The present study was designed to establish the possible curative effects of Dawa-ul-Qust (a Unani compound drug) by its crude as well as 50% hydro-alcoholic extract on Carbon tetra chloride (CCl4) induced liver injury in rats. Toxic control, standard (Silymarin) and test drug (crude and extract forms) treated rats were received a single dose of Carbon tetra chloride (CCl4) (2 ml/kg) on 2nd day of study. Antihepatotoxic potential was assessed by the estimation of biochemical markers; viz. serum transaminases [aspartate aminotransferase (AST) and alanine aminotransferase (ALT)] and Malondialdehyde (MDA) levels. The histopathological examinations of rat livers were also studied to authenticate the biochemical findings. In post-treatment groups, rats were treated with Silymarin at a dose of (10mg/100g) and test drug (Dawaul Qust) at doses of 500 mg/kg and 74.9 mg/kg of body weight respectively in crude as well as in extract forms after Carbon tetra chloride (CCl4) administration. Rats treated with the test drug after the establishment of Carbon tetra chloride (CCl4) induced liver damage showed significant (p=0.001) protection of liver as evidenced from the reduction in the serum transaminases [aspartate aminotransferase (AST), alanine aminotransferase (ALT) and lipid peroxide levels, as both the doses forms exhibited significant reduction in biochemical markers and Malondialdehyde (MDA) levels. Carbon tetra chloride (CCl4) poisoned rats showed significant elevation of biochemical markers and Malondialdehyde level compared to normal control group as well as post treatment groups. The histopathological study supported the hepatocurative activity of the test drug. In conclusion, the findings of the present study suggest that Dawa-ul-Qust possesses potent curative effect against Carbon tetra chloride (CCl4) rendered liver injury in rats.

Keywords: curative effect, hydro-alcoholic extract, biochemical markers, Malondialdehyde

INTRODUCTION

The liver is a complex vital organ of the body present in vertebrates and some other animals and has the gigantic task of maintaining the body’s metabolic homoeostasis. The hepatic injury therefore, is always associated with distortion of metabolic functions (Kumar and Robbins, 2003). Liver regulates internal chemical environment efficiently but it is susceptible to be affected by a wide variety of diseases; fortunately most are rare, but there are a few that are all too common, including hepatitis, cirrhosis, alcohol-related disorders and liver cancer. (Sharma et al., 2000). Jaundice and hepatitis are two major hepatic disorders that account for a high death rate (Gujrati et al., 2007). Antibiotics are largest cause of drug-induced liver damage, while 90% of the acute hepatitis is estimated to be caused by the viruses. Unani system of medicine possesses several single and compound drugs that are highly effective and safe for the management of hepatic disorders. There are some compound formulations such as Jigrine (Abul et al., 2004), Icterene (Nasreen, 1999), Majoon-Dabedul-ward (Naeem, 1995), Hepatogard, Biliarin, Livol (Tareque, 2001), Livergen (Naseem, 2003) etc, which have been proven scientifically for their activities against the liver injury. However, only a small portion of the hepatoprotective plants as well as formulation used in traditional medicine are pharmacologically evaluated for their efficacy and a number of drugs particularly compound drugs have still not been scientifically evaluated for their described effects. (Handa et al. 1989). Dawa-ul-Qust is one such compound preparation described to be effective in liver diseases (Khan, 1921), and prescribed commonly by the Unani physicians has not been investigated so far, for its effect in hepatic diseases. Qust (Sausuria lappa) is its chief ingredient combined with other ingredients viz. Dar cheeni (Cinnamomum zelaynicum), Saleekha (Cinnamomum tamala), Zafraan (Crocus sativus), Badyan (Foeniculum vulgare), Karafs (Apium graveolens,) Tagar (Aquillaria agallocha), Rewand chini (Rheum emodi) Shagoofa-e-Izkhar (Andropogan schaerranthus), and Murmakki (Dendron myrrh) (Khan, 1921). All the ingredients have been mentioned to possess properties that are effective directly or indirectly in liver disease (Ibne Sina, 1906, Ghani, 1921; Azam, 1313 H; Antaki, 1317 H; Sharif, 1280 H; Momin, 1272 H) and the combination has even more strong hepatoprotective effect. Murakkabat (compound drugs) with therapeutic effectiveness are given to achieve maximum and quick results to complex therapeutic objectives, but modern scientific research and even the study of Tibb-e-Unani have been devoted mainly to single drugs and compound drugs are generally ignored. Since liver diseases entail a lot of complexities and diverse clinical manifestations therefore, the use of compound formulation appears to be more appropriate.
In view of these points, the present study has been designed to investigate its curative effect in chemically induced hepatic damage in rats. The damage produced by Carbon tetra chloride (CCl4) is described to be similar to the pathological changes seen in infective and viral hepatitis and in many other liver diseases (Berger et al., 1986) therefore, it was used to produce liver damage in rats for evaluation of the curative effect of the test drug.

MATERIALS AND METHODS

Plant materials
Ingredients of Dawa-ul-Qust (Khan, 1921)
1. Dar cheeni (Cinnamomum zelaynicum) 85 gm
2. Saleekha (Cinnamomum tamala) 85 gm
3. Qust (Sassuria lappa ) 85 gm
4. Zafraan (Crocus sativius) 24 gm
5. Badiyan-e- Roomi (Foeniculum vulgare) 30 gm
6. Tukhm-e-Karafs(Apium graveolans) 30 gm
7. Tagar (Aquillaria agallocha) 4.5 gm
8. Rewande Cheeni(Rheum emodi) 30 gm
9. Shagoofae Izkhar (Andropogen schaerranthus) 70 gm
10. Murmakki (Dendron myrrh) 70 gm

Preparation of powder and extract
All the ingredients of Dawa-ul-Qust (DQ) were procured from the herbal market in Aligarh and New Delhi and after identification and authentication of the crude ingredients of the test drug, was crushed to make a powder and homogenized in water for crude administration in aqueous medium. A 50% ethanol extraction was made through Soxhlets Apparatus (Anonymous, 1968; Anonymous, 1987) and dissolved / suspended in water for oral administration to the animal. Both the forms of Dawa-ul-Qust (DQ) collectively were used for screening the curative effects against CCl4 induced liver damage. The doses for animals were determined by extrapolating the Unani human dose range by multiplying it by conversion factor of 7 (Dhawan, 1982). The doses of Dawa-ul-Qust (DQ) thus calculated for albino rats, was found to be 500 mg/kg and 74.9 mg/kg of body weight respectively in crude as well as in extract forms.

Animals
Albino rats weighing 175-200 gm of either sex were obtained from the Meerut animal house and they were housed in clean polypropylene cages. The rats had free access to standard diet and water ad libitum throughout the experiment with the exception in which the animals were deprived of food, but not water, 12 h before the experiments. The room temperature was maintained at 25 ± 10 C with 12 hour light and dark cycle. The rats were randomly selected and were divided into five groups with six animals in each group and were left for one week for acclimatization to experimentation room. The experimental protocol was approved by the Institutional Ethics Committee.

Experimental design
In the post-treatment, the animals were divided into five groups containing six animals in each group. Except normal control all other groups (CCl4 treated, standard, DQ crude and extract treated) received carbon tetrachloride (CCl4) 50% v/v in olive oil (2 ml/kg of body wt) intraperitoneally on 2nd day to induce hepatotoxicity along with their routine treatment. The normal control group received normal saline orally in equal volume of test drug. The standard group received Silymarin 100 mg/kg orally for 7 days. The animals kept in group IV & V received treatment of Dawaul Qust (test drug) suspended in water at doses of 500 mg/kg and 74.9 mg/kg of body weight respectively in crude as well as in extract forms orally. On the 8th day all the animals were sacrificed under the ether anesthesia and blood was collected from each animal for serum analysis and liver were removed and fixed in 10% formalin for histopathological studies of the liver to determine the degree of hepatic damage. (Devaraj et al, 2011).

Preparations of Samples for Biochemical studies
The blood and liver were collected after sacrificing the animals. The blood was kept for 30 minutes without disturbing and was centrifuged for 15-20 minutes at 5000 rpm to separate the sera and stored at 40C. The serum of each animal of all groups were estimated for, Serum transaminases [aspartate aminotransferase (AST) and alanine aminotransferase (ALT (Reitman and Frankel 1957), and )] and Malondialdehyde (Okhawa et al, 1979) which is an index of lipid peroxides (Lowry et al., 1951)

Histopathological Observation
The small pieces of liver of all groups were removed immediately and fixed in 10% formalin. Care was taken to keep the volume of the fixative (Mukherjee, 1988). The tissue was processed and sections were cut. The slides were prepared and stained with hematoxyline and eosin stain and observed the histopathological changes by a photomicroscope under various magnifications.

Statistical Analysis
Data was presented as mean ± standard error and analyzed using one way ANNOVA test, followed by pair-wise comparison of various groups by LSD. The analysis was carried out by using the software of the website, www. Analyse it.com. P<0.05 or less was considered significant. Table 1 shows biochemical parameters i.e. alanine aminotransferase (ALT), aspartate aminotransferase (AST) and Malondialdehyde (MDA) levels in different groups. The values of biochemical parameters (ALT and AST) and MDA level were found significantly higher in the Group II as compared to group I and other post treated groups (p< 0.001). The values of ALT, AST and MDA were found lower in the post-treated Silymarin and crude as well as extract treated Groups than the CCl4 control group II (p<0.001). While comparing group I with group III, IV and V, it was observed that values of all the biochemical parameters had significantly increased (p<0.001). However, while comparing group III, IV and V no statistically significant difference was observed (p>0.05).

RESULTS
Biochemical Parameters

Table 1. Curative effect of Dawaul Qust in CCl4 mediated hepatic damage. Click here to view full image

Histological study

Figure 1A. Slide shows central blood vessels and radiating cords of hepatocytes, vascular sinusoids with no evidence of fatty changes, necrosis or inflammation. (1B) Centrilobular (Acidophilic) necrosis and vascular congestion (1C) Mild vascular congestion and peri-vascular infiltrate of mono nuclear cells and fibroblast. No fatty changes (1D) Edema less, inflammatory cell fewer (1E) Architecture preserved, congestion in Portal Triad and edema minimal. Click here to view full image

The histopathological studies of the liver showed centrilobular necrosis and vascular congestion in CCl4 control rats. CCl4 treatment caused marked congestion of central vein and portal triads, indicating fibrosis (Figure-1B), in comparison with normal control where, central blood vessels and radiating cords of hepatocytes as well as the vascular sinusoids were observed with no evidence of fatty changes, necrosis or inflammation (Figure-1A). The animals treated with Silymarin showed mild vascular congestion and peri-vascular infiltrate of mono nuclear cells and fibroblast. No fatty changes were seen (Figure-1C). Animals administered with crude form exhibited some inflammatory cells and up to some extent of edema in vessels. The group received extract form of test drug showed minimal degree of edema, architecture was noted preserved, and some congestion in portal triad was noticed. The maximum protection against hepatic damage was achieved by the both forms of test drug (Figure. 1D and 1E).
The Silymarin and test drug preserved the CCl4-induced structural changes in liver and showed excellent protection and cure to liver architecture.

DISCUSSION
Hepatocurative study was conducted to investigate the efficacy of crude as well as extract forms of Dawa-ul-Qust in curing the liver damage caused by a single dose of CCl4. In this study, liver damage was induced prior to the application of standard drug and test drug. Hepatotoxicity of the CCl4 in the rats was determined by changes in serum parameters by estimating the levels of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) which are enzymes originally present at higher concentration in the cytoplasm (Mohamed et al, 2009).When there is hepatopathy, these enzymes leak into the blood stream in conformity with the extent of liver damage (Nkosi et al., 2005). The CCl4 damaged liver toxicity was also associated with marked increase in liver Malondialdehyde (MDA) level and the elevation of MDA has been well accepted reliable marker of lipid peroxidation. Hence, in the present study MDA level was also estimated to evaluate curative properties of test drug. A significant difference in liver marker enzymes and MDA level was observed (Table-1). Histological studies of the liver also showed severe damage to the hepatocytes. Necrosis of the hepatocytes is quite prominent in rats in CCl4 control group (Figures-1B) as compared to other post treated groups (Figure-1C, 1D and 1E). Crude as well as extract of Dawaul Qust showed significant hepatocurative effect by lowering the liver marker enzymes alanine aminotransferase (ALT), aspartate aminotransferase (AST) and a significant decrease in Malondialdehyde (MDA) level was also observed in both the forms of test drug treated groups as compared to the CCl4 control group. Histological examination of the liver sections of rats treated with CCl4 with the silymarin and test drug (crude and extract forms of DQ) significantly decreased the above serum elevated parameters and the normal architectural liver pattern was restored. CCl4 can cause damage to many tissues in the body. However, the most important primary target organ for CCl4 induced toxicity in many species is the liver. CCl4 when metabolized in the body is changed into very reactive free radicals (halogenated free radical) by cytochrome P450 mixed function oxidase system. These reactive species then induce hepatic damage. Many latest evidences show that oxidative stress caused by free radicals may induce peroxidation and damage to biomolecules (lipid protein and nucleic acids). This may further leads to aging, cancer, severe fatty changes in liver and many other diseases in human. (Afaf et al., 2008). Hepatocurative properties of plants or plants extracts are generally attributed to the presence of chemicals which act as antioxidants or inhibitor of the microsomal drug metabolizing enzymes (Gopinathan et al., 2004). As it is widely accepted that CCl4 is metabolically activated by hepatic microsomal cytochrome P450 mediated reactions to the trichloromethyl radical (Slater, 1984). Therefore, the inhibitors of cytochrome P450 can impair the bioactivation of CCl4 into its toxic species and thus provide protection against hepatocellular damage (Nelson et al., 1980). The hepatocurative activity in DQ may be due to the presence of certain antioxidants which act as scavengers and remove the free radicals formed. These antioxidants also have the ability to prevent the process of peroxidation and improve the health of hepatocytes. The result of serum biochemical parameters, level of MDA and histopathological studies in the post-treatment groups support the highly potent hepatocurative activity of the crude as well as extract of Dawaul Qust against the CCl4 induced liver injury.

CONCLUSION

It can be concluded that both the doses form of test drug (Dawaul Qust) possess significant hepatocurative activity against acute hepatic damage induced by CCl4. However, the effect produced by the extract was more marked, which was almost equal to that of Silymarin. Further it should be evaluated in the human studies in order to have the proper treatment for the liver diseases.

ACKNOWLEDGEMENT
We express our sincere thanks to Central Council for Research in Unani Medicine (CCRUM) Department of AYUSH, Ministry of Health and Family Welfare, Govt. of India, New Delhi for providing financial assistance for the research work. We are also thankful to Professor Nafees Ahmad Farooqi Department of Anatomy and Prof. Shaista Vasenwala Department of Pathology Jawahar Lal Nehru Medical College, Aligarh Muslim University, Aligarh who extended their generous support in histological studies and special thanks to Dr. Ghufran Ahmad, Department of Ilmul Advia, Ajmal Khan Tibbiya College, Aligarh Muslim University, Aligarh for his constant encouragement, guidance and critical suggestions.

CONFLICT OF INTEREST
None declared.

REFERENCES

Abul KN, Pillai KK, Pal SN, Aqil M. Free radical scavenging and Hepatoprotective activity of jigrine against Galactosamine induced hepatopathy in rats. Journal of Ethanopharmacology. 2003;97(3):521-525.
Afaf I, Abuelgasim, Nuha HS, Mohammed AH. Hepato-protective effect of Lepidium sativum against carbontetrachloride-induced damage in rats. Research Journal of Animal and Veterinary Sciences. 2008;3:20-23.
Anonymous. British Pharmacopoeia”General Medical Council. Pharmaceutical Press, Blumsberg Square, London, p. 872-73, 1276-77, 1285-88, 1968.
Anonymous. ”Physico-chemical Standards of Unani Formulations” Central Council for Research in Unani Medicine, New Delhi, Part II, pp.274, 277, 1987.
Antaki, DA. Tazkira-e-ulil-Albab (Arabic)” Azhari Press, Egypt, p. 133, 1317H.
Berger BML, Comber H, Esta BB. CCl4 induced toxicity in isolated hepatocytes, “The importance of direct solvent injury”. Hepatalogy. 1986;6:325-327.
Dhawan BN. “Organization of Biological Screening of Medicinal Plants” with special reference to C.D.R.I programmes. Appendix-1, Lectures UNESCO-CDRI workshop on the use of Pharmacological Techniques for Evaluation of Natural Products, CDRI, Lucknow, p. 61, 1982.
Devaraj V C, Gopala K B,Viswanatha GL, Jagadish KV, Kumar S. Hepatoprotective activity of Hepax- A polyherbal formulation. Asian Pacific Journal of Tropical Biomedicine. 2011;1:142-146
Ghani, N. Khazeenat-ul-Advia” Matba Munshi Nawal Kishore, Lucknow, Vol. II, p. 455-58, 1921.
Gopinathan N, Srinivasan KK, Mathew JE. Free radical scavenging properties of ethanol extract of Saccharum spontaneum. Indian Drugs. 2004;41:633-635.
Gujrati V, Patel N, Rao VN, Nandakumar K, Gouda TS, Shalam Md., Kumar SMS. Hepatoprotective activity of alcoholic and aqueous extracts of leaves of Tylophora indica Linn in rats Indian Journal of Pharmacology. 2007;39(1)43-47.
Handa SS, Sharma A, Chakraborty KK. Fitoterapia. 1989;57:307-51.
Ibne-sina. “Kitab-ul-Qanoon-Fil-Tibb” Mataba Nami, Lucknow, Vol. II, p.164, 164, 98-99, 1906.
Khan S. “Ilaj-ul-Amraz” Matba Munshi Naval Kishor, Lucknow, p. 212, 1921.
Khan, MA. Muheet-e-Azam” Mataba Nizami, Kanpur, Vol. II, Pp. 76-81, 56-59,Vol. I, 252-54, 1313H.
Kumar V, Cortan RS, Robbins SL. Robbin’s Basic Pathology. 7th Ed, Pennysylvania Saunders, p.11, 600, 2003.
Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ. Protein measurement with Folin phenol reagent. Journal of Biological Chemistry. 1951;193:265-275.
Mohamed RA, Ramadan RS, Ahmed LA. Effect of substituting pumpkin seed protein isolate for casein on serum liver enzymes, lipid profile and antioxidant enzymes in CCl4-intoxicated rats. Advanced Biological Research. 2009;3(1–2):9–15.
Momin MH. Tohfat-ul-Momineen” Matba Hasani, p. 125, 1272H.
Mukherjee KL. Medical Laboratory Technology. Tata McGraw Hill, Publishing Company, Vol. 3, Pp. 1111-1124, 1988.
Naeem A Khan, Rahman SZ, Amin KMY. Majoon Dabeedul Ward: A compressive study of its protective effect in experimental liver damage. International Seminar on Ilmul Advia, Faculty of Unani Medicine, Aligarh Muslim University, Aligarh, p. 4-5, 1990.
Naseem M. Qadri, Shamim Qureshi, Zakir-Ur-Rehman and Zahara Yaqeen. Studies on Beneficial Role and Subchronic Toxicity of Livergen A Polyherbal Hepatoprotective Drug. Hamdard Medicus. 2003;XLVI(2):127-140.
Nasreen Fatima. Hepatoprotective activity of Icterene. Hamdard Medicus. 1993;XXXVI(3):56-62.
Nelson D, Kamataki T, Waxman D, Guengerrich P, Estabrook R, Feyerisen R, Gozalcz F, Coon M, Gunsahts I, Cotoh O, Okuda K, Nebert D. The P450 Superfamily: update on new sequences, gene mapping, expression numbers, early trivial names of enzymes and nomenclature. DNA and Cell Biology 12:1-51, 1993.
Nkosi CZ, Opoku AR, Terblanche SE. Effect of pumpkin seed (Cucurbita Pepo) protein isolate on the activity levels of certain plasma enzymes in CCl4-induced liver injury in low protein fed rats. Phytotherapy Research. 2005;19(4):341–345.
Ohkawa H, Ohishi N, Yagi K. Assay for Lipid Peroxides in animal tissues by Thiobarbituric Acid Reaction. Analytical Biochemistry. 1979;95:351-358.
Reitman S, Frankel S. A colorimetric method for the determination of Serum Glutamic Oxaloacetic and Glutamic Pyruvic Transaminases. American Journal of Clinical Pathology. 1957;28:56-63.
Shareef K. “Taleef-e-Sharifi” Matba Kishore Darussalam, Delhi, p.110, 192, 33, 1280H.
Sharma SK, Ali M, Ansari SH, Gupta, J. Evaluation of Indian herbal Hepatoprotective drugs. Hamdard Medicus. 2000;XLIII (2):39-58.
Slater TF, Cheeseman KH, Davies MJ, Proudfoot K, Xin W. Free radical mechanisms in relation to tissue injury. Proceedings of the Nutrition Society. 1987;46:1-12.
Tareq Hasan Khan. Traditional Medicines and Plant Drugs in Hepatic Diseases. Hamdard Medicus. 2001;XLIV(1):14-16.