Evaluation of Hepatoprotective Activity of Whole Fruit Extracts of Luffa Acutangula. Var. Amara and Rhizome Extracts of Rheum Emodi in CCl4-Induced Chronic Hepatotoxicity

To investigate the hepatoprotective activity of whole fruit extracts of Luffa acutangula.var. amara and rhizome extracts of Rheum emodi in CCl4 treated rats. The dried powders of L. amara and R. emodi were extracted successively with petroleum ether, ethanol and distilled water. The hepatoprotective capacity of the extract of the whole fruits of L. amara and the rhizomes of R. emodi was analyzed in liver injured CCl4treated male rats. The present study explored the possibilities of using low doses of both plant extracts (150mg/kg, and 300mg/kg bw, po route) to treat CCL4 intoxicated albino rats in both acute and chronic models of hepatic damage, evident by increased serum levels of glutamate pyruvate transaminase (SGPT), glutamate oxaloacetate transaminase (SGOT), alkaline phosphatase (ALP), direct bilirubin, total bilirubin, cholesterol and triglycerides, all being implicated in considerable hepatic damage. Histopathological examination in CCl4 treated rats revealed collapse of liver parenchyma with early fibrosis and chronic inflammatory cell infiltration in patchy areas around central vein (Pic.group-2) Original Research Article Abid et al.; JPRI, 34(24B): 26-38, 2022; Article no.JPRI.78899 27 when compared to the control group. Histopathological and physical examinations also indicated their effectiveness with their dose tolerability and liver protection. Ethanolic and aqueous extracts of the whole fruits of L. amara and rhizomes of R. emodi were indicative of more hepatoprotective properties when compared to the petroleum ether extracts of both plants against CCl4 induced liver damage as confirmed from hepatic serum marker enzyme activities and histopathological studies.


INTRODUCTION
The liver is an important unique organ with considerable regenerative capacity, as even a moderate cell injury remains the same but there will be measurable changes in its metabolic functions. Apart from its significant role in the metabolism and disposition of the chemicals to which it is exposed directly or indirectly, it also aids in the metabolism of carbohydrates, proteins, fats, and immunomodulation. Conversely, some abnormalities start appearing in liver functions owing to their sensitivity towards the nature and the degree of initial damage.
Various factors such as nutritional, biochemical, bacteriological, viral, or environmental aberration affect the etiology of liver disorders. The liver function is generally impaired by xenobiotics, excessive exposure to various pharmacological and chemical agents, and viral or protozoal infections. The severity of cellular injury is detrimental in the pathogenesis of acute to chronic hepatitis, which eventually results in cirrhosis.
or malignant lesions if remained untreated. The possibility of altered liver functions is indicative of changes in its chemical composition of liver or its subcellular organelles. A slightly altered hepatic structure and function might cause portal hypertension, ascites, jaundice, increased bleeding; further, it can lead to multiple metabolic changes affecting other organs as well. Medical studies indicate high incidences of hepatic diseases are being adjudged as a serious public health problem [1,2]. Selective hepatotoxins like CCl 4 induce reactive free radical initiating cell damage via two different mechanisms of covalent binding membrane proteins precipitating lipid peroxidation [3]. Numerous investigations have been performed using CCl 4 as hepatotoxicant for inducing hepatic fibrosis and cirrhosis in experimental animals. Though advances in modern medicine has improved to treat many diseases, no effective drug has been established or discovered to improve liver function and aid or induce liver regeneration in hepatic fibrosis. Generally, its vital to prevent liver fibrosis and cirrhosis in hepatology [4]. The magnitude of derangement of liver by disease or hepatotoxins is normally measured by the level of glutamate pyruvate transaminase (ALT), glutamate oxaloacetate transaminase (AST), alkaline phosphatase (ALP), bilirubin, albumin, and whole liver homogenate.
With increased importance towards health and lifestyle changes, herbal medicine as is gaining momentum globally with herbal remedies prescribed along with allopathic drugs ranging from common cold to more organ specific diseases. Both Ayurveda and Unani systems assign much clinical importance to the pharmacological efficacy of medicinal plants. Plants with hepatoprotective action of some plants like Acacia catechu, Zingiber officinale, and Garcinia indica have been established [5]. Conversely, there is a lack of satisfactory plant formulations for treating hepatic diseases. Generally, chemically induced hepatic damage in animal models is used for the study of plants with hepatoprotective actions [6].
Luffa acutangula.var.amara (Cucurbitaceae) family, known as karvi turai, is an annual herb found in all parts of India and more towards the western peninsula and is used as a carminative, laxative, tonic for intestines, digestible, used to cure the vata and kapha, biliousness, liver complaints, leukoderma, piles, ascites, tumour useful in bronchitis and asthma and the seed kernals are used to treat dysentery [7,8]. It is also used in treating jaundice when taken in the form of very fine powder through nose, while its seeds show emetic, expectorant, and demulcent properties.The ancient literature also revealed that the plant is significantly used as abortifacient and antifungal agent. The reported chemical examination of Luffa acutangula showed the presence of carbohydrates, carotene, fat, protein, phytin, aminoacids, alanine, arginine, cystine, glutamicacid, glycine, hydroxyproline, leucine, serine, tryptophan, pipecolic acid, flavonoids and saponins [9].
R. emodi of family Polygonaceae, known as Indian or Himalayan Rhubarb, found in India, Rheum emodi is considered as purgative, stomachic, and astringent tonic, possesses aperient, emmenagogue and diuretic properties. Root is used as expectorant, appetizer, as powder applied on cuts, wound, and muscular swelling, toothache, tonsillitis, mumps and ulcers [10]. It is used to heal skin sores and scabs. While larger doses are used as laxative, small doses are used to treat dysenteric diarrhea [11] Chinese medicine uses rhubarb for ulcer remedy, as a bitter, cold, dry herb used to "clear heat" from the liver, stomach and blood, also to expel helminthes and to treat cancer, fever, upper intestinal bleeding (ulcers), and headache [12,13]. Its also used in of spring tonics or blood cleansing cures [14,15].
The extracts from both plants L. amara (whole fruit including seeds) and rhizomes of R. emodi were selected to evaluate the hepatoprotective potential of these plant extracts against CCl 4 induced liver damage liver in chronic model.

Plant Materials
Both plants, viz, the rhizomes of R. emodi and dried fruits of L. amara were identified and procured from by Dr. K.Madhava.Chetty, Assistant Professor, Dept.of Botany, Sri Venkateshwara University, Tirupati, Andhra Pradesh, India.

Animals
Wistar rats of 160-200 g were supplied from the animal house of Deccan College of Medical Sciences, Hyderabad and housed in cages with free access to standard pellet chow and water ad libitum. Rats, 6 in a group, were housed in clean polypropylene cages under standard conditions of humidity (60%-70%), temperature (25 ± 2°C) and light (12 hr light: 12 hr dark cycle) and free access to food and water ad libitum. Experiments were conducted after obtaining the approval from Institutional Animal Ethics Committee constituted as per CPCSEA guidelines. Protocols for all experiments described below were approved by the ethical board.

Extraction of the Plant Materials
For phytochemical analysis, approximately 100 g of dried whole fruits of L.amara and rhizomes of R. emodi were separately chopped, air dried at 35-40℃ and pulverized in electric grinder. The powders obtained were successively extracted with the following solvents, petroleum ether (PE) (50℃), ethanol, and distilled water respectively. The extracts obtained were powdered by using rotary evaporator under reduced pressure. The dried extracts of the fruits of L. amara and rhizomes of R. emodi were subjected to phytochemical screening.

Preliminary Phytochemical Investigations
The extraction and preliminary phytochemical investigations were carried out on the petroleum ether, alcoholic and aqueous extracts of fruits of Luffa amara for presence of phytoconstituents such as sterols, glycosides, saponins, flavonoids, amino acid proteins, tannins, carbohydrates and triterpenes using standard methods described in practical pharmacognosy by K.R. Khandelwal ,Dr. C.K. Kokate, and Trease G.E, Evans [16,17,18] .

Animals
Wistar rats weighing 160-200g, obtained from the Deccan College of Medical Sciences, Hyderabad, TS, India, were maintained by housing them under controlled temperature, humidity and 24-hr light and dark cycle before acclimatising them for seven days under controlled temperatures (23-25ºC), humidity (60-70%) and dividing them in groups of 6 animals each for carrying out hepatoprotective studies using crude extracts-after approval of all experimental protocols from the ethics committee.

Chronic Toxicity Induced by CCl 4
A 30 days study was carried out using CCL 4 (1 ml/kg b.w, p.o route) toxicant and silymarin (25mg/kg, b.w,p.o route) as reference [19,20] on both LA and RE plant extracts. Rats were divided into 15 groups of 6 each and treated in the following way: Group 1: control, received vehicle daily (10 ml/kg, po) for 30 days. All animals were sacrificed 24 hr after the last treatment. Blood samples were collected from orbital sinuses, and the separated serum from the centrifuged blood samples were used for assay of the marker enzymes. After isolating livers, they were washed in normal saline, blotted with filter paper and weighed [21,22,23]. The liver tissues were prepared for histopathological deductions.

Statistical Analysis
The statistical significance was assessed using one-way analysis of variance (ANOVA) followed by (Armitage, 1987) and students t-test for control and test groups. The two tailed unpaired student t-test using the computer programme Prism Pad. Changes in the Mean ± SEM serum hepatic parameters of the test (plant extracts) were compared with that of the control, the Pvalues expressed as P≤0.05 or less was considered significant.

Preliminary Phytochemical Investigation
The preliminary phytochemical investigation of the fruit extracts of L. amara showed that petroleum ether extract contained triterpeines and fixed oil; ethanolic extract had carbohydrates, glycosides, saponins, flavonoids, sterols, amino acids and proteins; aqueous extract tested positive for carbohydrates, glycosides, saponins, flavonoids, sterols, amino acids and proteins.

Hepatoprotective Activity
The serum test results of all the plant extracts petroleum further confirmed the aqueous and ethanolic extracts significant protective action against CCl 4 by reducing most of the elevated hepatic parameters (excluding serum HDL, albumin, and total proteins which were elevated following treatment with silymarin and the plant extracts) following CCl 4 treatment. Both alcoholic and aqueous extracts of both plants showed significant hepatoprotective activity than petroleum extract.

DISCUSSION
The present study explored the possibilities of using low doses of both plant extracts (150mg/kg, and 300mg/kg bw, po route) to treat CCL 4 intoxicated albino rats in both acute and chronic models of hepatic damage, evident by increased serum levels of glutamate pyruvate transaminase (SGPT), glutamate oxaloacetate transaminase (SGOT), alkaline phosphatase (ALP), direct bilirubin, total bilirubin, cholesterol and triglycerides, all being implicated in considerable hepatic damage.
As established earlier that accumulated CCl 4 in hepatic parenchymal cells get activated by cytochrome P450 dependant monooxygenases form free radicals of (. CCl 3 ), which simultaneously attacks polyunsaturated fatty acids in presence of oxygen producing lipid peroxides and further alkylates cellular proteins, inclusive of P450 and other macromolecules leading to hepatic damage and cirrhosis. Thus, CCl 4 is one of the most commonly used hepatotoxins in pharmacological experiments [24][25][26][27].
CCl 4 induced hepatotoxicity study, both alcoholic (LAEE150 and LAEE300, REEE150, REEE300 and aqueous extracts (LAEE150, LAEE300, REEE150, REEE300) of both plants Luffa amara (LA) (fruit pulp including seeds) and Rheum emodi (RE) rhizomes respectively, showed significant reduction in the elevated levels of all serum enzymes as afore mentioned (Tables 3  and 4). with subsequent reduction in body and liver weight and as well (Table 5). Though, the pet ether treated groups (LAPE150, LAPE300, REPE150 and REPE300) when compared to both alcoholic and aqueous groups, didn't show much significant hepatoprotection.
Histopathological examination in CCl 4 treated rats revealed collapse of liver parenchyma with early fibrosis and chronic inflammatory cell infiltration in patchy areas around central vein and with REAE300 showed no hepatocellular necrosis with liver architecture appearing towards normal (Pic.group-15). Thus, in low doses of (150 mg/kg, po route), the alcoholic and aqueous extracts of both plants showed less hepatoprotection when compared to high doses (300 mg/kg, po route) which showed significant protection against CCl 4 intoxication of hepatocytes. Body and liver weight changes were observed in the same manner with CCl 4 treated group showing maximum increase in body and weight, compared to the control group. The petroleum ether treated group showing least changes, while the alcoholic and aqueous groups showed significant restoration in body and wet liver weights when compared to CCl 4 treated group at the end of chronic CCl 4 induced hepatotoxicity study.

CONCLUSION
Thus, the curative and prophylactic hepatoprotective efficiencies of both alcoholic and aqueous extracts of both plant extracts of L. amara and R.emodi at 300mg/kg bw not only revealed in restoring normal hepatic functioning, evident by serum analysis, and further supported by histopathological and physical examinations, but also indicated their effectiveness with their dose tolerability and liver protection. The order of hepatoprotection of both LA and RE was observed with alcoholic extracts being significantly more compared to the aqueous extracts. While the petroleum ether extracts showing least or no significant changes. With conclusive findings of both plants providing good hepatoprotection individually, it can provide a window to further study their summative hepatoprotectiveness using either crude extracts or their isolated active constituents for providing more proficient formulations in treating liver disorders like cirrhosis.

CONSENT
It is not applicable.

ETHICAL APPROVAL
The approval from Institutional Animal Ethics Committee constituted as per CPCSEA guidelines. Protocols for all experiments described below were approved by the ethical board.

NOTE
The study highlights the efficacy of " herbal " which is an ancient tradition, used in some parts of India. This ancient concept should be carefully evaluated in the light of modern medical science and can be utilized partially if found suitable.