INTRODUCTION:

Liver is an important organ responsible for regulating the physiological and biological functions of human body and performs different roles in regulating metabolic, secretory and elimination process. It plays as a key organ for metabolism and detoxification of xenobiotic victim for several life-threatening injuries¹.

Bioactive secondary metabolites of plants mainly flavonoids and phenolic compounds proved their antioxidant activity and tremendous potential for correction of many forms of hepatic disorders².

In experimental animals CCl₄ is mainly used as a hepatotoxic chemical to induce hepatocellular injury which also helps to screen the activity of serum marker enzymes, AST, ALT and ALP³. The standard drug used in this study for hepatoprotective action is ‘Silymarin’⁴. Silymarin having antioxidant activity and helps to prevent lipid peroxidation in humans and animal models⁵.

This study was undertaken to study the hepato corrective activity of hydro ethanolic extract of Cocos nucifera L. and Borassus flabellifer sprout in carbon tetrachloride (CCl₄) induced hepatotoxic rats. Various studies were proved the antioxidant, antidiabetic and cardioprotective activity of Cocos nucifera L. sprout⁶. 'Borassus' is a Greek word means the leathery covering of fruit and "flabellifer", which means fan-bearer. Borassus flabellifer sprout having high antioxidant activity which can scavenge harmful active oxygen species such as O₂^.-, H₂O₂, OH^-etc⁷. Therefore, the aim of this study was included assaying the hepatocorrective effects of Cocos nucifera L. sprout and Borassus flabellifer sprout on antioxidant enzymes, liver marker enzymes, lipid peroxidation and biochemical factors in CCl₄induced hepatotoxic rats.

MATERIALS AND METHODS:

Phase I; Characterization of Sprouts:

Collection of sprout material:

The Sprouts were collected from the local areas of Kerala and authenticated at the Botanical survey of India, Tamil Nadu Agricultural University and Coimbatore.

1. Cocos nucifera L. sprout- (No: BSI/SRC/5/23/2019/Tech.3537)

2. Borassus flabellifer sprout- (No: BSI/SRC/5/23/2019/Tech.3538)

Preparation of extracts from sprout:

10g of the coarse powder each was dissolved in 100ml of water, ethanol, 50% ethanol and benzene for about 3 days. Filtered and the filtrates were used to various phytochemical analysis. Then, 450g of the coarse powder of each sprout were cold macerated in 1.5L 50% ethanol for 3 days (72 hrs) with occasional stirring. The suspension was filtered through a fine muslin cloth. The filtrates were evaporated to separate the distilled – alcohol fraction at a RT and the water portion was evaporated to dry at a low temperature (<40⁰C) under low pressure in a rotary evaporator. Finally, white coloured crystals of approximately 35g (7.4%) were obtained. They are stored separately in refrigerator and whenever is needed, the residual extracts were dissolved in distilled water and used for further study.

Phytochemical analysis:

Screened the alkaloids, flavonoids, phenols, steroids, carbohydrates, amino acids and proteins of Cocos nucifera L. sprout and Borassus flabellifer sprout qualitatively using various solvent extracts.

GCMS analysis:

GC-MS analysis of Cocos nucifera L sprout and Borassus flabellifer sprout was carried out using Gas Chromatograph, Shimadzu QP2010 system and interfaced to a Mass Spectrometer (GC-MS) equipped with Elite-1 fused silica capillary column. Comparison was done with the spectrum of unknown component with the spectrum of known components, stored in Wiley8 library⁸.

Phase II; Hepatocorrective Study:

Maintenance of animals:

Adult Wistar albino rats (male) were obtained from PSG Institute of Medical Sciences and Research, Coimbatore. Rats weighing between 200-300g were used for the present study. Ethical clearance for handling of the experimental animals was obtained (CPCSEA) Reg: number: (No. 425/IAEC/2019). The rats were housed in well ventilated room in the polypropylene cages (6 rats per each) and maintained under standard laboratory conditions (temperature 25±2⁰C) with dark and light cycle (14/10 hrs). They were allowed free access to standard dry pellet feed and water ad libitum. The rats were acclimatized to the laboratory conditions for 10 days before commencement of the experiment.

Acute oral toxicity study:

An acute toxicity study was performed as per Organisation for Economic Co-operation and Development guidelines (OECD, Test no. 423).

Hepatocorrective Activity Evaluation:

To evaluate the hepatocorrective study, hydroethanolic extract of Cocos nucifera L. sprout and Borassus flabellifer sprout were used. The rats were divided in to 7 groups of 6 animals each. Silymarin (standard drug) was the positive control and the whole study was conducted for 21 days.

Experimental design:

Healthy Wistar albino rats were divided in to seven groups consisting of six rats each.

Group I: Normal Control

Group II: Hepatotoxic Control administrated with CCl₄(1 ml/kg body weight CCl₄ twice in a week with

Olive oil 1:1) for 21 days.

Group III: CCl₄as in group II + Silymarin drug (100 mg/kg body weight) for 21 Days⁹.

Group IV: CCl₄ as in group II + Cocos nucifera L. sprout extract (100mg/kg bw, po). for 21 days.

Group V: CCl₄ as in group II + Cocos nucifera L. sprout extract (200mg/kg bw, po). for 21 days

Group VI: CCl₄ as in group II + Borasus flabellifer sprout extract (100mg/kg bw, po). for 21days.

Group VII: CCl₄ as in group II + Borassus flabellifer sprout extract (200mg/kg bw, po). for 21days.

Biochemical Estimations:

The animals were fasted 16 hours before the collection of blood. At the end of the experimental period, the rats were anaesthetized by light ether anaesthesia and blood was withdrawn through intra cardiac puncture. Serums separated are used for the estimation of various biochemical parameters. The liver samples are excised, rinsed in ice-cold normal saline and stored in a refrigerator for further study. Parameters such as Total protein¹⁰, Serum cholesterol¹¹, Blood Urea¹²and Serum Creatinine¹³,prothrombin Time¹⁴were estimated. Activities of Aspartate transaminase (AST), Alanine transaminase (ALT)¹⁵, Alkaline phosphatase (ALP)¹⁶ and Gamma glutamyl transferase (GGT)¹⁷were estimated in the liver and blood sample. Lactate dehydrogenase (LDH)¹⁸. Liver glycogen¹⁹. Lipid peroxidation²⁰, Superoxide dismutase (SOD) activity²¹, Catalase²², Glutathione peroxidase (GPx)²³, Reduced Glutathione (GSH)²⁴and ascorbic acid²⁵were determined in liver tissue.

Histopathological Analysis of Liver:

The part of Liver samples was excised immediately after sacrifice and then washed with normal Saline and dried using blotting paper. It was then fixed with 10% formalin. Then the microtome sections (0.5µ) stained with haematoxylin- eosin dye and observed under microscope (40x) to study the histopathological changes.

Statistical Analysis:

Data reported as mean ± standard deviation by using the Statistical Package for the Social Sciences (SPSS, Version 17.0 for windows) Data from the replicates of each Experiment conducted (n varying from 6) were analysed using one-way analysis of variance (ANOVA) and the means of the group were compared by Duncan’s Multiple Range test (DMRT) values were considered statistically significant when p<0.01 and p<0.05.

RESULTS:

Phase I:

Phytochemical Screening:

Phytochemical screening showed that the hydroethanolic extracts contains flavonoids, amino acids, phenols, steroids, carbohydrates, and saponins with therapeutic effect.

GCMS Analysis:

GC-MS analysis of Cocos nucifera L. sprout and Borassus flabellifer sprout revealed the presence of various components such as Squalene, Hexadecanoic acid, Stigmasterol, 9, 12 Octadecanoic acid.

Phase II:

Acute oral toxicity study:

The result of acute toxicity study of hydroethanolic extract of Cocos nucifera L. sprout and Borassus flabellifer sprout on Laboratory rats showed no lethality up to the dose of 2000mg/kg body weight. Hence the doses were fixed as dose 100mg/kg and 200mg/kg body weight and administrated to test the hepato curative effect according to the articles reviewed.

Effect of Cocos nucifera L. sprout (Cn) and Borassus flabellifer sprout (Bf) extract on AST, ALT and ALP in Liver of Experimental Rats:

Biochemical results suggested that the liver marker enzymes such as AST, ALT and ALP were significantly increased (p<0.05) in Group II (CCl₄) rats when compared with normal rats, and the levels of these enzymes were restored to normal level in serum and liver by administration of both Cocos nucifera L. sprout and Borassus flabellifer sprout extract (Fig: 1).

Fig.1: Effect of Cocos nucifera L. (Cn) and Borassus flabellifer Sprout (Bf) Extract on AST, ALT and ALP in Serum

Effect of Cocos nucifera L. sprout (Cn) and Borassus flabellifer sprout (Bf) extract on GGT in Liver of Experimental rats:

Evaluation of GGT activity in the liver of experimental animal is an important traditional marker for determining liver dysfunction. In this present study, the levels of GGT significantly increased in hepatotoxic rats (Group II) when compared with normal rats. The GGT levels of Cocos nucifera L. sprout and Borassus flabellifer sprout extract administrated rats showed significant (P<0.05) reduction when compared with hepatotoxic rats.

Effect of Cocos nucifera L. sprout (Cn) and Borassus flabellifer sprout (Bf) Extract on LDH in Serum of Experimental Rats:

The present findings reported that LDH activity was significantly increased (P<0.05) in CCl₄ administrated rats (GROUP II) when compared to normal rats (Group I) and the level of LDH returns to normal in Silymarin treated rats. In the Cocos nucifera L. sprout extract administrated Group IV and Group V the activities of LDH were showed the significant (P<0.05) reduction. The LDH activity of Borassus flabellifer sprout treated rats (Group VI and Group VII) was also significantly decreased. Results suggested that up on treatment with higher dose of Cocos nucifera L. sprout the levels of LDH was restored more effectively than Borassus flabellifer sprout treated rats.

Effect of Cocos nucifera L. sprout (Cn) and Borassus flabellifer sprout (Bf) Extract on LPO in Liver of Experimental Rats:

The present findings reported that Oral administration of Cocos nucifera L. sprout and Borassus flabellifer sprout show significant decrease (p<0.05) in LPO level similar to standard drug treated rats and normal rats. In hepatotoxic group lipid peroxidation increases because of excessive formation of free radicals and its level also increases in inflammatory condition. These results contributed to the antioxidant activity of both sprout extracts.

Levels of Total protein, Total cholesterol, Liver glycogen, Prothrombin time, Urea and Creatinine in Normal and Experimental rats:

Table 1: Levels of Total Protein. Prothrombin Time, Total cholesterol, Urea, Creatinine, Liver Glycogen In Normal and Experimental Rats,

Groups	Total Protein		Prothro Mbin Time	Total Cholesterol	Urea	Creatinine	Liver Glycogen
Groups	Serum (g/dl)	Liver (mg/g tissue)	PLASMA Sec.	SERUM (mg/dl)	SERUM (mg/dl)	SERUM (mg/dl)	LIVER (mg/g tissue)
Group 1	6.178 ± 0.542^a	198.203 ± 8.37^b	32 ± 3^e	81.330 ± 2.900^f	41.027 ± 1.91^e	0.902 ± 0.078^e	29.198 ± 8.565^b
Group 2	3.67 ± 0.44^f	157.25 ±3.68^e	56 ± 8^a	169.97 ±5.21^a	62.51± 0.86^a	1.64± 0.14^a	19.34 ± 6.17^c
Group 3	5.02 ± 0.57^cd	188.54 ± 2.98^b	37 ± 4^c	128.18 ± 4.88 ^e	49.87 ± 1.62^d	0.92± 0.11^e	28.93 ± 5.86^b
Group 4	5.65 ± 0.70^ab	168.71 ± 2.46^d	44 ± 6^b	147.89 ± 2.32^c	50.90± 1.06^cd	1.32±0.05^b	43.99 ± 1.91^a
Group 5	5.45 ± 0.30^bc	185.97 ± 2.65^b	38 ± 3^e	142.22 ± 4.49^d	49.40 ± 2.31^d	0.94± 0.03^e	29..01 ± 7.39^b
Group 6	4.39 ± 0.26^e	165.91 ± 2.09^d	46 ± 5^b	150.72 ± 3.50^c	53.25± 1.40^b	1.21 ± 0.09^c	23.92± 4.72^bc
Group 7	4.73 ± 0.36d^e	174.83 ± 3.40^c	42 ± 4^e	164.95 ± 3.34^b	52.00± 1.71^bc	1.06± 0.03^d	18.46 ± 3.04^c
SEd CD (P<0.05)	0.2750 0.5582	2.4051 4.8828	3.0 8.0	2.2889 4.6468	0.9350 1.8981	0.0489 0.0992	3.3497 6.8005

Values are mean ± SD of six samples in each group

^a-g Mean values with in a column no common superscript differ significantly at 5% by DMRT.

Levels of GSH, GPX and Vitamin C in Normal and Experimental Rats:

Table 2: Levels of GSH, GPX AND Vitamin C in Normal and Experimental Rats.

Groups	GSH	GPX	Vitamin C
	Liver (mg/gm protein)	Liver (units/mg protein)	LIVER (µg/gm protein)
Group 1	3.23 ± 0.29^e	5.80 ± 0.63^a	3.78 ± 0.25^a
Group 2	1.26 ± 0.13^a	3.02 ±0.42^d	1.22 ± 0.11^f
Group 3	2.85 ± 0.46^d	5.04 ± 0.47^bc	2.98 ± 0.16^b
Group 4	2.36 ± 0.24^b	4.71 ± 0.32^cd	2.13 ± 0.28^e
Group 5	2.11 ± 0.33^c	5.65 ± 0.39^b	2.42 ± 0.45^d
Group 6	1.60 ± 0.12^c	5.50 ± 0.57^cd	1.73 ± 0.31^cd
Group 7	2.47 ± 0.34^bc	4.37 ± 0.40^bc	2.74 ± 0.41^c
SEd CD (P<0.05)	0.1705 0.3461	0.2719 0.5520	0.1749 0.3551

Values are mean ± SD of six samples in each group

^a-g Mean values with in a column no common superscript differ significantly at 5% by DMRT

Effect of Cocos nucifera L. Sprout (Cn) and Borassus flabellifer Sprout (Bf) Extract on SOD and Catalase in Liver of Experimental Rats:

Higher dose of Cocos nucifera L. sprout administrated groups (Group V) show significant (p<0.05) increase in SOD and Catalase activity similar to normal level. Treatment with Borassus flabellifer sprout extract also show significant increase in SOD and Catalase activity when compared with normal rats. So, from this present findings, understand that Cocos nucifera L. and Borassus flabellifer sprout extract were help to prevent the accumulation of free radicals, maintain the antioxidant property and structural integrity of hepatic cell.

Histopathological Examination of Liver Section:

Liver tissue morphology of normal rats, hepatotoxic rats (Group II), and hepatotoxic rats treated with Cocos nucifera L. sprout and Borassus flabellifer sprout were shown in Plate. 1 (a – g).

Normal hepatic cells were found in Group 1 rats (Plate (a)). Plate (b), hepatotoxic rats (Group II) show fatty deposition and microvascular change. Plate (c), Histological study of Group III, drug control group shows moderate fatty accumulation and normal hepatocytes. Plate (d), Group IV, treated with Cocos nucifera L. (100mg/kg b.wt) sprout shows mild chronic granulomatous inflammation of hepatocytes with normal hepatocytes. Plate (e), of Group V treated with Cocos nucifera L. sprout (200mg/kg b.wt) shows less fatty change and minimal destruction of hepatocytes. Plate (f) Rats treated with Borassus flabellifer sprout, Group VI (100mg/kg b.wt) show minimal fatty changes. Plate (g), Group VII shows minimal lymphocyte infiltrate and mild hepatic change.

DISCUSSION:

For the prevention and management of hepatic illness, herbal medicines were have been used all over the world. Now a day’s clinical investigations proved the effectiveness of many plants in developing hepatic disorder therapies²⁶. The major phytoconstituents present are Carbohydrates, Proteins, Amino acids, Alkaloids, Flavonoids, Phenols, Steroids and Saponins. Phytochemicals such as flavonoids, carotenoids, alkaloids are having free radical scavenging activity²⁷. Flavonoids are involved in preventing the injury caused by free radicals and also having antioxidant and health promoting activities. Phenolic content is exhibits antioxidant activity and linked to the hepatoprotective effects²⁸. GC-MS analysis reveals the presence of bioactive compounds such as 1,1,3-D3-Trans,1, 2, Dihydroxy cyclopentane, Squalene, Hexadecenoic acid in both sprouts. Acute toxicity study suggested that Cocos nucifera L. sprout (Cn) and Borassus flabellifer sprout (Bf) was safe to a dosage of 2000mg/kg and it did not cause any mortality and any other changes to animals’ behavior. The activities of liver marker enzymes (AST, ALT, ALP) increased by oral administration of CCl₄and the leakage of enzymes in to blood stream confirms the extent of liver damage²⁹.

The levels of LDH and GGT in Cocos nucifera L. sprout and Borassus flabellifer sprout treated rats were significantly reduced as in standard drug treated group which was similar to normal group but increased in hepatotoxic group. Results reveals that higher dose of in Cocos nucifera L. sprout was much more effective than Borassus flabellifer sprout for treating liver damage. Evaluation of GGT in the liver of experimental animals is an important traditional marker for determining liver function³⁰. The increased activity of LDH in CCl₄ hepatotoxic liver is due to the damaged structural integrity of liver releases cytoplasamic LDH into circulation³¹.

The Cocos nucifera L. sprout and Borassus flabellifer sprout extracts treated groups and standard drug treated group showed significant decrease in the levels of urea, creatinine, total cholesterol and liver glycogen which was found to be increased in hepatotoxic rats. The liver injury cause reduction in serum total protein is due to the impairment of metabolic biotransformation of amino acid³². The urea level is elevated in hepatotoxic rats due to increased amino acid catabolism, impaired kidney function and liver damage³³.

The lipid peroxidative effects were decreased in Cocos nucifera L. sprout and Borassus flabellifer sprout treated rats as similar to standard drug treated rats and normal rats. These results contribute to the antioxidant activity of both sprout extracts. Administration of CCl₄ increases lipid peroxidation in hepatotoxic experimental animals which is responsible for the leakage of cytosolic enzymes³⁴. Levels of enzymatic antioxidants (SOD, Catalase, GP_X)and nonenzymatic antioxidants (Vitamin C and Reduced glutathione) were decreased in Group II (Untreated group) and increased in both sprout extracts treated group. These increased activities were found to be nearer as normal and standard drug treated rats. SOD, CAT and GSH in the cells are protect liver from reactive oxygen species including free radicals³⁵. The higher dose of Cocos nucifera L. sprout is more effective to restore all the levels of enzymatic antioxidants.

Histopathological reports showed improvement in the liver cell morphology by the oral administration of hydroethanolic extract of Cocos nucifera L. sprout and Borassus flabellifer sprout. The CCl4 administrated animals liver section produced extensive vascular degenerative changes and centrilobular necrosis in hepatocytes³⁶. In this present study it is confirmed that the sprout extracts possess significant hepatocorrective effect by decreasing the serum enzymes activity induced by CCl₄ which shows improvement in the functional status of liver. The recovery of liver architecture and serum enzyme levels caused by both sprouts were similar to that caused by silymarin³⁷. However, further study and biochemical investigations were needed to isolate and identify the active hepatoprotective constituents present in both sprouts.

ACKNOWLEDGEMENT:

Authors are thankful to PSG College of Arts and Science for providing the laboratory for carrying out Research works. And also thankful to PSG IMSR for maintaining the experimental animals.

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Received on 07.01.2021 Modified on 14.10.2021

Research J. Pharm. and Tech 2023; 16(2):483-488.

DOI: 10.52711/0974-360X.2023.00082