Toxicity Assessment, Evaluation of Antioxidant and Hepatoprotective Activity on Cordia obliqua Fruit Extracts

Liver is the second largest and vital organ in the body which plays a vital role in metabolism and maintenance of homeostasis in human system. Toxic liver injury produced by drugs and chemicals are similar to natural liver disease. Continuous use of agents like Rifampicin, Isoniazid, Paracetamol, Ethyl alcohol, Carbon tetrachloride, Ranitidine and Oral contraceptives of hormonal origin and chemicals used as food preservatives and agrochemicals are threatening the integrity of liver.1,2 The effects of liver disease on hepatic metabolism of drugs are complex and difficult to predict, particularly when multiple drugs are administered simultaneously.3 Undiscerning use of anti-malarial drugs4, Anti-tubercular drugs5, analgesics, oral contraceptives6, Pharmaceuticals (mainly antibiotics, chemotherapeutics and CNS active drugs).7 Toxins in food (especially aflatoxin8, Viruses, bacteria, mushrooms, parasites like amoebas etc. may also infect the liver.9 According to American Gastroentorogical institutes official journal “Gastroenterology” recent survey has reported that they are 13% deaths has reported due to antibiotics and stated has “Idiosyncratic Drug Induced Liver Injury”.


INTRODUCTION
Liver is the second largest and vital organ in the body which plays a vital role in metabolism and maintenance of homeostasis in human system. Toxic liver injury produced by drugs and chemicals are similar to natural liver disease. Continuous use of agents like Rifampicin, Isoniazid, Paracetamol, Ethyl alcohol, Carbon tetrachloride, Ranitidine and Oral contraceptives of hormonal origin and chemicals used as food preservatives and agrochemicals are threatening the integrity of liver. 1,2 The effects of liver disease on hepatic metabolism of drugs are complex and difficult to predict, particularly when multiple drugs are administered simultaneously. 3 Undiscerning use of anti-malarial drugs 4 , Anti-tubercular drugs 5 , analgesics, oral contraceptives 6 , Pharmaceuticals (mainly antibiotics, chemotherapeutics and CNS active drugs). 7 Toxins in food (especially aflatoxin 8 , Viruses, bacteria, mushrooms, parasites like amoebas etc. may also infect the liver. 9 According to American Gastroentorogical institutes official journal "Gastroenterology" recent survey has reported that they are 13% deaths has reported due to antibiotics and stated has "Idiosyncratic Drug Induced Liver Injury".
Herbal plants which contain wide variety of phytoconstituents which acts as a protective barrier for the liver and includes-monoterpenes, terpenoids, flavonoids, phenolic compounds, carotenoids, alkaloids, glycosides, organic acids, lipids 10 Several Indian medicinal plants have been extensively used in the Indian traditional system of medicine for the management of liver disorders. In India over 40 polyherbal commercial formulations reputed to have hepatoprotective action is being used. Scrutiny of the literature indicates that 160 phyto-constituents from 101 plants belonging to 52 plant families have antihepatotoxic activity. 11 In the present study, the fruits of Cordia obliqua were selected for evaluation of pharmacological activity and to identify the chemical constituents. Cordia obliqua Willd plant is a genus of flowering plants in the borage family, Boraginaceae. It contains about 300 species of shrubs and trees, which are found worldwide, mostly in warmer regions. It is a medium-sized deciduous tree and very vigorous in growth. Many of the species are commonly called manjack, Clammy Cherry, glue berries, sebesten, or snotty gobbles. According to traditional system, it possesses anthelmintic, purgative, diuretic, expectorant, antipyretic, hepatoprotective and analgesic action. The fruits are edible and used as pickle, vegetable, raw, cooked. They lessen thirst and the scalding of the urine, remove pain from the joints and the burning of the throat and are also effective in treating the diseases of the spleen. 12 It was found that fruits are traditionally used for the treatment of hepatic disorders. 13,14 The leaves of Cordia obliqua were investigated for hepatoprotective. 15 Analgesic, antipyretic and antiinflammatory activities. 16 Since the fruits were not investigated and reported for its hepatoprotective activity, this prompted us to select the fruits of Cordia obliqua.

Collection and authentification of plant
Cordia obliqua fruits were collected from Chidambaram in month of September, herbarium was prepared & submitted. Identification (Specimen voucher-HPU:102/2019) has been done by Dr P Shivakumar Singh, Head Department of Botany, Palamuru University, Mahabubnagar, Telangana, India.

Preparation of extracts
The fruits of Cordia were collected; shade dried, segregated, pulverized by a mechanical grinder and passed through a 40 mesh sieve. The powdered plant materials were stored in an air-tight container. Extraction of dried coarse powder of fruits was carried out by Soxhlet apparatus with different solvents such as Petroleum ether, Benzene, Chloroform, Acetone, Ethanol and finally macerated with Chloroform water by increasing order of polarity. The solvents were evaporated under reduced pressure to get semisolid masses and placed in desiccator for further use. The percentage yield of ethanol and water extract was found 12.76 and 34.78 w/w and it was dark brown in color. As per our previous studies the ethanolic and water extracts has exhibited in vitro antioxidant activity due to maximum number of Phyto-constituents as well as maximum total phenol content. So, the ethanol and aqueous extracts were selected for screening of biological activity.

Preliminary phytochemical screening
Several phytochemical tests were performed for testing different chemical groups present in the extracts. The chemical tests for various phytoconstituents present in the extracts were carried out as described. Alkaloids were tested by Mayer's, Wagers, Hager's and Dragendroffs reagent, phenols were identified by lead acetate and ferric chloride solution, saponins by foam test, steroids were identified by Libbermann-Burchard and Salkowski test, cardiac glycosides by keller-kilianis and legal's test, tannins by ferric chloride solution, Sugars were identified by Molischs and Fehling's reagent, glycosides by acid hydrolysis followed by Molischs test. [17][18][19] Experimental animals Wistar rats of either sex with 8-12 weeks age, weighing 120-140g, were procured from the Sai Nath agency, Hyderabad, Telangana, India. The animals were set aside in cages, 2 per cage, with twelve: twelve hours light and dark cycle at 25 ± 2 ºC. The rats were maintained on their particular diets and water ad libitum. All experimental Procedure done with standard procedure prescribed by CPCSEA, India and protocol approved by Institutional Ethical Committee with IAEC/COP/19-20/02).

Chemicals and drugs
The chemicals used in study were Silymarin and Paracetamol which were collected from Micro labs, Bangalore. 2-thiobarbituric acid and Ellman's reagent was purchased from Sigma Aldrich, Bangalore. All other solvents and chemicals of all the drugs and chemicals used were of Pharmaceutical grade.

Determination of acute oral toxicity (LD 50 )
The acute oral toxicity study 20 of fruit extracts of Cordia obliqua was determined in female albino mice (120-140 g) maintained under standard husbandry conditions. The animals were fasted 4 h prior to the experiment and Up and Down procedure (OECD Guidelines No. 425) method was adopted for acute oral toxicity studies. Animals were administered with single doses of each extract and observed for their physical behavior and mortality during 48 h study period (short term toxicity). Based on the short term profile of extracts the doses for the next animals were determined. All the animals were observed for long term toxicity (7 days). The LD 50 studies of the test extracts were conducted with 180 mg/ kg and use dose progression factor 3.2 single animals dose till body weight change or mortality and we met our criteria at dose level of 3000 mg/kg body weight.

Paracetamol induced hepatotoxicity
Wistar rats weighing between (120-140 g) were divided into five groups of six rats in each group. Group A was served as normal control which was given with vehicle only, Group B with Paracetamol (2000 mg/Kg p.o), Group C with Silymarin (25 mg/Kg p.o) that serves as standard, Animals in groups D, E treated with 300 mg/kg of aqueous and ethanolic extracts respectively. B, C and D were intoxicated with Paracetamol (2 gm/Kg p.o) for 3 days and from 4 th -10 th day with different doses of Cordia obliqua in related groups respectively. On the 11 th day, after recording thiopentone sodium sleeping time in all groups of animals and they were anaesthetized with ether. Blood was collected through cardiac puncture, later sacrificed by overdose of ether. Livers removed were washed with saline, weighed and stored in 10 % Formaldehyde for histological studies. 21

Histopathological studies
For light microscopic evaluation, liver tissues were fixed in 10% buffered formalin and then thin sections (4 μm) were subsequently stained with hematoxylin/eosin for general histological features determination. 22 Sections were studied under light microscope Leica DM 500.

In Vivo antioxidant activity on paracetamol induced hepatotoxicity in rats
The antioxidant potential of the plant extracts were tested using three tests viz, Reduced GSH, Catalase and SOD activity. Accurately weighed 1g of the liver tissue was taken and homogenized in 9 ml of 1.15% KCl with a Homogenizer. After successful homogenization, homogenized liver was kept in a freezer until it got frozen and later kept at room temperature to become liquefied. The same procedure was repeated for two freeze-thaw cycles. The homogenated liver was centrifuged at 3000 rpm at 4°C for 30 min. The supernatant of the liver homogenate was separated and used for assays. The activity of GSH and Catalase was determined by the method of Sinha 23 and Superoxide dismutase (SOD) activity by Markuland. 24

Statistical analysis
The obtained values in all five groups were expressed as mean with SD from 6 animals, subjected to statistical analysis using one-way analysis of variance followed by Dunnett's t test to verify significant difference if any among the groups. P < 0.05*, 0.01**, and 0.001*** was considered statistically significant.

Preliminary phytochemical analysis
Preliminary phytochemical investigation of Cordia obliqua fruit extracts was carried out in our previous study to identify different primary and secondary metabolites. Petroleum ether, benzene, chloroform and acetone extracts showed the presence of steroids and cardiac glycosides; alkaloids in ethanolic extract, saponin; phenols and tannins in ethanolic and aqueous extracts, carbohydrates in chloroform, acetone, ethanolic and aqueous extracts, gums and mucilage in aqueous extract.

The acute oral toxicity study of fruit extracts of Cordia obliqua
Acute toxicity studies showed the non-toxic nature of the Cordia obliqua fruit extract upto dose of 3000 mg/kg body weight which is 10 times more than the dose used in the study. The studies followed OECD guidelines. Based on the observation there was no change in physical behavior and mortality of any animals when observed for 48 hr. There was no lethality or any toxic reactions found at any of the doses selected until the end of the study period.

Effect of aqueous and Ethanolic fruit extracts of Cordia obliqua on serum biochemical parameters
ALT is an enzyme found in the liver that helps convert proteins into energy for the liver cells. When the liver is damaged, ALT is released into the bloodstream and levels increase. Aspartate transaminase (AST) is an enzyme that helps metabolize amino acids. Like ALT, AST is normally present in blood at low levels. An increase in AST levels may indicate liver damage, disease or muscle damage. Alkaline phosphatase (ALP). ALP is an enzyme found in the liver and bone and is important for breaking down proteins. Higher-than-normal levels of ALP may indicate liver damage or disease, such as a blocked bile duct, or certain bone diseases. Bilirubin is a substance produced during the normal breakdown of red blood cells. Bilirubin passes through the liver and is excreted in stool.
Elevated levels of bilirubin (jaundice) might indicate liver damage or disease or certain types of anemia. 25 The serum levels of Aspartate aminotransferase (AST), Alanine aminotransferase (ALT) and alkaline Phosphatase (ALP) was significantly increased and the levels of total bilirubin, direct bilirubin and total protein were significantly decreased in paracetamol intoxicated rats when compared to normal control group (Tables 1 and 2). The group which received fruit extracts of Cordia obliqua at 300mg/kg body weight of both the extracts showed a significant increase in the mean levels of AST, ALT, ALP, TP, total bilirubin and direct bilirubin.

Histopathology of liver
In normal group (control) liver sections showed the normal lobular architecture of the liver (Figure1). Histological studies revealed that liver show moderate vacuolar degeneration, severe congested vessels. (Paracetamol) (Figure2). Histology of liver showed no micro vascular fatty changes and mild central venous congestion. (Sylimarin) (Figure3). In this group mild perivascular edema, moderate inflammatory cell infiltration was observed, (Ethanolic Extract) (Figure 4). Liver section showed very mild perivascular edema and very mild vacuolar degeneration and hemorrhage (Aqueous Extract) ( Figure 5).

In Vivo antioxidant activity on paracetamol induced hepatotoxicity in rats
The activities of GSH, SOD and CAT in liver were significantly lower in Paracetamol induced hepatotoxicity rats compared to control rats. Administration of Cordia obliqua fruit extract at both doses showed a significant increase in GSH, SOD and CAT activity when compared to paracetamol intoxicated group. The in vivo antioxidant assay showed that the extract increased the activity of serum superoxide dismutase (SOD) and catalase and glutathione (GSH). Catalase is a ubiquitous enzyme that catalyzes the decomposition of hydrogen peroxide, a reactive oxygen species, which is a toxic product of both normal aerobic metabolism and pathogenic ROS production. 26,27 The SOD catalyzes the dismutation of superoxide to hydrogen peroxide and oxygen, thereby reducing the likelihood of superoxide anion reacting with nitric oxide to form reactive peroxynitrite. 27 Glutathione is largely known to minimize the lipid peroxidation of        cellular membranes and other such targets that is known to occur with oxidative stress. N-acetyl-cysteine is a by-product of glutathione and is popular due to its cysteine residues and the role it has on glutathione maintenance and metabolism. 28 The increased serum activities of Catalase, GSH and SOD as observed in this study suggest that the extract has an in vivo antioxidant activity and is capable of ameliorating the effect of ROS in biologic system. 29,30

DISCUSSION
The study was carried out to evaluate the hepatoprotective and in vivo antioxidant activity of both aqueous and ethanolic fruit extracts of Cordia obliqua in paracetamol induced hepatotoxicity in Wistar Albino Rats. The results of present study showed that the extracts safe dose was 3000mg/kg. Liver injury is manifested in terms of increase in levels of serum aminotransferases, modest hepatic infiltration by both lymphocytes and eosinophils and slight focal hepatocellular necrosis also causes liver cholestasis associated with increased plasma bilirubin and alkaline Phosphatase. Paracetamol is a non-steroidal anti-inflammatory drug (NSAID), most commonly indicated in fever and mild to moderate pain like headache and myalgia. It is one of the safest NSAID in therapeutic dose and is well tolerated. PCM induces liver injury through the action of its toxic metabolite, N-acetyl-pbenzoquinone imine, produced by the action of Cytochrome P-450. Depletion of GSH causes the remaining quinone to bind to cellular macromolecules leading to cell death. Damage induced in the liver is accompanied by the increase in the activity of some serum enzymes. However, toxic doses of paracetamol cause severe hepatotoxicity characterized by abnormality of hepatic function and degeneration, necrosis or apoptosis of hepatocytes that leads to liver failure in both human and animals. Paracetamol has been taken as test model to screen the anti-hepatotoxicity of indigenous drugs.
In the present study it was observed that Chronic administration of Paracetamol to rats increased the levels of marker enzymes like ALT, AST and ALP as these are stored in the liver cells and increase in the levels of these marker enzymes in serum indicate damage to the liver cells. Pretreatment with aqueous and ethanolic fruit extracts of Cordia obliqua decreased the levels of ALT, AST, ALP and increased levels of total protein, total bilirubin and direct bilirubin is an indication for the hepatoprotective activity of these extracts against drug induced hepatotoxicity. He has written more than 08 articles in leading journals and presented more than 17 research papers in national and international conferences.