INTRODUCTION:
In 1909, Paul Ehrlich developed a targeted delivery when he investigated a drug carriers are utilized to carry the drug at the target organ/tissue which include hemoglobin, serum, proteins, liposomes, microspheres, niosomes etc. Among these different carriers liposomes and niosomes [1] are well suitable for drug delivery. Drug targetting is defined as the ability of a drug molecule to accumulate in the target organ or tissue selectively such that the concentration of the drug at the diseased site is high, while its concentration in non-target organs and tissues is low. However niosomes are similar to liposomes in functionality. In both basic unit of assembly is Amphiphiles, but phospholipids in liposomes and non-ionic surfactants are niosomes. Niosomes has higher chemical stability than liposomes. Niosomes also increase the bioavailability of the drug and reduce the clearance like liposomes [2].
As with liposomes, the properties of niosomes depend both on the composition of the bilayer and the method of the production used. Niosomes can be prepared by hydration of the surfactant to obtain colloidal dispersion that entrapped by the desired compound. The compound remains in the hydrating solution is separated from the vesicles by gel chromatography centrifugation or dialysis. Hence vesicle membrane is influenced by method [3] of preparation. The most common methods used in the preparation are sonication and reverse phase evaporation method. Therefore formulations of niosomes were first developed by “LOREAL" cosmetic laboratories.
Niosomes (non-ionic surfactant vesicles) are defined as microscopic vesicles containing an aqueous core enclosed by a membrane of non-ionic [4] surfactants that forms closed bilayer structures based on their amphiphilic nature. The liphophilic groups are located in interior of the membrane. The hydrophilic groups mainly exposed to the aqueous medium. This is shown that in general liposome and niosome structures do not form spontaneously and they need some energy output (e.g physical agitation or heat) which leads to different preparation techniques. Then vesicles entrapped the active compound that can be released at a controlled rate.
Structure of niosome:
Structurally, niosomes are similar to liposomes. Both are made up of bilayer, which is made up of non-ionic surfactant [5] in the case of niosomes and phospholipids in case of liposomes. Both hydrophilic and hydrophobic drugs can be incorporated into niosomes. The niosomes are ampiphillic in nature, which allows entrapment of hydrophilic drug in the core cavity and hydrophobic drugs in the non-polar region present within the bilayer[6]. Niosomal gels contains a nanometric systems embedded in a gel. Nanometric systems have a greater surface area, which renders them highly satisfactory for the application of drug substances promoting a homogenous drug release. Such structures have been investigated as alternatives to classical formulations based on chemical skin permeation enhancers. Additionally, the nanostructure systems have nano size so it is easy for application to skin in dermatological product.
Advantages of niosomes [7-9]:
1. Niosomes may act as a depot, releasing the drug in a controlled manner.
2. It increases the oral bioavailability of drugs.
3. It provides targeted drug delivery, enhanced cellular uptake and protection to drugs.
4. It improves permeability of drugs through skin.
5. It can be administered through parenteral route.
6. It offers greater patient compliance over oil based systems.
7. It can accommodate drug molecules with a wide range of solubilities.
8. They are osmotically active and stable, hence they increase the stability of entrapped drug.
9. They improve the therapeutic performance of the drug molecules by delayed clearance from the circulation.
10. Niosomal formulation in aqueous phase can be emulsified in a non aqueous phase to regulate the delivery rate of drug.
11. Niosomes are biodegradable, biocompatible and non immunogenic.
3. Disadvantages of niosomes [10]:
1. Niosomes may undergo fusion,aggregation,leaking of entrapped drugs and hydrolysis of encapsulated drugs.
2. It has limited shelf life.
3. The methods of preparation of multilamellar vesicles such as extrusion, sonication are time consuming.
4. It requires specialized equipments for processing.
Components of niosomes [11]:
Non-ionic surfactants:
The nonionic surfactants form a closed bilayer vesicle in aqueous media based on its amphiphilic nature using some energy for instance heat, physical agitation to form this structure. In the bilayer structure hydrophobic parts are oriented away from the aqueous solvent whereas the hydrophilic heads remain in contact with the aqueous solvent. Selection of surfactant should be done on the basis of hydrophilic lipophilic balance(HLB) value. HLB number between 4 to 8 was found to be compatible with vesicle formation. The surfactants are amphiphilic, biodegradable and non immunogenic in nature. The following types of non ionic surfactants are used for the formation of niosomes.
Alkyl ethers:
L’Oreal explained about some surfactants for the preparation of niosomes such as
1.Surfactant-I(Mol.Wt.473)
It is C16 monoalkyl glycerol ether with average of three glycerol units.
2. Surfactant-II(Mol.Wt.972)
It is diglycerol ether with average of seven glycerol units.
3. Surfactant-III(Mol.Wt.393)
It is ester linked surfactant.
Alkyl esters:
Sorbitan esters are mostly used surfactant for the preparation of niosomes among these category of surfactants. Polyoxyethylene sorbitan monolaurate is more soluble than other surfactants. Sorbitan monoesters are a series of mixtures of partial esters of sorbitol and its mono and dianhydrides with fatty acids. Sorbitan monolaurate(span 20), Sorbitan monopalmitate(span 40), Sorbitan monostearate (span 60), Sorbitan monooleate(span 80) have HLB values 8.6,6.7,4.7 and 4.3 respectively. They show higher entrapment as their chain length increases. The final entrapment order may be as follows span 80 <span 20<span 40<span 60 at moderate concentration of cholesterol.
Polyoxyethylene sorbitan fatty acid esters:
Polyoxyethylene sorbitan fatty acid esters(polysorbates) are a series of partial fatty acid esters of sorbitol and its anhydrides copolymerized with ethylene oxide. Tween is the most common name of polyoxyethylene sorbitan fatty acid esters.
Polyoxyethylene alkyl ether:
These are a series of polyoxyethylene glycol ethers of n alcohols(lauryl, oleyl, myristyl, cetyl and stearyl alcohol.
Alkyl amides:
Alkyl amide (e.g. galactosides and glucosides) have been utilized to produce niosomal vesicles.
Cholesterol:
Cholesterol is the integral part of biological membrane where it influences several membrane properties such as aggregation, ion permeability, fusion process, size and shape. It acts as fluidity buffer in bilayer membrane providing stability and rigidness to vesicle membrane. It results a change in transition temperature producing change in stability and change in vesicle entrapment and release. The high concentration of cholesterol in noisome decreases the drug entrapment and cometes with drug bilayers. The low concentration of cholesterol in noisome increases the drug entrapment. Cholesterol prevents vesicle aggregation by inclusion of molecules that stabilize the system against the formation of aggregates by repulsive steric or electrostatic forces that leads to the transition from the gel to the liquid phase noisome systems. Hence the noisome becomes stable and less leaky in nature.
Charged molecule:
Charged molecules are introduced to niosomes to increase the stability of niosomes by electrostatic repulsion which prevents coalescence.Diacetyl phosphate (DCP) and phosphotidic acid are used as negatively charged molecules for niosomal formulation. Stearyl amine and stearyl pyridinium chloride are used as positively charged species for niosomal preparation. Use of these charge inducing agent in bilayers induce the hydrophilicity. Hence the water uptake is increased. The charged species in the bilayer induces repulsion among adjacent layers to produce large size vesicles in niosomes.
Methods of preparation of niosomes [12-15]:
Ether injection method:
It involves injecting the immiscible organic solution (containing a particular ratio of cholesterol and surfactant) very slowly into an aqueous phase through a narrow needle at the temperature of vaporizing the organic solvent. The solution of mixtures dissolved in diethyl ether or ether/methanol mixture is slowly injected to an aqueous solution of the material to be encapsulated at 55-65°C or under reduced pressure. The subsequent removal of ether under vacuum leads to the formation of niosomes. The main drawbacks of the method are that the population is heterogeneous (50-1000 nm) and the exposure of compounds to be encapsulated to organic solvents or high temperature. Alternatively fluorinated hydrocarbons may be used as a substitute for ether for thermolabile drugs as they vaporize at much lower temperature.
Hand shaking method (thin film hydration technique):
This is the most widely used method for the preparation of MLV. The mixture of vesicles forming ingredients like surfactant and cholesterol are dissolved in a volatile organic solvent(chloroform, methanol, diethyl ether) in a round bottom flask. In this method the mixtures are casted as stacks of film from their organic solution using flash rotary evaporator under reduced pressure. The method involves drying a solution of mixtures so that a thin film is formed at the bottom of round bottom flask and then hydrating the film by adding aqueous buffer and vortexing the dispersion for some time. The hydration step is done at a temperature above the gel-liquid crystalline transition temperature Tc of the lipid or above the Tc of the highest melting component in the lipid mixture. Due to hydration the mixtures swell and peel off from the wall of the round bottom flask and vesiculate forming mulilamelar vesicles. The compounds to be encapsulated are added either to aqueous buffer or to organic solvent containing lipids depending upon their solubilities.MLV are simple to prepare by this method and a variety of substances can be encapsulated in these niosomes. The drawbacks of the method are low internal volume, low encapsulation efficiency and the size distribution is heterogeneous
Sonication:
The principle of sonication involves the use of pulsed, high frequency sound waves(sonic energy) to agitate a suspension of the MLVs. At high energy level the average size of the vesicles is further reduced. This was first achieved on exposure of MLVs to ultrasonic irradiation and most widely used for producing small vesicles. There are two methods of sonication based on use such as bath sonicator and probe sonicator.
Bath sonication:
Bath sonicators are most widely used for the preparation of SUVs. Sonication of an MLV dispersion is accomplished by placing a test tube containing the dispersion in a bath sonicator and sonicating for 5-10min (100000g)above the Tc of the constituent lipid. The lipid dispersion should begin to clarify to yield a slightly hazy transparent solution. The haze is due to light scattering induced by residual large particles remaining in the dispersion. These particles can be removed using centrifugation to yield a clear SUV dispersion.
Probe sonication:
The probe is employed for dispersions which require high energy in small volume (e.g high concentration of lipids, viscous aqueous phase).Probe tip sonicators supply high energy input to the lipid dispersion but suffer from overheating of the niosomal dispersion causing lipid degradation. Sonicator tip tends to release titanium particles to liposome dispersion that can be removed by centrifugation process prior to use.
Micro fluidization method:
It is a technique used to prepare unilamellar vesicles of defined size. 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 a way that the energy supplied to the system remains within the area of niosomes formation. The niosomes which are formed have greater uniformity, smaller size and better reproducibility.
Multiple membrane extrusion method:
In membrane extrusion method the size of niosomes is reduced by gently passing them through membrane filter of defined pore size. This can be achieved at much lower pressure (<100psi).In this process the vesicle contents (mixture of surfactant, cholesterol and additives) are exchanged with the dispersion medium during breaking and resealing of mixtures as they pass through the polycarbonate membrane. In order to high entrapment the water soluble compounds should be present in suspending medium during extrusion process.
Reverse phase evaporation technique (REV):
First water in oil emulsion is formed by bath sonication of a two phase system containing cholesterol and surfactant in organic solvent (diethylether or chloroform) and aqueous buffer. The organic solvents are removed in rotary evaporator under reduced pressure, resulting in the formation of a viscous gel. The niosomes are formed when residual solvent is removed by continued rotary evaporation under reduced pressure. With this method high encapsulation efficiency up to 65% can be obtained in a medium of low ionic strength. The method has been used to encapsulate small, large and macromolecules. The main disadvantage of the method is the exposure of the materials to be encapsulated to organic solvents and to brief periods of sonication. These conditions may possibly result in the denaturation of some proteins or breakage of DNA strands. The heterogeneous sized dispersion of vesicles is formed by this method.
The bubble method:
It is a technique for the one step preparation of 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 phosphate buffer at 700C,the dispersion mixed for 15 seconds with high shear homogenizer and immediately afterwards bubbled at 700C using nitrogen gas.
Separation of unentrapped drug:
The removal of unentrapped solute from the vesicles can be accomplished by various techniques such as dialysis,gel filtration and centrifugation.
Dialysis:
Niosomal dispersion are highly soluble in both aqueous and organic media and there is equilibrium between the detergent molecules in the water phase and in the mixture of the micelle. The critical micelle concentration can give an indication to the position of this equilibrium. Upon lowering the concentration of detergent in the bulk aqueous phase the molecules of detergent can be removed from mixed micelle by dialysis. A higher CMC indicates that the equilibrium is strongly shifted towards the bulk solution, so that removal from the mixed membrane by dialysis becomes easy. The samples are withdrawn from the medium at suitable time intervals, centrifuged and analyzed for drug content using suitable method (U.V. spectroscopy, HPLC etc).
Gel filtration:
The unentrapped drug is removed by gel filtration of niosomal dispersion through a sephadex-G-50 column and eluted with suitable mobile phase and analyzed with suitable analytical technique.
Centrifugation:
The niosomal formulation is centrifuged and supernatant is separated. The pellet is washed and then resuspended to obtain a niosomal dispersion free from unentrapped drug.
Characterization of niosomes [16-18]:
Angle of repose:
The angle of repose test is very sensitive method used to create the heap. Angle of repose may be determined by heap shape measurement. By using classical method angle of repose can be measured. The granules were allowed to flow through funnel freely onto the clean surface. Funnel was placed in such a height that bottom tip of funnel should not touched apex of heap of granules. Angle of repose is calculated using the following equation.
tanϴ=h/r…………...…………………………………(1)
ϴ=tan-
1(h/r)…………………………………………………..(2)
Where ϴ is the angle of repose, h is the height of heap in cm and r is the radius of the circular support (cone) in cm.
Size, shape and surface morphology:
Particle size of niosomes is very important characteristic. The surface morphology (roundness, smoothness and formation of aggregates) and the size distribution of niosomes were studied by Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM), Freeze Fractured Microscopy(FFM) and Optical Microscopy Technique.
Scanning Electron Microscopy(SEM):
Niosomes were sprinkled on to the double sided tape that was affixed on aluminum stubs. The aluminum stub was placed in the vacuum chamber of a scanning electron microscope. The samples were observed for morphological characterization using a gaseous secondary electron detector(working pressure 0.8torr,voltage 3000KV).
Transmission Electron Microscopy (TEM):
TEM is used to determine the size, shape and lamellarity of noisome. A suspension is prepared and mixed with 1% phosphotungstic acid (in sufficient amount).A drop of resultant was then used on carbon coated grid, draining off the excess and then the grid was observed and images are taken under suitable magnification under TEM after complete drying.
Freeze Fractured Microscopy (FFM):
Vesicles are generally freeze thawed and then visualized under freeze fractured microscope for the determination of size. Liquid propane is generally used for the cryofixation of the vesicular suspension at low pressure (10-2Pa).The cryofixed vesicles are fractured at a specified angle. The resultant surface is then shadowed using platinum or carbon vapors at angle of 45°. Carbon coating used in this method strengthen the formed replica. Replica is cleaned and then observed.
Optical Microscopy Technique:
This technique is used for observation of noisome size and shape. In this method size of stage micrometer coinciding with the eye piece micrometer is recorded and size of the noisome is then recorded.
Number of lamellae:
Number of lamellae of niosomes are determined by nuclear magnetic resonance (NMR), X-Ray scattering and electron microscopy.
Entrapment efficiency:
It is defined as the percentage amount of drug which is entrapped by the noisome. For the determination of entrapment efficiency the unentrapped drug is first separated by using suitable method such as dialysis, centrifugation or gel filtration. The drug entrapped in niosomes is determined by complete vesicle disruption using 50% n propanol or 0.1% Triton X-100 and the collected supernatant is then diluted and analyzed by appropriate assay method for the drug as specified monograph. The drug entrapment efficiency is determined using the following formula
Percentage entrapment efficiency = W × 100………(3)
W0
Where
W is total amount of drug
W0 is amount of drug entrapped
Osmotic shock:
The change in vesicle size can be determined by osmotic studies. Niosomes formulations are incubated with hypotonic, isotonic, hypertonic solutions for 3 hrs. Then the change in the size of vesicles in the formulations are viewed under optical microscopy.
Zeta potential analysis:
The surface charge plays a vital role in the stability of niosomes. Generally the charged niosomes is more stable as compared with uncharged vesicles. Zeta potential analysis is done to determine the colloidal properties of the prepared formulations. The charge of vesicles are determined by estimating zeta potential. It is estimated by using micro electrophoresis, zeta potential analyzer based on electrophoretic light scattering and laser doppler velocimetry method.
In-vitro drug release:
In vitro drug release study can be done by dialysis membrane method. In this method small amount of niosomes are taken in the dialysis bag and tied at both the ends. The beaker containing suitable dissolution media is maintained at 370C and the dialysis bag is put into it and stirred by magnetic stirrer. A sample solution is taken from the beaker at specified time intervals and replaced with fresh dissolution media. The samples were analyzed for the concentration of drug at specified wavelength reported in respective monograph of that particular drug by using UV-Visible spectrophotometer or HPLC.
Stability study:
Stability studies can be done by storing noisome at
two different conditions usually 4
1°C and 25 2° C. Formulation size, shape and number of
vesicles can be determined before and after storing 30days.After 15 and 30 days
residual drug can be measured for drug content, vesicle size, number of
vesicles using specified equipments.
Pharmaceutical applications of niosomes [19-21]:
Targeting of bioactive agents:
The reticulo endothelial system(RES) has tendency to take the niosomes. The uptake of niosomes by the cells can be circulated by serum factors known as opsonins which mark them for clearance. The localized drug accumulation can be used to treat animal tumours known to metasize to the liver, spleen and in parasitic infection of liver.
To organs other than RES:
Niosomes act as a carrier system that can be directe to specific sites in the body by use of antibodies. Immunoglobulins have found to bind with lipid surface thus offers a convenient means for targeting of drug carrier system. Many cells possess the intrinsic ability to recognize and bind particular carbohydrate determinants and this can be used to direct carrier system to particular cells.
Immunological application of niosomes:
Niosomes can be used to study the nature of antigen antibody response provoked by antigens. Niosomes may act as potent adjuvant in terms of immunological selectivity, low toxicity and maximum stability.
Niosomes as carriers for haemoglobin:
Niosomes can be delivered as a carrier for haemoglobin. Niosomal suspension shows a visible spectrum super imposable onto that of free haemoglobin. Vesicles are permeable to oxygen and haemoglobin dissociation curve can be modified similarly to non encapsulated haemoglobin.
Ophthalmic drug delivery:
Niosomes can be used for ophthalmic drug delivery by using various bioadhesive polymers. Bioadhesive coated niosomal formulation of acetazolamide prepared from span 60,cholesterol stearylamine or dicetyl phosphate exhibits more tendency for reduction of intraocular pressure. The chitosan coated niosomal formulation timolol maleate(0.25%) exhibits more effect for reduction intraocular pressure as compared to marketed formulation with less chance of cardiovascular side effects. The water soluble antibiotic gentamicin suiphate showed maximum drug drug release in the form of niosomal drug delivery rather than normal drug solution.
Transdermal delivery of drugs by niosomes:
The penetration of drug through skin by transdermal drug delivery system is slow. An increase in the penetration rate of drug can be achieved by transdermal drug delivery system through incorporation of niosomes in TDDS. Bola niosomes were able to promote the intracellular uptake of ammonium glycerrhizinic acid.
Diagnostic imaging with niosomes:
Niosomes can be used as a carrier for radiopharmaceuticals and showed site specificity for spleen and liver for their imaging studies using 99mTc labelled DTPA containing noisome. Conjugated niosomal formulation of gadobenate with (N-palmitoyl glucosamine), PEG 4400 and PEG and NPG can be used to increase tumour targeting of a paramagnetic agent.Niosomes are considered as a carrier of iobitridol, a diagnostic agent for X-ray imaging. \
Niosomes used in cosmetic formulations:
Various cosmetic preparations are prepared as niosomes because both hydrophilic and hydrophobic drugs in topical formulations are prepared. N-acetyl glucosamine is used in the treatment of thyrosinase enzymes in melanocytes. Many formulations are used for the treatment of hyperpigmentation disorders. Ellagic acid permeation rate is increased by administration through niosomes.
CONCLUSION:
In niosomes provide as a drug carrier to achieve better bioavailability and targetting properties and as well as reducing the toxicity and side effects of the drugs. So far only animal experimentation of this targeted drug delivery system is reported but further clinical investigations in human volunteers, pharmacological and volunteers may help to exploit niosomes as prosperous drug carriers for targetting drugs more efficiently for treating various diseases.
REFERENCES:
1. Malhotra M, Jain NK. Niosomes as drug carriers. Indian Drugs 1994; 31:81-6.
2. Allen TM. Liposomal drug formulations: Rationale for development and what we can expect for the future. Drugs 1998; 56:747-56.
3. Udupa N. Niosomes as drug carriers. In: Jain NK, editor. Controlled and novel drug delivery. 1st edition. New Delhi: CBS Publishers and Distributors; 2002.
4. Baillie AJ, Florence AT, Hume LR, Muirhead GT, Rogerson A. The Preparation and propereties of Niosomes-Non ionic surfactant vesicles. J Pharm Pharmacol 1985; 37:863-8.
5. Khan A, Sharma PK, Visht S, Malviya R. Niosomes as colloidal drug delivery system: a review. Journal of Chronotherapy and Drug Delivery, 2011; 2(1):15-21.
6. Hu C, Rhodes DG. Proniosomes: A Novel Drug Carrier Preparation. Int J Pharm 1999; 185:23-35.
7. Jain C.P., Vyas S.P., Dixit V.K., Niosomes: A Controlled and Novel Drug delivery System, Indian J Pharm Sci. 2006; 68: 575-578.
8. Chandraprakasha K. S., Udupaa N., Umadevib P., Pillaic G. K., Niosomes: A Controlled and Novel Drug delivery System,Journal of Drug Targeting. 1993; 1: 143-145.
9. Raja Naresh R.A., Pillal G.K., Udupa N., Chandrashekar G., Niosomes: A Controlled and Novel Drug delivery System, Indian Journal of Pharmacology. 1994; 26: 46 – 48.
10. Priyanka B., Suresh K, Yogesh C, Manisha B. Niosomes: A Novel approach for novel drug delivery systems. Int. J. of Ph. Sci. 2013; 5(2):2071-2080.
11. Verma AK, Bindal MC. A vital role of niosomes on Controlled and Novel Drug delivery. Indian Journal of Novel Drug delivery 2011; 3(4): 238-246.
12. Khandare JN, Madhavi G and Tamhankar BM. Niosomes Novel Drug Delivery System. The Eastern Pharmacist. 1994; 37:61-64.
13. V.Shakya , B.K.Bansal. Niosomes : a novel trend in drug delivery Int. J. Res. Dev. Pharm. L. Sci. 2014; 3(4): 1036-1041.
14. Stafford S., Ballie A.J., Florence A.T., Drug effects on the size of chemically defined non- ionic surfactant vesicles, J. Pharm. Pharmacol. 1988; 40: 26.
15. Yoshida H., Lehr C.M., Kok W., Junginger H.E., Verhoef J.C., Bouwistra J.A., Niosomes: A Controlled and Novel Drug delivery System, J.Control Rel. 1992; 21: 145-153.
16. Biswal S., Murthy PN., Sahu J., Sahoo P., Amir F.,Vesicles of Non-ionic Surfactants (Niosomes) and Drug Delivery Potential. Int J Pharm Sci Nanotech 2008; 1:1-8.
17. Azmin MN., Florence AT., HandjaniVila RM., Stuart JF., Vanlerberghe G., Whittaker JS., The effect of non-ionic surfactant vesicle (niosome) entrapment on the absorption and distribution of methotrexate in mice. J Pharm Pharmacol 1985; 37: 237-42.
18. Manosroi A., Wongtrakul P., Manosroi J., Sakai H.,Sugawara F., Yuasa M., Characterization of vesicles prepared with various non-ionic surfactants mixed with cholesterol. Colloids Surf B 2003; 30:129-38.
19. Khanam N, Alam MI, Sachan AK, Gangwar SS, Sharma R. Recent trends in drug delivery by niosomes: a review. Asian Journal of Pharmaceutical Research and Development 2013; 1(3):155-122.
20. Suzuki K, Sokan K. The Application of Liposomes to Cosmetics. Cosmetic and Toiletries 1990; 105:65-78.
21. Mayer LD, Bally MB, Hope MJ, Cullis PR. Uptake of antineoplastic agents into large unilamellar vesicles in response to a membrane potential. Biochem Biophys Acta 1985; 816:294-302.
Received on 15.04.2018 Modified on 10.05.2018
Accepted on 14.06.2018 © RJPT All right reserved
Research J. Pharm. and Tech 2018; 11(11): 5205-5211.
DOI: 10.5958/0974-360X.2018.00950.2