Histopathological examination

Ventilago calyculata Tul. (Bark) ameliorates the effect of isoniazid and rifampicin induced hepatotoxicity in rats

Abstract

Aim: Drug-induced liver injury (DILI) is the major cause of acute liver failure throughout the world. Ventilago calyculata has a potential use in folk, Ayurveda and Siddha.

The study aimed to investigate the hepatoprotective effect and possible mechanism of Ventilago calyculata bark extracts against a combination of two antitubercular drugs-Isoniazid and Rifampicin (INH+ RIF) which are highly hepatotoxic in rats.

Materials and methods: Oxidative stress is considered as the main factor responsible for hepatotoxicity.  In vitro, DPPH assay of various extracts was performed to investigate the extract with the most potent antioxidant activity. The chloroform extract (CEVC) was chosen for in vivo studies. The CEVC at a dose of 200 mg/kg was administered orally once daily for 10 days in albino Wistar rats.

Results: The elevated levels of SGOT, SGPT, SALP, total bilirubin and cholesterol were effectively reduced by CEVC co-administration, as compared to INH+ RIF-treated rats (p< 0.01). The protective effect of CEVC was associated with restoration of hepatic levels of glutathione (GSH), lipid peroxidase (LPO), superoxidase (SOD), albumin and protein. The biochemical changes were supported by histological observations.

Conclusion: CEVC has a definite protective property against INH+RIF-induced acute hepatic injury which may mainly be associated with its antioxidant effect. Therefore, it could be suggested that supplementation with the CEVC might be able to minimize the side effects of anti-TB drugs.

KEYWORDS: Isoniazid, rifampicin, hepatoprotective, antioxidant activity, tuberculosis, silymarin, Ventilago calyculata Tulasne, Ventilago denticulata.

Short-term chemotherapy containing isoniazid and rifampicin (INH+RIF) in combination

Has proved to be highly effective in the treatment of tuberculosis but is known to be highly hepatotoxic and cause drug- induced liver injury. In India, the cases of hepatotoxicity are 11.6%, when compared to western countries where it is found to be 4.3%. INH produces toxic metabolite hydrazine by CYP450 which is hepatotoxic. RIF is also toxic to hepatocytes and aggravates INH- induced hepatotoxicity by enhancing the production of toxic metabolites [1]. Moreover, hydrazine depletes the stored glutathione (GSH) level in the liver, resulting in oxidative stress and cell death.  Once this occurs, the antituberculosis regimen must be altered or discontinued, which can result in relapse, drug resistance, and tuberculosis-related death [2, 3].

Histopathological examination

Since, oxidative stress is regarded as one of the major mechanisms of antitubercular drug- induced hepatotoxicity, antioxidants might be used in supplementation with antitubercular therapy (ATT). This may provide effective protection against liver dysfunction and enable TB infected patients to continue the entire course of ATT that may result in lower morbidity and mortality rates.

Ventilago calyculata Tul. (Rhamnaceae) commonly known as ‘kevati, ‘raktavalli’ in Sanskrit and ‘pappili’ in Siddha. The plant is large much-branched woody climber [4]. Traditionally, the powder of stem bark is used as a tonic and externally for the treatment of skin diseases and sprains. Sap obtained from the bark is utilized for the treatment of deafness. The root bark is used for the atonic dyspepsia, diabetes, mild fever and debility [5]. In Ayurveda, it is an ingredient of “maha syonaka taila” which is used in the form of drink, massage, inhalation and enema. This medicated oil is used in looseness of joints, chronic fever, gout, insanity, dysuria, vomiting, trembling etc. [6]. It is reported that stem bark of Ventilago leiocarpa possess potent hepatoprotective property, which is an allied species of V. calyculata [7].

Therefore, the present study was aimed to find out the antioxidant activity of various extracts of V. calyculata bark for the selection of most bioactive extract and to investigate the hepatoprotective activity of the most potent extract in Wistar albino rats.

MATERIALS AND METHODS

The stem bark of Ventilago calyculata was collected from the local area of Bhopal, Madhya Pradesh, in the month of October 2010 and authenticated by Dr. Zia Ul Hassan, Botanist, Safia College of Science and Education, Bhopal, Madhya Pradesh. The voucher specimen (248/Bot/Safia/11) was deposited in Department of Pharmacognosy, Technocrats Institute of Technology, Pharmacy, Bhopal, Madhya Pradesh. The bark was cleaned thoroughly, shade dried, pulverized to a coarse powder and stored in airtight container for further use.

Chemicals

1,1-diphenyl-2-picrylhydrazyl (DPPH) was purchased from Sigma Chemicals Co. (St. Louis, MO,U.S.), and ascorbic acid obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China), purity was >98%. Drugs like Isoniazid (INH), Rifampicin (RIF) and Silymarin (Micro labs, Bangalore) were used. Other chemicals and reagents used for extraction were of LR grade obtained locally. For estimation of biochemical parameters; biochemical diagnostic kits like AST, ALT, ALP, albumin, total protein, total cholesterol, direct bilirubin and total bilirubin were procured from Span Diagnostics Ltd., Surat, India.

 Preparation of the extracts

About 1 kg dried powder of stem bark was evenly packed in the soxhlet apparatus. The drug was extracted with petroleum ether (40-60°C) for about 36 hrs. The drug was removed from the soxhlet apparatus and air dried to remove last traces of petroleum ether. Similarly, the drug was subjected to extraction with chloroform, ethyl acetate and ethanol. The process was carried out for about different timings for different solvents. The aqueous extraction was carried out by cold maceration process after above extractions. The extracts were concentrated by vacuum distillation to reduce the volume up to 1/10th. The extract were packed and labelled in air tight containers for the further studies.

Preliminary phytochemical analysis

In order to detect the various constituents present, the petroleum ether, chloroform, ethyl acetate, ethanolic and aqueous extracts of Ventilago calyculata stem bark were subjected to the phytochemical analysis [8, 9].

Determination of in vitro antioxidant activity by DPPH method

Briefly, 1 mM solution of DPPH in ethanol was prepared, and 4 mL of this solution was mixed with 1 mL of extract solution at various concentrations immediately and then incubated for 30 min at room temperature. The absorbance of the sample was measured at 517 nm. Radical scavenging activity was expressed as the inhibition percentage (IP) of free radical and was calculated using the formula:

IP (%) = ([Acontrol − Atest)/Acontrol]) × 100%

Where Acontrol is the absorbance of the control reaction (containing all reagents except the tested extracts), and Atest is the absorbance of the test extract. Scavenging activity of the plant extracts was also estimated based on the percentage of the DPPH reduction by calculating the IC50 values (concentration in μg/mL that caused 50% inhibition of DPPH radicals) using regression analysis [10].

Animals

Swiss albino mice (20-25g) and Wistar albino rats (150-200g) of either sex and of approximate 9-12 weeks old, used in the present studies were procured from National Central Laboratory for Animal Science, Hyderabad. They were maintained in the animal house of Technocrats Institute of Technology, Bhopal for experimental purpose. Then all the animals were acclimatized for seven days under standard husbandry conditions, i.e. room temperature of 25 ± 10C; relative humidity 45-55% and a 12:12h light/ dark cycle in clean polypropylene cages. The animals had free access to standard rat pellet (Pranav Agro Industries Ltd, Bangalore, India), with water supplied ad libitum under strict hygienic conditions. The experiment was carried out as per the guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) and approved by the Institution Animal Ethics Committee.(Reg. No.TIT/IAEC/831/P’cog/2011/11)

Acute oral toxicity study

A safe oral dose of the extract was determined by acute toxic method of organization of   economic co-operation and development (OECD) as per 423 guidelines. Animals fasted prior to dosing. An increasing dose of 100-2000 mg/kg body weight was administered p.o. to the rats. Animals were observed individually after dosing at least once during the first 30 minutes, periodically during the first 24 hours, special attention was given during the first 4 hours and daily thereafter, for a total of 72 hours [11].

Experimental design

INH (100 mg/kg bw) and RIF (100 mg/kg bw) solution were prepared separately in sterile distilled water. Rats were treated with INH+RIF at the dose of (100 mg/kg bw, i.p.) to the experimental animals for 10 days. Silymarin (100 mg/kg bw) was used as a standard drug in the study and was given orally.  In order to study the effect of the most potent extract (based on in vitro antioxidant study) in rats, CEVC (200 mg/kg bw) was administered by oral route. Rats were divided into four groups. Each group contained six animals and treatment was followed as per the protocol given below [12].

Group I (Normal): The animals of this group were administered 1% sodium carboxymethyl cellulose (CMC) (100 mg/kg, p.o.) for 10 days.

Group II (Negative control): This group was administered INH + RIF (100 mg/kg bw, i.p.) in 1% CMC for 10 days.

Group III (Standard): This group was administered the standard silymarin (100 mg/kg bw, p.o.) for 10 days an hour before receiving the toxicant INH + RIF (100 mg/kg bw, i.p.) in 1% CMC for 10 days.

Group IV (Treatment): This group received CEVC (200 mg/kg, p.o.) for 10 days an hour before receiving the toxicant INH + RIF (100 mg/kg bw, i.p.) in 1% CMC for 10 days. Rats were treated as per the treatment protocol for 10 days.

Biochemical assay

On the 10th day, the rats were sacrificed by cervical decapitation one hour after administration of the last dose under light ether anaesthesia and the blood was obtained from animals by puncturing retro-orbital plexus. The serum was separated by centrifugation at 2500 rpm at 30°C for 15 min and utilized for the estimation of various biochemical parameters including Serum glutamate oxaloacetate transaminases (SGOT), [13] Serum glutamate pyruvate transaminase (SGPT), [13] Alkaline phosphatase (ALP), [14] total bilirubin (TB), [15] total protein (TP), [16] total albumin (TA) [16] and total cholesterol (TC) [17] using standard kits.

Antioxidant assay

The liver was dissected out, washed with ice-cold saline, and a homogenate was prepared in 0.1 M Tris-HCl buffer (pH 7.4). The homogenate was centrifuged at 4ºC at 2500 rpm for 10 mins. and the supernatant was used for the assay of marker enzymes, glutathione (GSH), superoxide dismutase (SOD), catalase (CAT) and lipid peroxidase (LPO) [18].

Histopathological observations

The animals were sacrificed and the liver of each animal was isolated. The isolated liver was cut into small pieces and preserved and fixed in 10% formalin for two days. The clearing was done by using chloroform. After clearing, the liver pieces were subjected to paraffin infiltration in automatic tissue processing unit.  The blocks were cut using a microtome to get sections of the thickness-5µm.The sections were taken on a micro slide on which an egg albumin (sticking substance) was applied. The sections were allowed to remain in an oven at 60°C for 1 hour. Paraffin melts and egg albumin denatures, thereby fixing the tissues to slide and stained with Hematoxylin-Eosin (H-E) for photomicroscopic assessment [19].

Data analysis

The mean (SEM) and standard deviation (SD) values were calculated for each group. The results of the study were expressed as mean ± SEM, n = 6. ANOVA (Analysis of Variance) was used to analyze and compare the data, followed by student’s t-test for multiple comparisons.

RESULTS

Acute oral toxicity study

The acute toxicity study of the chloroform extract of V. calyculata (CEVC) showed no mortality at all doses administered up to 2000 mg/kg indicating that the bark extracts were safe even at higher doses. Therefore, on the basis of acute toxicity study, 200 mg/kg dose of CEVC was chosen for further study.

Preliminary phytochemical analysis

The preliminary phytochemical screening of various extracts was carried out and it was found that petroleum ether extract showed the presence of phytosterols; chloroform extract showed the presence of glycosides, phytosterols; ethyl acetate extract showed the presence of alkaloids, glycosides and carbohydrates; ethanol  showed the presence of carbohydrates, glycosides, phenolic compounds and flavonoids and aqueous extract showed the presence of alkaloids, carbohydrates, phenolic compounds, saponins and flavonoids (Table 1).

In-vitro DPPH radical scavenging activity

In this study, in vitro DPPH assay of all the extracts were performed to investigate the antioxidant activity of various extracts of the bark of V. calyculata. Petroleum ether fraction showed IC50 35.07 µg/mL, chloroform fraction showed IC50 9.54 mg/mL, ethyl acetate extract showed IC50 24.53 mg/mL, ethanol fraction showed IC50 18.35 mg/mL, aqueous fraction IC50 was 26.57 mg/mL and the standard ascorbic acid showed IC50 11.10 µg/mL (Figure 1). Thus, antioxidant assay revealed that the chloroform fraction was having best antioxidant activity and on this basis it was decided that hepatoprotective activity of this fraction will be assessed.

   Effect of CEVC on lipid peroxidation, cell glutathione and liver antioxidant enzymes

Compared with the normal group, the level of LPO was significantly (p<0.05), elevated      reduction in CAT, GSH and SOD in INH+RIF control group (Table 2 & Figure 2). On treatment with CEVC (200mg/kg) the level of LPO decreased and the levels of CAT, GSH and SOD remarkably increased. The CEVC (200 mg/kg) was found to be comparable to the standard drug silymarin at 100mg/kg.

  Effect of CEVC on serum biomarkers of liver toxicity

The results of the hepatoprotective effect of extracts are summarized in Table 3 & Figure 3. In comparison to the normal group, there was a significant increase in the levels of SGPT, SGOT and ALP in INH+RIF intoxicated rats, reflecting the liver injury by INH+RIF. The elevated levels of SGPT, SGOT and ALP were significantly (p<0.05) reduced in the animal groups treated with the extract.

  Effect of CEVC on total bilirubin, total protein and total cholesterol levels

The results of the hepatoprotective effect of extracts are summarized in Table 3 & Figure 3. In               comparison with normal group, there was a significant increase both in the levels of TB and TC in INH+RIF intoxicated rats and a significant (p<0.05) decrease in TP and TA levels, reflecting the liver injury by INH+RIF. The normal levels of TB, TP, TA and TC were almost restored in the animal groups treated with extract.

    Histopathological studies

The histopathological examinations of the group I (normal control) rat liver tissues showed a   normal lobular architecture of liver with hepatocytes arranged in single cords (Figure 4A). But, the group II (INH+RIF treated group) liver sections showed perivenular necrosis and steatosis with a degree of steatosis being variable, from ballooning degeneration to necrosis (Figure 4B). Standard drug treated group: This group which is treated with silymarin one hour before INH+RIF treatment shows normal lobular architecture (Figure 4C). The histopathological study of liver tissue treated with CEVC (200 mg/kg) showed minimal fatty changes with normal architecture indicating the protective action of chloroform extract (Figure 4D).

   DISCUSSION

The observed decrease of superoxide dismutase may be due to hepatocellular damage by        INH+RIF. The reduction of activity of this enzyme may lead to deleterious effects as a result of superoxide and hydrogen peroxide assimilation. However, an increase in the level of SOD after plant extract administration implies an efficient protective mechanism of this plant. The ability of extracts to reactivate the hepatic glutathione reductase was reflected by decreasing the level of lipid peroxidation [20].

The increased levels of SGOT, SGPT, ALP, TB and TC in INH+RIF treated rats in the  present study  was interpreted as  a result  of  the  liver  cell  destruction or  changes  in the membrane  permeability  indicating  the severity of  hepatocellular damage induced by INH+RIF.

An abnormal increase in the levels of bilirubin in the serum indicates hepatobiliary disease and severe disturbance of hepatocellular function [21]. Hepatocellular damage causes a modest hypertriglyceridemia [22]. The mechanism of INH+RIF and protective effects of CEVC is shown in the Figure 5.

INH+RIF hepatitis is associated with ballooning degeneration, focal hepatocyte necrosis, with minimal cholestasis [23]. Another study reported diffuse microvesicular fatty infiltration with mild portal triaditis. Similar changes were seen in our study, confirming the validity of our animal model. The model is summarized schematically in Figure 6.

In conclusion, the CEVC seems to be relatively safe, even at higher doses when taken orally. When administered at 200 mg/kg bw orally, it has a protective effect against INH+RIF induced toxicity. This hepatoprotective effect may be mediated through a mechanism such as antioxidant activity. Flavonoids, triterpenoids and alkaloids [24, 25] were reported as active substances for hepatoprotective substance induced by various chemicals In vitro and In vivo. The phytoconstituents such as glycosides, flavonoids, present in the extract are considered to be responsible for the hepatoprotective activity of CEVC in INH+RIF induced hepatotoxicity in rats [8]. It provides a support for the traditional use of Ventilago calyculata in liver disorders. Chloroform extract of other plants has been found to possess potent hepatoprotective properties [26, 27].

Apart from liver damage, INH+RIF also causes gastrointestinal intolerance, anaemia, drug fever, rashes, headache, general malaise and weakness [28]. These side-effects can be very well managed by Ventilago calyculata as it has also been reported for the following pharmacological properties – analgesic, anti-inflammatory, [29] antioxidant, [30] anthelmintic, [31] anti-herpes, [32] antibacterial [33] etc.

 Finally, it was concluded that the administration of CEVC combined with antitubercular chemotherapy could be effective and a safe adjuvant. Further studies should be conducted to determine the active compound responsible for the hepatoprotective effect.

  Acknowledgement

Authors are thankful to the Department of Pharmacognosy, Technocrats Institute of Technology,              Bhopal for providing the facilities to carry out the project.

  REFERENCES

  1. Gajanan SG, Alisha C, Jyothi H. Drug-induced hepatitis and the risk factors for liver injury in pulmonary tuberculosis patients. J Family Med Prim Care 2015; 4: 238-243.
  2. Tasduq SA, Peerzada K, Koul S, Bhat R, Johri RK. Biochemical manifestations of anti-tuberculosis drugs induced hepatotoxicity and the effect of silymarin.Hepatol Res 2005; 31, 132-135.
  3. Li F,Lu J, Cheng J, Wang L, Matsubara T, Csanaky IL, Klaassen CD. Human PXR modulates hepatotoxicity associated with rifampicin and isoniazid co-therapy. Nat Med 2013; 19(4):418-20.
  4. Kirtikar KR, Basu BD. Indian Medicinal Plants. 2nd Allahabad, India: Lalit Mohan Basu; 1987. p. 585-586.
  5. Pawar RS, Kumar S, Balakrishnan N, Lakshmi PK, Toppo Pharmacognostical and phytochemical evaluation of Ventilago calyculata Tul. (bark). Pharmacogn J 2015; 7: 271-275.
  6. Das B, Kashyap L. In: Diagnosis and treatment of diseases in Ayurveda: Todarnanda ayurvedic saukhya series no. 5, New Delhi. Concept publishing company; 1996. p. 700-718.
  7. Lin CC, Lin WC, Chang CH, Namba T.Antiinflammatory and hepatoprotective effects of Ventilago leiocarpa. Phytotherapy Research 1995; 9(1): 11-15
  8. Ansari HS. Essentials of Pharmacognosy: Nirali Prakashan; 2001. p. 588- 591.
  9. Khandelwal KR. Practical Pharmacognosy. 9th Nirali Prakashan; 2002. p. 149-153.

10.  Liang T, Yue W, Qingshan, L. Comparison of the phenolic content and antioxidant     activities of Apocynum venetum L. and two of its alternative species. Int J Mol Sci 2010; 11: 4452–4464.

11.  Ecobichon D J. In: The basis of toxicity testing. New York: CRC press; 1997. p. 43-49.

  1. Ravi V, Patel SS, Verma NK, Dutta D & Saleem TSM. Hepatoprotective activity of Bombax ceiba against Isoniazid and Rifampicin-induced toxicity in experimental rats. International J Applied Research Nat Pro. 2010; 3: 19-26.

13.  Reitman S, Frankel S. Colorimetric method for the determination of serum glutamic   oxaloacetic and glutamic pyruvic transaminases. Am J Clin Pathol 1957; 28: 56-63.

  1. Kind PRN, King EJ. Estimation of plasma phosphatase by determination of hydrolysed phenol with amino-antipyrine. J Clin Pathol 1954; 7: 322-326.
  2. Malloy HT, Evelyn KA. The determination of bilirubin with the photoelectric colorimeter. J Biol Chem 1937; 119: 481- 490.
  3. Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951; 193: 265-275.

17.     Allain CC, Poon LS, Chan CSG, Richmond W. Enzymatic determination of total serum cholesterol. Clin Chem 1974; 20: 470.

  1. Gupta AK. Ganguly P, Majumdar UP, Ghosal S. Hepatoprotective and antioxidant effects of total extracts and steroidal saponins of Solanum xanthocarpum and Solanum nigrum in paracetamol induced hepatotoxicity in rats. Pharmacol online 2009; 1: 757-768.
  2. Luna LG. In: Manual of histology and staining methods of armed forces, Institute of Pathology, 3 rd ed. New York: Mc Graw Hill Book Co; 1986. p. 43.
  3. Saraswathy SD, Suja V, Gurumurthy P, Shymaladevi CS. Effect of Liv.100 against antitubercular drugs induced hepatotoxicity in rats. Indian J Pharmacol 1998; 30: 233-238.
  4. Thresiamma KC, Kutton Inhibition of liver fibrosis by ellagic acid. Indian J Physiol Pharmacol. 1996; 40: 363.
  5. Rajesh MG, Latha MS. Preliminary evaluation of the anti-hepatotoxic activity of kamilari, a polyherbal formulation. J Ethnopharmacol 2004; 91: 99.
  6. Kang EH, Kown TY, Oh GT, Park WF, Park SI, Park SK, Lee YI. The flavonoid ellagic acid from a medicinal herb inhibits host immune tolerance induced by the hepatitis B virus-e antigen. Antiviral Res 2006; 72(2):100-106.
  7. Khalid HJ, Sheikh AS, Anwar HG. Protective effect of rutin on paracetamol and CCl4 induced hepatotoxicity in rodents. Fitoterapia 2002; 73(7): 557-563.
  8. Lal VK, Kumar P, Kumar A, Yadav KS. Screening of leaves and roots of Eclipta alba for  hepatoprotective activity. Scholars Res Lib 2010; 1: 86-94.

 

Table 1: Qualitative chemical tests performed in the various extracts of Ventilago  calyculata

Phytochemical tests Petroleum Ether Chloroform Ethyl acetate Ethanolic Aqueous
Alkaloids _

 

_

 

+

 

+

 

_
Carbohydrates _ _

 

+

 

+

 

+

 

 Glycosides _

 

+

 

+

 

_

 

_

 

 Phytosterol +

 

+

 

_

 

_

 

_

 

Saponins _

 

_

 

_

 

_

 

_

 

 Flavonoids _ _ + + +
 Mucilage _ _ _         _ +
 

Tannins/Phenols

 

_

 

 

+

 

 

+

 

 

+

 

 

_

 

 

Table 2: Effect of administration of INH+RIF on LPO, CAT, GSH and SOD antioxidant enzymes with or without concomitant administration of CEVC/Silymarin in liver homogenate of rats

 

Group /Treatment

LPO

( nmol/mg protein)

CAT

(Units/mg protein)

GSH

(nmoles/gm wet tissue)

SOD

(U/mg protein)

I  (CMC) 0.716 ±.2042 9.02±1.013 15.01±1.9879 29.9±1.3
II (INH+RIF 100 g/kg) 3.96±0.9935# 5.9±1.413# 3.7879±0.5442# 11.81±1.68#
III (Sily + INH+RIF 100 mg/kg) 0.85±0.1674* 11.18±1.021* 14.79±0.9349* 24.71±2.4*
IV (CEVC + INH + RIF 200 mg/kg 1.113 ±0.4776* 8.665±1.2907* 11.24±0.4601* 19.08±0.771*
Values are in the form of mean ± SD, n=6; (*p<0.05) vs INH+ RIF, (#p<0.05) vs CMC, (one-way ANOVA followed by Dunnett’s multiple comparision tests),  SD: standard deviation, CMC:  sodium  carboxy methyl cellulose, Sily: silymarin, INH: isoniazid, RIF: rifampicin, CEVC: chloroform extract of  Ventilago calyculata (stem bark)

Table 3: Effect of stem bark extracts of Ventilago calyculata on biochemical parameters of     blood serum in INH+RIF induced hepatotoxicity in rats

Group/Treatment     SGOT

(IU/L)

  SGPT

  (IU/L)

   ALP

  (IU/L)

     TB

  (mg/dL)

     TP

   (g/dL)

    TA

  (g/dL)

     TC

(mg/dL)

I (CMC) 71.64±6.3 38.97±6.1 85.66±2.325 0.263±0.14 8.48±0.305 4.76±0.24 60.78±2.43
II (INH+RIF 100 g/kg) 133.7±8.1# 124.1±6.42# 201±10.01# 0.978±0.164# 4.73±0.149# 3.02±0.35#  141.18±5.3#

 

III (Sily + INH+RIF 100 mg/kg) 90.42±5.3* 47.9±4.3* 124.8±3.26* 0.297±0.011* 7.78±0.201* 4.02±0.165* 80.92±3.15*
IV (CEVC + INH + RIF 200 mg/kg ) 113.1±5.5* 63.64±3.56* 139.6±5.32* 0.445±0.112* 7.0±0.13* 2.98±0.124* 90.014±4.9*
Values are in the form of mean ± SD, n=6; (*p<0.05) vs INH+ RIF, (#p<0.05) vs CMC, ( one-way ANOVA followed by Dunnett’s multiple comparision tests),  SD: standard deviation, CMC:  sodium carboxy methyl cellulose, Sily: silymarin, INH: isoniazid, RIF: rifampicin, CEVC: chloroform extract of  Ventilago calyculata (stem bark)

 List of titles for figures

Figure 1:  Effect of various extracts of Ventilago calyculata (stem bark) on DPPH scavenging activity

Figure 2: Effect of CEVC on lipid peroxidation, cell glutathione and liver antioxidant enzymes. Values are mean ± SD, n=6; (*p<0.05) vs Negative control, (#p<0.05) vs Normal, (one-way ANOVA followed by Dunnett’s multiple comparision test), Normal: carboxy methyl cellulose, Negative control: INH+RIF, INH: isoniazid, RIF: rifampicin, CEVC: chloroform extract of  Ventilago calyculata (stem bark)

Figure 3: Effect of CEVC on biochemical parameters of  blood serum in INH+RIF induced hepatotoxicity in rats. Values are mean ± SD, n=6; (*p<0.05) vs Negative control, (#p<0.05) vs Normal, (one-way ANOVA followed by Dunnett’s multiple comparision test), Normal: carboxy methyl cellulose, Negative control: INH+RIF, INH: isoniazid, RIF: rifampicin, CEVC: chloroform extract of Ventilago calyculata (stem bark)

Figure 4: a) Liver section of normal rats b) Liver section of INH+RIF treated rats c) Liver section of rats treated with INH+RIF and silymarin d) Liver section of rats treated with INH+RIF and chloroform extract of Ventilago calyculata (stem bark)

Figure 5: Figure 5: Schematic diagram depicting mechanism of protective action of chloroform extract of Ventilago calyculata (CEVC) against toxicity caused by anti- tubercular drugs, INH+RIF. The drugs INH+RIF increase the expression of cytochrome P450 enzymes. These enzymes generate reactive metabolites and free radicals which in turn bind to macromolecules, cause membrane lipid peroxidation and increase cellular toxicity. As concluded by the results, CEVC increases the level of intracellular (GSH) and antioxidant enzymes. It also restores the elevated serum amino transaminases

Figure 6: Antioxidant and hepatoprotective potential of the extracts from stem bark of Ventilago

Figure 1:  Effect of various extracts of Ventilago calyculata (stem bark) on DPPH scavenging activity

Figure 2: Effect of CEVC on lipid peroxidation, cell glutathione and liver antioxidant enzymes. Values are mean ± SD, n=6; (*p<0.05) vs Negative control, (#p<0.05) vs Normal, (one-way ANOVA followed by Dunnett’s multiple comparision test), Normal: carboxy methyl cellulose, Negative control: INH+RIF, INH: isoniazid, RIF: rifampicin, CEVC: chloroform extract of  Ventilago calyculata (stem bark)

Figure 3: Effect of CEVC on biochemical parameters of  blood serum in INH+RIF induced hepatotoxicity in rats. Values are mean ± SD, n=6; (*p<0.05) vs Negative control, (#p<0.05) vs Normal, (one-way ANOVA followed by Dunnett’s multiple comparision test), Normal: carboxy methyl cellulose, Negative control: INH+RIF, INH: isoniazid, RIF: rifampicin, CEVC: chloroform extract of Ventilago calyculata (stem bark)

Figure 4: a) Liver section of normal rats b) Liver section of INH+RIF treated rats c) Liver section of rats treated with INH+RIF and silymarin d) Liver section of rats treated with INH+RIF and chloroform extract of Ventilago calyculata (stem bark)

 

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