Ameliorative effect of Ficus dalhousiae Miq. (Moraceae) methanolic leaf extract on carbon tetrachloride induced hepatic and renal..

Introduction There are various organic compounds, environment and drugs which causes cellular damage. Carbon tetrachloride (CCl4) is a potent environmental hepatotoxins [1 hepatic problems, CCl4 also causes disorder in kidneys, lungs, testis and brain as well as in blood by generating free radicals Reactive oxygen species (ROS) are chemically reactive molecules containing oxygen and are formed as a natural byproduct of the normal metabolism of oxygen and have important roles in signaling and homeostasis [9]. This may result in significant damage to cell structures. Cumulatively, this is known as stress. Studies also revealed that there are various herbal extracts could protect organ against CCl4 induced oxidative stress by enhancing the decreased level of catalase (CAT), superoxidase dismutase (SOD), reduce glutathione (GSH) decrease the level of ROS. Natural plant products have been widely used for medical purpose and according to Ayurveda, these medicine have less side effect when compared with allopathy medicine.In Ayurveda, plants are widely used for medicinal purpose. Compare with allopathy DOI:10.5138/09750185.1992 This article is distributed under the terms of the and redistribution provided that the origina Phytomedicine 9 (2017) 33 Original Research Article Miq. (Moraceae) methanolic leaf induced hepatic and renal toxicity 1, Sunil Kumar CR1, Anand Barapatre2, Geetha N1* Ficus dalhousiae Miq. (Moraceae) methanolic single oral dose of CCl 4 administration, rats were sacrificed and biochemical markers like Serum ALT), aspartate aminotransferase (AST), alkaline phosphatase and reactive oxygen species (ROS). The data were analysed by one way in the reduced level of SOD, CAT and GSH. The ROS activity also found down but also manifested by restoring antioxidant enzymes. With this pilot study words: superoxide dismutase, catalase, glutathione and reactive oxygen species.


Introduction
There are various organic compounds, environment and drugs which causes cellular damage. Carbon tetrachloride (CCl 4 ) is a potent environmental hepatotoxins [1 hepatic problems, CCl 4 also causes disorder in kidneys, lungs, testis and brain as well as in blood by generating free radicals Reactive oxygen species (ROS) are chemically reactive molecules containing oxygen and are formed as a natural byproduct of the normal metabolism of oxygen and have important roles in signaling and homeostasis [9]. This may result in significant damage to cell structures. Cumulatively, this is known as stress. Studies also revealed that there are various herbal extracts could protect organ against CCl 4 induced oxidative stress by enhancing the decreased level of catalase (CAT), superoxidase dismutase (SOD), reduce glutathione (GSH) decrease the level of ROS. Natural plant products have been widely used for medical purpose and according to Ayurveda, these medicine have less side effect when compared with allopathy medicine.In Ayurveda, plants are widely used for medicinal purpose. Compare with allopathy DOI: 10

A b s t r a c t
Developing traditional medicine in the field of hepatology and nephrology research is the key to pharmacology. This study demonstrates the mechanism of hepatoprotective and renal protective activity of Ficus dalhousiae Miq. (Moraceae) methanolic tetrachloride induced hepatic and renal toxicity. Shade dried powder was subjected to shoxlet extraction with methanol and assessed for hepatoprotective and renal protective activities. Hepatotoxicity and renal toxicity were induced in rats by single oral dose of CCl olive oil (1:1 v/v; ml/kg body weight) after pretreatment of methanolic extract for seven days. Sixteen hrs after CCl 4 administration, rats were sacrificed and biochemical markers like Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) estimated followed by the measurement of liver and kidney cytosolic antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione (GSH) and reactive oxygen species (ROS). The data were analysed by one way analysis of variance (ANOVA). The extract at the doses of 150 and mg/kg b.w. significantly reduces elevated levels of ALT, AST, ALP and LDH. The extracts also showed significant increase in the reduced level of SOD, CAT and GSH. The ROS activity also found down regulated. The activity of methanolic extracts were comparable with the standard Silymarin. These findings not only showed potential hepatoprotective and renal protective activitie Ficus dalhousiae but also manifested by restoring antioxidant enzymes. With this pilot study we can justify the medicinal importance of this plant. Keywords: superoxide dismutase, catalase, glutathione and reactive oxygen species.
(ROS) are chemically reactive molecules containing oxygen and are formed as a natural byproduct of the and have important roles in cell . This may result in significant damage to cell structures. Cumulatively, this is known as oxidative Studies also revealed that there are various herbal extracts induced oxidative stress by enhancing the decreased level of catalase (CAT), superoxidase dismutase (SOD), reduce glutathione (GSH) [10,11] and also Natural plant products have been widely used for medical purpose and according to Ayurveda, these medicine have less side effect when compared with allopathy medicine.In Ayurveda, plants are pose. Compare with allopathy medicine it have less side effects or no side effects.
dalhousiae Miq belong to family Moraceae, which is reported to have various medicinal properties like in the treatment of liver, skin diseases [12] and its root possesses very good anti activity [13]. The present study was undertaken to evaluate protective effect of Ficus dalhousiae induced toxicity in rat liver and kidney. Miq. (Moraceae) methanolic leaf induced hepatic and renal toxicity * Developing traditional medicine in the field of hepatology and nephrology research is the key to pharmacology. This study demonstrates the mechanism of hepatoprotective and renal Miq. (Moraceae) methanolic leaf extract on carbon tetrachloride induced hepatic and renal toxicity. Shade dried powder was subjected to shoxlet extraction with methanol and assessed for hepatoprotective and renal protective activities.

Materials and method
single oral dose of CCl 4 diluted with olive oil (1:1 v/v; ml/kg body weight) after pretreatment of methanolic extract for seven days.
administration, rats were sacrificed and biochemical markers like Serum ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) estimated followed by the measurement of liver and kidney cytosolic antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), and reactive oxygen species (ROS). The data were analysed by one way analysis of variance (ANOVA). The extract at the doses of 150 and mg/kg b.w. significantly reduces elevated levels of ALT, AST, ALP and LDH. The extracts also showed significant in the reduced level of SOD, CAT and GSH. The ROS activity also found down regulated. The activity of methanolic extracts were comparable with the standard Silymarin. These findings not only showed potential hepatoprotective and renal protective activities of but also manifested by restoring antioxidant enzymes.

Extraction procedure and conditions
Thirty grams of the powdered leaf samples were extracted using methanol by soxhlet apparatus. The solid to liquid ratio was kept at 1:10 and the process was run for a total of 16 hours. After extraction, the resultant extracts were allow to concentrat to dryness under reduced pressure in a rotary evaporator (Heidolph rotary evaporator) to yield methanolic extract (FdLM) and dried material were kept in air tight tubes in dark at 4 °C. The rats were kept in separated animal rooms with a 12 h light-dark cycle, and the temperature and humidity were kept at 23 ± 2 o C and 55%-65%, respectively. The animal care and experimental procedures performed were in compliance with the Regulations for Animal Research and Animal Ethical Committee of the UOM. Rats were acclimatized for a week and randomly divided into five groups (n = 6, per group). FdLM extracts were dissolved in sterile distilled water and two different concentrations were administered orally. The scheme of the treatment was as follows: Group I -(normal control) received neither plant extract nor CCl 4 but they received only food and water. Group II -serve as a toxic control and received CCl 4 (1 mg/ml); only once on 8 th day. Group III -FdLM (150 mg/kg b.w.) followed by CCl 4 (1 mg/ml); only once on 8 th day. Group IV -FdLM (300 mg/kg b.w.) followed by CCl 4 (1 mg/ml); only once on 8 th day. Group V-Silymarin (100 mg/kg b.w.) followed by CCl 4 (1 mg/ml); only once on 8 th day.

Animal and Treatments
On the eighth day, the rats of Group II to V were administered a single oral dose of CCl 4 in olive oil (1:1) at 1 ml of body weight after the last dose of extracts. Sixteen hours after CCl 4 administration, animals were sacrificed by anaesthesia. The liver was perfused with saline and processed immediately for biochemical assay.

Estimation of Serum enzymes
Blood samples were collected through retro-orbital plexus and allowed to clot for 30 min at 28 °C. The serum was collected by centrifugation for 10 min at 2,000 rpm and stored at 4 °C. Liver damage was assessed by estimating serum activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) using commercially available test kits. The results were expressed as units/liter (U/L).

Assessment of GSH
The level of reduced Gultathione (GSH) in the liver and kidney was measured [15]. The Liver and kidney tissue (0.5 g) was homogenized in 5 ml of 1:1 cold buffer (10% TCA, 10 mM EDTA, pH 7.4) at 4 °C. The homogenate was centrifuged at 5,000 rpm for 10 min at 4 °C and the supernatant was used for the determination of GSH level. A 100 µl of supernatant was added to 3.0 ml of 0.2 M Tris-HCl and 50 µl of Ellman's reagent (0.02% of DTNB). Blank was without DTNP. The yellow colour developed was measured immediately at 412 nm.

Assessment of Catalase activity
Assessment of catalase activity in liver and kidney was according to the method [16] and [17]. Liver and kidney tissue was homogenized (10% w/v) in ice cold 50 mM phosphate buffer (pH 7.4), centrifuged at 2,500 rpm for 10 min at 4 °C and the supernatant was used for determining catalase activity. The activity was measured using 3% H 2 O 2 (v/v) as the substrate in phosphate buffer and the change in absorbance was measured at 240 nm for two min at 30 s intervals. Units of catalase were expressed as the amount of enzyme that decomposes 1 µM of H 2 O 2 per min at 25 °C and the activity was expressed as units/mg of protein.

Assessment of superoxide (SOD) activity
For the assessment of SOD activity liver and kidney tissue was homogenized (10% w/v) in ice cold 50 mM phosphate buffer (pH 7.4), centrifuged at 2500 rpm for 10 min at 4 °C and the supernatant was used for assessing SOD activity. The activity was measured using pyrogallol (2 mM) auto oxidation in Tris buffer (pH 8.2). The reaction mixture contained 0.1 Tris buffer and tissue homogenate. Reaction was started by adding 2 mM pyrogallol. The absorbance was read at 420 nm for two min at an interval of 30 s Involvement of the superoxide anion radical in the autoxidation of pyrogallol and a PAGE | 334 | convenient assay for superoxide dismutase. European Journal of Biochemistry Involvement of the superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase. European Journal of Biochemistry [18].
Reactive oxygen species (ROS) assay The ROS activity was measured by the fluorometric assay by the use of DCF-DA reagent [19]. Liver and kidney tissue was homogenized (10% w/v) in ice-cold 50 mM phosphate buffer (pH 7.4), centrifuged at 5,000 g for 10 min at 4 °C. Supernatant was used to measure relative fluorescence unit in a fluorescence spectrophotometer at the excitation and emission wavelengths of 488 and 525 nm, respectively.
Histopathological studies of liver tissues For histopathological examination, a small portion of liver and kidney was transferred to 4% formalin solution (v/v) for fixation and later on processed [20] The microtome sections were cut, processed and stained with hematoxylin and eosin. The sections of liver and kidney of both control and treated rats were scanned and analysed by Carl-Zeiss microscope (Axio Imager 2).
Functional group characterization of FdLM by FTIR FTIR analysis was performed to detect the characteristic functional groups in the FdLM using FTIR spectrophotometer (Perkin Elme, Version 10.03.09). Dry fraction of the sample (FdLM) was ground properly with spectrophotometric grade KBr (1:100) and spectrum was obtained in the range of 600-4000 cm −1 .

Statistical Analysis
Proper evaluations were performed by using SPSS 22 software in which multiple groups were analysed by one way analysis of variance (ANOVA). The experiments were carried out in triplicates and presented as mean ± SD and the significance was considered at p < 0.05. IC 50 values were calculated from logarithmic dose response curve by the use of Graphpad Prism 5.0.

Results and Discussion
Many previous reports demonstrate that carbon tetrachloride (CCl 4 ), is a typical hepatotoxic compound and tit cause significant damage to the liver after a single intraperitoneal dose [3,21]. Generation of free radical reactions is the main reason of the CCl 4 mediated liver injury, which produced during its metabolism inside the liver. This free radical is another outcome in the form of the initiation of lipid per oxidation [22].

Serum Enzyme
The effect of F. dalhousiea bark extract (FdLM) on serum AST, ALT, ALP, and LDH levels in treated wistar rats along with the control rats presented in Table.1. The increase levels of AST, ALT, ALP and LDH in CCl 4 treated group serum as compared to the control normal group clearly indicated the toxicity of the CCl 4 and the level of increment of the above marker enzymes in CCl 4 intoxicated rats were 2.06, 3.83, 1.97 and 1.71 time respectively. After administration of extract to the CCl 4 intoxicated rats, the levels of AST, ALT, ALP and LDH were significantly (p <0.05) reduced as compared to the CCl 4 toxic control groups. The reduction in the level of enzyme was significantly high in group IV (FdLM 300 mg) as compare to group III (FdLM 150 mg). The standard drug Silymarin (300 mg) also produced almost the similar results when the CCl 4 intoxicated rats were treated with equal amount of FdLM (300 mg, group IV) indicating that the FdLM extract was almost equally effective in the reduction of CCl 4 induced toxicity.

Effect of FdLM treatment on rat liver and kidney antioxidant enzymes in CCl 4 intoxicated rats
The effect of F. dalhousiea bark extract (FdLM) pre-treatment on liver antioxidant enzymes (GSH, SOD and CAT) in CCl 4 intoxicated rats are presented in Figure. 1-3. It was found that in control group all three enzyme activities werehigh from other groups.
The quantity of GSH enzyme in control rat liver (group found 0.99 ± 0.02 μg/mg protein, while in the CCl the production was 4.3 fold lower i.e. 0.23 ± 0.04. On the other hand, in the FdLM and the control drug supplemented group (Silymarin), GSH activity was recovered after the CCl In the group where 300 mg FdLM (group IV) was s display a better GSH activity (0.77 ± 0.02 μg/mg) compared to Figure. 1: GSH activity in the rat liver and kidney tissues before and after CCl 150 mg/kg b.w. + CCl 4 ; Group IV-FdBL 300 mg/kg b.w. + CCl n = 6; bars with different alphabets differ significantly at In the case of rat kidney the GSH level was highest 1.09 ± 0.07 μg/mg in the control rats, The intoxication of CCl considerable inhibition effect on the production of GSH and among all tested rats the GSH level was found significantly lower (0.29 ± 0.01 μg/mg) in CCl 4 intoxicated rats. The treatment of CCl intoxicated rats with FdLM produce slightly lower recovery of GSH production in kidney tissue compared to liver tissue. The highest GSH activity was recovered in case of the drug Silymarin (100 mg/kg b.w.) and it was ~75.23% (0.82 ± 0.05 μg/mg) after the CCl intoxication in comparison to control, whereas the rats treated with test samples recovered about ~44.03 and 55.05% GSH activity for 150 and 300 FdLM mg/kg b.w. Superoxide dismutase (SOD) activity in liver and control and CCl 4 intoxicated rats is shown in Fig  tissue, there was a huge difference in the SOD activity in control and CCl 4 intoxicated rat which was 2.95 ± 0.16 and 0.12 ± 0.01 IU/mg protein respectively ( Figure. 2A). CCl 4 almost ceased the SOD activity and produced oxidative stress conditions. In the CCl intoxicated rat, it was observed that the SOD activity dropped upto ~93%. The treatment of CCl 4 intoxicated rat silymarin recover the SOD activity and helped in the reduction in oxidative stress mediated damage. The highest recovery of SOD activity was found in the case of Silymarin drug (group Kujur et al. International Journal of Phytomedicine 9 The quantity of GSH enzyme in control rat liver (group -I) was g/mg protein, while in the CCl 4 intoxicated rat the production was 4.3 fold lower i.e. 0.23 ± 0.04. On the other hand, in the FdLM and the control drug supplemented group (Silymarin), GSH activity was recovered after the CCl 4 intoxication. In the group where 300 mg FdLM (group IV) was supplemented, g/mg) compared to positive control drug (group V; 0.74 ± 0.09 recovered up to ~71% when compared to CCl while rat supplemented with 150 mg FdLM (group III) recovered GSH activity in significantly lower level i.e. only ~26% (0.43 ± 0.04 μg/mg).
GSH activity in the rat liver and kidney tissues before and after CCl 4 intoxicated. Group I -control; Group II FdBL 300 mg/kg b.w. + CCl 4 ; Group V -silymarin 100 mg/kg b.w. + CCl 4 .Each bar represents the mean ± SE, n = 6; bars with different alphabets differ significantly at p < 0.05 level.
ney the GSH level was highest 1.09 ± 0.07 g/mg in the control rats, The intoxication of CCl 4 produce a inhibition effect on the production of GSH and among all tested rats the GSH level was found significantly lower (0.29 ± intoxicated rats. The treatment of CCl 4 intoxicated rats with FdLM produce slightly lower recovery of GSH production in kidney tissue compared to liver tissue. The highest GSH activity was recovered in case of the drug Silymarin (100 g/mg) after the CCl 4 intoxication in comparison to control, whereas the rats treated with test samples recovered about ~44.03 and 55.05% GSH activity for Superoxide dismutase (SOD) activity in liver and kidney tissues of intoxicated rats is shown in Figure. 2. In the liver tissue, there was a huge difference in the SOD activity in control intoxicated rat which was 2.95 ± 0.16 and 0.12 ± 0.01 almost ceased the SOD activity and produced oxidative stress conditions. In the CCl 4 intoxicated rat, it was observed that the SOD activity dropped upto with FdLM and nd helped in the reduction in oxidative stress mediated damage. The highest recovery of SOD activity was found in the case of Silymarin drug (group -V) where the activity was found 78.31% of control. The treatment of CCl intoxicated rat with FdLM recover activity respectively for 150 and 300 mg/kg b.w. FdLM. It was observed that the high dose of FdLM (300 mg/kg b.w.) was more effective than lower dose. In rat kidney tissue, the SOD activity drastically decreased after intoxication with CCl 4 ( Figure. 2B) and the significant reduction was observed after 8 th day of CCl 4 intoxication, from ± 0.05 units/mg protein. Among all the three treated groups, the group -V in which Silymarin drug treated CCl shows the highest recovery for SOD activity, and it shows about 84.36% activity (1.78 ± 0.03 units/mg protein control. In comparision to liver tissue, the recovery of SOD activity in kidney was less and it was obtained about 0.84 ± 0.1 an 0.09 units/mg protein which is about 39.81 and 50.71% compared to control. In the case of CAT activity, there was a drastically different pattern was obtained in the rat liver and kidney tissues. In the control rat (group -1), the CAT activity in liver and kidney tissue was found to 4.06 ± 0.03 and 2.10 ± 0.056 IU/ml respectively, while in CCl intoxicated rats the reduction in the CAT was 27.34 (1.11 ± 0.05 IU/ml) and 18.09% (0.38 ± 0.05IU/ml) in liver and kidney tissue respectively. In FdLM treated rat liver the CAT activity was high in 9 (2) 332-341 [2017] PAGE | 335 | positive control drug (group V; 0.74 ± 0.09 μg/mg), and activity was recovered up to ~71% when compared to CCl 4 intoxicated rat, while rat supplemented with 150 mg FdLM (group III) had recovered GSH activity in significantly lower level i.e. only ~26% control; Group II -CCl 4 ; Group III-FdBL Each bar represents the mean ± SE, the activity was found 78.31% of control. The treatment of CCl 4 intoxicated rat with FdLM recovered 56.27% and 65.08% SOD activity respectively for 150 and 300 mg/kg b.w. FdLM. It was observed that the high dose of FdLM (300 mg/kg b.w.) was more In rat kidney tissue, the SOD activity drastically decreased after . 2B) and the significant reduction was intoxication, from 2.11 ± 0.14 to 0.37 . Among all the three treated groups, the V in which Silymarin drug treated CCl 4 intoxicated rat shows the highest recovery for SOD activity, and it shows about units/mg protein) compared to the control. In comparision to liver tissue, the recovery of SOD activity in kidney was less and it was obtained about 0.84 ± 0.1 and 1.07 ± which is about 39.81 and 50.71% compared In the case of CAT activity, there was a drastically different pattern was obtained in the rat liver and kidney tissues. In the control rat liver and kidney tissue was found to 4.06 ± 0.03 and 2.10 ± 0.056 IU/ml respectively, while in CCl 4 intoxicated rats the reduction in the CAT was 27.34 (1.11 ± 0.05 IU/ml) and 18.09% (0.38 ± 0.05IU/ml) in liver and kidney tissue ted rat liver the CAT activity was high in group IV (treatment with 300 mg/kg d.w. FdLM) whereas in case of kidney tissue, higher activity about ~91% (1.91 ± 0.08 IU/ml).was in Figure Reactive oxygen species (ROS) activity in liver and kidney tissues intoxicated rats is shown in Figure.   Group VII -Silymarin 100 mg/kg b.w. + CCl 4 . Each bar represents the mean ± SD, n = 6; bars with different alphabets differ significantly at p < 0.05 level.

Histopathology Kidney
The histological study of the kidney tissue sections of the control group rats (Groups I) did not show any abnormal microscopic findings. The variations in kidney section of CCl 4 intoxicant rat (Fig.  5B) displayed the major signs of extensive tubular necrosis (blue arrow) along with the high degree loss of their brush border (black arrow), with less intact renal tubules (yellow arrow), many cast formation (black asterisk), tubular dilation (green arrow) and a thickened basement membrane. An increased sized Bowman's capsules were occupying the whole glomeruli spaces. The epithelial lining cells were found highly disrupted with pyknotic nuclei. Hemorrhage and lymphocytic infiltrate in the interstitial area and periglomerular lymphocytic infiltration were noticed. Some of the tubules having protein casts. A segmental sclerosis with basement membrane thickening and interstitial fibrosis was found in CCl 4 intoxicated kidney but most of the glomeruli showed atrophy. These changes were found to be significantly reduced in kidneys of the experimental group treated with extract. The group of rats which received lower dosages of extract (150 mg/kg) showed moderate improvement in the tubular and glomerular morphology. Post treatment with the plant extract at 300 mg/kg dose showed considerable improvement in glomeruli and tubules. Bowman's capsule showed comparatively less hypertrophy. Glomeruli were compact with wide spacing in the Bowman's capsule. Tubules were organized but debris was was seen. Lumens were maintained. Epithelium was intact in most of the tubules and hence improved histoarchitecture of the kidney (Figure. 6). The thickening of the walls was decreased by the extract (150 and 300 mg/kg) treatment ( Figure. 6).

Liver
The histopathological section of liver from control, CCl 4 intoxicated, standard drug silymarin and extract treated rats were collected at the end of the experimental periods (i.e., 8 th day after CCl 4 treatment Figure. 3). Untreated rat liver showed normal lobular architecture with intact central vein and sinusoids, normal portal tracts and preserved hepatocytes. While, CCl 4 intoxicated rats liver tissue depicted the necrosis, surrounding fibrosis, perivenular inflammation and vacuole formation. Rats treated with the FdLM (group II and IV) and standard drug revealed the reduced vacuole formation and inflammation and almost normal hepatocellular architecture in the liver section. Overall histopathological examination showed a protective effect of FdLM on CCl 4 induced hepatotoxicity.  (23). It was reported that, the phenolic mobile hydrogen atom (Ar-OH) plays the significant role in the scavenging of reactive free radicals (like SOD and ROS) (24). Absorption peak obtained at 2938 and 2842 cm −1 , primarily due to the C-H stretching in aromatic methoxyl groups and in methyl and methylene groups of side chains. The characteristic vibration in aromatic ring skeleton was obtained at 1605, 1518 and 1442 and also 1360 cm −1 recognized for the aromatic skeleton vibration and O-CH 3 , C-H deformation symmetry. The band near 1250 and 1050 cm −1 appeared mainly due to aromatic C-O, C-H and C=O out of plane deformation (23). The obtained characteristic peaks in FdLM also suggest that this fraction contains aromatic moiety having a strong free radical scavenging capacity.

Conclusion
Overall, our study concluded that Ficus dalhousiae Miq.
(Moraceae) leaf methanolic extract prevented CCl 4 induced hepatic and renal toxicity by scavenging free radicals, which suggested that antioxidant molecule is present in FDLM extract which accelerate the antioxidant capacity of liver and kidney.

Authors Contribution
Geetha N supervised the project, Swati Kujur performed the experiments, drafted the manuscript and interpreted the results. Sunil K.C.R. assisted in handling animals, and experiments Anand Barapatre helped in analyzing FTIR spectra. All the authors involved in drafting and revision of manuscript and approved the final manuscript.