Hepatotoxicity means damage to the liver caused by drugs and other factors resulting in problems in its functioning. Chemicals or drugs that because hepatotoxicity are called hepatotoxins. The liver is the largest solid organ in the upper abdomen that aids in digestion and removes waste products and worn out cells from the blood. Itis considered to be one of the most vital organs that functions as centre of metabolism of nutrients such as carbohydrates, proteins and lipids and excretion of waste metabolites. Additionally, it is also handling the metabolism and excretion of drugs and other xenobiotics from the body thereby providing protection against foreign substances by detoxifying and eliminating those (Figure 1) [1-6].

The seeds of Celastrus Paniculatus are acrid, bitter, thermogenic, emollient, stimulant, intellect promoting, digestive, laxative, emetic, expectorant, appetizer, aphrodisiac, cardiotonic, antiinflammatory, diuretic, emmenagogue, diaphoretic, febrifuge and tonic, abdominal disorders, leprosy, pruritus, skin diseases, paralysis, cephalalgia, arthralgia, asthma, leucoderma, cardiac debility, inflammation, nephropathy, amenorrhoea. The seed oil is useful in abdominal disorders, beri-beri and sores (Table 1-4) [7,8].

Figure 1: Picture of Celastrus Paniculatus (Malkangani) Seeds

Table 1: Botanical Description of Celastrus Paniculatus

Table 2: Taxonomical Classification of Celastrus Paniculatus

Table 3: Vernacular Names of Celastrus Paniculatus

Table 4: Chemical Constituents of Celastrus Paniculatus

Medicinal Properties and Traditional Uses

• Tuberculosis

• Kidney disorders

• Blood tonic

• Improvement in retention ability

• Anti-spermatogenic action

• Anti-inflammatory activity

• Anti-tumour activity

• Analgesic activity

• Diuretic

• Anti-dysentric activity

• Anti-malarial activity

Pharmacological Activity

Central Nervous System: The crude CP seed oil administered orally, intramuscularly (i.m.) and intraperitoneal (i.p.) in a dose of 1 g/kg produced sedation in rats. The oil administered orally (100 mg/kg) as an emulsion showed no sedative effect in rabbits. The same emulsion (1 g/kg i.p.) to mice produced mild sedation. Administration of oil (1 g/kg i.m.) to mice showed a significant reduction of movement. CP seed oil given as emulsion showed tranquilizing effect on adrenaline and amphetamine-induced excitement in mice [9,10].

Cardiovascular Activity

The crude CP seed oil administered as emulsion (50-100 mg/kg) produced a gradual fall in cardiac output, bradycardia and marked increase in pulse pressure on isolated heart lung preparation in cat. A similar action with 1 g of emulsified oil was also observed in dogs. The aqueous extract of CP seed showed 50% Angiotensin Converting Enzyme (ACE) inhibition; ethanol extract showed mild activity while the acetone extract was devoid of it [11,12].

Antifertility Activity

The seed oil when given in a dose of 0.2 mL/animal/48 h to adult albino rats for 30 days showed antispermatogenic effects as evidenced by vacuolization of seminiferous tubules, germ cell depletion and exfoliation culminating into an arrest in spermatogenesis. The shrunken tubules revealed only sertoli cells and spermatogonia in the final stage of impairment of spermatogenesis. The livers revealed focal necrosis in animals receiving 0.2 mL (i.p.) seed oil for 30 days, but 45 days’ post treatment these lesions were absent. These results indicate that CP oil may have useful antifertility effects and that the degenerative changes seen in the liver are reversible with time [13].

Selection of Plant and Authentication

Seeds of Celastrus paniculatus collected from the cultivated farm and the open field of Kolhapur, Maharastra and authenticated by botanist from the Department of Botany, Safia Science College, Bhopal. Four hundred gram of the powder prepared from shade dried seeds was subjected to Soxhlet extraction for 16 h using Petroleum ether (40 - 60ºC) as nonpolar solvent at first. Exhausted plant material (marc) was dried and then extracted with ethanol. Colourless solvent was collected from siphon tube and evaporated for residue. Absence of residue confirmed the completion of extraction. Obtained extracts were evaporated using rotary vacuum evaporator (Bucchi type) at 40°C. The percentage yield of the extract was 2.41%, based on the starting quantity. The doses of 200 mg/kg body weight (b.w.) were prepared by suspending the dried extract in water and administering it to the rats by the per os (p.o.) route.

Extraction of Plant

In the present study, extraction was performed using continuous hot percolation ‘Soxhlation’. Dried seeds of Celastrus paniculatus willd. were placed in thimble of Soxhlet apparatus. Soxhlation was performed at 60°C using Petroleum ether (40 - 60ºC) as non-polar solvent at first. Exhausted plant material (marc) was dried and then extracted with ethanol. For confirmation of exhausted plant marc (i.e. completion of extraction), colourless solvent was collected from siphon tube and evaporated for residue. Absence of residue confirmed the completion of extraction. Obtained extracts were evaporated using rotary vacuum evaporator (Bucchi type) at 40°C.

Dried extract was weighed and percentage yield for each extract was determined using the following formula:

Preparation of Ethanolic Extracts

About 400 grams of the powder prepared from shade dried seeds of Celastrus Paniculatus was subjected to Soxhlet extraction for 16 h using Petroleum ether (40 - 60ºC) as non-polar solvent at first. Exhausted plant material (marc) was dried and then extracted with ethanol. Colorless solvent was collected from siphon tube and evaporated for residue. Absence of residue confirmed the completion of extraction. Obtained extracts were evaporated using rotary vacuum evaporator (Bucchi type) at 40°C.

Phytochemical Screening

Qualitative photochemical investigation. The phytochemical investigation was carried out by procedure given in Kokate et al.

Tests for Carbohydrates

• Molish Test: About 2 mL of aqueous extract was treated with 2 drops of alcoholic α-naphthol solution in a test tube and then 1 mL of concentrated sulphuric acid was added carefully along the sides of the test tube. Formation of violet ring at the junction indicates the presence of carbohydrates

• Benedict’s Test: Equal volume of Benedict’s reagent and extract were mixed in a test tube and heated in the water bath for 5-10 minutes. Solution appears green, yellow or red depending on the amount of reducing sugar present in the test solution which indicated the presence of reducing sugar

Tests for Protein and Amino acids

Biuret’s Test: The extract was treated with 1 mL of 10% sodium hydroxide solution in a test tube and heated. A drop of 0.7% copper sulphate solution was added to the above mixture. The formation of violet or pink colour indicates the presence of proteins

Tests for Glycosides

Borntrager’s Test: To 3 mL of test solution, dilute sulphuric acid was added, boiled for 5 minutes and filtered. To the cold filtrate, equal volume of benzene or chloroform was added and shake it welled. The organic solvent layer was separated and ammonia was added to it. Formation of pink to red colour in ammonical layer indicates presence of anthraquinone glycosides.

Tests for Alkaloids

To the extract, dilute hydrochloric acid was added, shake it well and filtered. With the filtrate, the following tests were performed.

• Mayer’s Test: To 2-3 mL of filtrate, few drops of Mayer’s reagent were added along sides of tube. Formation of white or creamy precipitate indicates the presence of alkaloids

• Hager’s Test: To 1-2 mL of filtrate, few drops of Hager’s reagent were added in a test tube. Formation of yellow colour precipitate indicates the presence of alkaloids

• Wagner’s Test: To 1-2 mL of filtrate, few drops of Wagner’s reagent were added in a test tube. Formation of reddish brown precipitate indicates the presence of alkaloids. Tests for Saponins: Froth Test: The extract was diluted with distilled water and shaken in graduated cylinder for 15 minutes. The formation of layer of foam indicates the presence of saponins

Tests for Flavonoids

• Lead Acetate Test: The extract was treated with few drops of lead acetate solution. Formation of yellow precipitate may indicate the presence of flavonoids

• Alkaline Reagent Test: The extract was treated with few drops of sodium hydroxide separately in a test tube. Formation of intense yellow colour, which becomes colour less on addition of few drops of dilute acid, indicate presence of flavonoids

Tests for Triterpenoids and Steroids

Salkowski’s Test: The extract was treated with chloroform and filtered. The filtrate was added with few drops of concentrated sulphuric acid, shaken and allowed to stand. If the lower layers turn red, sterol are present. Presence of golden yellow layer at bottom indicates the presence of triterpenes.

Tests for Tannin and Phenolic Compounds

• Ferric Chloride Test: Some amount of extract was dissolved in distilled water. To this solution 2 mL of 5% ferric chloride solution was added. Formation of blue, green or violet colour indicates presence of phenolic compounds

• Dilute Iodine Solution Test: To 2-3 mL of extract, few drops of dilute iodine solution were added. Formation of transient red colour indicates presence of phenolic compounds

Preparation of Ethanolic Extracts

About 400 grams of the powder prepared from shade dried seeds of Celastrus Paniculatus was subjected to Soxhlet extraction for 16 h using Petroleum ether (40 - 60ºC) as non-polar solvent at first. Exhausted plant material (marc) was dried and then extracted with ethanol. Colourless solvent was collected from siphon tube and evaporated for residue. Absence of residue confirmed the completion of extraction. Obtained extracts were evaporated using rotary vacuum evaporator (Bucchi type) at 40°C.

Pharmacological Screening Model Design

CCl4 induced toxicity model.

CCl4- induced Hepatotoxicity in Rats

CCl4-induced hepatotoxicity was done by intraperitoneally injecting 1 mL/kg b. w. CCl4 dissolved in liquid paraffin in ratio 1:1 after animals are being denied food for 18 hours. The rats were randomLy divided into four groups of six rats each.

• Group I: Normal control and were administered 1 mL/kg normal saline

• Group II: CCl4 intoxicated (0.7mL/kg by Intraperitonial injection)

• Group III: Standard drug treated, Silymarin, 100 mg/kg, orally

• Group IV: CCl4 - induced hepatotoxic rats treated with 100 mg/kg b.w Ethonolic extract of Celastrus Paniculatus

Extracts and Standards Used

• Extracts Used: Ethanolic extract of dried seeds of Celastrus Paniculatus

• Standard Drug: Silymarin 100mg/kg

Treatment of Animals

Healthy male Albino Wistar rat of age 8-10 weeks and weight 150 to 200gm were selected after physical and veterinary examination. All experiments involving animals comply with the ethical standard of animal handling and are approved by Institutional Animal Ethics Committee (IAEC). All the selected animals were kept under acclimatization on the same day. The animals were acclimatized one week prior to dosing. Identification of animals by cage number and marking on animal. The rats were housed in separate standard polypropylene cages with stainless steel top grill. Clean autoclaved paddy husk was used as bedding. The paddy husk was changed at least thrice in a week. The animals were kept in a clean environment with 12-hour light and 12-hour dark cycles. The air was conditioned at 22±2°C and the relative humidity was maintained between 30-70% with 100% exhaust. Standard rat pellet feed was provided ad libitum throughout the study, except overnight fasting prior to blood collection and was offered the feed immediately after completion of blood collection of all the animals. Drinking water was provided ad libitum in polypropylene bottles with a stainless steel sipper tube throughout study period.

Plant Extraction

The plant material was extracted by continuous hot percolation Soxhlation and the percentage yield calculated by the following formula was found to be 2.41 % by ethanol.

Acute Oral Toxicity (OECD)

The acute oral toxicity study was carried out according to OECD 423 guidelines. Four ranges of dose were used for toxicity studies, i.e 5mg/Kg, 50 mg/Kg, 300 mg/Kg, 2000 mg/Kg. animals were observed individually for next 4 hours after dosing for the presence of mortality during this period and 72 hours after sample administration.

Histology

Hepatotoxicity in rat as Shown in Figure 2.

Figure 2: Hepatotoxicity in Rat

According to the World Health Organization (WHO), herbal medicines are being used by about 60% of world population primarily in developing countries for primary health care. The objective of the biological screening of plants is to find the sources of the biologically active chemical compounds which can be developed as a drug or to discover the lead molecules which can be modified through chemical procedures into useful drugs.It has been suggested that aqueous and ethanolic extracts from plants are potential source of antiviral, antitumoral and antimicrobial agents [14].

Historically, plants have provided a good source of anti-infective agents; emetin, quinine and berberin, which remain highly effective in the fight against microbial infections. Phytomedicines derived from plants have shown great promise in the treatment of intractable infectious diseases [15].

CCl4 is commonly used hepatotoxin for the experimental study of liver disorder. CCl4 induced liver cell injury involves CCl4 biotransformation caused by cytochrome P450 leading to the production of trichloromethyl free radical which causes peroxidative degradation in adipose tissue causing hepatocyte fatty infiltration. Lipid peroxidation is caused by trichlo- romethyl free radicals in the presence of oxygen produced by metabolic leakage from mitochondria. All these changes inhibit the damage of hepatic tissue and loss of integrity of cell membrane. CCl4 increases SGPT, SGOT and induced hepatotoxicity.

In the present study, Celastrus paniculatus was selected for hepatoprotective activity owing to its traditional use. The liver function is assessed by determining the activities of SGOT, SGPT, SALP, i.e. enzymes present in the cytoplasm. In the case of hepatotoxicity, the enzymes move into the bloodstream and their amount confirms the liver damage extent.

The plant material was extracted by continuous hot percolation Soxhlation and the percentage yield calculated by the following formula was found to be 2.41 % (by ethanol). On phytochemical screening of ehanolic extract of Celastrus paniculatus showed the presence of carbohydrate, alkaloid, protein, absence of glycoside. In case of acute oral toxicity study when animals were treated with 5, 50, 300 and 2000 mg/kg b.w. of dose for 72 hours. There was no mortality and any behavioural changes so they decided dose will be choosen as, 200 mg/kgb.w. On the basis of LD50.

Phytochemical tests of ethanolic extract of leaves of Celastrus paniculatus indicated the existence of saponins, flavonoids and phenolic compounds. Administration of ethanolic extract showed a substantial decrease in SGPT, SGOT, SALP level (p<0.05) at a dose of 200mg/kg. Similarly, treatment with hepatoprotective drug such as silymarin showed a significant decrease in SGPT, SGOT and SALP levels. Therefore, it was reported that Celastrus paniculatus extract treatments normalize few morphological features of the liver in rats with CCl4 induced hepatitis.

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