Niosomal Drug Delivery for Oral Route

Abstract

Niosomes are artificial vesicles with potential technological advantages. They are non-ionic surfactant vesicles. As efficient drug delivery systems with a wide range of uses, niosomes have the same potential benefits as phospholipid vesicles (liposomes), including the capacity to hold both water- and lipid-soluble pharmaceutical molecules. Niosomes can also be thought of as more cost-effective, chemically stable, and occasionally physically stable alternatives to liposomes. Simple preparation techniques and commonly used surfactants in pharmaceutical technology can be employed to create niosomes. Numerous studies have covered noisome physicochemical characteristics and their uses as drug delivery vehicles. In this report, a brief and simplified summary of different theories of self-assembly are discussed. Furthermore, manufacturing methods, physical characterization techniques, bilayer membrane additives, unconventional niosomes (discomes, proniosomes, elastic and polyhedral niosomes), their recent applications as drug delivery systems, limitations and directions for future research will be discussed.

Introduction

I. INTRODUCTION

Niosomes are non ionic surfactant vesicles which can entrap both hydrophilic and lipophilic drugs, either in aqueous layer or in vesicular membrane made of lipid materials. Niosomesare either unilamellar or multilamellar vesicles that have a better stability than liposomes. Niosomes are formed on admixture of nonionic surfactant, cholesterol with subsequent hydration in aqueous medium. The addition of cholesterol, gives the rigidity to the bilayer which results in less leaky niosomes. Niosome behave invivo like liposomes prolonging the circulation of entrapped drug.{1}

Niosomes offer a versatile vesicle delivery concept with potential for delivering drug via transdermal route. Topically applied niosomes can increase residence time of drug in the stratum corneum and epidermis, while reducing the systemic absorption of the drug. It also improves the horny layer properties both by reducing transepidermal water loss and by increasing smoothness via replenishing lost skin lipids.{2}

Paul Ehrlich, in 1909, initiated the era of development for targeted delivery when he investigate a drug delivery mechanism that would target directly to diseased cell. Since then, numbers of carriers were utilized to carry drug at the target organ/tissue, which include immunoglobulins, serum proteins, synthetic polymers, liposomes, microspheres, erythrocytes, & niosomes.{3,4,5}

A. Defination of Niosome

Niosomes are non-ionic surfactant vesicles obtained on hydration of synthetic nonionicsurfactants, with or without incorporation of cholesterol or other lipids. The sizes of niosomesare microscopic and lie in nanometric scale. The particle size ranges from 10nm-100nm.{2}

B. Structure of Niosome

Structurally, niosomes are similar to liposomes, in that they are also made up of a bilayer. However, the bilayer in the case of niosomes is made up of non-ionic surface active agents rather than phospholipids as seen in the case of liposomes. Most surface active agents when immersed in water yield micellar structures however some surfactants can yield bilayervesicles which are niosomes. Niosomes may be unilamellar or multilamellar depending onthe method used to prepare them. The niosome is made of a surfactant bilayer with its hydrophilic ends exposed on the outside and inside of the vesicle, while the hydrophobic chains face each other within the bilayer. Hence, the vesicle holds hydrophilic drugs within the space enclosed in the vesicle, while hydrophobic drugs are embedded within the bilayer itself.{7}

The properties of the vesicles can be changed by varying the composition of the vesicles, size, lamellarity,tapped volume, surface charge and concentration. Various forces act inside the vesicle like Vander Waals forces among surfactant molecules, repulsive forces emerging from the electrostatic interactions among charged groups of surfactant molecules, entropic repulsive forces of the head groups of surfactants, short-acting repulsive forces etc. These forces are responsible for maintaining the vesicular structure of niosomes. But, the stability ofniosomes are affected by type of surfactant, nature of encapsulated drug, storage temperature,detergents, use of membrane lipids, the interfacial polymerization of surfactant monomers in situ, inclusion of charged molecule. The geometry of vesicle to be formed from surfactants is affected by its structure, which is related to critical packing parameters. On the basis of critical packing parameters of surfactants, we can predicate geometry of vesicle to be formed.Critical packing parameters can be defined using following equation, CPP (Critical Packing Parameters) v/lc xa0

Where v hydrophobic group volume, le= the critical hydrophobic group length, a0 the area ofhydrophilic head group. From the critical packing parameter value type of miceller structure formed can be ascertained as given, If CPP < ½, then formation of spherical micelles, If ½ < CPP<1, then formation of bilayer micelles, If CPP> 1, then formation inverted micelles.{3,14}

C. Advantages of Niosomes

1. They are osmotically active and stable.
2. They increase the stability of the entrapped drug
3. Handling and storage of surfactants do not require any special conditions
4. They increase the oral bioavailability of drugs
5. They enhance the skin penetration of drugs
6. They be used for oral, parenteral as well topical route.
7. The surfactants are biodegradable, biocompatible, and non-immunogenic.
8. Improve the therapeutic performance of the drug by protecting it from the biologicalenvironment and restricting effects to target cells, thereby reducing the clearance ofthe drug
9. The niosomal dispersions in an aqueous phase can be emulsified in a non-aqueous phase tocontrol the release rate of the drug and administer normal vesicles in external non-aqueous phase

D. Disadvantages Of Niosomes

1. Physical instability
2. Aggregation
3. Fusion
4. Leaking of entrapped drug
5. Hydrolysis of encapsulated drugs which limiting the shelf life of the dispersion {1}

In niosomes the surface charge determines the behaviour of niosomes in vivo and in vitro uncharged vesicles sometimes caused aggregation and fusion. Vesicle aggregation of niosomes may be prevented by the inclusion of compounds that introduce repulsive steric or electrostatic forces. An example of steric stabilization is the inclusion of Solulan C24 (a cholesteryl poly-24- oxyethylene ether) noisome formulations. Examples of electrostatic stabilization are the inclusion of dicetyl phosphate in 5(6)-carboxyfluorescein loaded Span 60based niosomes.[46]

HLB value in the range 14-17 is not suitable to produce niosomes so cholesterol must be added to the surfactant in order to form a bilayered vesicle and for lower HLB values,cholesterol enhances stability of vesicles. It is also seen that the addition of cholesterol enables more hydrophobic surfactants to form vesicles, suppresses the tendency of the surfactant to form aggregate

II. COMPOSITIONS OF NIOSOMES

The major components used for the preparation of niosomes are, Nonionic surfactants

The role of surfactants play a major role in the formation of niosomes. The following non-ionic surfactants are generally used for the preparation of niosomes.

E.g

Spans (span 60, 40, 20, 85, 80)

Tweens (tween 20, 40, 60, 80) and

Brijs (brij 30, 35, 52, 58, 72, 76).

The non ionic surfactants possess a hydrophilic head and a hydrophobic tail.HLB value in therange 14-17 is not suitable to pruduce niosomes so cholesterol must be added to the surfactant in order to form a bilayered vesicle.{1}

Ether linked surfactants These are polyoxyethylene alkyl ethers which have hydrophilic and hydrophobic moieties are linked with ether. The general formula of this group is (CnEOm), where n can be 12-18 andm can be 3-7. Surfactants with polyhydroxyl head and ethylene oxide units are also reported to be used in niosomes formation. Single alkyl chain surfactant C16 mono alkyl glycerol ether with an average of three glycerol units is one of the examples of this class of surfactantsused for the preparation of niosomes. Polyoxyethelene 4 lauryl ether (Brij30) has an HLB value of 9.7, phase transition temperature <10°C cannot be used to formulate some drugs and iodides mercury salts, phenolic substances, salicylates, sulfonamides and tannins as it cause ethers (Brij58) and Polyoxyethylene stearyl ethers (Brij72and76) are also used in preparation of niosomes{9}

A. Types Of Niosomes

The niosomes are classified as a function of the number ofbilayer (e.g. MLV, SUV) or as a function of size. (e.g.LUV, SUV) or as a function of the method of preparation (e.g.REV, DRV). The various types of niosomes are described below:

1. Multilamellar Vesicles (MLV)

It consists of a number of bilayer surrounding the aqueous lipid compartment separately. The approximate size of these vesicles is 0.5-10 µm diameter. Multilamellar vesicles are the most widely used niosomes. It is simple to make and are mechanically stable upon storage for long periods. These vesicles are highly suited as drug carrier for lipophilic compounds.thin film hydration method and transmembrane ph gradient method was used for the preparation of multilamellar vesicles.{5,7}

2. Large Unilamellar Vesicles (LUV)

Niosomes of this type have a high aqueous/lipid compartment ratio, so that larger volumes of bio-active materials can be entrapped with a very economical use of membrane lipids the sizeof large unilamellar vesicles is more than 100 nm, Ether injection method and reverse phase evaporation method was used for the preparation of large unilamellar vesicles. {5,7}

3. Small Unilamellar Vesicles (SUV)

These small unilamellar vesicles are mostly prepared from multilamellar vesicles by sonication method, French press extrusion.the size of small unilamellar vesicles is upto 100 nm i.e 20-50 nm{7

B. Factors Affecting Niosomes Formulation

1. Drug

Entrapment of drug in niosomes increases vesicle size, probably by interaction of solute with surfactant head groups. In polyoxyethylene glycol (PEG) coated vesicles; some drug isentrapped in the long PEG chains, thus reducing the tendency to increase the size. The hydrophilic lipophilic balance of the drug affects degree of entrapment. The physicochemical properties of encapsulated drug affect charge and rigidity of the niosome bilayer. Here drug interacts with surfactant head group and develops the charge which creates mutual repulsion between surfactant bilayer and hence increase vesicle size [1,14] Another factor to be considered is whether the drug to be encapsulated is amphiphilic. The best example of such a drug is doxorubicin. When encapsulated in niosomes,aggregation occurred and was overcome by the addition of asteric stabilizer. The increase in encapsulation of a drug that occurs when more is added could be the result of saturation of the medium. This suggests that thesolubility of certain poorly soluble drugs can be increased by formulation in niosomes but only upto acertain limit above which drug precipitation will occur. An increase in the encapsulation of flurbiprofen due to saturation of drug in the hydration medium has been reported. However, when niosomes were prepared using higher amount sof minoxidil,optica Imicro-scopy revealed minoxidil crystals dispersed in between the niosomal particles{11}

2. Amount and Type of Surfactant

The mean size of niosomes increases proportionally with increase in the HLB surfactants likeSpan 85 (HLB 1.8) to Span 20 (HLB 8.6) because the surface free energy decreases with an increase in hydrophobicity of surfactant. The bilayers of the vesicles are either in the so- called liquid state or in gel state, depending on the temperature, the type of lipid or surfactant and the presence of other components such as cholesterol. In the gel state, alkyl chains are present in a well ordered structure, and in the liquid state, the structure of the bilayers is moredisordered. The surfactants and lipids are characterized by the gel-liquid phase transition temperature (TC). Phase transition temperature (TC) of surfactant also effects entrapment efficiency i.e. Span 60 having higher TC, provides better entrapment. HLB value of surfactants affects entrapment efficiency, such as HLB value of 14 to 17 is not suitable for niosomes but HLB value of 8.6 has highest entrapment efficiency and entrapment efficiency decreases with decrease in HLB value from 8.6 to 1.7 [1.4] The maximum amount of surfactant/lipid used to prepare niosomes is generally 10-30mmol/L (1-2.5%, w/w). Alterations in the surfactant:water ratio during the hydration step may affect the structure andproperties of the niosomes produced. As the surfactant/lipid level increases, the amount of drug to been capsulated also increases leading to an increase in the viscosity of the system.{11}

3. Cholesterol Content and Charge

Inclusion of cholesterol in niosomes increased its hydrodynamic diameter and entrapment. In general, the action of cholesterol is two folds; on one hand, cholesterol increases the chain order of liquid-state bilayers and on the other, cholesterol decreases the chain order of gel state bilayers. At a high cholesterol concentration, the gel state is transformed to a liquid- ordered phase. An increase in cholesterol content of the bilayers resulted in a decrease in the release rate of encapsulated material and therefore an increase of the rigidity of the bilayers obtained. Presence of charge tends to increase the interlamellar distance between successive bilayers in multilamellar vesicle structure and leads to greater overall entrapped volume.{1}

4. Resistance to Osmotic Stress

Addition of a hypertonic salt solution to a suspension of niosomes brings about reduction in diameter. In hypotonic salt solution, there is initial slow release with slight swelling of vesicles probablys due to inhibition of eluting fluid from vesicles, followed by faster release, which may be due to mechanical loosening of vesicles structure under osmotic stress.{1}

5. Effect of Additives

The stable niosomes can be prepared with addition of different additives along with surfactants and drugs. Niosomes formed have a number of morphologies and their permeability and stability properties can be altered by manipulating membrane characteristicsby different additives. In case of polyhedral niosomes formed from C16G2, the shape of these polyhedral noisome remains unaffected by adding low amount of solulan C24 (cholesterol poly-24- oxyethylene ether), which prevents aggregation due to development of stearic hydrance. In contrast spherical Niosomes are formed by C16G2: cholesterol: solution (49:49:2). The mean size of niosomes is influenced by membrane composition such as Polyhedral niosomes formed by C16G2: solution C24 in ration (91:9) having bigger size (8.0+0.03 mm) than spherical/tubular niosomes formed by C16G2: cholesterol: solution C24 in ratio (49:49:2) (6.6+0.2 mm). Addition of cholesterol molecule to niosomal system provides rigidity to the membrane and reduces

III. METHOD OF PREPARATION

A. Ether Injection Method
This method provides a means of making niosomes by slowly introducing a solution of surfactant dissolved in diethyl ether into warm water maintained at 60°C. The surfactant mixture in ether is injected through 14-gauge needle into an aqueous solution of material. Vaporization of ether leads to formation of single layered vesicles. Depending upon the conditions used, the diameter of the vesicle range from 50 to 1000 nm. {3,4,15}

D. Micro fluidization

Micro fluidization is a recent technique used to prepare unilamellar vesicles of defined size distribution. This method is based on submerged jet principle in which two fluidized streams interact at ultra high velocities, in precisely defined micro channels within the interaction chamber. The impingement of thin liquid sheet along a common front is arranged such that the energy supplied to the system remains within the area of niosomes formation. The resultis a greater uniformity, smaller size and better reproducibility of niosomes formed.{3,4,}

E. Multiple Membrane Extrusion Method

Mixture of surfactant, cholesterol and dicetyl phosphate in chloroform is made into thin film by evporation. The film is hydrated with aqueous drug solution and the resultant suspension extruded through polycarbonate membranes, which are placed in series for upto 8 passages. Itis a good method for controlling niosome size.{3,4

F. Reverse Phase Evaporation Technique (REV)

Cholesterol and surfactant (1:1) are dissolved in a mixture of ether and chloroform. An aqueous phase containing drug is added to this and the resulting two phases are sonicated at 4-5°C. The clear gel formed is further sonicated after the addition of a small amount of phosphate buffered saline (PBS). The organic phase is removed at 40°C under low pressure. The resulting viscous niosome suspension is diluted with PBS and heated on a water bath at 60°C for 10 min to yield niosomes. have reported the preparation of Diclofenac Sodium niosomes using Tween 85 by this method.

G. Trans Membrane pH Gradient (inside acidic) Drug Uptake Process (remote Loading)

Surfactant and cholesterol are dissolved in chloroform. The solvent is then evaporated under reduced pressure to get a thin film on the wall of the round bottom flask. The film is hydratedwith 300 mM citric acid (pH 4.0) by vortex mixing. The multilamellar vesicles are frozen andthawed 3 times and latersonicated. To this niosomal suspension, aqueous solution containing 10 mg/ml of drug is added and vortexed. The pH of the sample is then raised to 7.0-7.2 with IM disodium phosphate. This mixture is later heated at 60°C for 10 minutes to give niosomes.{3,4,}

H. The "Bubble" Method

It is novel technique for the one step preparation of liposomes and niosomes without the use of organic solvents. The bubbling unit consists of round-bottomed flask with three necks positioned in water bath to control the temperature. Water-cooled reflux and thermometer is positioned in the first and second neck and nitrogen supply through the third neck. Cholesterol and surfactant are dispersed together in this buffer (pH 7.4) at 70°C, the dispersion mixed for 15 seconds with high shear homogenizer and immediately afterwards "bubbled" at 70°C using nitrogen gas.{3,4,}

I. Formation of Niosomes from Proniosomes

Another method of producing niosomes is to coat a watersoluble carrier such as sorbitol with surfactant. The result of the coating process is a dry formulation. In which each water-solubleparticle is covered with a thin film of dry surfactant. This preparation is termed "Proniosomes". The niosomes are recognized by the addition of aqueous phase at T > Tm andbrief agitation.{3,4,}

T=Temperature

Tm= mean phase transition temperature

IV. EVALUATION OF NIOSOMES

V. EXPERIMENTAL WORK

Pre-Formulation Studies Drug Characterization

A. Glimepiride Drug Authentication

Preformulation studies were performed to determine the physiochemical properties of thedrug that affect the development and efficacy of new drug formulation.

1. Descriptions: The sample of the drug was visually observed for color, odor, and appearance.
2. Melting Point Determination: The melting point of Glimepiride was determined by Thiele’s tube method i.e., by taking a small amount of the drug in one end closed capillary tube placed in the melting point apparatus and taking the temperature at which, the drug melts was noted. The average of triplicate readings was noted and compared with standard values.
3. Solubility Study: The solubility of the drug was determined by adding an excess amount of the drug in 10 ml ofdistilled water in glass tubes sealed with aluminum foil. Resultant suspension shaking for 48 hr. on the mechanical shaker and filtered. The filtrate obtained was diluted with distilledwater and analyzed spectrophotometrically using a UV- spectrometer.

B. Spectroscopic Studies

1. UV Spectroscopic

a. Preparation of Standard stock Solution: Accurately weigh 10 mg of glimepiride dissolved in ethanol to make 50ml (100 up/ml. (stocksolution A) 1 ml of solutions separately diluted with methanol up to 10 ml to obtain the concentration of 10 up/ml (stock solution ) and scanned in the wavelength range of 200-400 nm.

2. Selection of Analytical Wavelength

From the standard stock solution ‘B’, various dilution ranging between 2-10 up/ml was prepared and scanned in the wavelength range 400-200 nm using a UV spectrometer.

3. Preparation of Calibration Curve for Glimepiride in Ethanol

By using the standard stock solution, the concentration ranges from 2,4,6,8, and 10 up/ml. The absorption of the above solution was measured at 301 nm and a calibration curve of absorbance against concentration was plotted.

4. Fourier Transform Infrared Spectroscopy (FTIR)

One of the most important advantages of IR over the usual methods of structural analysis is that it provides useful information about the structure of the molecule quickly, withouttiresome evaluation methods. The technique is based on the fact that a chemical substance shows marked selective absorption in the IR region. The FT-IR spectrum of chlorpromazine HCl was recorded to confirm its purity on the FTIR spectrophotometer using the KBr powderpress technique. The baseline correction was done using dried potassium bromide. The instrument was operated under a dry air purge with a resolution of cm-1 over the region 4000-400 cm-1. The scans were evaluated for the presence of principal peaks of the drug. Theidentified peaks were compared with the principal peaks of the reported IR spectrum. The FTIR spectra of Glimepiride are depicted in FIG and observed peaks are reported in table.

C. Compatibility Studies of Drug and Excipients

1. Fourier Transform Infrared Spectroscopy (FTIR) Study: FTIR spectra of pure glimepiride and excipients were obtained on an FTIR spectrometer using KBr powder. The instrument was operated under dry air purge and the scans were collected at the scanning speed of 2mm/sec with a resolution of 4 cm-1 over the region of 4000-400 cm-1. The scans were evaluated for the presence of principal peaks of the drug, shifting and disappearance of drug peaks, and appearance of the new peak due to conformer interaction. The result is reported in Fig.

VI. PREPARATION OF NIOSOMES

Niosomes loaded with glimepiride were formulated by ether injection method based on 32 factorial design . 32 Factorial design is a three-level and two factorsbased factorial design as reported by Webb . In total, there are nine runs or combinations. In designing niosomes, cholesterol and span 60 were selected as two independent variables, both at three different levels (1, 0, +1). In the design, the lower concentration of cholesterol, i.e. 1.0%w/v, has been assigned at 1 level, whereas 2.0% w/v concentration was assigned the level 0. The concentration at highest level +1 was 3.0% w/v. In case of span 60, concentrations at three levels from lowest to highest (1, 0, +1) were 0.5, 1.0 and 3.0% w/v, respectively. Based on this factorial design, nine niosomal formulations were developed. Span 60, cholesterol and drug were dissolved in a mixture of diethyl ether and methanol as per the composition given in and was stirred continuously on a magnetic stirrer . Distilled water was heated to 55 ± 1C. Organic phase was then injected into an aqueous phase.

A. Purification of drug-loaded niosomes

Drug-loaded niosomes were purified by dialysis membrane technique to remove the free drug from niosomal suspension. For this, Hi-media dialysis membrane was kept in saline solution for 2 h before dialysis to ensure complete wetting of the membrane. Niosomal vesicles loaded with glimepiride were placed in a dialysis bag, which was transferred into 200 ml of phosphate buffer pH 7.4. The receiver medium was stirred with a magnetic stirrerat 500 rpm. Five millilitres of sample was withdrawn at appropriate time intervals and replaced with an equal volume of fresh media and analyzed spectrophotometerically for the amount of free drug. Purification time was optimized by applying statistical paired t test at5% level of significance.

Table 1 32 Factorial design of niosomal formulations of glimepiride

Conclusion

1) Oral formulation has been preferred and most common route of drug delivery. 2) In the begining of the work, drug authentication was performed. The authentication study showed that the obtained sample of Glimepiride was pure and complied with IP limit. 3) Niosomes are versatile in structure, morphology and size; they can entrap hydrophilic drugs in aqueous compartments to decrease the degradation of durg. 4) Glimepiride niosmes were prepared with an provide the drug prolonged period of timein the stomach. . Glimepiride was targeted to stomach because it has the absorption window in upper part of GIT so it has been provided for prolonged period of time in stomach for better therapeutic activity by increasing its bioavailability. 5) Glimepiride is an Antidibetic drug of BCS class II having low solubility and high permeability. It has biological half life of 5 to 8 hours , log P value is 3.81, and molecular weight of 490.617 g/mol, these characteristics makes Glimepiride drug model for formulating it into niosomes. 6) Glimepiride niosomes were prepared using surfactants span 60 and Cholesterol in twodifferent ratios by three methods. 7) Niosomes were prepared by ether injection method. Span 60 used as the Surfactant.The Cholestrol was used as carrier and diethyl ether and ethanol used asthe solvent. 8) The shape of Glimepiride niosomes are spherical in nature and the size ranges between 50 and 200 nm. 9) The niosomal formulations were characterized for their vesicle size, entrapment efficiency and invitro release study. 10) The formulated and optimized niosomes batch (F9) shows 90.04 % entrapment efficiency. 11) From factorial design study, it was concluded that Cholesterol concentration increase entrapment efficiency ani partical size alo increase. 12) The present study concluded that the aqueous solubility of poorly water soluble drug Glimepiride can be enhanced by the formulation by Cholestrol surfactant by using ether injection method.

References

[1] Mohamad PU, Mohamed S, Wahab SU. Formulation and Evaluation of miconazole Niosomes, IJPRIF.2011;3 (2):1019-1022 [2] Chandu VP, Arunachalan A, Jaganath S. Niosomes A Novel Drug Delivery Sistem INTJ Of Novel Trends In Pharmaceutical Sciences.2012;2(1):25-31. 3. Kaur Diljyot. Niosomes: A New Approach to Targeted Drug Delivery.IJPPR.2012;2(1):53-59. [3] Madhav NV, SainiA, Niosome: A Novel Drug Delivery System JJRPC 2011;1(3):498-511. 5. Horsimran K, Sonia D, Sandeep A, Niosomes: A Novel Drug Delivery Ststem IJPSRR.2012,15 (1):113-120. [4] Naresh A, Vipin S, Vijay KB,Formulation and Evaluation of lansoprazole Niosomes 2008;3(2): 561-563. [5] Makeshwar KB, Wasankar SR, Niosome:A novel Drug Delivery System. Asian J.Pharm. 2013;3(1):16-20 [6] Agarwal S, Londhe V, Gaud R.Niosomes:Layered Delivery System for Drug targeting USR.2014;3(1):413-417. [7] Sankhyan A, Pawar P,Recent trends in Niosomes ;as vesicular drug delivery system Journal of applied pharm.science.2012;2(6):20-32. [8] Nasir A, Harikumar SL, Kaur A,Niosomes: An excellent tool for drug delivery.JJRPC.2012;2 (2):479-487. [9] Kumar GP, Rajeshwarrao P, Nonionic surfactant vescicular systems for effective drug delivery-an overview.Acta pharmaceutical sinica(Elasevier).2011;1(4):208-219. [10] Monosroi A, Jantrawut P, Novel elastic nonovesicles for cosmeceutical and parmaceuticals applications.chiang mai.J.sci.2009;36(2):169-178.

Copyright

Copyright © 2023 Prashant S. Bhosale, Jitendra V. Shinde, Rajashri S. Chavan, Pavan P. Kondewad, Prasanna K. Gaware. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.