HEPATOPROTECTIVE ACTIVITY OF BERRY EXTRACTS OF DIOSPYROS KAKI LINN. AGAINST CARBON TETRACHLORIDE INDUCED LIVER DAMAGE MODEL

HEPATOPROTECTIVE ACTIVITY OF BERRY EXTRACTS OF DIOSPYROS KAKI LINN. AGAINST CARBON TETRACHLORIDE INDUCED LIVER DAMAGE MODEL

S. N. V. L. Sirisha ^{* 1}, K. Prabhu ² and A. Sambasiva Rao ³

JNTU Institute of Science and Technology ¹, Centre for Pharmaceutical Sciences, Hyderabad - 500085, Telangana, India.

Vivekananda School of Pharmacy ², Batasingaram, Hayath Nagar, Ranga Reddy District, Greater Hyderabad - 501505, Telangana, India.

Sri Indu Institute of Pharmacy ³, Sheriguda, Ibrahimpatnam, Ranga Reddy District - 500030, Telangana, India.

ABSTRACT: Currently, nutrition and health linkages focused on the emerging strategy of diet based regimen to combat various physiological threats including cardiovascular disorders, oxidative stress, diabetes mellitus, etc. Berry / Fruit part of Diospyros kaki Linn. was extracted using petroleum ether, chloroform, ethanol consecutively and the obtained extracts were screened for hepatoprotective activity using CCl₄ induced liver damage model. The activity was assessed by comparing the serum enzyme levels such as serum glutamate pyruvate transaminase, serum glutamate oxalate transaminase, total bilirubin, alkaline phosphatase of fruit extracts treated group with carbon tetrachloride treated animals and results showed dose-dependent activity, ethanolic extract treated group showed highly significant activity, whereas chloroform extract treated group has shown the significant action but less compare to ethanolic extract, petroleum ether treated group showed moderate action and petroleum ether extract at a dose of 100 mg/kg b.w showed least significant action. The results were further supported by histopathological studies.

Diospyros kaki, Serum glutamate oxaloacetate, Serum glutamate pyruvate transaminase, Total bilirubin, Alkaline phosphatise

INTRODUCTION: The liver is the largest organ of the body. It is located between the portal and the general circulation, between the organs of the gastrointestinal tract and the heart. The main function of the liver is to take up nutrients, to store them, and to provide nutrients to the other organs ¹. The liver is not only an important power and sewage treatment plant of the body.

The liver is probably the best example of a cheap recycling system. The function of the liver as a clearance organ, however, harbors the danger that the substances that should be degraded and eliminated lead to tissue damage ². Thus, effective defense mechanisms are necessary. During the process of elimination, there is a chance of accumulation of different kinds of toxic materials inside the hepatocytes, and there is a chance of liver infection, and hepatic disorders such as hepatitis.  Amongst, persimmon (Diospyros kaki L.) is one of these nutritious fruits bestowed with strong antioxidant activity ^{3, 4}. Persimmon is fleshy fibrous tropical, deciduous fruit belonging to Ebenaceae family.

It is commonly cultivated in warm regions of the world including China, Korea, Japan, Brazil, Turkey, and Italy ^{5, 6}. In 2007, the global production of persimmon reached over 3.3 million tons, with 70.0 % from China, 10.0 % from Korea and 7.0 % from Japan. The persimmon is not so popular in European Communities, but its demand is increasing owing to consumer's awareness regarding its hidden health-promoting potential.

Generally, over 400 species of persimmon are planted globally. Among these, Diospyros kaki, Diospyros virginiana, Diospyros oleifera, and Diospyros lotus ⁷ are of significant importance. It is interesting for the readers that D. kaki (Japanese persimmon) is the most promising species. Fruit is used to treat Ischemia stroke, angina, internal hemorrhage, hypertension, atherosclerosis, and some infectious diseases. Folklore usage of D. kaki revealed to be used as an anti-diarrhoeal, improve eye movement, aid digestion, lower B.P, bleeding hemorrhoids, reduce cholesterol level, strengthen bones, boosts cognitive function, fight intestinal disorders and boosts body's immune system, bitter astringent, antiviral, antibacterial, astringent, styptic, treat various respiratory abnormalities such as Influenza, cold, cough ⁸. Even though different kind of allopathic molecules are available in the market all of them are suffering from some are the other toxic effect, so an urgent need of developing a herbal medicine which has got both liver protecting and nutritional value is required hence an attempt has been made to screen the hepatoprotective activity of fruit extracts of Diospyros kaki Linn.

MATERIALS AND METHODS:

Plant Materials: D. kaki berries were collected in October 2016 from the market of Missouri and were authenticated by Prof. D. Ramakanth Raju retired botanist Acharya Nagarjuna University, a voucher specimen (Snvl/jntu/2017-05) has been deposited in the Viswabharati College of Pharmacy, Guntur, Andhra Pradesh.

Preparation of Plant Extracts: Obtained plant material has been dried under shade and made into coarse powder passed though sieve# 20 and has been successively soxhelated using solvents like petroleum ether, chloroform, and ethanol for 72 h. Obtained extracts were made solvent free using rota evaporator and stored in a vacuum desiccator. Yield was found to be 0.28%, 0.3142%, 3.62% respectively. Obtained extracts were tested for preliminary phytochemical screening ⁹. Oral suspensions of the extracts were prepared at a dose of 200 mg/ml and 100 mg/ml using 5% aqueous gum acacia ¹⁰.

Acute Toxicity Studies: Adult Swiss albino mice 20-25gm were taken for acute toxicity tests. The mice were divided into control and test groups containing 6 animals each. The control group received vehicle (5% of normal saline’s), and the test group receives graded doses of extracts. The animals were observed up to 4 h then occasionally up to 48 h for the sign of any behavioral changes and motility, and LD₅₀ values were calculated ^11.

Determination of Hepatoprotective Activity: The institutional animal ethical committee approved the experimental protocol (Reg. no. 1963/po/Re/s/17/ CPCSEA).

Selection of Animals: Wister albino rats about 150-200g were chosen for study. The animals were fed with a balanced diet and tap water ad libitum. The animals were maintained at room temperature and 40 - 70% RH with 12 h light / 12 h dark cycle. They were allowed free access to a standard dry pellet diet. The food was withdrawn 18 h before the experiment but allowed free access to water.

Group I received vehicle 5% aqueous gum acacia,  Group II to Group IX received CCl₄ for 7 days at a dose of  0.25 ml/100gm ⁹, Group II serves as toxic group receives only CCl₄, Group III serves as standard receives silymarin 25 mg/kg b.w and group IV received D. kaki petroleum ether extract (D.K.P.E) at a dose of 100 mg/kg b.w, group V received D. kaki petroleum ether extract (D.K.P.E) at a dose of 200 mg/kg b.w, Group VI received D. kaki chloroform (D.K.C.E) at a dose of 100 mg/kg b.w, Group VII received D. kaki chloroform extract (D.K.C.E) at a dose of 200 mg/kg b.w, Group VIII received D. kaki ethanolic extract (V.M.E.E) at a dose of 100 mg/kg b.w, and group IX received D. kaki ethanolic extract (V.M.E.E) at a dose of 200 mg/kg b.w. Each group consisting of 6 animals. The vehicle and the test samples were administered orally for 7 days, and the liver damage was induced in rats on the 7^th day after 6 h of administration of a drug, by giving a single oral dose of CCl₄ in olive oil (1:1 ratio). On the 8^th day, the blood samples were withdrawn by puncturing retro-orbital plexus ⁸. The blood samples were allowed to clot for 30 min, and serum was separated by centrifuging at 2500 rpm for 10 min.

Assessment of Liver Function: Assessment of liver function was done by studying changes in biochemical parameters viz Reitman and Frankel method 12 estimated serum glutamic oxaloacetate transaminase (SGOT)/ (AST) and serum glutamic pyruvic transaminase (SGPT)/ (ALT).  Total bilirubin ¹³, Alkaline phosphatase was also estimated ¹⁴.

Statistical Analysis: ¹⁵ The results are expressed as mean ± S.E.M and the statistical significance of the difference between groups was analyzed by one-way ANOVA followed by Dunnett’s multiple comparison tests. P<0.05 was considered significant. The percentage reduction was calculated by considering the difference between the mean values of toxicant and control as 100% reduction.

Histological Study: For the histopathological study, liver from each animal was removed after dissection and preserved in 10% formalin. Then representative blocks of liver tissues from each lobe were taken and possessed for paraffin embedding using the standard microtechnique ¹⁶. Sections (5 μm) of livers stained with hematoxylin and eosin, were observed microscopically for histopathological studies.

TABLE 1: RESULTS OF PRELIMINARY PHYTOCHEMICAL TESTS OF THE D. KAKI BERRY EXTRACTS

TABLE: 2: EFFECTS OF D. KAKI BERRY EXTRACTS ON SERUM BIOLOGICAL PARAMETERS IN CCl₄ INDUCED LIVER DAMAGE MODEL

Values are mean ±SEM n=6, **P<0.01 When compared with the control, *P<0.01 when compared with the toxic control

TABLE 3: PERCENTAGE DECREASE IN BIOCHEMICAL PARAMETERS DUE TO TREATMENT WITH D. KAKI BERRY EXTRACTS

RESULTS AND DISCUSSION: This work is an attempt made for the validation of rational usage of D. kaki berry as a hepatoprotective agent in liver infections. In acute toxicity study, no mortality was found up to 2000 mg/kg p.o of D. kaki berry extracts treated animal group. The LD₅₀ was not determined, and 1/10^th of the tested proven safe concentration is taken as our experimental dose.

CCl₄ is a hepatoxin commonly used for the production of experimental liver toxicity ¹⁷. The serum transaminase level is most widely used as a measure of hepatic injury, due to its ease of measurement and a high degree of sensitivity.  It is useful for the detection of early damage of hepatic tissue and requires less effort than that required for histological analysis, moreover without sacrifice of the animal. From the results, it was observed that there is a significant increase in the levels of SGOT, SGPT & total bilirubin in the toxicant group. Pretreatment with plant extracts and silymarin in test groups and standard group respectively daily for seven days showed significant (p<0.01) protective effect against CCl₄ induced hepatotoxicity when compared to the toxicant group.

From the results, it was observed that the percentage reduction in silymarin pretreated group in the biochemical parameters, SGOT, SGPT, ALKP, TB were found to ­­­­­­­­be 99.42, 95.68, 99.04, and 86.02 respectively, whereas ethanolic extract treated group was showing a highly significant reduction of biochemical parameters at a dose of 200 mg/kg b.w whereas chloroform extract has shown moderated activity and petroleum ether extract showed the least significant hepato-protective activity

GRAPH 1: GRAPHICAL REPRESENTATION OF THE % RESPONSE OF BIOCHEMICAL PARAMETERS WITH D. KAKI BERRY EXTRACTS TREATMENT

Hence the ethanolic extract, chloroform extract of D. kaki at the dose of 200 mg/kg and 100 mg/kg were found to have significant hepatoprotective activity. The hepatoprotective activity of D. kaki could be due to the presence of alkaloids ¹⁷, proteins, and mucilage in case of chloroform extract. Whereas ethanolic extract possesses amino-acids, carbohydrates, flavonoids, and mucilage ¹⁸ which also are reported to have hepatoprotective and anti-oxidant properties.

Histopathological Sections of the Liver in Rats: Results of histopathological studies provided supportive evidence for biochemical analysis.

FIG. 1: HISTOPATHOLOGICAL SECTIONS OF THE LIVER IN RATS

Histology of liver section of normal control animal exhibited normal hepatic cells each with well defined cytoplasm, prominent nucleus, and nucleolus and well brought out central vein whereas that of CCl₄ intoxicated group animal showed total loss of hepatic architecture with centrilobular hepatic necrosis, fatty changes, vacuolization and congestion of sinusoids, kupffer cell hyperplasia ²⁰ crowding of central vein and apoptosis. Treatment with petroleum extract of D. kaki at a dose of 200 mg/kg b.w. showed moderate to weak activity in protecting the liver cells from CCl₄-injury. Among the plant extract, treatment with chloroform extract returned the injured liver to quite normal. And in the case of animal treated with ethanolic extract almost, it is equivalent to the standard group liver. Now, it could be decided that the hepatoprotective activity was dose and time-dependent. Out of three different extracts, the ethanol extract of D. kaki had shown very high significant potential hepatoprotective activity at a dose of 200 mg/kg. b.w. Even chloroform extract had shown significant protection against ccl₄ induced liver toxicity.

Results of histopathological studies provided supportive evidence for biochemical analysis. Histology of liver section of normal control animal exhibited normal hepatic cells each with well defined cytoplasm, prominent nucleus, and nucleolus and well brought out central vein whereas that of CCl₄ intoxicated group animal showed total loss of hepatic architecture with centrilobular hepatic necrosis, fatty changes, vacuolization and congestion of sinusoids, kupffer cell hyperplasia ²⁰ crowding of central vein and apoptosis. Treatment with petroleum extract of D. kaki at a dose of 200 mg/kg b.w. showed moderate to weak activity in protecting the liver cells from CCl₄-injury. Among the plant extract, treatment with chloroform extract returned the injured liver to quite normal. And in the case of animal treated with ethanolic extract almost, it is equivalent to the standard group liver. Now, it could be decided that the hepatoprotective activity was dose and time-dependent. Out of three different extracts, the ethanol extract of D. kaki had shown very high significant potential hepatoprotective activity at a dose of 200 mg/kg. b.w. Even chloroform extract had shown significant protection against Ccl₄ induced liver toxicity.

CONCLUSION: From this work, it can conclude that the folklore usage of D. kaki as a hepatoprotective drug has been validated, it is useful in treating different liver infections and diseases.

ACKNOWLEDGEMENT: Authors are thankful to the Management of Viswabharathi College of pharmacy for their support in the form of providing facilities.

CONFLICT OF INTEREST: The Authors declare no conflicts of interest

REFERENCES:

1. Cederbaum I: Molecular mechanisms of the microsomal mixed function oxidases and biological and pathological implications. Redox Biology 2015; 4: 60-73.
2. Alqahtani S and Mahmoud A: Gamma-glutamyl cysteine ethyl ester protects against cyclophosphamide-induced liver injury and hematologic alterations via up-regulation of PPARA and attenuation of oxidative stress, inflammation, and apoptosis. Oxidative Medicine and Cellular Longevity 2016; 2016(4016209): 14.
3. DI Meo S, Reed TT, Venditti P and Victor VM: Role of ROS and RNS sources in physiological and pathological conditions 2016; 12: 1-44.
4. Jung ST, Park YS, Zachwieja Z, Folta M, Barton H and Piotrowicz J: Some essential phytochemicals and the antioxidant potential in fresh and dried persimmon. Int J Food Sci Nutr 2005; 56: 105-113.
5. Igual M, Castello ML, Ortola MD and Andres A: Influence of vacuum impregnation on respiration rate mechanical and optical properties of cut persimmon. J Food Eng 2008; 86: 315-23.
6. Cichon-Lach H and Michalk A: Oxidative stress as a crucial factor in liver diseases. World J gastro enterol 2014; 20: 8082-91.
7. Dewanjee S, Das AK, Sahu R and Gangopadhyay M: Antidiabetic activity of Diospyros peregrina fruit: effect on hyperglycemia hyperlipidemia and augmented oxidative stress in experimental type 2 diabetes. Food Chem Toxicol 2009; 4710: 2679-2685.
8. Bibi N, Khattak AB and Mehmood Z: Quality improvement and shelf life extension of persimmon fruit Diospyros kaki. J Food Eng 2007; 79: 1359-63.
9. Asgar MA, Yamauchi R and Kato K: Modification of pectin in Japanese persimmon fruit during the sun-drying process. Food Chem 2003; 81: 555-560.
10. Rukhsana A, Hussin A and Ismail S: In-vitro effect of mitragynine (a major alkaloid of mitragyna speciosa Korth) on aminopyrine metabolism in rat hepatocytes, International Journal of Pharmaceutical Sciences and Research 2012; 3(7): 2241:2246.
11. Jalapure SS: Hepatoprotective activity of fruits of piper longum L Indian J Pharm Sci 2003; 65: 363.
12. Gosh, MN: Fundamentals of experimental pharmacology, Kolkata, scientific book publishers, Edition 2^nd, 1984: 327.
13. Reitman S and Frankel S; A colorimetric determination of serum glutamate oxalo acetatic acids, glutamic pyruvate transaminase. A J Clin Pathol 1957; 28: 56.
14. Mohamed EZ, Amani SA, Monerah RAO and Sahar KAD: Phytochemical analysis and antimicrobial activity of medicinal plants against pathogenic microorganisms. Life Sci J 2014; 11: 350-354.
15. Bessy OA: Method for rapid determination of alkaline phospotase five cubic millimeters of serum. J Biol Chem 1964; 164-321.
16. Richmond SB: Textbook of statistical analysis, Ronald press 1964; 569.
17. Galiger AE and Kaloff EN: In Essentials of Practical microtechniques. Philadelphia; Lea and Febiger 1976; 77.
18. Chowdhury SS and Uddin GM: In-vitro antioxidant and cytotoxic potential of a hydroethanolic extract of Averrhoa bilimbi Fruits. International Journal of Pharmaceutical Sciences and Research 2012; 3(7): 2263-2268.
19. Rukhsana A, Hussin AH, Ismail S and Mansor SM: In-vitro Effect of Mitragynine A Major Alkaloid of Mitragyna Speciosa Korth on Aminopyrine Metabolism in Rat Hepatocytes. Int J Pharm Sci & Res 2012; 3(7): 2238-2242.
20. Kavitha M: Hepatoprotective activity of a Polyherbal extract in Carbon tetrachloride intoxicated hepatotoxicity in Male albino rats. An International Journal of Pharma and Biosciences 2011; 2(3): 307-313.
21. Ahmed RA: The Hepatoprotective and antiapoptotic role of aged black garlic against hepatotoxicity induced by cyclophosphamide. The Journal of Basic and Applied Zoology 2018; 79: 8.
    

    ---

    How to cite this article:

    Sirisha SNVL, Prabhu K and Rao AS: Hepatoprotective activity of berry extracts of Diospyros kaki Linn. Against carbon tetrachloride induced liver damage model. Int J Pharm Sci & Res 2019; 10(1): 381-86. doi: 10.13040/IJPSR.0975-8232.10(1).381-86.

    ---

    All © 2013 are reserved by International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.

    ---