Evaluation of Hepatoprotective and Antioxidant Activity of Classical Ayurvedic Formulation Punarnavashtak Kwath Against Paracetamol Induced Hepatotoxicity in Rats

 

V.N. Shah1*, D.B. Doshi1, M.B. Shah2 and P.A. Bhatt2

1APMC College of Pharmaceutical Education and Research, Himatnagar-380001, Gujarat, India

2L.M. College of Pharmacy, Ahmedabad -380009, Gujarat, India

ABSTRACT:

Objective: Punarnavashtak (PN) kwath a classical Ayurvedic formulation mentioned in Ayurvedic literature “Bhaishyajyaratnavali” for hepatic disorders and asthma. The present study investigated the hepatoprotective activity of PN kwath to validate the traditional use of this formulation.

 

Materials and methods: PN kwath was prepared in the laboratory according to the method given in Ayurvedic literature. Preliminary phytochemical screening was performed to determine the presence of phytoconstituents. Hepatoprotective and antioxidant activity was evaluated against paracetamol induced hepatotoxicity in rats

 

Results: Preliminary phytochemical screening revealed the presence of alkaloids, tannins, flavonoids, saponins and bitter principal in PN kwath. Administration of PN kwath produced significant hepatoprotective effect as demonstrated by decrease level of serum liver marker enzymes like AST, ALT, ALP, SBRN and increase protein level. It also showed antioxidant activity by increase in activity of GSH, SOD, CAT and decrease in TBARS level compare to paracetamol treated group. A comparative histopathological study of liver exhibited almost normal architecture, as compared to paracetamol treated group.

 

Discussion and Conclusion: It can be concluded that PN kwath protects hepatocyte from paracetamol-induced liver damages due to its antioxidant effect on hepatocytes

 

KEYWORDS: Punarnavashtak kwath, Hepatoprotective, Antioxidant, Paracetamol.

 

INTRODUCTION

Liver is an important organ actively involved in many metabolic functions and is a frequent target for a large number of toxicants1. Hepatic damage is associated with distortion of these metabolic functions2. Liver disease is still a worldwide health problem. Unfortunately, conventional or synthetic drugs used in the treatment of liver diseases are inadequate and sometimes can have serious side effects3. In the absence of a reliable liver protective drug in modern medicine, there are a number of medicinal preparations in Ayurveda recommended for the treatment of liver disorders4. Plants are complex mixtures of compounds and no single compound can provide the desired activity. Some compounds potentiate a desired therapeutic action, while others reinforce the same and yet others interact to neutralize and counteract any possible side effect that may exist5.

 

 

In the traditional system of Indian medicine, plant formulation and combined extracts of plants are used as drug of choice rather than individual and these herbal formulations are used for the treatment of a wide variety of diseases6. This  therapeutic approach is often ignored by many and considered to be an alternative to conventional medicine by others due to lack of scientific validation of efficacy and safety7. An article published in JAMA (Journal of American Medical Association) emphasizes that the fundamental issue is not traditional medicine versus alternative medicine, but medical practice supported by clinical and scientific evidence8. Hence, there is a requirement for a scientific proof (biological assays, animal models, clinical trials, and chemical standardization).

 

Number of reports indicate that overdose of paracetamol can produce centrizonal hemorrhagic hepatic necrosis in human and experimental animals9,10. Paracetamol-induced hepatotoxicity in rodents is a widely used animal model to assess hepatoprotective activity of new compounds11,12. So In the present investigation Punarnavashtak kwath, a classical ayurvedic polyherbal (Table 1) formulation mentioned in Ayurveda13, consisting of Boerhaavia diffusa Linn, Picrorhiza Kurroa Royle ex Benth, Tinospora cordifolia (Willd.) Miers, Zingiber officinalis Rosc, Berberis aristata DC, Terminalia chebula Retz, Azadirachta indica A. Juss and Tricosanthes dioica Roxb. plants has been evaluated for its hepatoprotective action against paracetamol induced hepatotoxicity. Traditionally this formulation is used in treatment of hepatic disorders and asthma. Many of the individual ingredients of the formulation were reported earlier for their protective activity against different models of experimental hepatotoxicity. An aqueous extract of thinner roots of Boerhaavia diffusa at a dose of 2 ml /kg exhibited marked protection of various enzymes such as serum glutamic oxaloacetic transaminase, serum glutamic pyruvic transaminase and bilirubin in serum against hepatic injury in rats14. The active constituents of Picrorhiza kurroa were effective in preventing liver toxicity and the subsequent biochemical changes caused by numerous toxic agents15,16. The hepatoprotective action of Tinospora cordifolia was reported in one of the experiment in which goats treated with Tinospora cordifolia have shown significant clinical and hemato-biochemical improvement in CCl4 induced hepatopathy. Extract of Tinospora cordifolia has also exhibited in vitro inactivating property against Hepatitis B and E surface antigen in 48-72 h17. The aqueous ethanol extract of Zingiber officinalis showed hepatoprotective effect against acetaminophen-induced acute toxicity, mediated either by preventing the decline of hepatic antioxidant status or due to its direct radical scavenging capacity18. Berberis aristata and berberine (an alkaloid from Berberis aristata) were found to be protective against both paracetamol and CCl4-induced liver damage and also showed MDME (microsomal drug metabolizing enzymes) inhibitory activities19. Terminalia chebula extract was found to prevent the hepatotoxicity caused by the administration of rifampicin (RIF), isoniazid (INH) and pyrazinamide (PZA) (in combination) in a sub-chronic model20. The aqueous extract of Azadirachta indica leaf was found to offer protection against paracetamol induced liver necrosis in rats21. Tricosanthes dioica was reported as a hepatoprotective agent in ferrous sulphate (FeSO4) intoxicated rats22.

 

Silymarin is a mixture of flavonolignans from the fruits of Silybum marianum that has been known since ancient time and recommended in traditional European and Asian medicine mainly for the treatment of liver disorder23, 24. Therefore in the present study silymarin was used as positive control to compare the efficacy of PN kwath against paracetamol induced hepatotoxicity.

 

MATERIALS AND METHODS:

Collection of plants and preparation of formulation:

Punarnava root, Galo stem, Tricosanthes leaves, Neem bark were collected from medicinal garden of APMC College of Pharmaceutical Education and Research (January 2008) while other plants Picrorhiza stem, Berberis stem, Harde fruit and Ginger rhizome were purchased from market. All the plants were authenticated by Dr Mukesh Prajapati, botanist, H.N.S.B Science College, Himatnagar and voucher specimen of all plants were kept in department of Pharmacognosy, APMC College of Pharmaceutical Education and Research, Himatnagar. (APMC 0801 to 0808). Kwath (decoction) was prepared by boiling powder of all drugs (Table 1) in equal quantity in proportion of 16 times of water reduced to one fourth and strained in cloth. Filtrate was evaporated and dried under reduced pressure13.Yield of extract was 10% w/w.

 

Table 1: Composition of Punarnavashtak (PN) kwath

No

Botanical name

Family

Part used

1

Boerhaavia diffusa Linn.

Nyctaginaceae

Root

2

Picrorhiza  Kurroa  Royle ex Benth

Scrophulariaceae

Root

3

Berberis aristata DC.

Berberidaceae

Stem

4

Tinospora cordifolia (Willd.) Miers.

Menispermaceae

Stem

5

Terminalia chebula Retz,

Combretaceae

Fruit

6

Azadirachta indica A. Juss.

Meliaceae

Bark

7

Zingiber officinalis Rosc.

Zingiberaceae

Rhizome

8

Tricosanthes dioica Roxb.

Cucurbitaceae

Leaf

 

Phytochemical screening:

The dried extract of kwath was subjected to the preliminary phytochemical analysis for the presence of different phytoconstituents25,26.

 

Acute toxicity study:

Swiss albino mice of either sex weighing between 25-30 g were divided into ten groups of six animals in each. The control group received normal saline (2 ml/kg p.o) while other groups received 100, 200, 300, 600, 800, 1000, 2000, 3000, 5000 mg/kg of the test extract, respectively. Immediately after dosing, the animals were observed continuously for the first 4 h for any behavioral changes. They were then kept under observation up to 14 days after drug administration to find out the mortality if any. The observations were made twice daily, one at 7 a.m. and another at 7 p.m.27.

 

Hepatoprotective effect of PN kwath in paracetamol induced liver damage:

Healthy albino Wister rats of either sex weighing between 150-200 g were used. They were collected from animal house, Zydus Cadila Pharmaceuticals, Ahmedabad. The animals were grouped and housed in poly acrylic cages, with not more than two animals per cage and maintained under well-controlled conditions of temperature (27 ± 20°C), humidity (55 ± 5%) and 12/12 h light-dark cycle. Conventional laboratory diet and tap water were provided ad libitum. The protocol of the experiment was approved by the Institutional Animal Ethical Committee as per the guidance of the Committee for the Purpose of Control and Supervision of Experiments on Animals (Proposal No APMC 08/02, Himatnagar), Ministry of Social Justice and Empowerment, Government of India.

 

The animals were divided into four groups of 6 animals each.

Group I served as control, received vehicle (distill water) for 8 days.

Group II received vehicle for 7 days.

Group III received PN kwath (100 mg/kg p.o) for 7 days.

Group IV received silymarin (50 mg/kg p.o) for 7 days.

Paracetamol at 3 g/kg body wt was administered orally to all groups except for control on the day eight28,29. Animals in all the groups were sacrificed 48 h after paracetamol administration. Liver was isolated and used for antioxidant activity and histopathological analysis.

 

Biochemical studies:

Blood was collected from retro orbital plexus after 48 h of paracetamol treatment. Serum was separated by centrifugation at 2500 rpm at 30°C for 15 min and utilized for the estimation of various bio-chemical parameters viz. aspartate transaminase (AST), alanine transaminase (ALT)30, serum alkaline phosphatase (ALP)31, serum bilirubin32 and total protein33.

 

Antioxidant activity:

For estimating antioxidant activity, animals were sacrificed and liver was excised, rinsed in ice-cold normal saline followed by 0.15 M Tris-HCl (pH 7.4) blotted dry and weighed. A 10 % w/v of homogenate was prepared in 0.15 M Tris-HCl buffer and processed for the estimation of lipid peroxidation (TBARS)34. A part of homogenate after precipitating proteins with trichloroaceticacid (TCA) was used for estimation of glutathione35. The remaining homogenate was centrifuged at 1500 rpm for 15 min at 4°C. The supernatant thus obtained was used for the estimation of super oxide dismutase36 and catalase37.

 

 

Histopathological studies:

Paraffin sections (7 µm thick) of buffered formalin-fixed liver samples were stained with hematoxylin-eosin to study the histological structure of control and treated (Toxicant, PN kwath, Silymarin) rats liver

 

Statistical analysis:

Results are expressed as mean ± S.E.M. The statistical difference was analyzed by one way analysis of variance followed by Tukey-Kramer multiple comparison test, significance was calculated as the P value and P values of less than 0.05 were regarded as statistically significant.

 

RESULTS:

Preliminary phytochemical screening showed the presence of alkaloids, tannins, carbohydrate, glycosides, flavonoids and saponins in PN kwath.

 

In acute toxicity study, it was observed that there was no mortality at any of the tested doses up to end of 14 days of observation.

 

Hepatic damage induced by paracetamol caused significant increase (P<0.01) in marker enzymes ALT, AST, ALP and bilirubin levels as compared to normal animals. Parcetamol toxicity also showed significant decrease (P<0.01) in protein level compare to normal animal. Oral administration of PN kwath (100 mg/kg) significantly (P<0.001) lowered ALT, AST and ALP compare to paracetamol treated rats. PN kwath treatment also showed significant decrease (P<0.05) in bilirubin levels compare to paracetamol treated animals. Level of serum protein was also significantly (P<0.05) increased in rats, which received PN kwath as compared to paracetamol treated group (Table 2).

 

Hepatic damage induced by paracetamol also caused significant increase in thiobarbituric acid reactive substance (TBARS) level and significantly (P<0.01) decrease the activity of antioxidant enzymes SOD and CAT in liver when compared with normal rats (Table 3). Intracellular antioxidant GSH level was also significantly (P<0.01) depleted. Treatment with PN kwath significantly (P<0.01) prevented the increase in TBARS levels and brought them near to normal levels compared to paracetamol treated rats. PN kwath treatment also showed significant (P<0.01) increase in GSH activity. SOD and CAT activity were significantly (P<0.05) increased in PN kwath treated groups compare to paracetamol treated group. The effects of PN kwath were comparable to that of standard reference drug silymarin.

 

Histopathological study of paracetamol treated rat livers showed marked necrosis, severe fatty degeneration and extensive vacuolization with disappearance of nuclei compared to normal untreated rats. (Fig1a, 1b) PN kwath and silymarin treated liver were structurally normal as compare to the paracetamol treated group (Fig 1c, 1d).

 

 

 

DISCUSSION:

Paracetamol (N-acetyl-p-aminophenol, acetaminophen), a widely used analgesic and antipyretic drug are known to cause hepatotoxicity in experimental animals and humans at high doses38-41. It is well established that following an oral therapeutic dose, a fraction of paracetamol is converted via the cytochrome P450 pathway to a highly toxic metabolite, N-acetyl-p benzoquinoneimine (NAPQI)42, which is normally conjugated with glutathione and excreted in the urine as conjugates. Overdoses of paracetamol deplete glutathione stores, leading to accumulation of NAPQI, mitochondrial dysfunction43 and the development of acute hepatic necrosis44. Paracetamol-induced hepatotoxicity in rodents is a widely used animal model to assess hepatoprotective activity of new compounds8,45. Paracetamol is a powerful inducer of cytochrome P450 and produces a highly reactive quinoneimine, which combines with sulphydryl groups of protein and cause rapid depletion to intracellular GSH46,47. Normally GSH contributes significantly to the intracellular antioxidant defensive system as it is a powerful consumer of superoxide, singlet oxygen, and hydroxyl radicals48. The breakdown of the GSH-dependent antioxidant defensive

 

system increases the intracellular flux of oxygen free radicals49 creating an oxidative stress and initiating apotopsis.

 

In the assessment of liver damage by paracetamol the determination of enzyme levels such as ALT and AST are largely used. Necrosis or membrane damage releases the enzyme into circulation; therefore, it can be measured in serum. High levels of AST indicate liver damage, such as that due to viral hepatitis as well as cardiac infarction and muscle injury. ALT catalyses the conversion of alanine to pyruvate and glutamate and is released in a similar manner. Therefore, ALT is more specific to the liver, and is thus a better parameter for detecting liver injury50. Elevated levels of serum enzymes are indicative of cellular leakage and loss of functional integrity of cell membrane in liver 51. Serum ALP and bilirubin level on other hand are related to the function of hepatic cell. Increase in serum level of ALP is due to increased synthesis, in presence of in-creasing biliary pressure52.

 

Pretreatment with PN kwath at a dose of 100 mg/kg significantly reduced the elevated levels of these enzymes indicating stabilization of plasma membrane as well as repair of hepatic tissue damage caused by paracetamol. The above changes can be considered as an expression of the functional improvement of hepatocytes, which may be caused by an accelerated regeneration of parenchyma cells. Effective control of alkaline phosphatase (ALP) and bilirubin levels point towards an early improvement in the secretary mechanism of the hepatic cell.

 

The histopathological pattern of the liver treated with PN kwath showed minimal necrosis in centrilobular and regeneration of hepatocytes as compared to control animals.

Lipid peroxidation has been postulated as being the destructive process in liver injury due to paracetamol administration52. In present study; level of TBARS was elevated in rats treated with paracetamol. The increase in TBARS level in liver suggest enhanced lipid peroxidation leading to tissue damage and failure of antioxidant defense mechanisms to prevent formation of excessive free radicals. Treatment with PN kwath significantly reversed these changes. Hence it may be possible that the mechanism of hepatoprotection of PN kwath is due to its antioxidant effect.

 

Glutathione is one of the most abundant tripeptide, non-enzymatic biological antioxidant present in liver. Its functions are concerned with the removal of free radical species such as hydrogen peroxide, superoxide radicals, alkoxy radicals, and maintenance of membrane protein thiols and as a substrate for glutathione peroxidase (GPx) and glutathione transferase (GST)53. In our present study, decreased level of GSH has been associated with an enhanced lipid peroxidation in paracetamol treated rats. Administration of PN kwath significantly increased the level of glutathione.

 

Increase in serum activity of superoxide dismutase (SOD) is a sensitive index in hepatocellular damage and is the most sensitive enzymatic index in liver injury54. SOD has been reported as one of the most important enzymes in the enzymatic antioxidant defense system. It scavenges the superoxide anion to form hydrogen peroxide, hence diminishing the toxic effect caused by this radical. In the present study, PN kwath significantly increased hepatic SOD activity possibly reducing reactive free radicals that might lessen oxidative damage to the tissues improving activities of hepatic antioxidant enzymes like SOD.

 

Catalase (CAT) is an enzymatic antioxidant widely distributed in all animal tissue and the highest activity is found in red cells and liver. CAT decomposes hydrogen peroxide and protects the tissue from highly reactive hydroxyl radicals55. Therefore, the reduction in the activity of these enzymes may result in a number of deleterious effects due to accumulation of superoxide radicals and hydrogen peroxide. Administration of PN kwath increased the activities of SOD and CAT in paracetamol induced liver damage in rats to prevent the accumulation of excessive free radicals and protected the liver from paracetamol intoxication. All the results were comparable to that of silymarin. These biochemical restorations may be due to the inhibitory effects on cytochrome P450 or/and promotion of its glucuronidation56,57 PN kwath showed significant hepatoprotective activity due to antioxidant activity. Acute toxicity study showed its LD50 was more than 5gm/kg showed this formulation is non toxic which may be due to the polyherbal formulation because some compounds potentiate a desired therapeutic action, while others reinforce the same and yet others interact to neutralize and counteract any possible side effect that may exist5. It concludes that results of present investigation support the traditional use of this formulation in hepatic disorder.

 

ACKNOWLEDGEMENTS:

The author express their sincere thanks to Dr. Ghanshyam R Patel , M.D. Ayurveda, B.A.P.S Swaminarayan Herbal Care, Shahibaug, Ahmedabad and Dr Tapan Vaidya, MD Ayurveda, Shri Dardi Narayan Sevamandal Hospital, Paldi, Ahmedabad, India for their generous help.

 

Table 2: Effect of Punarnavashtak kwath on different biochemical parameter in Paracetamol induced hepatotoxicity.

Group

AST(U/L)

ALT(U/L)

ALKP (U/L)

Bilirubin(mg%)

Protein(mg/dl)

I  (Normal)

33.3 ± 2.18

122.5 ±11.7

149.16 ± 13.61

0.58  ±   0.03

5.6 ±  0.15

II (Toxicant)

174 ± 6.3††

315 ± 16.3††

391 ±  20.7††

1.11 ± 0.11†

3.95 ± 0.35†

III (PN kwath)

75.3 ± 8.5***

154.6 ± 7.4***

216 ± 19.3***

0.91 ± 0.10*

5.3 ± 0.31*

IV (Silymarin)

54.5 ± 7.9***

131.6 ± 7.6***

162.83 ± 14.9***

0.67  ±  0.08*

5.45 ± 0.31*

Values are mean ± SEM of 6 animals in each group

†† P<0.001 relative to Normal group, † P<0.01 relative to Normal group, ***P<0.001 relative to Toxicant group, **P<0.01 relative to Toxicant group, *P<0.05 relative to Toxicant group.

 

Table 3: Effect of Punarnavashtak on lipid peroxidation (TBARS), superoxide dismutase (SOD), catalase (CAT) and total glutathione (GSH) level in Paracetamol induced hepatotoxicity

Group

TBRAS

SOD

Catalase

Gutathione

 

nmol/ mg protein

U/mgprotein

u/min/mgprotein

nmol/mg protein

I  (Normal)

1.54 ± 0.26

12.50 ± 0.83

31.12 ± 3.63

106.18 ± 12.41

II (Toxicant)

11.50 ± 1.90††

4.16 ± 0.70††

7.87 ± 2.19†

28.40 ± 7.09†

III (PN kwath)

3.12 ± 0.43**

7.78 ± 0.67**

26.78 ± 2.01*

91.27 ± 18.80*

IV (Silymarin)

2.44 ± 0.29**

8.09 ± 0.37**

27.04 ± 5.66*

94.78 ± 11.41*

Values are mean ± SEM of 6 animals in each group

†† P<0.001 relative to control group, † P<0.01 relative to control group, ** P<0.01 relative to Toxicant group, *P<0.05 relative to Toxicant group.

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Received on 09.05.2010

Accepted on 31.05.2010     

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Research J. Pharmacology and Pharmacodynamics. 2(4): July-August 2010, 283-288