Comparative Analysis in Hepatoprotective Activity of Crude Extracts of important Medicinal Plants

 

Jitendra Patel¹, G. Shiva Kumar¹, Khemkaran Ahirwar², Mukesh K. Gupta³,

Sandeep K. Singh⁴, Saket S. Chandel⁵, Vijay K. Patel⁵*

¹Gitam School of Pharmacy, GITAM Deemed to be University, Rudraram, Hyderabad, Telangana 502329.

²Sant Gahira Guru Vishwavidyalaya, Sarguja, Ambikapur, Chhattishgarh, India, 497001.

³Sai College of Pharmacy, Shekhpura, Bihar, India, 811101.

⁴Central Ayurveda Research Institute, Jhansi, Uttar Pradesh, India, 284003.

⁵Dr. C. V. Raman Institute of Pharmacy, Dr. C. V. Raman University, Bilaspur, C.G. 495113, India.

 

ABSTRACT:

According to the World Health Organization, chronic liver diseases account for approximately 46% of all diseases and 59% of all deaths worldwide. It is critical to look for hepatoprotective crucial findings because they are important for human health. The aim of this study was to compare the hepatoprotective activity of six medicinal plants based on biochemical reduction on the animal study. The increased levels of ALT, AST, ALP, total bilirubin, direct bilirubin, and cholesterol in the blood have been marked to the damaged structural virtue of the liver. When the plant extract was given to rats after they were administered a hepatotoxicant and the level of marker enzymes was discovered to be lower than normal, it meant that the particular dose of the plant extract was considerably supporting the hepatoprotective action. The comparative study of hepatoprotective efficacy of six medicinal plants was determined by a biostatistical analysis of data received from an animal investigation. The Terminalia coriacea and Artocarpus hirsutus at 250 and 500mg/kg respectively revealed that the *P<0.05; **P< 0.01; ***P<0.001; when compared to the CCl4 group. The Cucumis melo revealed P<0.05 and P<0.01 as compared with the liver-damaged control to the drug-treated animal at doses of 100, 250, and 500mg/kg. Buchanania lanzan revealed significant at P<0.05*, 0.01** and 0.001*** at 100 mg/kg, 200mg/kg and 400 mg/kg for aqueous and alcoholic extract both. The Diospyros melanoxylon (Roxb) and Solanum americanum Mill at doses 100, 200, and 400 mg/kg and 200, 400, and 600 mg/kg respectively. The T. coriacea and A. hirsutus revealed a 5%, 1%, and less than 1% chance of false-positive, which is statistically significant at all levels.

 

 

 

INTRODUCTION: 

The liver is the most important glandular organ, which plays a pivotal role in regulating, synthesizing, and restoring the various physiological processes in the body. It is involved in several vital functions, such as metabolism, secretion, and storage. The liver is the key organ regulating homeostasis in the body.

 

It is involved in almost all the biochemical pathways related to growth, fighting against diseases, nutrient supply, energy provision, and reproduction. It has a great capacity to detoxicate toxic substances and synthesize useful principles. Liver diseases are mainly caused by toxic chemicals, excess consumption of alcohol, infections, and autoimmune disorders. The majority of hepatotoxic chemicals damage liver cells mainly by inducing lipid peroxidation and other oxidative damages^1-5. In general, the liver is the only organ that can regenerate itself. Despite tremendous advances in modern medicine, there are no effective drugs available that stimulate liver function, offer protection to the liver from damage, or help to regenerate hepatic cells^6-8.

The goal of this study was to compare the hepatoprotective effects of six medicinal plants, using biochemical reduction in animals. The liver protection was measured by two parameters i.e., biochemical analysis and histopathology of treated rat’s liver. Different plant extracts reveal the different levels of significance at different doses of extracts. It is crucial to compare the relevance of hepatoprotective activity among six plants that are known for having different pharmacological effects. Terminalia coriacea (Roxb.) Wight and Arn. (Combretaceae) have been used traditionally in Indian medicine for the treatment of various ailments⁹. Artocarpus hirsutus belongs to the family Moraceae used for healing effects¹⁰. Cucumis melo Linn. (Cucurbitaceae) also called as musk melon fruits have been used, traditionally in the Indian traditional system of medicine, for the treatment of various disorders¹¹. Buchanania lanzan Spreng (locally called as Chironji)¹², a member of the family Anacardiaceae is a commercially useful tree species found in several areas of India¹³. Diospyros melanoxylon (Roxb) is a native and endemic tree of India and is widely found in forests of central, western, and northern India¹⁴. Solanum Americanum is commonly known as American nightshade or Glossy nightshade¹⁵.

 

MATERIALS AND METHODS:

Plant material collection:

The plant materials were collected from the Tirumala Hills, Chittoor district, Andhra Pradesh, India, and the forest of Basana, Chhattisgarh, India. The plant materials were identified and authenticated by the Botanical Survey of India (BSI) at Attapura, Hyderabad, India.

 

Extraction of Plant Materials:

All the plant parts used were subjected to extraction under the soxhlet apparatus. The extracts are prepared from 15g of leaves of Terminalia Coriacea, Artocarpus hirsutus, and Diospyrus melanoxylon.  The extracts are prepared from 15g of Cucumis melo fruit and Buchania lanzaan bark. The solvent was run in apparatus by increasing order of polarity, started from the petroleum ether after 3-4 syfen removed plant materials and again subjected the same leave powder to the next solvent. The order of solvent was pet ether, chloroform, methanol and hydro alcoholic. After completion of the extraction, we had 4 extracts of each plant such as pet ether extract, chloroform extract, methanolic extract, and hydro alcoholic extract¹⁶.

 

Preliminary Phytochemical Screening:

After extraction, each extract from each plant was subjected to examination for the presence of phytochemicals. All the extracts were checked for tests for alkaloids, glycosides, tannins, flavonoids, sterols, and steroids etc. The methanolic or alcoholic extract was found to have the maximum presence of expected phytochemicals¹⁷.

 

Hepatoprotective activity:

The albino rats were flakily divided into five classes (n=6). The Class-I and Class-II are the negative control (- control) and positive control (+control) groups respectively, and they administer 1 mL of distilled water orally for 5 days. Class-III consisted of standard silymarin at 50 mg/kg, Class-IV of 70% methanolic extract at 250 mg/kg, and Class-V of 70% methanolic extract at 500 mg/kg, all of which were administered orally for 5 days. On the 2nd and 3rd day, Class-I was given olive oil (1ml/kg) administered subcutaneously. On the 2nd and 3rd day, Class-II, Class-III, Class-IV, and Class-V were given CCl₄ : olive oil (1:1) at a dose of 2ml/kg subcutaneously just after thirty minutes of vehicle, 50 mg/kg silymarin, 250 mg/kg 70% METC and 500 mg/kg 70% methanolic extract administration. The animals were sacrificed on the sixth day under mild anaesthesia of chloroform. The blood sample was collected through the retro-orbital plexus route for biochemical analysis such as ALT, AST, ALP, Total bilirubin, Direct bilirubin, and cholesterol.

 

Table 1:  Comparative reduction of Marker Enzymes of Six Plants Significance Extracts:

ALT: Alanine aminotransferase, AST:  Aspartate aminotransferase, ALP: Alkaline phosphatase, BILT: Serum total bilirubin, BILD: Serum direct bilirubin, CHO: Cholesterol, TP:  Total proteins, NE: Not Estimated.

Each value characterizes by Mean ± SEM, (n=6). p-value is the probability *P<0.05; **P< 0.01; ***P<0.001; compared to Toxicant group. One way ANOVA proceed by Dunnett’s multiple comparison tests. (Graph Pad Prism).

 

Graph 1: Comparative reduction of marker enzymes of different plant extracts.

 

The isolated livers were carefully dissected out and gently washed with normal saline and stored in a 10% formalin solution, and then further processed for histopathology to evaluate the hepatic structure microscopically^18-22.

 

RESULTS AND DISCUSSION:

The hepatoprotective activity of methanolic extract of Terminalia coriacea leaves²³, methanol extract of the leaves of Artocarpus hirsutus²⁴, methanolic extract of fruits of Cucumis melo²⁵, methanolic and aqueous extracts of bark of Buchanania lanzan Spreng²¹, ethanolic extract of leaves of Diospyros melanoxylon²⁷, and aqueous extract of S. americanum leaves²⁸, was performed based on the phytochemical investigation. The resultant data was subjected to bio-statistical analysis (Table 1). The graph pad prism software is used for all statistical analyses (figure 1). This study reveals the effect of the plant extracts with respect to their dose, toxicant, and model.

 

In statistical significance testing, the p-value is the probability of obtaining a test statistic at least as extreme as the one that was actually observed, assuming that the null hypothesis is true. Hence, the sample data agree with some hypotheses. The result calculated that p>0.1 it means that there is less than a 0.1 probability (i.e., a 10% chance) that the sample data only agree with our hypothesis due to random chance. There’s at least a 90% chance that the sample data agree with our hypothesis because the “whole” data support the hypothesis. If there is p>0.5 (i.e., 5% chance of false positive), P<0.01 (i.e., 1% chance of false positive), or even stricter. P<0.5 is a very weak standard for an experiment, almost worthless. P<0.1 is stronger and p<0.01 is stronger, but is still probably the bare minimum for trying to show anything. At P<0.1, there's a 1-in-10 chance of a false-positive. Most serious researchers will try to show that the sample data support their hypothesis with p<0.05 (i.e. 5% chance of false positive), P<0.01 (i.e. 1% chance of false positive)^29-31.

 

In the present study for Terminalia coriacea and Artocarpus hirsutus at 250 and 500mg/kg respectively revealed that the *P<0.05; **P< 0.01; ***P<0.001; when compared to the CCl₄ group. Which indicates the 5%, 1%, and less than 1% chance of false positive. The probability is supporting the activity significantly. The study for Cucumis melo revealed P<0.05 and P<0.01 as compared with liver-damaged control to the drug-treated animal at doses 100, 250, and 500mg/kg. The study for Buchanania lanzan revealed significant at P<0.05*, 0.01** and 0.001*** at 100 mg/kg, 200mg/kg, and 400 mg/kg for both aqueous and alcoholic extract both. The study for Diospyros melanoxylon (Roxb) and Solanum americanum Mill at doses 100, 200, and 400 mg/kg and 200, 400, and 600 mg/kg respectively. This study reveals the level of significance at P<0.05 which is a weak standard for an experiment^{32, 33}.

 

CONCLUSION:

The six important medicinal plants from Indian sources have been screened for their property on the protection of hepatocytes. There are various parameters used to analyze the level of significance. All the six plants supporting the hepatoprotective activity. The plants Terminalia coriacea and Artocarpus hirsutus leaves extracts revealed excellent results compare to the other four plants. Therefore, these extracts have been taken for the GC-MS analysis to find out the possible and responsible moiety present in the plants. The results of the phytochemical analysis suggested that these plants are composed of certain constituents which prove hepatoprotection. Further phytochemical analysis and characterization can be continued to know the exact structure of the moiety.

REFERENCES:

1.     Sahu SK. Das D. Tripathy NK. Hepatoprotective activity of aerial part of Glinus oppositifolius L. against Paracetamol-induced Hepatic Injury in Rats. Asian Journal of Pharmacy and Technology 2012; 2(4):154-6.

2.     Dubey SK. Batra A. Hepatoprotective Activity from Ethanol Fraction of Thuja occidentalis Linn. Asian Journal of Research in Chemistry 2008; 1(1):32-35.

3.     Selvanayaki R. Ananthi T. Hepatoprotective Activity of Aqueous Extract of Lawsonia inermis against Paracetamol Induced Rats. Asian Journal of Pharmaceutical Research 2012; 2(2):75-7.

4.     Kumar SK. Rao AS. Evaluation of hepatoprotective and invitro cytotoxic activity of leaves of Chrozophora plicata Linn. Res. J. Pharmacology and Pharmacodynamics. Research Journal of Pharmacognosy and Phytochemistry 2017; 9(1): 19-26.

5.     Shukla A. Bigoniya P. Hepatoprotective Effect of Lepidium Sativum Linn (Cruciferae) Total Alkaloid Fraction against CCl₄ Induced Hepatotoxicity on Rats. Research Journal of Pharmacognosy and Phytochemistry 2013; 5(2): 94-99.

6.     Mishra LC. Scientific basis for Ayurvedic therapies, chapter 14: Hepatic Disorder, CRC Press, Special Indian Edition. 2004, 231.  

7.     Shanmugasundaram P. Venkataraman S. Hepatoprotective and antioxidant effect of Hygrophila auriculata (K.Schum) Heine Acanthaceae root extract. Journal of Ethnopharmacology 2006; 104:124-128.

8.     Ward FM. Daly MJ. Hepatic Disease. In: Clinical Pharmacy and Therapeutics, Walker R. and C. Edwards Eds. Churchill Livingstone, New York; 1999 195-212.

9.     Khare C. Terminalia coriacea Wight & Arn.. In: Khare C. (eds) Indian Medicinal Plants. Springer, New York, NY 2007. https://doi.org/10.1007/978-0-387-70638-2_1618

10.   Khare C. Artocarpus lacucha Buch.-Ham.. In: Khare C. (eds) Indian Medicinal Plants. Springer, New York, NY 2007. https://doi.org/10.1007/978-0-387-70638-2_158

11.   Khare C. Cucumis melo Linn. var. utilissimus Duth. & Fuller.. In: Khare C. (eds) Indian Medicinal Plants. Springer, New York, NY 2007. https://doi.org/10.1007/978-0-387-70638-2_422

12.   Nigel Groom, The Perfume Handbook, Second Edition, Springer International Edition. 2^nd Edition, Springer India, 2011, 45.

13.   Khare C. Diospyros melanoxylon Roxb.. In: Khare C. (eds) Indian Medicinal Plants. Springer, New York, NY 2007. https://doi.org/10.1007/978-0-387-70638-2_515

14.   Khare C. Buchanania lanzan Spreng. In: Khare C. (eds) Indian Medicinal Plants. Springer, New York, NY 2007. https://doi.org/10.1007/978-0-387-70638-2_250

15.   Khare C. Solanum tuberosum Linn. In: Khare C. (eds) Indian Medicinal Plants. Springer, New York, NY 2007. https://doi.org/10.1007/978-0-387-70638-2_1524

16.   Houghton PJ. Raman A. Laboratory Handbook for the Fractionation of Natural Extracts. Basic techniques in handling extracts, Springer Internation Edition. First edition, 1998, 139.

17.   List PH. Schmidt PC, Phytopharmaceutical Technology, Heyden & Son Published, Wiley-Blackwell 1991, 352.

18.   Sathish R. Sravan PK. Natarajan K. Sridhar N. Hepatoprotective and Anti Pyretic Activities of Methanolic Extract of Butea Monosperma Lam Stem Bark In Wister Rats. Asian Journal of Pharmaceutical Research 2011; 1(4): 130-133.

19.   Selvanayaki R. Ananthi T. Hepatoprotective Activity of Aqueous Extract of Lawsonia inermis against Paracetamol Induced Rats. Asian Journal of Pharmaceutical Research 2012; 2(2): 75-77.

20.   Malathi R. Ahamed S.Cholarajan JA. Tylophora asthmatica L. Prevents Lipid Peroxidation in Acetaminophen Induced Hepato Toxicity in Rats. Asian Journal of Research in Pharmaceutical Sciences 2011; 1(3): 71-73.

21.   Doorika P. Ananthi T. Antioxidant and Hepatoprotective properties of Terminalia arjuna Bark on Isoniazid Induced Toxicity in Albino rats. Asian Journal of Pharmacy and Technology 2012; 2(1): 15-18.

22.   Dubey SK. Batra A. Hepatoprotective Activity from Ethanol Fraction of Thuja occidentalis Linn. Asian Journal of Research in Chemistry 2008; 1(1): 32-35.

23.   Patel J. Reddy AV. Kumar GS. Satyasai D. Bajari, B. Nagarjuna V. Hepatoprotective activity of methanolic extract of Terminalia coriacea leaves. Research Journal. Pharmacy. and Technology.  2017; 10(5): 1313-1316.

24.   Patel J. Reddy AV. Kumar GS. Preliminary Phytochemical Screening and Hepatoprotective activity of methanolic extract of Artocarpus hirsutus leaves. International Journal of Phytomedicines. 8 (2016) 379-383.

25.   Patel J. Reddy AV. Kumar GS. Phytochemical Evaluation and Hepatoprotective Activity of Methanolic Extract of Fruits of Cucumis Melo Linn against Isoniazid and Rifampicin Toxicity in Rats; International Journal of Applied Biology and Pharmaceutical Technology; 2016; 7 (2) :182-189.

26.   Patel J. Reddy AV. Kumar GS.; Phytochemical Evaluation and Hepatoprotective Activity of Methanolic and Aqueous Extracts of Bark of Buchanania lanzan spreng. against Paracetamol Induces Hepatotoxicity in Rats. World Journal of Pharmacy and Pharmaceutical Sciences2015; 5 (1):1055-1066.

27.   Patel J. Reddy AV. Kumar GS. Satyasai D. et al Evaluation of hepatoprotective activity of ethanolic extract of Diospyros melanoxylon (Roxb) leaves against CCl₄ induced hepatotoxicity in albino rats; Research Journal Pharmacy and Technology 2015; 8(5): ; 571-574.

28.   Patel J. Reddy AV. Kumar GS.  Evaluation of Hepatoprotective activity of Aqueous extract of Solanum americanum against Ally Alcohol induced Hepatotoxicity in Albino rats. Advance Research in Pharmaceuticals and Biologicals; 2014; 4(4): 798-801.

29.   Arumugam N. Basic Concepts of Biostatistics. Saras Publication. 2015; 20-25.

30.   Patel VK. Rajak H. Significance of amino group substitution at Combretastatin A-4 and phenstatin analogs. Letters in Drug Design & Discovery 2016; 13:943-951.

31.   Patel VK. Singh A. Jain DK. Patel P. Veerasamy R. Sharma PC. Rajak H. Combretastatin A-4 based thiophene derivatives as antitumor agent: Development of structure activity correlation model using 3D-QSAR, pharmacophore and docking studies, Future Journal of Pharmaceutical sciences 2017; 3:71-78.

32.   Parikh MN, Gogtay N. Abc of Research methodology and Applied Biostatistics. A primer for clinicians and researchers. Jaypee Brothers Medical Publishers Pvt Ltd 2009; 36-39.

33.   Mahajan BK. Methods in Biostatistics for medical students and research workers. 7th edition, Jaypee publications 2012; 95-112. 

 

 

 

 

Accepted on 14.05.2022        © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(2):659-662.