STUDY ON FRESH LEAF AQUEOUS EXTRACT OF FLACOURTIA INDICA FOR HEPATOPROTECTIVE, ANTI-ANEMIC AND HYPOGLYCEMIC ABILITIES IN CCL4 INDUCED HEPATOTOXICITY IN ALBINO WISTAR RATS

Objective: Hepatic injury and its associated conditions have been reportedly shown to be managed through herbal remedies. In this study, investigation of the fresh leaf aqueous extract of Flacourtia indica (of the family of Salicaceae) as hypoglycemic, antianemic and hepatoprotective agent in albino wistar rats induced CCl4 hepatotocxicity was done. Methods: Fifteen rats of either sex, weighing 175-295g, divided into five groups (I-V) of three rats each, were used. Group-I is negative control, II-positive control and III –V test groups. Groups II-V were induced 200mg/Kg/bodyweight CCl4, for 3-days, for hepatic injury and anemia. Groups III-V were respectively treated orally with 400, 600 and 800 mg/Kg/bodyweight of fresh leaf aqueous extracts (FLAE) of Flacourtia indica, for 7-days. Results: Activities of alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, concentrations of bilirubin, albumin, total protein, blood glucose and packed cell volume (PCV) and hemoglobin were assayed. Results after induction showed significant (p˂0.05) decrease in heamoglobin and PCV, significant (p˂0.05) increase in the liver function enzymes and blood glucose compared with results of liver function enzyme and blood glucose having significant (p˂0.05) decrease, and significant (p˂0.05) increase of PCV and hemoglobin after treatment with FLAE of Flacourtia indica. Body weight of rats induced CCl4 was found to increase with FLAE of Flacourtia indica treatment. Conclusion: It may be concluded that the potentials exhibited by FLAE of Flacourtia indica to manage hyperglycaemia, hepatic injury and anemia induced by CCl4 are associated with its antioxidant activity and the presence of phytochemicals, minerals and nutrients value.


INTRODUCTION
Plants have been largely used in the quest of managing health challenges as alternative of nature's providence. Traditional medicine practice is an age long practice common in developing countries 1 . Several bioactive compounds in plants are known with their antioxidant and scavenging abilities 2 . Phytochemicals are chemicals in plants with no nutritive value but highly effective in disease prevention and protection when consumed. Most of these bioactive plants' compounds have been implicated in the treatment, management and prevention of ailments. Several studies have demonstrated the use of plants' extracts as hepatoprotective and gluco-stabilizer in Albino wistar rats induced aluminium chloride hepatic toxicity 3 and 4 reported various methods of extraction, isolation, identification and purification of bioactive compounds in plants. Flacourtia indica's leaf, stem bark, fruits and root like other plants, is not exempted from these beneficial characteristics. These have been shown to possess biological, medicinal and pharmacological potentials in the prevention and treatment of hepatic disease 3 , cardiovascular diseases, cancer 5 , diabetes 6 , bacterial infection 1 , and other conditions like anemia hyperglycemia and hypercholesterolemia 7, 8 . Various researchers have demonstrated that plants are rich sources of antioxidant vitamins such as vitamins A, C and E 4 , minerals such as Fe, Mg, Mn, N, P, Ca, Na and K 9 and phytochemicals such as phenolics (tannins, flavonoids), carotenoids, anthocyanins, coumarin glycosides 1 . The liver is an organ with multiple functions. It is involved in circulation of blood, plays major role in metabolic reactions, seen in conversion of excess blood glucose to glycogen, carries out detoxification by secretion of bile, involved in production of blood clotting factor by production of fibrinogen, heparin and prothrombin 10, 11 . Hepatic diseases pose a universal concern to Humans and other animals, contributing a large cause of mortality and morbidity. These include fatty liver, cirrhosis; hepatitis (A, B, C, D and E), drug/chemical induced hepatic injury, hepatic cancer and alcohol induced hepatic injury 11 . Chemical/drug induced liver toxicity is reported to be the paramount cause of hepatotoxicity. This has been linked to life style, abuse and misuse of drug, occupational, laboratory and industrial exposure to substances and chemicals like carbon tetrachloride, aluminum chloride, alcohol etc 10 . The mechanism through which carbon tetrachloride CCl 4 and these other chemical substances exert liver damage is understood to be linked to production of reactive oxygen species. This result in lipid peroxidation of liver tissues as a consequence of the high put of free radicals generated which subdues the liver's defense system, degenerating to inflammation, hepatic apoptosis, liver cirrhosis and fibrosis 12, 13 . Anemia sets in due to lack of adequate and healthy red blood cells (RBC) and hemoglobin, the oxygen binding component of the blood. Anemia is a condition that is commonly affected by infants, child bearing age women/pregnant women, the young and the elderly 14 . Different types of anemia arise from their causes. Anemia is considered to be caused by abnormal RBC production (iron deficiency anemia, vitamin deficiency anemia, aplastic anemia thalasemia etc), destruction of red blood cell (sickle cell anemia, clotting disorder, hemolytic disease etc) and loss of blood through uncontrolled bleeding 15 . Anemia has been reported to be induced by several chemical substances such as AlCl 3 and phenylhydrazine 14, 16 , reported the prevalence of anemia among the elderly with value of hemoglobin Hb < 12g/dl in women and Hb <13g/dL in men. This study was carried out to evaluate the potential of fresh leaf aqueous extracts (FLAE) of Flacourtia indica as a hepatoprotective, anti-anemic and hypoglycemic agent in CCl 4 induced hepatic injury in Albino wistar rats.

MATERIALS AND METHODS Collection and Preparation of Plant Samples
Fresh leaf materials of Flacourtia indica (Governor's plum) were collected from around staff quarter of Caritas University, Amorji-Nike, Enugu state, Nigeria. The required plant leaf was authenticated and a voucher number of PSB/109-12.A was given by Mr. Okafor, C.U., a botanist in plant tissue culture and biotechnology department, Faculty of Biological Science, University of Nigeria, Nsukka. The aqueous plant extracts were prepared selecting fresh leaf aerial part, weighed and squeezed in a bowl of containing water and filtered and filtrate was used for oral treatment. The volumes of the extracts to be administered were calculated according to the body weight of the rats using the formula:

Collection and Preparation of Blood Sample
Three milliliter (3ml) of blood was collected from the rats by capillary pressure insertion into the side of the eye using capillary tubes into a plain bottle, for the collection of serum used for biochemical assay (liver function test) and about 3mls collected in an EDTA sample bottle for hematological assay (PCV and hemoglobin). The samples in bottles were stored at room temperature.

Study Animals
Albino Wistar rats of 175-294g weight, of either sex were obtained from university of Nigeria Nsukka. Animals were housed at an ambient temperature and relative humidity in the animals' house of department of Biochemistry, natural sciences, Caritas University, Amorji-Nike Enugu. The rats were allowed to acclimatize for one week prior to the experiment and had access to standardized pelletized finisher feed and clean water within the period of the acclimatization. The principle of laboratory animals' care and ethical guidelines for investigation of experimental pain in conscious animals were followed respectively 17,18 . Design and Animal Grouping A total of fifteen (15) Wistar albino rats, divided into five groups (Groups I-V) of three rats each was used for this study. Group I: Negative control consist of 3 rats, no carbon tetrachloride CCl 4 and FLAE of Flacourtia indica were administered.

Group II:
Test control (positive control) consist of 3 rats, were administered orally with 200mg/Kg/ body weight CCl 4 without FLAE of Flacourtia indica.

Group V:
Consist of 3 rats, administered orally with 200mg/Kg/body weight CCl 4 and 800mg/kg/body weight FLAE of Flacourtia indica. At the end of induction (three days), blood sample was collected from each group for biochemical and hematological assays before treatment with FLAE of Flacourtia indica. After treatment with FLAE of Flacourtia indica for seven days, blood sample was also collected for biochemical and hematological assays.

Induction of Liver Injury and Anemia
Rats of groups II-V were induced with liver injury and anemia by single oral administration with 200mg/kg body weight of CCl 4 respectively. A confirmatory test was carried out after induction of anemia by assaying the plasma hemoglobin percentage to show that the rats were anemic.

Liver Function Assay
After collection of blood sample from rats, serum was collected by clot retraction. Serum ALT, AST, ALP, Albumin, Total protein and Bilirubin were assayed by ISSN: 2456- 8058 19 CODEN (USA): UJPRA3 the standard method as described by 19 with the use of kits from Randox Laboratories Ltd, 55 Diamond Road, Crumlin, country Antrim, BT29 4QY, United Kingdom.

Figure 1: Liver function test after induction with 200 mg/kg body weight CCl 4
Letters a and b indicates significant difference (P < 0.05) when group I was compared with groups II, III, IV and V, respectively after CCl4 induction of liver damage to rats in these groups. Graphs with same letters are not significantly (P < 0.05) different.

Hematological Assay
The Haemoglobin (Hb) and packed Cell Volume (PCV) values were determined by standard method as described by 20

Statistical Analysis
Results were expressed as mean ± standard deviation and analyzed using one-way ANOVA (analysis of variance), p value (<0.05) was considered significant. A component of graph pad instat 3 software version 3.05 and graphpad prism version 7.04 by graph pad Inc. was employed 21 .

Figure 3: Alkaline phosphatase level of CCl4 induced rat's liver damage
Letters a, b, c, d and e indicates significant difference (P < 0.05) when group I was compared with groups II, III, IV and V, respectively after CCl4 Table 1 shows the liver function parameters of rats after induction with 200mg CCl 4, for liver injury. There was an observed significant (p<0.05) increase in TP, ALB, BIL, ALP, ALT and AST of group I (negative control) compared to group II (positive control) and test groups (III, IV and V).    There were significant differences (P < 0.05) observed in comparing group I with groups III, IV and V. Also, significant differences (P < 0.05) were observed when the treated groups (III, IV and V) are compared with one another, not necessary in a dose dependent manner. As shown in Figure 1, the results of induction with 200mg/kg CCl 4 reveal significantly higher (P < 0.05) levels of groups II (positive control), III, IV and V compared to group I (negative control). Results in Figure 4 also reveal significantly higher (P < 0.05) differences in the concentration of Alkaline Phosphatase (ALP) when group I (negative control) is compared with group III (400mg/kg FLF. indica), group IV (600mg/kg FLF indica) and group V (800mg/kg FLF. indica). When the concentrations of ALP in groups III, IV and V were compared with one another, significant differences (P < 0.05) were observed in a dose dependent pattern.

DISCUSSION
Carbon tetrachloride (CCl 4 ) induced hepatic injury is shown in Table 1 and (Figure 1 and Figure 3). The rise in the level of ALT, AST, ALB, ALP, BIL and TP of groups II, III, IV and V when compared to group I indicates a CCl 4 induced liver damage. This is consistence with 22 , who reported the use of 1.5ml/kg body weight of CCl 4 orally administered to rats to induce liver damage. These liver function enzymes are found to be located in the cytosol of the liver cell and thus, are easily released into the serum after cellular liver damage 23 . The mechanism of action involved in CCl 4 hepatic injury is understood to be linked to the liver phase II detoxification action. The liver, in the process of detoxification transforms Carbon tetrachloride (CCl 4 ) in the presence and action of cytochrome P 450 enzyme component to produce peroxy trichloromethyl and trichloromethyl free radicals 13 . These free radicals results in lipid peroxidation by reacting covalently with biomolecules (proteins, nucleic acids, lipids etc) in the presence of oxygen. Thus the liver becomes damaged and obviously its cell membrane becomes degenerate, permeable and licks out its cellular contents of AST, ALT, TP, ALP, BIL and ALB 24 .  After induction with CCl 4 , the level of blood glucose was raised (Table 4), hemoglobin and PCV (Table 3) levels were decreased. This could suggest that CCl 4 induced anemia was possible owing from the destruction of red blood cells and shortage of circulating mineral iron and vitamins 15 . Hyperglycemia induced by CCl 4 could be due pancreatic injury caused by generation of free radical, cell membrane lipid peroxidation and subsequent destruction of pancreatic βislet cells 3 . Administration of CCl 4 induced rats with FLAE of Flacourtia indica shows reduction in the concentrations of liver function enzymes (TP, AST, ALT, ALP, and BIL) in the serum and blood glucose ( Table 2 and Table 4 and Figure 2 and Figure 4). Similarly, after treatment with FLAE of Flacourtia indica the levels of ALB, Hb and PCV increased as shown in Table 2 and . Antioxidant phytochemicals such as saponins, flavonoids and alkaloids have been reported for their ant-anemic abilities, prevent thrombosis and aggregation of platelet and promote enhanced blood circulation 25, 26 . Thus, FLAE of Flacourtia indica was able to increase the levels of PCV and Hb because of these phytochemicals and mineral elements contents. This action could be made possible by its enhanced removal of the toxic effects caused by CCl 4 and creating flourishing iron utilization for the production of heme and subsequent release of new red blood cells 14 . FLAE of Flacourtia indica could have exert its effects in the improved production of Hb and PCV by enhancing the production of erythropoietin in the bone marrow stem cells and subsequent synthesis of new blood cells 27 . In addition, there was an observed reduced body weight of anemic rats (CCl 4 induced groups) when compared to the groups treated with FLAE of Flacourtia indica (Table 5). This reduction in body weight of anemic rats and increase or weight gain in the treated group with FLAE of Flacourtia indica is consistent with the report of previous study 14 . The association of weight loss with anemia is not very clear. However, it appears to be related to defect in carbohydrates digestion in the small intestine of anemic rats due to insufficient amount of the enzyme, disaccharidases, thus leading to undigested carbohydrates 28 .

CONCLUSION
The use of fresh leaf aqueous extract of Flacourtia indica in this study reveals that the plant possesses anti-anemia, hypoglycemic and hepato-healing potentials. This is obviously seen in the reduced levels of blood glucose, liver function assay, and in the raised levels of the hematological parameters, coupled with weight gain after treating CCl 4 induced groups with FLAE of Flacourtia indica.