetric flask and volume made up to the mark with diluent.

 Sample preparation

10 tablets were weighed and crushed to fine powder. Powder equivalent to 20 mg OLM and 25mg of MET was transferred into 100 mL of clean, dry volumetric flask. 50 mL of diluent (Water: Acetonitrile (1:1)) added and sonicated for 15 min and volume made up to the mark with diluent. 5mL of standard stock transferred into 50mL of volumetric flask and volume made up to the mark with diluent. It was filtered using 0.45µ Filter discarding 3mL of filtrate.

Forced degradation study

Forced degradation study on formulation was carried out in solution state. For acid stress, 5 mL of sample stock solutions were transferred into 50 mL of volumetric flask and 2 mL of 0.1N HCl added. Sample solution was kept at 100°C for 1 hour. After 1 hour sample was neutralized with 2 mL of 0.1N NaOH and volume made up to the mark with diluent. Similarly solution for base stress was prepared. For oxidation stress, 0.1% hydrogen peroxide was used and sample was kept at 100°C for 1 hour. Thermal and photo stress were carried out on solid state. For thermal stress, tablet formulation was kept at 105°C for 24 hours, for photo stress tablets were exposed to 1 ICH cycle.

Robustness

Robustness study was performed with deliberate changes in method parameters with respect to flowrate, column oven temperature, detection wavelength and pH of buffer.

Results and Discussion

The method has been employed successfully for quantitative determination of OLM and MET by Reverse Phase High Performance Liquid Chromatographic method and validated according to ICH Q2 (R1) guidelines.

Identification by UV

A 10ppm solution of Olmesartan Medoximil and Metoprolol Succinate individually was scanned in the range of 200 to 400 nm and maximum absorbance observed at 257 nm for Olmesartan Medoximil and 222 nm for Metoprolol Succinate. Result of identification by UV is given in Fig-2a and 2b.

Identification by IR

Identification by IR was carried out for all APIs. Samples were scanned in the range of 400-4000cm-1. IR peaks observed in sample preparation were matched with the reference spectra available in pharmacopeia. Result of identification by IR is given in Fig-3a and 3b

Melting point determination

Melting point was determined by using capillary method. Test results were compared to reference results available in COA and met acceptance criteria. Result of identification by melting point is given in Table-1.

Table-1: Melting Point Determination

 Chromatographic conditions

The optimized UHPLC conditions are given in Table-2.

Table-2: Optimized Chromatographic Condition

Method Validation

The results for various validation parameters viz. system suitability, specificity, linearity, accuracy, precision, forced degradation are depicted below.

System suitability

Results for various system suitability parameters for analytical method was checked throughout and reported in Table-3.

Table 3: Result of System Suitability Test

 Specificity

Table-4: Results of Forced Degradation Study

Interference from blank, placebo (excipients of formulation) at the retention time of both these active peaks was checked. No interference was observed at the retention time of Olmesartan and Metoprolol. (Fig. 4). Peak purity of Olmesartan and Metoprolol were passing by total point method for specificity as well as forced degradation study (Table-4).

Filter study

Results of Filter study performed to select suitable filter against centrifuged sample solution are shown in Table-5.

Table 5: Result of Filter Study

Linearity

Results of Linearity for both the analytes evaluated from 50% level to 150% level are depicted in Table-6. The correlation co-efficient value was greater than 0.999.

Accuracy

Accuracy of analytical method was evaluated by recovery study.

Known amount of API was spiked in placebo mixture preparation at 50%, 100% and 150% level. The % recovery obtained for the analytes is shown in Table-7.

Table-7: Recovery Results

System precision

The five replicate injections of standard preparation were injected to determine the reproducibility of the instrument and %RSD was reported in Table-8.

Method precision

The six different sample sets were prepared and injected. Results obtained for %Assay of 6 different sample preparations is depicted in Table-9.

Table-9: Results of Method Precision

Robustness

Robustness results obtained with deliberate change as shown in below Table-10.

Table-10: Results of Robustness

Conclusion

The short chromatographic time makes this method suitable for processing of multiple samples in short time. The method shows no interference by the excipients.

The statistical parameters and recovery data reveals the good accuracy and precision. This method can be useful and suitable for the estimation of the OLM & MET in bulk and pharmaceutical formulations.

Acknowledgement

The authors are thankful to Spinco Biotech Private Limited, Ahmedabad (India), for providing facility to do work

REFERENCES

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Sonekar College of Pharmacy, Mahadula, Devi Road, Koradi, Nagpur-441111. (MS) India. Smt. Kishoritai Bhoyar College of Pharmacy, New Kamptee, Nagpur-441002. (MS) India

Abstract

The IPN microspheres were prepared by using emulsion crosslinking method. In that the chitosan is used as crosslinking polymer while Hydroxy propyl cellulose(HPC), Hydroxy propyl methyl cellulose (HPMC) and sodium carboxy methyl cellulose (Na CMC) were used as neutral polymer by using glutaraldehyde as crosslinking agent and span 80 as emulsifying agent. Drug was scanned under UV spectroscopy and maximum absorbance was found at 288.2 nm in both pH 1.2 and pH 6.8 medium. Both drug and polymer were investigated for interaction by FTIR spectroscopy. In the present work nine batches were prepared by varying the different ratio of polymer concentration in each batch. First three batches (P1-P3) contain chitosan:

Keywords

Interpenetrating polymer network

Cite This Article

Ms. J. D. Walde. Mr. H. S. Kanhere. (2020). Formulation and Evaluation of Interpenetrating polymer network Microspheres containing ritonavir. International Journal for Pharmaceutical Research Scholars, 9(1); 01-17.

INTRODUCTION

An ideal dosage regimen in the drug therapy of any diseases is the one which immediately attains the desired therapeutic concentration of drug plasma (or at site of action) and maintains it constant for entire duration of treatment. This is possible through administration of conventional dosage form in particular dose and at particular frequency. The frequency of administration or the dosing interval of any drug depends upon its half-life or mean residence time and its therapeutic index. In most cases, the dosing interval is much shorter than the half-life of the drug resulting in a number of limitations associated with such a conventional dosage form.

The oral sustained release formulation has been developed in an attempt to release the drug slowly into the GIT and maintain an effective drug concentration in serum for longer period of time.¹

1.1 INTERPENETRATING POLYMER NETWORK

Over the past decades, blends have been investigated to satisfy the need of specific sectors of polymer industry. Such polymeric blends showed superior performances over the conventional individual polymers and consequently, the range of applications have grown rapidly for such class of materials. In the recent years, carbohydrate and biodegradable polymers have been extensively used to develop the controlled release formulations of drugs having short plasma life. Among the various polymers employed, hydrophilic biopolymers are quite suitable in oral applications due to their inherent advantages over the synthetic polymers.² The importance of biocompatible and biodegradable polymers is continuously increasing in pharmaceutical applications because of their propensity to form crosslinked three‐dimensional network hydrogels that tend to swell in water or biological fluids. Such systems have been considered as a the potential candidate to deliver bioactive molecules, particularly in controlled release applications³

Interpenetrating polymer network (IPN) is regarded as one of the most useful novel biomaterial. The excellent biocompatibility and safety due to its physical characteristics such as impart stability of the drug in the formulations, improves solubility of hydrophobic drugs, excellent swelling capacity and its biological characteristics, like biodegradability, impart bioavailability, drug targeting in a specific tissue and very weak antigenecity. IPN offers novel way to address delivery of hydrophobic and low bioavailable drug. Interpenetrating polymer networks are the polymeric blends showed superior performances over the conventional individual polymers and consequently, the range of applications have grown rapidly for such class of material.

IPN is defined as a combination of at least two polymers chains each in network form, of which at least one is synthesized and/or cross‐linked in the immediate presence of the other without any covalent bonds between them. If only one component of the assembly is cross linked leaving the other in a linear form, the system is termed as semi‐ interpenetrating polymer networks^3,4

1.1.1 Classification of IPN

Based on Chemical Bonding

Covalent Semi IPN:

A covalent semi IPN contains two separate polymer systems that are crosslinked to form a single polymer network.

NonCovalent Semi IPN:

A non‐covalent semi IPN is one in which only one of the polymer systems is crosslinked

NonCovalent Full IPN:

A non‐covalent full IPN is one in which the two separate polymers are independently crosslinked.

1.2 DRUG PROFILE:

Ritonavir^5,6,7

Ritonavir is a member of the group of drugs as Anti-Retroviral. It is a protease inhibitor drug used against infection.

Structure

Chemical name: 10 hydroxy-2-methyl -5(1-methyl ethyl )-1-[2-(1-methyl ethyl)-4-                                   Thiazolyl]-3-6-dioxo-8,11bis (phenymethyl)- 2,4,7,12-tetraazatrid -ecan-13-oic acid, 5-thiazolymethyl ester.

Molecular formula: C₃₇H₄₈N₆O₅S₂

Molecular weight: 721.0

Melting point: 119-123⁰ C.

Description: Ritonavir is an almost white to light –tan powder, odour slight and Characteristic.

Category: Antiretroviral.

Solubility: Practically insoluble in water, freely soluble in methanol and ethanol.

Pka: 3.48

Log P: 5.28

Half-life: 3-5 hr

Storage: Store protected from light and moisture.

Mechanism of Action : Ritonavir is a peptidomimetic inhibitor of both the HIV-1 and                                              HIV-2 proteases. Inhibition of HIV protease renders the enzyme incapable of processing the gag-pol polyprotein precursor which leads to production of non-infectious immature HIV particles.

2.0 MATERIALS AND INSTRUMENTS

2.1 MATERIALS:

Ritonavir , Chitosan  , Hydroxy Propyl Cellulose (HPC) ,Hydroxy Propyl Methyl, Cellulose K100M (HPMC K100M),Polyvinyl Pyrrolidone (PVP),

Glutaraldehy ,Hydrochloric acid (HCl),Potassium Dihydrogen Phosphate (KH₂PO₄) ,Sodium Hydroxide (NaOH) and Light Liquid Paraffin

2.2 INSTRUMENTS:

Double Beam UV Spectrophotometer, FTIR Spectrophotometer, Electronic Weighing Balance ,Dissolution Test Apparatus,Peristaltic Pump,

Differential Scanning Calorimetry,Imaging System, Magnetic Stirrer, Heating Humidity Chamber and  Scanning Electron Microscope

3. XPERIMENTAL AND RESULTS

3.1 PREFORMULATION STUDY

3.1.1 Characterization of Ritonavir: ^8,9

1. i) Description: Visual inspection of drug evealed that drug is a white colored crystalline solid.
2. ii) Melting Point: The melting point of the drug sample was determined by capillary method and found to be 119-123^oC, which complies with melting point reported in Merck Index (119-122⁰C)

Solubility: Practically insoluble in water, Freely soluble in methanol and ethanol, soluble in isopropanol.

3.1.2 Identification Tests for Propranolol HCl ⁸:

3.1.2.1 UV Absorption Spectrum of Ritonavir in Acid Buffer pH 1.2:

1. I) Preparation of 0.1 N HCL:

Acid buffer pH 1.2 was prepared by placing 8.5 ml of conc. HCl into 1000 ml volumetric

flask and making the volume upto the mark using distilled water. The pH was adjusted to 1.2 on the digital pH meter.

1. II) Scanning of Ritonavir in 0.1 N HCL:

Ritonavir (20mg) was accurately weighed and dissolved in10 ml 0.1 N HCL (pH 1.2) and clear solution was obtained. To this sufficient amount of the medium was added to make the volume to 100 ml. The resultant solution was diluted with the same medium (pH1.2) to obtained a concentration of  20 ug/ml and scanned between 200-400nm.

Observation: The λmax was found to be at 246 nm as shown in Figure No. 8

3.1.2.2 Preparation of Standard Calibration Curve of Ritonavir in Acid Buffer pH 1.2:

Procedure:

50 mg of Ritonavir was weighed accurately and dissolved in 50 ml of acid buffer pH 1.2, from this 1ml solution was withdrawn and diluted to 50 ml to get standard stock solution of 20 ug/ml. The stock was suitably diluted to get concentrations from 2 – 20 µg/ml and was analyzed at 246 nm to plot the standard calibration curve.

Table no. 1: Standard Calibration Curve of Ritonavir in Acid Buffer pH1.2 at 246nm.

*Each value represent the mean ± standard deviation (n=3)

Observation:

Equation of Regressed Line: y = 0.0535x+0.1533

Correlation Coefficient: (R²) = 0.997

3.1.2.3 UV Absorption Spectrum of Propranolol HCl in Phosphate Buffer pH 6.8

I) Scanning of Ritonavir in pH 6.8 :

20 mg of Ritonavir was taken in 100ml volumetric flask. To that 5 ml of methanol was added and shaken well to dissolve the drug. The solution was made up to the mark with 6.8 pH phosphate buffer solutions. From the above solution, 10 ml is diluted to 100ml with, 6.8 pH phosphate buffer solution to give 20 ug/ml concentration. From the above solution 1 ml is diluted to 10ml with, 6.8pH phosphate buffer solutions to give 2 ug/ml concentration. The prepared solution i.e., 2 ug/ml concentration was scanned for 200-400nm UV/ Visible spectrophotometer.

3.1.2.4 Standard Calibration Curve of Ritonavir in Phosphate Buffer pH 6.8 at 243 nm.

Accurately measured 50.0 ml of 0.2 M KH₂PO₄ solution and 22.4 ml of 0.2 M NaOH solution was added into 200.0 ml volumetric flask. The volume was made up to the mark with distilled water. The pH was adjusted to 6.8 on the digital pH meter.

Procedure:

50 mg of Ritonavir was weighed accurately and dissolved in 50 ml of acid buffer pH 6.8, from this 1ml solution was withdrawn and diluted to 50 ml to get standard stock solution of 20 ug/ml. The stock was suitably diluted to get concentrations from 2 – 20 µg/ml and was analyzed at 243 nm to plot the standard calibration curve.

Table No 02: Standard Calibration Curve of Ritonavir in Phosphate Buffer pH6.8 at 43nm.

*Each value represents the mean ± standard deviation (n=3)

Observation:

Equation of Regressed Line: y = 0.0496-0.048

Correlation Coefficient: (R²)

3.3 PREPARATION OF MICROSPHERES¹⁰

IPN microspheres were prepared using different ratios of Chitosan: HPC, Chitosan: HPMC K100M and Chitosan: PVP by using emulsion crosslinking method. Briefly, 2% (w/v) of Chitosan solution was prepared by dissolving in 2% (w/v) acetic acid in double-distilled deionized water and stirring it continuously until the attainment of a homogeneous solution. Different ratio HPC was then dispersed in different ratio of Chitosan solution and stirred. The drug Ritonavir was dissolved in the above polymer blend solution, which was added slowly to light liquid paraffin (100 g, w/w) containing 2% (w/w) span-80 under constant stirring at 1200 rpm speed for about 60 min. To this w/o emulsion, 5 ml of GA as a crosslinking agent containing 0.5mL of 1N HCl were added slowly and stirred for 3 h. The hardened microspheres were separated by filtration, wash repeatedly with n-hexane and distilled water to remove the unreacted GA. Similar Procedure for chitosan: HPMC K100M and chitosan: PVP was repeated. Solid microspheres obtained were vacuum dried at 40˚C for 24 h and stored in a desiccator until further use. Totally, nine formulations were prepared as per the formulation codes assigned in Table No. 12.

Table No. 03: Composition of IPN Microspheres.

4.4: EVALUATION OF MICROSPHERES

4.4.1: Drug Content and Entrapment Efficiency^{11, 12, 13}

Ritonavir microspheres 50 mg from each batch were digested in 50 ml of pH 6.8 phosphate buffer solution for overnight, and then sonicated for 15 min. After complete dissolution of microspheres drug content was determined by UV-visible spectrophotometer at 243 nm taking 6.8 phosphate buffer solution as a blank. The percent drug loading of microspheres was calculated using following equation

Table No. 04: Theoretical Drug Content (%), Actual Drug Loading (%) and Entrapment Efficiency (%) of Propranolol HCl Microspheres

* Each value represent the mean ± standard deviation (n=3)

4.4.2 Particle size analysis and morphological studies: ^14,15,16

The particle size and shape analysis of propranolol microspheres was done by Metzer optical microscope enabled with camera. About 200 particles were measured for particle size analysis and it was expressed as volume mean diameter in microns (SD), results are shown in Table No. 9

Table No 05: Mean Particle Size of Propranolol HCl microspheres.

*Each value represent the mean ±± standard deviation (n=3)

4.4.3 Swelling Study^17,18

Water uptake of the cross-linked microspheres loaded with the drug was determined by measuring the extent of swelling of the matrix in pH 1.2 and phosphate buffer 6.8 solutions. The samples were allowed to swell in pH 1.2 buffer solution for 2 hr and then at pH 6.8 phosphate buffer for 10 hr. The excess surface adhered liquid drops were removed by blotting with soft tissue papers and the swollen microspheres were weighed to an accuracy of 0.01 mg using an electronic microbalance. The hydrogel microspheres were then dried in an oven at 50 ⁰C for 5 hr until there was no change in the dried mass of the samples.

Table No. 06: Percent Water Uptake of Microspheres.

*Each value represent mean (n=3) observation ± S.D.

4.4.4   In Vitro Drug Release Studies: ^{10, 19, 20}

Drug release from the IPN microspheres with different % drug loading and different polymer composition was investigated in 0.1 N HCl for the initial 2 h, followed by phosphate buffer pH 6.8 until the completion of dissolution. These experiments were performed using a fully automated dissolution tester coupled with a UV system (Double Beam UV Spectrophotometer   Model -UV 2401 PC   Shimadzu Corporation, Koyto, Japan.) equipped with six baskets at the stirring speed of 100 rpm. A weighed quantity of each sample was placed in 500 ml of dissolution medium maintained at 37 ˚C. Dissolution study is conducted initially 2hrs in acid  buffer pH 1.2 and remaining 10 hr in phosphate buffer pH 6.8 During dissolution study 1 ml aliquot was withdrawn at different time intervals of 1 hr upto 12 hrs and same was replaced with equal volume of fresh medium. The withdrawn samples were filtered through Whatmann filter paper no.42 and Propranolol HCl concentration was determined by UV spectrophotometer at λ max of 245.4 nm.

Table No. 07: Percent Cumulative Drug Release of Batch F-1 to F-5.

Each value represent the mean ± standard deviation (n=3)

Table No. 08: Percent Cumulative Drug Release of Batch P-6 to P-7.

4.4.5 Duration of Mucoadhesion:^21,22,23

This is an important factor in the formulation of bioadhesive dosage forms capable of being retained on mucosal surfaces for extended period of time and must be given careful consideration.

Method: A freshly cut 5 cm long piece of pig nasal mucosa obtained from local abattoir within 1 hr of sacrificing the animal was cleaned by washing with isotonic saline solution. An accurate weight of the microspheres was mixed with sudan red and applied on mucosal surface which was attached over a polyethylene plate fixed at an angle at 40^o relative to the horizontal plane. Phosphate buffer saline pH 7.4 warmed at 37^oC was pumped peristatically, over the tissue at the rate of 5ml/min. The duration of complete washing of the microspheres was recorded.

Table No. 09: Duration of Mucoadhesion for formulations P1-P9.

4.6: CHARACTERIZATION OF MICROSPHERE

4.6.1: Differential Scanning Calorimetry (DSC): ^24,25,26

A differential scanning calorimeter was used for thermal analysis of drug and physical mixture. Drug and its physical mixture were weighted directly in was weighed directly in the pierced DSC aluminum pan (Aluminum Standard 40 µl) and scanned at the temperature range of 25-400 °C and at heating rate of 10 °C/min. in nitrogen atmospheres at flow rate of  20 ml/min, thermogram obtained were observed for any interaction Observation:

4.6.2 Scanning Electron Microscopy:^27,28,29

The surface photography of the microparticles was examined using scanning electron microscopy. Microspheres were spread on a double sided adhesive plate, one side of which was stuck to glass slide. Excess microspheres were removed and the slide was kept on the sample holder and scanning electron micrograph was taken using an electron microscope. (JEOL, JSM-5200, Japan 15kv).

4.6.3 Treatment of In vitro Drug Release Data with Different Kinetic Equations

Drug release kinetics was assumed to reflect different release mechanisms of controlled release drug delivery systems. Therefore, five kinetics model were applied to analyze the in vitro data to find the best fitting equation.²⁶

Zero order release equation;

F_t =K₀t

Where Ft represents the fraction of drug released in time t and K₀ is the apparent rate constant of zero-order release constant.

First-order equation;

In (l-F) = -K₁t

Where F represents the fraction of drug released in time t and K₁ is the first-order release constant.

Higuchi equation;

F = K₂t^1/2

Where F represents the fraction of drug released in time t and K₂ is the Higuchi constant.

Hixson – Crowell equation;

Q₀^1/3 – Qt^1/3 = kHC t

Where Q₀ = Amount of drug released or dissolved at time t=0

& Qt = Amount of drug released or dissolved at time t.

Pappas equation;

Mt/M^∞ = K₃tⁿ

In Korsmeyer – Peppas equation Mt and M^∞ are the amount of drug released at time t and^∞, respectively and n is the diffusion coefficient. In spherical matrices, if n<0.5, a Fickian (case -l), 0.5 <n<1.0, a non-Fickian, and n> 1.0 a case-II (zero order) drug release mechanism dominates.

Table No. 10: Kinetic Treatment of Dissolution Data of Formulations P1-P9.

4.6.4. Stability study

Formulation P3 selected as optimized formulation as it gave desirable drug release hence it was kept for stability study. Stability study of an optimized formulation was carried out by storing the microspheres (wrapping in aluminum foil) at 40± 2 ⁰C and 75 ± 5% relative humidity for 3 months. At an interval of 1 month, the microspheres were examined for % Drug Loading, % Entrapment Efficiency and in vitro release data.

Table No. 11 Percent Drug Loading and Percent Entrapment Efficiency study of  Batch P3 kept for stability at 40 ± 2˚C/75 ± 5 % RH