Acute Hepatotoxicity and Nephrotoxicity Study of Orally Administered Aqueous and Ethanolic Extracts of Carica papaya Seeds in Adult Wistar Rats

About 80% of the population in many third world countries rely on traditional medicine as the sole means of therapy against diseases. Carica papaya is a medicinal plant which has been proven to contain substances that and is being used for such purposes. This study was designed to evaluate the acute hepatotoxic and nephrotoxic effects of orally administered aqueous and ethanolic extracts of Carica papaya seed in adult Wistar rat in low and high doses. The OECD (2003) guideline for testing of chemicals was used for the acute oral toxicity study to determine LD50 and was found to be above 2000 mg/kg. The acute studies were done using five adult Wistar rats per group. Group one was the control, group two and three received 100 mg and 1000 mg/kg body weight of aqueous extract respectively while groups four and five received 100 mg and 1000 mg/kg body weight of ethanolic extract respectively. The animals were dosed for 5 days orally. The haematological indices for both extracts were within normal range and so also were the renal function test and liver function test. The histological studies of the liver kidneys and spleen did not reveal any pathologic changes when compared to the control group. In Conclusion it can be said that aqueous and ethanolic extracts of Carica papaya seed extract are not hepatotoxic and nephrotoxic to adult wistar rats in the tested doses and duration.


INTRODUCTION
The history of medicinal plants is intimately connected with the history of civilization.Records of early civilization in all parts of the world reveal that a considerable number of drugs used in modern medicine were in use even in ancient times.According to the world health organization, about 80% of the population in many third world countries still use traditional medicine (medicinal plants) for their primary health care due to poverty and lack of access to modern medicine (Silva, 1997).WHO therefore approved the use of herbal products for national policies and drug regulatory measures in order to strengthen research and evaluation of the safety and efficacy of these products (Saxena, 2001).Farnsworth et al. (1985) his co-worker reported that of the 119 plant derived drugs listed by WHO study, 74% were discovered as a result of chemical studies to isolate the active compounds responsible for the use of original plant in traditional medicine.Carica papaya (Pawpaw) is a tree like herbaceous plant in the family caricaceae.It is believed to have its origin from the low lands of Eastern Central America, from Mexico to Panama (Nakasone and Paul, 1998).Papaya is cultivated mainly for their ripe fruit used as fruit desert.Carica papaya is a medicinal plant in that it contains substances that can be used for therapeutic purposes.These substances are precursors for chemopharmaceutical synthesis as such this plant has been used traditionally in cases of kidney failure, low sperm count, dental care, heart problems, natural memory enhancer and remedy for fibroids in uterus (Krishna et al., 2008).From previous studies, papaya leaves have been reported to contain carpaine which has high antioxidant content that may be helpful for the prevention of atherosclerosis, diabetic heart disease, heart attacks and strokes.Pawpaw (C.papaya L.) is also said to improve the immune system and prevents illnesses such as recurrent ear infections, colds and flu (Okeniyi et al., 2007).This plant has been recommended as an anti-ulcerogenic, anti-amoebic, anti-fungal, anti-microbial, anti-tumour, hypolipidaemic and employ in wound-healing activity, free radical scavenging activity, diuretic activity, uterotonic activity and antifertility activity (Krishna et al., 2008).The powdered seeds of Carica papaya have numerous applications worldwide; some includes its use in Northern India as an antihelminthic and their extract also used as anti-inflammatory and analgesic agents (Villegas, 1997).Carica papaya seeds is also said to possess Antimicrobial properties (Nester et al., 1998).Some other uses of Carica papaya includes, for example, the use of dead leaves of Carica papaya that fall off the tree as abortifacient (Sofowora, 1985).Carica papaya seed extract is currently being marketed as a nutritional supplement with purported ability "to rejuvenate the body condition and to increase energy".According to Mojica-Henshaw et al. (2003), the product is said to improve immunity against common infection and body functioning.This study was designed to evaluate the acute hepatotoxic and nephrotoxic effects of orally administered aqueous and ethanolic extracts of Carica papaya seed in adult Wistar rat in low and high doses.

MATERIALS AND METHODS
Experimental animals: A total of sixty healthy, adult Wistar rats weighing 160-220 g were acclimatised to the laboratory conditions for 7 days prior to the experiment.Ten animals were used for LD50 while twenty five were used for acute studies.The animals were bred and housed in polypropylene cages in the animal house of the Department of Human Anatomy, Ahmadu Bello University, Zaria-Nigeria.The animals were fed rat pellet diet and layers mesh, exposed to approximately 12 h light: 12 h dark cycle and water was provided ad libitum.Animals were treated humanely; Veterinary care and supervision were provided throughout the period of study.
Phytochemical screening tests: Desirable amount of Carica papaya extract was used for phytochemical tests.The extract solution was tested for alkaloids, glycosides, flavonoids, saponins, sugars and tannins according to the protocol described by Trease and Evans (1983).
Extract preparation: Ripe C. papaya fruits of Homestead variety were obtained from a local market in Zaria Kaduna State between the months of November and December and authenticated at the Department of Biological Sciences, Ahmadu Bello University, Zaria-Nigeria.The voucher number 0911 was obtained.The seeds were removed, air dried under shade and coarsely powdered.A measure of 200 grams each of the powdered material was used for each extraction.There were soxhleted with ethanol and distilled water respectively in the Department of Pharmacognosy and Drug Development of Ahmadu Bello University, Zaria.The soxhleted material was concentrated under reduced pressure and the obtained residue was weighed to calculate the yield and the extracts were used for the study.

Acute oral toxicity studies (LD50):
The up-and-down method as outlined in the OECD ( 2003) guideline for testing of chemicals was used for the acute oral toxicity study.The animals were randomly selected, marked for permit individual identification.They were aged between 8 and 12 weeks and the weights of those used were kept within an interval of 20% of the mean weight of any previously dosed animals.Five nulliparous, nonpregnant female Wistar rats weighing between 135-160 grams were used to test each extract.The animal were fasted (only food was withheld) for 12 hours and weighed prior to dosing.Varying single doses of the Carica papaya extracts was administered orally using intubation cannula to one animal at a time and food withheld for another 4 h after dosing.The dosing was initiated at 175 mg/kg a 3.2 progression factor was used and the subsequent doses were 550 mg/kg and 2000 mg/kg.The limit was set at 2000 mg/Kg and dosing was stopped after 3 animals survived.This procedure was done for both extracts.After each dose, the animals were observed for 14 days and the toxicity signs checked out included changes in skin and fur, eyes, mucus membranes, tremors, convulsion, salivation, diarrhea, lethargy, sleep and coma (OECD, 2003).
Acute studies: Twenty five adult Wistar rats were randomised and five (5) assigned to each of the treatment groups.Group one was the control and received 2 ml/100 g body weight of normal saline with 2% Tween 80, group 2 and 3 had 100 mg and 1000 mg/kg body weight of aqueous extract respectively while Groups 4 and 5 had 100 mg and 1000 mg/Kg body weight of ethanolic extract respectively.They were dosed orally for 5 days.The test extracts were dissolved in a suitable vehicle that is aqueous in distilled water and ethanolic extract in 2% tween 80.After treatment the rats were sacrificed and the blood obtained for haematological indices and biochemical analysis for liver and renal function test.The tissues i.e., liver, kidneys and spleen were carefully dissected out, cleaned of any fats and weighed.The harvested tissues were fixed in 10% formal saline for routine histological processing and stained with haematoxylin and eosin.

Statistical analysis:
The data obtained from the studies are represented as Mean ± SEM.The data were analyzed by one way analysis of variance (ANOVA), 'P' value less than 0.05 was considered as statistically significant.EZAnalyze 3.0 and Microsoft Excel 2007 were used for analysis and production of charts.

Phytochemical screening tests:
The yields obtained were 7.8% for aqueous extract and 7% for ethanol extract.The results of phytochemical screening of aqueous and ethanolic extracts of Carica papaya revealed the presence of saponins, alkaloids, tannins,  1).None of the extracts contained glycosides.

Acute oral toxicity studies (LD50):
No mortality or morbidity was recorded in any of the animals used throughout the 14-day observation period following the oral administration of the different doses of the aqueous and ethanolic extracts of C. papaya seeds.There were no signs associated with oral toxicity.Animals did not show any sign of aggression or unusual behaviour during handling.The LD50 of the aqueous and ethanolic extract of C. Papaya seed was found to be above 2000 mg/kg.

Acute oral administration:
The duration of administration was five days at doses of 100 mg/kg and 1000 mg/kg for the two extracts.There were no significant changes were observed in the treatment group compared to the control group.The animals were not aggressive and did not exhibit any unusual behaviour during handling.

Haematological indices:
The haematological indices observed after 5 days extract administration is as presented in Table 2.It indicated no significant difference within and between the groups.

Biochemical studies:
The Liver function test results obtained after 5 days of administration of the different extracts as presented in Table 3 it showed the differences that exist between the groups are statically insignificant.The results obtained are expressed in international unit per liter (IU/L) for serum Aspartate aminotransferase (AST), Alanine aminotransferase (ALT) and Alkaline Phosphatase (ALP).The renal function test results after 5 days of extract administration is as shown in Table 3.The renal function test results obtained after 5 days of administration of the different extracts showed the differences that exist between the groups are statically insignificant when compared to the control.

HISTOLOGICAL ANALYSIS HISTOLOGICAL ANALYSIS OF THE LIVER
Light microscopic examination of liver sections obtained from animals in the study groups showed appearance of normal hepatic lobules consisting of branching and anastomosing plates of hepatic cells in between which are clear sinusoids showing branching and anastomosing pattern (Fig. 1 to 3).The interlobular septa with the portal area consisting of branches of portal veins and hepatic arteries are also visible and clearly demarcated.Endothelial cells lining the central vein and sinusoids were also visible in all the groups and red blood cells were also visible within the central vein and sinusoids.Also within the sinusoids were Kupffer cells.No necrosis or fatty changes were observed in all the section examined.Figure 1 represents control group while Fig. 2 and 3 represent sections from animals in the extract treated groups.

HISTOLOGICAL ANALYSIS OF THE KIDNEY
Light microscopic examination of kidney sections obtained from animals the study groups showed normal renal cytoarchitecture.Sections obtained from the renal cortex of the animals from all the groups exhibits normal renal corpuscles and renal tubules which are surrounded by basement membrane.The glomeruli and Bowman's capsule all appear normal.The glomerular epithelium and the capsular space also appear normal for both the control groups (Fig. 4) and the treated groups Fig. 5 and 6 as illustrated on the micrograph.Section of the medullary areas also exhibited normal collection of papillary ducts and ducts of loops of Henle.centres where also visible along with the connective tissue trabeculae and all appeared normal for both control group Fig. 7 as well as for the treated groups Fig. 8 and 9.

DISCUSSION
Assessment of haematological parameters can be used to determine the extent of deleterious effect of extracts on the blood of an animal.It can also be used to explain blood relating functions of a plant extract or its products (Yakubu et al., 2007).Such analysis is relevant to risk evaluation as changes in the haematological system have higher predictive value for human toxicity, when the data are translated from animal studies for use in man (Olson et al., 2000).According to Kohnke (2009), blood tests can also be used to determine the dehydration state, degree of anaemia, infection and immune challenge, physical stress as well as metabolic conditions.Some of these are undesirable effects that may be attributable to xenobiotics such as plant extracts.The result of this study showed that, the levels of RBC, Hb, PCV and WBC (Lymphocytes, neutrophils, monocytes, eosinophils and basophils) at all the doses were not altered in doses and duration used, again this may suggest therefore, the extracts at doses was not toxicologically significant on these parameter.The absence of significant effect of the extract on RBC, Hb and PCV, could also mean that neither the incorporation of haemoglobin into red blood cells nor the morphology and osmotic fragility of the red blood cells was altered (Adebayo et al., 2005).The nonsignificant effect of the extract on the RBC may also be an indication that the balance between the rate of production and destruction of the blood corpuscles (erythropoiesis) was not altered.This shows that it is relatively safe when used for long term in both high and low doses as done in the study.However, a contrary result was obtained by Ikpeme et al. (2011), the outcome of their study showed ethanolic seed and leaf extracts to be a blood boosters as the increased both Hb and PCV after 30 days oral administration.The result of the renal function test indicated that none of the assayed parameters was significantly different from the control group.According to Schmidt et al. (2007) it may imply that the extract did not affect the secretory ability of the organs and the homeostatic function of the kidney is still intact.It can thus be said that the different extracts are not nephrotoxic at the dose and duration used in the study.Serum enzyme measurements are valuable tool in clinical diagnosis, providing information on the effect and nature of pathological damage to any tissue (Wills, 1985).Therefore, the increase in serum Alanine aminotransferase (ALT), alkaline phosphatase and Aspartate aminotransferase (AST) activities may indicate liver tissue damage probably by altered cell membrane permeability leading to the leakage of the enzymes from the tissues to the serum.Alanine and aspartate aminotransaminases are considered to be sensitive indicators of hepatocellular damage and within limit can provide a quantitative evaluation of the degree of damage to the liver (Al-Habori et al., 2002).In the study all three enzymes were assayed for and where all within normal range and did not significantly differ from the control group, it could there for be interpreted that the extract was not injurious to the hepatocytes and the secondary organs which produce some of these enzymes.
In conclusions, this study has been able to establish that the Aqueous and Ethanolic extract of C. papaya of the homestead variety is non toxic in Wistar rats following acute oral administration.The result of the renal function test has also established that the extract lacks nephrotoxic property at 100 and 1000 mg/kg body weights in acute administration while the result of the liver function test established that the extract lacks hepatotoxicity property at 100 and 1000 mg/kg body weights in acute administration.Its consumption in various forms for medicinal purposes maybe is indeed harmless to the various organs and tissues studied.Further studies are being done to determine the effects on chronic administration.

Table 1 :
Phytochemical components seed extracts of Carica papaya Linn

Table 2 :
Mean haematological indices of adult wistar rats after 5 Days of C. Papaya seed extracts administration

Table 3 :
Mean serum ALAT, ASAT, ALP and Urea and electrolytes of Wistar rats after 5 Days of C. Papaya seed extracts administration