Templates by BIGtheme NET

ADMINISTRATION OF BANANA GREEN PEEL ETHANOLIC EXTRACT TO CARBONTETRACHLORIDE-INDUCED ALBINO WISTAR RATS OFFERS SLIGHT HEPATO-PROTECTION

Download File

Arit J. Ekpo1, Innocent A. Edagha2, Henry D. Effiong1

1Department of Biochemistry, Faculty of Basic Medical Sciences, Uyo, Nigeria
2Department of Anatomy, Faculty of Basic Medical Sciences, Uyo, Nigeria

ABSTRACT
Effect of banana green peels ethanolic extract on the liver function and histology of albino Wistar rats treated with intraperitoneal administration of 4 ml/kg body weight of Carbontetrachloride (CCl4) was investigated. 30 Wistar rats were divided into 6 groups of 5 each as follows: Group 1 (normal control) and 2 (CCl4 group) received normal feed and 4 ml/kg body weight CCl4 plus olive oil respectively; Group 2 were CCl4-induced on day 21; Group 3 received 1 ml/kg body weight tween 80 plus olive oil; Group 4, 5 and 6 were also CCl4-induced at 4 ml/kg body weight, and then administered 500mg/kg body weight of green banana peels, 1000 mg/kg body weight of green banana peels, and 100 mg of Vitamin C respectively. Animals were sacrificed after 21 days of treatment. Serum samples and liver organs were collected for liver function profile, and haematoxylin and eosin (H&E) histological analyses. Result showed a significant (p<0.05) decrease in levels of albumin, aspartate transaminase (AST) and alkaline phosphatase (ALP) (except alanine transaminase (ALT) that was significantly (p<0.05) increased when compared with positive control group). The histology of extract treated groups 4 and 5 showed prominent derangement of hepatocytes but was less severe when compared to CCl4-induced group. Hepatotoxic effect of CCl4 on the liver was positively modulated in the Vitamin C treated Group 6. In conclusion, the ethanolic green banana peels extract possess very mild hepato-protective effect on the histomorphology and altered liver function enzymes.
Key words: Green Banana Peel, Liver function, Histology, Wistar Rat

INTRODUCTION
Herbs are indispensable source of medicinal preparations, used both as preventive and curative agents and are known to contain phytochemicals such as flavonoids, alkaloids, steroids, tannins, and lignin (Shivanand et al., 2010) which are secondary metabolites; and primary metabolites, sugars and fats.
Musa sapientum belongs to the family Musaceae and there are various types of species in the Musa genus, and their pharmacologic studies have been studied (Agarwal et al., 2009). Musa sapientum has been shown to possess antioxidant activity in rats (Zenab et al., 2015). Peel of vegetables and fruits are of the most important part that helps the body during infection by getting rid of the free radicals, they contain vitamins and minerals, in addition to phenols (Zenab et al., 2015).
Carbon tetrachloride (CCl4) is an industrial chemical which has been found to be pretty toxic to human health and  the primary routes of potential human exposure to CCl4 are inhalation, ingestion, and dermal contact (Stephen et al., 2007). A number of reports clearly demonstrated that in addition to hepatic toxicity, CCl4 also causes disorders in kidneys, lungs, testis, hearts, brain and blood by generating free radicals (Jayakumar et al., 2008; Feral et al., 2003). The production of carbon tetrachloride has steeply declined since the 1980’s due to environmental concerns and the decreased demands for chlorofluorocarbons (CFCs) which were derived from carbon tetrachloride (Odabasi, 2008). CCl4 is reported to be resistant to aerobic biodegradation (Odabasi, 2008). This highly toxic industrial solvent has pronounced effects on the liver and brain (Rood et al., 2001).
The aim of this study is to investigate the ameliorative effect of the oral administration of Musa sapientum green peel ethanolic extract on the liver function and histology of albino Wistar rats induced with CCl4.

2.0. MATERIALS AND METHODS
2.1.1 SOURCING AND AUTHENTICATION OF THE GREEN BANANA PEEL
Banana was purchased from farms within the locality of Abak in Akwa Ibom State in the month of July, 2016. The banana was identified and authenticated as Musa sapientum by the Herbarium section of the Department of Botany, University of Uyo, with voucher number: UUH3158 (Abak).

2.1.2 CHEMICALS/REAGENTS AND EQUIPMENT
Randox Reagents kits was used, manufactured by Randox laboratories Limited Ardmore, Co. Antom UK. Chemicals used were manufactured by Sigma Chemical Company St. Louis USA, all of analytical grade.

2.1.3 EXPERIMENTAL ANIMALS
A total of thirty (30) male albino Wistar rats weighing between 180 – 220g were purchased from the College of Health Sciences Animal House of the University of Uyo, Uyo. The rats were housed in plastic cages with wire gauge for adequate ventilation and with provision for feed and water ad libitum. Room temperature and relative humidity were 26 ± 2ºC and 46% respectively, and were acclimatized for a period of two weeks prior to the commencement of the experiment. They were administered with of carbon tetrachloride and banana peel ethanolic extract. All protocols adhered to guidelines on care and use of laboratory animals after institutional ethical committee approval of the University of Uyo (National Institute of Health, 2011).

2.2 EXPERIMENTAL PROCEDURE
2.2.1 PHYTOCHEMICAL SCREENING
The green banana peels obtained was then taken to the laboratory to analyses the phytochemicals present in the sample. Samples of green banana peels were analyzed for the following established methods by (Trease and Evans, 2009; Sofowora, 2008).

2.2.2 PREPARATION OF SAMPLE AND ETHANOLIC EXTRACT
The peels were removed from the flesh, washed with water and cut into small pieces. The peels were dried in the sun for two weeks and grounded to obtain size particle of less than 1.0 mm using a milling machine. The powdery samples weighed 620 g, packed into labelled screwed bottle and stored for later use.
The ethanolic extract of the green banana peels were prepared by soaking the dried powdery sample (50 g) in 500 ml ethanol for 48 hours, during which the mixture was stirred intermittently. The extract was first filtered with cheese cloth then with a Whitman filter paper (125 mm).  Crude extract was obtained by evaporating the ethanol in a water bath at 40oC and stored in a refrigerator at about 8oC.

2.2.3 ACUTE TOXICITY – UP-AND-DOWN METHOD
This method involves the sequential dosing of single animals with the test substance within a time interval of 48 hours. After the administration of the first dose, the next is determined by the outcome of the previous dose administered. If the animal survives the previous dose, the subsequent dose is adjusted upward, but when mortality is recorded at previous dose, it is adjusted downward. The adjustment of dose either upward or downward is by a constant factor. Testing is terminated when the upper limit (2000-5000 mg/kg) have been reached without mortality or when the LD50 have been established from the test (Bruce, 1985).

2.2.4 DOSAGE FORMULATION AND ADMINISTRATION OF BANANA GREEN PEELS EXTRACT
The albino Wistar rats were randomly selected on the basis of their body weights into six (6) groups of five, and the rats in all groups were given water and rat diet. The green banana peels extract, feed, Tween80 and Vitamin C were given orally. Group III, IV and V were induced with CCl4 (50% solution of CCl4 in olive oil intra-peritoneally (i.p.). However, the positive control (group III) was induced with CCl4 on the 21st day. 24 hours after CCl4 administration which is day 22, the rats were sacrificed.

ADMINISTRATION-OF-BANANA-GREEN-PEEL-T1

2.2.5 COLLECTION OF BLOOD SAMPLE
The rats were sacrificed under chloroform anesthesia twenty-four (24) hours after the last administration. Blood sample was collected through cardiac puncture using sterile needles and syringes into a labeled sample bottle. Sample for each animal was divided into two; one was put in an EDTA sample bottle and used to evaluate haematological indices (data not shown), while the other was centrifuged at 3000 revolution per minute (rpm) for 10 minutes. The serum was obtained and used for the determination of liver function. The liver organs were also dissected and preserved in buffered formalin for histological studies.

2.2.6 BIOCHEMICAL ANALYSIS
Plasma transaminases as aspartate aminotransferase (AST) and alanine aminotransferase (ALT) as well as albumin and alkaline phosphatase (ALP) were measured using commercially available diagnostic kits supplied by Randox laboratories (Ardmore, Northern Ireland, UK)

2.2.7 HISTOLOGICAL ANALYSIS
The liver tissues were fixed in 10% buffered formalin and then subsequently processed 48 hrs later by the method as described by Bancroft and Gamble, 2002 stained with H&E, and observed under Olympic microscope, thereafter photomicrograph of desired section were obtained with each figure shown, indicating a study group.

2.3 STATISTICAL ANALYSIS
One way analysis of variance (ANOVA) was applied using the Primer software (version 3.01). Post-hoc analysis using the Newman-Keul test was applied to test the significant levels. Data with probability level (P < 0.05) was regarded as significant.

3. RESULTS

ADMINISTRATION-OF-BANANA-GREEN-PEEL-T2

ADMINISTRATION-OF-BANANA-GREEN-PEEL-T3

3.1 HISTOLOGICAL FINDINGS
The liver organs of the rats were collected, each from a group, and using H&E staining were viewed under the microscope with each figure presented, indicating a study group. Fig. 1 showed normal cytoarchitecture, Figs. 2 and 3 were severely and mildly affected respectively, Figs. 4 and 5 were severely affected, while Fig. 6 showed normal cytoarchitecture as shown in the photomicrographs below:

Historical-Findings1
Fig. 1. Photomicrograph showing normal liver cytoarchitecture with normal array of hepatocytes (HC) and average     sized central vein (CV) and sinusoid (SS), stained with H&E at magnification of x100
Inference – Normal cytoarchitecture (Not affected)

Historical-Findings2
Fig. 2. Photomicrograph showing abnormal liver cytoarchitecture of Hepatocytes (HC), central vein (CV), areas of necrosis (NC) and widely distributed steatosis (fatty tissue- Ft), stained with H&E at magnification of x400
Inference – Severely affected

Historical-Findings3
Fig. 3. Photomicrograph show near normal Liver cytoarchitecture with array of Hepatocytes (HC), central vein     (CV) and sinusoid (SS) with mild inflammation (IF) stained with H&E at magnification of x100
Inference: Mildly affected

Historical-Findings4
Fig. 4. Photomicrograph showing abnormal Liver architecture with normal array of Hepatocytes (HC), and peri-portal inflammation (PI) and Haemorrhagic pool (Hp), stained with H&E at magnification of x100
Inference: Severely affected

Historical-Findings5
Fig. 5. Photomicrograph of Liver section shows Hepatocytes (HC), congested Central vein (CV), with areas of     inflammation (IF) and few foci areas of fatty changes (Ft) stained with H&E, at magnification of x100
Inference: Severely affected

Historical-Findings66
Fig. 6. Photomicrograph of mildly inflamed Liver section showing Hepatocytes (HC), Central vein (CV), and sinusoid (SS) stained with H&E at magnification of x100
Inference: Mildly affected

3.1 HISTOLOGICAL FINDINGS
The liver organs of the rats were collected, each from a group, and using H&E staining were viewed under the microscope with each figure presented, indicating a study group. Fig. 1 showed normal cytoarchitecture, Figs. 2 and 3 were severely and mildly affected respectively, Figs. 4 and 5 were severely affected, while Fig. 6 showed normal cytoarchitecture as shown in the photomicrographs below:

4. DISCUSSION
The result of phytochemical screening in Table 2 indicates that Musa sapientum peels contain carbohydrates, proteins and some secondary metabolites. Secondary metabolites have been reported to be responsible for therapeutic activities (Rabe, 2000). The presence of terpenes demonstrates the antibacterial properties of plants (Amit and Shailandra, 2006). The presence of glycosides and carbohydrates (monosaccharides) components displays significant antioxidant activities (Matook and Fumio, 2005).

Table 3 shows that serum albumin, AST, were lower in group 5 than the positive group, while serum ALP were lower in groups 4, 5, and 6 than the positive control. The result proved that the liver enzymes were not significantly increased except AST that was significantly increased (p < 0.05) in the hepatotoxic, positive control, group. The liver enzymes, AST and ALT, function in amino acids metabolism (Chatterjea and Shinde, 2012). Hence, an increase level of both enzymes may indicate hepatocellular disease, active cirrhosis, and metastatic liver. Conversely, there was a reduction of AST in the non-hepatotoxic group fed with 500 mg and 1000 mg of ethanolic green banana peels extract. This shows that ethanolic green banana peels extract may possess antioxidant effect on the biochemical function of the liver. Conjugated bilirubin, AST, ALP and ALT are released into the blood whenever the liver cells are damaged, increase serum ALT therefore reflects hepatic damage more specifically (Gowda et al., 2009).
Figs. 4 and 5 had prominent morphologic derangement of the hepatocytes with presence of inflammation, necrosis, and haemorrhagic pool indicative of sinusoidal or vascular leakage compared to Fig. 1 which presented normal hepatic cytoarchitecture. Patterns of hepatic injury from morphologic viewpoint include; degeneration and intracellular accumulation, inflammation, necrosis and apoptosis, regeneration, and fibrosis (Kumar et al., 2005), from toxic or immunologic insult evident with swelling of hepatocytes and hyperplastic cells. Vascularized tissues provoke a host response; inflammation which is a complex reaction to injurious agents such as drugs and microbes; these reactions may be vascular or cellular and are mediated by chemical factors. Necrotic cells or tissues can also trigger the elaboration of inflammatory mediators and any significant insult to the liver can cause hepatic necrosis (Kumar et al., 2005) as seen in Figs. 2, 4 and 5. However, this effect was positively modulated in Fig. 6, a group administered with Vitamin C at 100 mg per kilogram body weight of the Wistar rat compared to group 2 as seen in Fig. 2 which was severely affected with prominent necrosis, steatosis and hypertrophy, as its been reported that CCl4 is a widely known hepatotoxin (Seifert et al., 1994). Vitamin C is widely reported as an effective antioxidant and plays a role in anti-inflammatory activity (Zenab and Ayman, 2015), and the bioavailability of this antioxidant therefore ameliorated the hepatotoxic effect of the CCl4. The liver resides between the digestive system and circulatory system and is very critical in maintaining the body’s metabolic homeostasis (Kumar et al., 2005).  Hepatocyte integrity, billary excretory function and hepatocyte function are credible tests/criteria for evaluation of liver disease (Kumar et al., 2005). Evident manifestation of liver damage is the oxidative damage of liver cells, and polyphenols exert marked antioxidant effect (Kang et al., 2012). As a result, polyphenols may be able to protect liver cells against oxidative damage and promote the repair of oxidatively-damaged cells in order to maintain health (Sebai et al., 2010). Polyphenols present in green banana peels may function in a similar manner. The presence of glycosides and carbohydrates components displays significant antioxidant effect on the liver (Matook and Fumio, 2005). Active biomolecules from the ethanolic green peel extract may not have had significant bioavailability perhaps a dose dependent factor to be able to moderate the cytotoxic effect of the CCl4 in the treated groups.

5. CONCLUSION
From this study we conclude that 500 mg/kg and 1000 mg/kg of green banana peels does possess only mild antioxidant effect on CCl4-induced hepatotoxicity on the biochemical function of the liver and cytoarchitecture compared strong antioxidant activity of Vitamin C in CCl4 induced Wistar  rats.

CONFLICT OF INTEREST
There was no conflict of interest.

ACKNOWLEDGEMENT
We acknowledge the personnel at the Animal House Facility for their guidance, and to Dr. Item Ekaidem for technical assistance in the biochemical analysis.

References:

  1. Agarwal, P. K., Singh, A., Gaurav, K., Goel, S., Khanna, H.D. and Goel, R. K. (2009). Evaluation of wound healing activity of extract of banana (Musa sapientum) in rats. Indian Journal of Experimental Biology; 47: 32-40.
  2. Amit, R. and S. Shailandra, (2006). Ethnomedicinal approach in biological and chemical investigation of phytochemicals as antimicrobials. Indian Journal of Pharmaceutical Science, 41:1-13.
  3. Bancroft, J. D., and Gamble, M. (2002). Theory and Practice of Histological Techniques, 5th edition. London:     Churchill- Living stone. Pp 56-78
  4. Bruce, R. D. (1985). An Up-and-down Procedure for Acute Toxicity Testing. Fundamental and Applied Toxicology, 5(1):151-157
  5. Chatterjea, M. N., and Shinde, R. (2012). Liver Function Enzymes. Textbook of Medical Biochemistry, 8th edition. London: Jaypee Brothers Medical Publishers (P) Limited. P 659.
  6. Feral, O., Muharram, U., Ozturk, I. C., Nigar, V., Kadir, B. (2003). Carbon tetrachloride-induced nephrotoxicity and protective effect of betaine in Sprague-Dawley rats. Urology 62:(2) 353-356
  7. Gowda, S., Desai, P. B., Hull, V. V., Math, A. K., Verekar, S. N., Kulkarni S. S. (2009). A review on laboratory     liver function tests. PanAfrican Medical Journal. 3: 17
  8. Jayakumar, T., Sakthivel, M., Thomas, P. A., Geraldine, P. (2008). Pleurotus ostreatus, an oyster mushroom decreases the oxidative stress induced by carbon tetraoxide in rat kidneys, heart and brain. Chem. Biol. Interact., 176: 108 -120
  9. Kang, J., Thakali, K. M., Xie, C., Kondo, M., Tong, Y., Ou, B. (2012). Bioactivities of Acai (Euterpe precatoria Mart) Fruit Pulp Superior antioxidant and anti-inflammatory properties to Euterpe oleracea Mart. Food Chemistry, 133:671-677
  10. Kumar, V., Abbas, A. K., and Fausto, N. (2005). Robbins and Cotran Pathologic Basis of Disease, seventh edition. Elsevier, Pennyslvania. Pp 878-883
  11. Matook, H., and Fumio, J. P. (2005). Antibacterial and antioxidants activities of banana (Musa AAA CV. Cavendish) fruits peel. American Journal of Biochemical Biotechnology, 1(3): 125-131.
  12. National Institute of Health, (2011). Guide for the Care and Use of Laboratory Animals, 8th edition, Washington     (DC): National Academies Press (US) pp 1 – 2
  13. Odabasi, M. (2008). Halogenated volatile organic compounds from the use of chlorine-bleach-containing household products. Environmental Science and Technology, 42(5): 1445-1451.
  14. Rabe, T. S. J. (2000). Isolation of an Antimicrobial sesquiterpenoid from Warbugie salutaris. Journal of Ethnopharmacology. 93:171-174.
  15. Rood, A. S., McGavran, P. D., Aanenson, J. W., and Till, J. E. (2001). Stochastic estimates of exposure and cancer risk from Carbon tetrachloride released to the air from the rocky flats plant. Risk Analogy, 21(4): 675-695.
  16. Sebai, H., Sani, M., Yacoubi, M. T., Aouani, E., Ghanem-Boughanmi, N., and Ben-Attia, M. (2010). Resveratol, a red wine polyphenol attenuates Lipopolyssaccharide-induced Oxidative Stress in Rat Liver. Ecotoxicology and Environmental Safety, 73:1078-1083
  17. Seifert, W. F., Bosma, A., and Van, L. (1994). Vitamin A deficiency potentiates Carbon tetrachloride-induced liver fibrosis in rats. Hepatology, 19(1): 193-201.
  18. Shivanand, P., Nilam, M., and Viral, D. (2010). Herbs play an important role in the field of cosmetics. International Journal of Pharmacological Technology Research, 2(1): 632-639.
  19. Sofowora, E. A. (2008). Phytochemical screening of Nigerian medicinal plants part II. Lloydia. 41(1): 234-235
  20. Stephen, O. A., Abdulkadir, A. S., Oladepo, W. D., Thajasvarie H. (2007). Effect of Melatonin on Carbontetrachloride induced kidney injury in Wistar Rats. African Journal of Biomedical Research 10:153-164
  21. Trease, G. E., and Evans, W. C. (2009). Pharmacognosy. Macmillan publishers Limited. Pp 213-832
  22. Zenab, M. M., and Ayman, F. K. (2015). The effect of banana peels supplemented diet on acute liver failure in rats. Annuals of Agricultural Sciences, 60(2): 373-379