In-Vivo Animal Models For Evaluation Of Anti-Inflammatory Activity
Anupama A. Suralkar
Inflammation is protective and defense mechanism of the body. During inflammatory conditions various pathological changes are take place. The production of active inflammatory mediators is triggered by microbial products or by host proteins, such as proteins of the complement, kinins and coagulation systems that are themselves are activated by microbes and damaged tissues.
In preclinical studies, these changes can be induced by administration of the agents causing inflammation. For purpose of evaluation of anti-inflammatory activity we have focused on some in vivo animal models which are commonly used in laboratory practice.
Numerous reports have been demonstrated in increase incidence of inflammatory condition. It is one of the most important natural defence mechanisms. Its main purpose is to destroy the injurious agent and/or to minimize its ill effects by limiting its spread. Though inflammation is protective in some situations if untreated can lead to serious complications. Inflammation is the dynamic pathological process consisting of a series of interdependent changes.
Pathophysiology of inflammation-
Inflammation is body’s response to disturbed homeostasis caused by infection, injury or trauma resulting in systemic and local effects. The Roman writer Celsus in 1st century AD named the famous four Cardinal Signs of inflammation as Rubor (redness), Tumor (swelling/ edema), Calor (heat) and Dolor (pain) 1
The main symptoms of the body against an inflammatory stimulation are increased body temperature and pain. Inflammation constitutes the body’s response to injury and is characterized by a series of events that includes the inflammatory reaction, a sensory response perceived as pain, and a repair process. Some causes of an inflammatory reaction are Infection (invasion and multiplication within tissues by various bacteria, fungi, viruses and protozoa, which in many instances, cause damage by release of toxins that directly destroy host cells), Trauma penetrating injury, blunt trauma, thermal injury, chemical injury, and Immunologically mediated injury (humoral or cellular) and as a result of the loss of blood supply (ischemia). 2
Inflammation may be acute and chronic inflammation. Inflammatory response occurs in three distinct phases. The first phase is caused by an increased in vascular permeability resulting in exudation of fluids from the blood into the interstitial space, the second phase involves the infiltrations of leukocytes from the blood into the tissue and in third phase granuloma formation and tissue repair. Mediators of inflammation originate either from plasma (e.g. complement proteins. Kinins) or from cells (e.g. histamine, Prostaglandins, Cytokines). The production of active mediators is triggered by microbial products or by host proteins, such as proteins of the complement, kinins and coagulation systems that are themselves are activated by microbes and damaged tissues. Generally the mediators of inflammation are Histamine, Prostaglandins (PGs), Leukotrienes (LTB4), Nitric oxide (NO), Platelet-activation factor (PAF), Bradykinin, Serotonin, Lipoxins, Cytokines, Growth Factors.
Large amount of experimental studies and detailed knowledge that arises of mediators of inflammation has been carried out. This review will address the commonly used animal models for the evaluation of anti-inflammatory activity in laboratory practice. It is also giving the principle and procedure behind using each animal model. This review hopefully fills the expectation to provide the in vivo models in area of inflammation.
1. Acute Inflammation
1.1 Carrageenan-induced Paw Edema in Rats 3
This model is based on the principle of release of various inflammatory mediators by carragenan. Edema formation due to carrageenan in the rat paw is biphasic event. The initial phase is attributed to the release of histamine and serotonin. The second phase of edema is due to the release of prostaglandins, protease and lysosome. 4 & 5 Subcutaneous injection of carrageenan into the rat paw produces inflammation resulting from plasma extravasation, increased tissue water and plasma protein exudation along with neutrophil extravasation, all due to the metabolism of arachidonic acid.6 The first phase begins immediately after injection of carrageenan and diminishes in two hours. The second phase begins at the end of first phase and remains through third hour up to five hours.
Procedure: Animals are divided into three groups (n=6) starved overnight with water ad libitum prior to the day of experiment. The control group receives vehicle orally, while other group receives test drug and standard drug respectively. Left paw is marked with ink at the level of lateral malleolus; basal paw volume is measured plethysmographically by volume displacement method using Plethysmometer (UGO Basile 7140) by immersing the paw till the level of lateral malleolus. The animals are given drug treatment. One hour after dosing, the rats are challenged by a subcutaneous injection of 0.1ml of 1% solution of carrageenan into the sub-plantar side of the left hind paw. The paw volume is measured again at 1, 2, 3, 4 & 5 hours after challenge. The increase in paw volume is calculated as percentage compared with the basal volume. The difference of average values between treated animals and control group is calculated for each time interval and evaluated statistically. The percent Inhibition is calculated using the formula as follows.
% edema inhibition = [1- (Vt / Vc)] X 100
Vt and Vc are edema volume in the drug treated and control groups respectively.
1.2 Histamine Induced Paw Edema in Rats7
Histamine induced paw edema is said to occur in earlier stage in mounting of vascular of the vascular reaction in the chemically induced inflammation. In this, swelling occurs primarily due to action of histamine. Generally histamine is released following the mast cell degranulation by number of inflammatory mediators including substances P interleukin-1 (IL-1). This is likely to evoke the release of neuropeptide as well as release of prostaglandins and monohydroxy eicosatetranoic-acid from endothelial cell leading to hyperalgesia and other pro-inflammatory effects. 8
Procedure: The procedure for this is same as that like of Carrageenan-induced paw edema, only instead of carrageenan the rats are challenged by a subcutaneous injection of 0.1ml of 1% solution of histamine into the sub-plantar side of the left hind paw. The paw volume is measured. The percent inhibition of the inflammation is calculated using the formula and compared with control group.
% Inhibition = Vc - Vt / x 100
Vt and Vc edema volume in the drug treated and control groups respectively.
1.3 Acetic Acid-Induced Vascular Permeability 9
The test is used to evaluate the inhibitory activity of drugs against increased vascular permeability which is induced by acetic acid by releasing inflammatory mediators.10 Mediators of inflammation, such as histamine, prostaglandins and leukotrienes are released following stimulation of mast cells. This leads to a dilation of arterioles and venules and to an increased vascular permeability. As a consequence, fluid and plasma protein are extravaseted and edemas are formed.
Procedure:Animals are divided into three groups (n=6). The control group received vehicle orally, while other groups received test drug and standard drug respectively followed by the injection of 0.25 ml of 0.6% solution of acetic acid intraperitoneally. Immediately after administration, 10 mg/kg of 10% (w/v) Evan’s blue is injected intravenously through the tail vain. Thirty minutes after Evan’s blue injection, the animals are hold by a flap of abdominal wall and the viscera irrigated with distilled water over a Petri dish. The exudate is then filtered and makes the volume up to 10 ml. The dyes leaking out into the peritoneal cavity measured spectophotometrically using visible spectra at 10 nm and compared with the control group.
1.4 Xylene Induced Ear Edema (Thickness and weight parameter) 11
In xylene induced ear edema model, the application of xylene induces neurogenous edema. It is partially associated with the substance P. Substance P is an undecapeptide, which is widely distributed in the central and peripheral nervous system and it functions as a neuro-transmitter or neuro-modulator in variety of physiological processes. Substance P is released from the neurons in the midbrain in response to stress, where it facilitates dopaminergic neurotransmission from sensory neurons in the spinal cord in response to noxious stimuli where it excites dorsal neurons. In the periphery, release of substance P from sensory neurons causes vasodilatation and plasma extravasations suggesting its role in neurogenous inflammation. Thus it can cause the swelling of ear in the mice.
1.4.1Xylene Induced Ear Edema (Thickness parameter)
The animals used in this method are mice divided into five groups (n=6), fasted overnight and allowed free access to water. The animals are administered with drugs to respective groups. One hour later, each animal received 30ml of xylene using micropipette on anterior and posterior surfaces of the right ear. The left ear is considered as control. Again after one hour later, the thickness of the ear is determined using Digimatic Caliper. The percentage of ear edema is calculated based on the left ear without xylene.
1.4.2Xylene Induced Ear Edema (weight parameter).
The animals used in this method are mice divided into five groups (n=6), fasted overnight and allowed free access to water. The animals are administered with drugs to respective groups. One hour latter, each animal received 30ml of xylene using micropipette (Lab Teck) on anterior and posterior surfaces of the right ear lobe. The left ear is considered as control. Again after one hour later the animals were sacrificed by excess ether anesthesia and both the ear are removed. Circular sections are taken, using a cork borer with diameter of 7 mm, and weighed. The percentage of ear edema is calculated based on the left ear without xylene (Junping Kou et al., 2005)).
1.5 Arachidonic Acid-Induced Ear Edema 12
This model is based on the principle of metabolism of arachidonic acid by COX (Cyclooxigenase) leads to the generation of PGs and thromboxanes that mediate pain and edema associated with inflammation and inhibition of these mediators by test drug is evaluated.
Procedure:Inflammation is induced by topical application of arachidonic acid (2 mg in 20µ ml of acetone) of both surfaces of the right ear of each mouse. Rest procedure is same as that like of xylene induced ear edema (Thickness and weight parameter).
1.6. Phorbol Myristate Acetate-Induced Ear Edema in Mice 13
Phorbol myristate acetate (PMA) is a protein kinase C (PKC) promoter, which induces the formation of free radicals in vivo. It has been also demonstrated that pre-treatment of mouse skin by antagonists of PKC suppresses inflammation and ROS (reactive oxygen species). This species involved in the synthesis of mediators and regulate the production of TNFα this in turn stimulate PLA2 activity, which releases arachidonic acid from phospholipids and stimulate the activity of COX and LOX (Lipoxygenase) these enzyme involved in release different inflammatory mediators.
Procedure:Four µg per ear of PMA, in 20 µl of acetone, is applied to the both ear of each mouse. The left ear (control) receives the vehicle. Test drug is administered 1 h before PMA application. Two control groups are used, a group with application of PMA on the right ear and a positive control group that receive standard drug. Six hours after PMA application, the mice are killed by cervical dislocation and a 6 mm diameter disc from each ear is removed with a metal punch and weigh. Ear edema is calculated by subtracting the weight of the left ear (vehicle) from the right ear (treatment), and is express as a reduction in weight with respect to the control group.
1.6.1 Myeloperoxidase (MPO) Assay 14
MPO is an enzyme present in neutrophil and much lower concentration in monocytes and macrophages. It is well know that the level of MPO activity is directly proportional to the neutrophil concentration on the inflamed tissue. Inhibition of MPO activity by the drug preventing the generation of oxidants such as hypochlorous acid.
Procedure: Tissue samples of each ear, from the PMA model, are assessed biochemically with neutrophil marker enzyme MPO. All the ear tissue is homogenized in 50 mM K2PO4 (pH 6) containing 0.5 % hexadecyl trimethylammonium bromide (HTAB) using a Polytron (Ultra-turax T-25) homogenizer. After freeze-thawing for three times, the samples is centrifuged at 2500 rpm for 30 min at 4 C and the resulting supernatant is assayed spectrophotometrically for MPO determination. In brief 40 µl sample is mixed with 960 µl of 50 mM phosphate buffer pH 6, containing 0.167mg/ml O-dianisidine dihydrochloride and 0.0005% hydrogen peroxide. The change in absorbance at 460 nm is measured with spectrophotometer. MPO activity data is presented as units per mg of tissue. One unit of MPO activity is defined as that degrading 1 µmol of peroxide per minute at 25 C.
1.7 Oxazolone-induced Ear Edema in Mice15
The oxazolone-induced ear edema model in mice is a model of delayed contact hypersensitivity that permits the quantitative evaluation of the topical and systemic anti-inflammatory activity of a compound following topical administration.
Procedure- Animals use in this model are divided into various groups (n=6). Before each use a fresh 2% solution of oxazolone (4- ethoxymethylene-2-phenyl-2-oxazolin-5-one) in acetone is prepared. The mice are sensitized by application of 0.1 ml on the shaved abdominal skin or 0.01 ml the inside of both ears under halothane anesthesia. The mice are challenged 8 days later again under anesthesia by applying 0.01 ml 2% oxazolone solution the inside of the right ear (control) or 0.01 ml of oxazolone solution, in which the test compound or the standard is solved. Special pipettes of 0.1 ml or 0.01 ml are used. Groups of 10 to 15 animals are treated with the irritant alone or with the solution of the test compound. The left ear remains untreated. The maximum inflammation occurs 24 h later. At this time the animals are sacrificed under anesthesia and a disc of 8 mm diameter is punched from both sides. The discs are immediately weighed on a balance. The weight difference is an indicator of the inflammatory edema.
2. SUB-ACUTE MODEL
2.1 Carrageenan Induced Granuloma Pouch Model 16
Carrageenan induced granuloma pouch model is an excellent sub acute inflammatory model in which fluid extravasations, leukocyte migration and various biochemical exudates involved in inflammatory response can be detected readily. The air pouch has the advantage of supplying a suitable space for the induction of inflammatory responses. The injection of irritants such as carrageenan into subcutaneous air pouch on the dorsal surface of rats initiates an inflammatory process.
Procedure: The animals used in this method are rats divided into five groups (n=6), fasted overnight and allowed free access to water, fasted overnight and allowed free access to water. The animals are administered with vehicle, standard drug and test drug. One hour after dosing, the back of the animal is shaved and disinfected. With a very thin needle subcutaneous dorsal granuloma pouch is made in ether anaesthetized rats by injecting 6 ml of air, followed by injection of 4 ml of 2 % Carrageenan in normal saline into it to avoid any leakage of air and the treatment continued for seven consecutive days as follows.
On day 8, the pouch is opened under anesthesia and the amount of exudates was collected with a syringe. The average volume of exudates, total WBC count and weights of granuloma is determined.
2.2 Formalin-induced Paw Edema 17
This model based upon the ability of test drug to inhibit the edema produced in the hind paw of the mice after injection of formalin. The nociceptive effect of formalin is biphasic, an early neurogenic component followed by a later tissue-mediated response. In the first phase there is release of histamine, 5-HT and kinin, while the second phase is related to the release of prostaglandins.
Procedure: The animals are divided into three groups (n=6). In animals of all groups, inflammation is produced by subplanter injection of 20 ml of freshly prepared 2% formalin in the right hind paw of mice. The paw thickness is measured by Plethysmometrically 1 h before and after formalin injection. The drug treatment is continued for 6 consecutive days. The increase in paw thickness and percentage inhibition are calculated and compared with control group.
3. Chronic Model
3.1 Cotton Pellet-Induced Granuloma in Rats 18
This model is based on the foreign body granuloma which is provoked in rats by subcutaneous implantation of pellets of compressed cotton. After several days, histologiclly gaint cells and undiffentiated connective tissue can be observed beside the fluid infiltration. The amount of newly formed connective tissue can be measured by weighing the dried pellets after removal. More intensive granuloma formation has been observed if the cotton pellets have been impregnated with carrageenan.
Procedure: The animals used in this method are rats divided into five groups (n=6), fasted overnight and allowed free access to water. The animals are administered with vehicle, standard drug and test drugs. One hour after the first dosing, the animals are anesthetized with anesthetic ether and 20 mg of the sterile cotton pellet is inserted one in each axilla and groin of rats by making small subcutaneous incision. The incisions are sutured by sterile catgut (Crunkhon et al., 1971). The animals are sacrificed by excess anesthesia on the 8th day and cotton pellets are removed surgically. Pellets are separated from extraneous tissue and dried at 60°C until weight become constant. The net dry weight, i.e. after subtracting the initial weight of the cotton pellet will be determined. The average weight of the pellet of the control group as well as of the test groups is calculated. The percent change of the granuloma weight relatively with vehicle control is determined and statistically evaluated. The percentage inhibition increase in the weight of the cotton pellet is calculated.
% Inhibition = Wc - Wd / Wc C 100
Wd = difference in pellet weight of the drug treated group.
Wc = difference in pellet weight of the control group.
3.2 The Glass Rod Granuloma 19
The glass rod granuloma as first described by Vogel reflects the chronic proliferative inflammation. Of the newly formed connective tissue not only wet and dry weight, but also chemical composition and mechanical properties can be measured.
Procedure: Glass rods with a diameter of 6 mm are cut to a length 40 mm and the ends rounded off by flame melting. They are sterilized before implantation by boiling in water. Male Sprague-Dawley rats with an initial weight 130 g are anaesthetized with ether, the back skin shaved and disinfected. From an incision in the caudal region a subcutaneous tunnel is formed in cranial direction with a closed blunted forceps. One glass rod is introduced into this tunnel finally lying on the back of the animal. The incision wound is closed by sutures. The animals are kept in separate cages. The rods remain in situ for 20 or 40 days. Treatment with drugs is either during the whole period or only during the last 10 or 2 days. At the end the animals are sacrificed under CO2 anesthesia. The glass rods are prepared together with the surrounding connective tissue which forms a tube around the glass rod. By incision at one end the glass rod is extracted and the granuloma sac inverted forming a plain piece of pure connective tissue. Wet weight of the granuloma tissue is recorded. The specimens are kept in a humid chamber until further analysis. Finally, the granuloma tissue is dried and the dry weight is recorded.
The above mentioned models have given broad spectrum for the evaluation of the anti-inflammatory activity. In different models, the inflammation has produced by different inducers by releasing inflammatory mediators. Each is having different mechanism of action for producing inflammation either by increased in vascular permeability, the infiltrations of leukocytes from the blood into the tissue or granuloma formation and tissue repair.
Among the many methods used for screening of anti-inflammatory drugs, one of the most commonly employed techniques is based upon the ability of such agents to inhibit the edema produced in the hind paw of the rat after injection of a phlogistic agent. Many phlogistic agents (irritants) have been used, such as brewer’s yeast, formaldehyde, dextran, egg albumin, kaolin, Aerosil®, sulfated polysaccharides like carrageenan or naphthoylheparamine. For producing edema, histamine, xylene, arachidonic acid, phorbol myristate acetate, oxozolone, croton oil and formalin are also used.
For evaluating the most effective and widely used model for inflammation is carrageenan-induced paw edema, Carrageenan is a mixture of polysaccharides composed of sulfated galactose units and is derived from Irish Sea moss, Chondrous crispus. Its use as an endemogen was introduced by Winter et.al. Carrageenan initially releases histamine and serotonin followed by release of prostaglandins, protease and lysosomes producing edema. ..
In Histamine induced paw edema, histamine causes vasodialation and increase in vascular permeability followed by edema which is one of the phases of. Inflammation. Xylene releases of substance P from sensory neurons causes vasodilatation and plasma extravasations. Arachidonic acid administration produces metabolism of arachidonic acid by COX (Cyclooxygenase) leads to the generation of PGs and thromboxanes that mediate pain and edema associated with inflammation. Phorbol myristate acetate (PMA) synthesizes mediators and regulate the production of TNFα this in turn stimulate PLA2 activity.
In acetic acid induced vascular permeability, acetic acid causes dilation of arterioles and venules and increased vascular permeability by releasing inflammatory mediators such as histamine, prostaglandins and leukotrienes are released following stimulation of mast cells. Myeloperoxidase (MPO) in neutrophils indicates intensity of inflammation.
By producing air pouch in air pouch model, formation of exudates is there with migration of leukocytes and interleukins. Angiogenesis, nitric oxide synthesis and Kinin release are said to be the main causes of the granuloma. Angiogenesis in a chronic inflammatory state, which facilitates migration of inflammatory cells to the inflammatory site and supplies nutrients and oxygen to tissue to granulation tissue. Therefore the suppression of Angiogenesis in granulation tissue is important to suppress the development of chronic granulation tissue (Ghosh et al., 2000). Nitric oxide synthesis (NO), by inducible nitric oxide synthase (iNOS), increases in inflammation and leads to cellular injury.
Kinins cause vasodilatation, increase vascular permeability and WBC migration in the early stages of the inflammation and are also responsible for, collagen formation in the later stages of inflammation. It may also be responsible for the vascular flushing that occurs in the carcinoid syndrome. The kinins formation is also implicated in the endotoxin shock, hereditary angioneurotic edema, anaphylaxis, arthritis and in acute pancreatitis.
Kinins degranulate mast cells to release histamine as well as other mediators of inflammation and also cause plasma extravasation by contraction of vascular endothelial cells. Kinins are potent algogenic substances, which induce pain by directly stimulating nociceptors in skin joint, and muscles. (Dray et al., 1995).
In cotton pellet induced granuloma and in glass rod granuloma, the foreign body like cotton or glass rod when implanted in the skin of animal is producing undiffentiated connective tissue around it indicating state of inflammation. The amount of newly formed connective tissue is measured by weighing the dried pellet after removal as an index of the extended severity of the inflammation (Hicks, 1969). This model is the indication of the proliferative phase of the inflammation of the microphages, neutrophils, fibroblasts and collagen formation which are basic source for the granuloma formation; therefore decrease in the granuloma formation indicates the suppression of the proliferative phase (Kavimani et al., 1999).
It has been therefore given a clear idea about in vivo models of inflammation which are widely used in laboratory. We hopefully expect that it is fulfilling the student’s criteria in area of inflammation.
- Harsh Mohan, Inflammation and Healing. Textbook of Pathology, Ed Jaypee Publication, New Delhi , 2002, 114-121.
- Robbins and Cortran, Acute and chronic inflammation. Pathologic Basis of Disease, Elsevier Publication, Ed 7, 2004, 47-87.
- Winter CA, Risley E, Nuss G, Carrageenan-induced edema in hind aw of the rat as an assay for anti-inflammatory drugs. Proc Soc Exp Biol Med. 111 , 1962, 544-547.
- Vinegar R, Schreiber W, Hugo R, Biphasic development of carrageenan oedema in rats, Journal of Pharmacological Experimental Therapeutics, 66, 1969, 96-103.
- Crunkhon P, Meacock S, Mediators of the inflammation induced in the rat paw by carrageenan. British Journal of Pharmacology, 42, 1971, 392-402.
- Chatpaliwar VA, Johrapurkar AA, Wanjari MM, Chakraborty RR, Kharkar VT, Anti-inflammatory activity of martynia diandra glox, Indian Drugs, 39, 2002, 543- 545.
- Amann R, Schuligoi R, Lanz, I., Donnerer J, Histamine induced edema in the rat paw-effect of capsaicin denervation and a cgrp receptor antagonist, Europian Journal of Pharmacology, 279 , 1995, 227-31.
- Dray A, Inflammatory mediators of pain. British Journal of Anesthesia, 75, 1995, 25-131.
- Whittle BA, The use of changes in capillary permeability in mice to distinguish between narcotic and non-narcotic analgesic, British Journal of Pharmacology Chemother. 22 , 1964, 24-253.
- Miles AA, Miles E, Vascular reactions to histamine, histamine-liberator and leukotaxine in the skin of guinea-pigs, Journal of Physiology, 118, 1992, 228-257.
- Junping K, Yun N, Wang N, Liang L, Zhi-Hong H, Analgesic and anti-inflammatory activities of total extract and individual fractions of Chinese medicinal plants Polyrhachis lamellidens, Biological Pharmaceutical Bulletin , 28, 2005, 176-180.
- Romay C, Ledon N, Gonzalez R, Further studies on anti-inflammatory activity of phycocianin in some animal models of inflammation, Inflammation Research, 47 , 1998, 334-338.
- Griswold DE , Martin L, Badge A, Evaluation of the cutaneous anti-inflammatory activity of azapiranes, Inflammation Research, 47, 1998, 56-61.
- Bradley PB, Pribat D, Christensen R, Rothstein G, Measurement of cutaneous inflammation: estimation of neutrophil content with an enzyme marker, Journal of Investigational Dermatology, 78 , 1982, 206-209.
- Evans PD, Hossack, M, Thomson DS, Inhibition of contact sensitivity in the mouse by topical application of corticosteroids, British Journal of Pharmacology, 43 , 1971, 403.
- Selye H, An experimental study with the granuloma pouch technique, JAMA 152, 1953, 1207-1213.
- Turner R, Screening Method in Pharmacology. Anti-Inflammatory agent, Academic Press New York , London , 1965, 13, 158Crunkhon P, Meacock S, Mediators of the inflammation induced in the rat paw by carrageenan. British Journal of Pharmacology, 42, 1971, 392-402.
- Goldstein SA, Shemano L, Daweo R, Betler J, Cotton pellet ganuloma pouch method for evaluation of anti-inflammatory activity, Arch Pharmacodyamic Ther, 165, 1976, 294-301.
- Vogel H, Anti-Inflammatory Activity. Drug Discovery and Evaluation. 1996, 725-771.
- Kavimani S, Llango R, Loganathan C, Karpagam S, Jaykar B, Anti-inflammatory activity of benidipine hydrochloride, Indian Drugs, 36, 1999, 147-149.
About Authors :
Ms.Anupama A. Suralkar
Lecturer, Dept.of Pharmacology.
Prashant S. Sarda
Lecturer, Dept.of Pharmacology.
Mahesh M. Ghaisas
Asst.Prof., Dept.of Pharmacology.
Vishnu N. Thakare
Dr. Avinash D. Deshpande
Director and Principle,Padm. Dr. D. Y. Patil Institute of Pharmaceutical Sciences & Research, Pimpri, Pune.