Abstract: Background and Objective: Celosia trigyna is from the family Amaranthaceae and highly consumed by human beings and animals. The leaves are rich in antioxidants and used medicinally to treat wounds, diarrhea and mouth ulcers. The aim of this study was to evaluate the healing potential of Celosia trigyina leave extracts on the liver and ileum in ethanol-induced toxicity in adult Wistar rats. Materials and Methods: Thirty adult wistar rats were procured, acclimatized and randomly divided into 6 groups of 5 animals each. Rats in groups 2-6 were administered with 5 mL kg–1 of 90% ethanol while animals in group 1 were administered with equivalent volume of distilled water. After 24 h, animals in groups 3-6 were administered with leave extracts of Celosia trigyna (methanol, dichloromethane, ethyl acetate and hexane, respectively). The therapies went on repeatedly for 14 days in all animal groups. Twenty four h after the last administration, the liver and ileum were excised and fixed in 10% formol saline for histopathological analysis. Results: The result showed a significant decrease (p<0.05) in the weight difference of animals in group 2 when compared to the Celosia trigyna treated groups. Furthermore, the histopathological evaluation showed a remarkable recovery of the necrotized hepatocytes by the leave extracts of Celosia trigyna with the dichloromethane and hexane extracts having better remediation against ethanol induced toxicity in both the liver and ileum than the methanol and ethyl acetate extracts. Conclusion: In conclusion, the leave extracts of Celosia trigyna showed promising protection of the liver and ileum against ethanol-induced toxicity in adult Wistar rats. This protection may be due to the antioxidant potential of Celosia trigyna.
INTRODUCTION
Ethanol toxicity has been on the increase in recent times1. Globally, there has been reported abuse of alcohol consumption. Such abuses often lead to severe health conditions such as pancreatitis, diabetes mellitus, gastrointestinal damage, liver disease and cancer2. Ethanol toxicity can cause infiltration of inflammatory cells, necrosis of the hepatocytes and in some cases, initiate the development of tumor in rats and mice3.
Liver damage is a gradual process and as such may not be noticeable at the onset4. Ethanol has caused more harm than good as available reports showed that 20% of all liver transplants in United States are traceable to liver alcohol disease4. Ethanol consumption is attributed to 6.5% of all deaths in Europe5,6. Alcohol dependence syndrome a disorder associated with preoccupation with alcohol, adaptability to the psychotropic effects of alcohol consumption and continuous consumption of alcohol despite its deleterious effects is responsible for the alcohol-related organ damage in most patients5. Direct interaction of ethanol with mucosa of the gastrointestinal tracts can induce functional and metabolic changes. This interaction often leads to mucosal damage culminating into diarrhea and gastrointestinal bleeding. Functional changes and cellular damage to the mucosal affect digestion and assimilation of nutrients leading to weight loss due to malnutrition7. Alcohol ingestion reduces the absorption of sodium in the jejunum and ileum8,9.
Ethanol consumption has been linked with free radical generation which can react with cellular macromolecules to cause cellular damage10. Antioxidants can protect the body against free radicals inflicted injury. There are basically two types of antioxidants: Enzymatic and non-enzymatic5. Enzymatic antioxidants such as catalase and glutathione peroxidase are capable of enhancing non enzymatic antioxidants such as vitamin E and ascobate5. Herbal therapies rich in antioxidants can mop up free radicals generated by alcohol thus, protecting body organs from oxidative damage.
Celosia trigyna is from the family Amaranthaceae. It is found in some African countries such as South Africa, Democratic Republic of Congo and Nigeria. Investigation has shown that both human being and animals consume the leaves of Celosia trigyna. The leaves can be used medicinally to treat wounds, diarrhea, mouth ulcers and intestinal worms11,12. Just very recently, Ofusori et al.13 reported the antiulcerogenic effects of Celosia trigyna plant extracts on ethanol induced gastric ulcer in adult Wistar rats.
In view of the high consumption rate of alcohol, various health challenges that accompany its use as well as the need to search for relatively more affordable and tolerable plant-derived alternatives, it was imperative to study the histopathological effects of different leave extracts of Celosia trigyna on the liver and ileum following ethanol toxicity with a view to ascertaining their ameliorative potentials.
MATERIALS AND METHODS
This study was conducted between May-October, 2018 at the Department of Anatomy and cell Biology, College of Health Sciences, Obafemi Awolowo University, Ile-Ife, Nigeria.
Plant materials: Celosia trigyna leaves were procured and authenticated by a taxonomist in the herbarium unit of the Department of Botany, Obafemi Awolowo University, Ile-Ife where a voucher specimen (IFE- 17466) was deposited.
Extraction process: The extraction processes were conducted in accordance to standard procedures14. All the leaves were air-died and grinded into powder using the squeezing and crushing machine (Daiki Rika Kogyo Co-ltd, Japan). The powdered product was percolated with methanol, dichloromethane (DCM), ethyl acetate and hexane and subjected to continuous shaking for a period of 48 h. The solution was filtered with a (Whatman No. 1) filter paper, concentrated with rotary evaporator and stored at 4°C until needed. The 4 extracts (methanol, DCM, ethyl acetate and hexane) were later constituted at a dose of 50 mg kg–1 b.wt., with 20% Tween 80.
Animal care: Handling and care of the animals conform to the rules and guidelines of the ethical committee of the Institute of Public Health, Obafemi Awolowo University, Nigeria. Rats were procured from Soyebo farm, Nigeria and acclimatized for one week before randomization and grouping into plastic cages at the Animal Holdings of the Department of Anatomy and Cell Biology, Obafemi Awolowo University, Ile-Ife, Nigeria.
Study design: Thirty adult female Wistar rats were used for the study. The rats were assigned into 6 groups with 5 animals10 each: 1, 2, 3, 4, 5 and 6:
| Group 1: | Normal control (DDH2O-1 mL kg–1 b.wt.,/day for 14 days) |
| Group 2: | (Negative control) was given 90% ethanol (5 mL kg–1) and 1 mL kg–1 b.wt.,/day of 20% tween 80 for 14 days |
| Group 3: | Was given 90% ethanol (5 mL kg–1) and MeOH extract (50 mg kg–1 b.wt.,/day in 20% tween 80 for 14 days) |
| Group 4: | Was given 90% ethanol (5 mL kg–1) and DCM extracts (50 mg kg–1 b.wt.,/day in 20% tween 80 for 14 days) |
| Group 5: | Was given 90% ethanol (5 mL kg–1) and EtOAC extract (50 mg kg–1 b.wt.,/day in 20% tween 80 for 14 days). |
| Group 6: | Was given 90% ethanol and hexane extract (50 mg kg–1 b.wt.,/day in 20% tween 80 for 14 days) |
Ethanol toxicity: At the start of the research, all the rats were fasted for 24 h and then a single dose of 90% ethanol (5 mL kg–1) was administered orally to the test groups (2-6)10. Twenty four hours after this, the rats in the test groups (3-6) were commenced on their respective treatment as stated above and administered orally and repeatedly for 14 days before sacrifice.
Sacrifice of animals: Twenty four hours after the last administration, all the rats were sacrificed under slight chloroform anesthesia. Midline incisions were performed and the liver and ileum excised and washed in normal saline.
Histopathological analysis: Histological procedures as documented by Bancroft and Gamble15 was adopted. A portion of the liver and ileum were fixed in 10% formol saline for histopathological analyses using haematoxylin-eosin (H and E) (for the demonstration of the general histoarchitecture), Verhoeff-van Gieson (VVG) (for the demonstration of elastic fibres) and Goddon and Sweets (for the demonstration of reticular fibres) staining procedures. The tissues were dehydrated through ascending grades of alcohol (50, 70, 90% and absolute), cleared in xylene and infiltrated with paraffin wax before embedding. Five micron sections were obtained on rotary microtome. The sections were subjected to different staining procedures. Thereafter, sections were dehydrated in ascending grades of alcohol, cleared in xylene and mounted in DPX. Digital images were achieved using Leica DM 750 microscope interfaced with Leica ICC50 digital camera.
Image analysis: The density of the reticular fibers per group was performed by measuring the stained area per 1600 mm2 on 5 random spots per animal. The software quantifies staining intensity by measuring the stained area in each of the images. Values for each of the photomicrographs per group which represents the staining intensity were generated which was then run through a statistical package (SPSS 22).
Statistical analysis: Data were expressed as Mean±SEM. Statistical significance between the groups were determined by one way analysis of variance (ANOVA). p<0.05 was considered statistically significant.
RESULTS
Weight difference: There was a significant decrease in the body weight difference of animals in group 1 when compared with all other groups (Table 1). There was no significant difference when the weight differences of animals in groups 4-6 were compared across groups. Out of the four groups administered with extracts (groups 3, 4, 5 and 6), only animals in group 3 (administered with methanolic extracts) exhibited a significant (p<0.05) decrease in body weight difference when compared with the other three groups (4, 5 and 6) as shown in Table 1.
Histopathological analysis: The histopathological results showed evidence of necrosis of the hepatocytes of animals in group 2. Also, noticeable were neutrophils infiltration and fat droplets when compared with the normal control (Fig. 1). The ileum of rats in group 2 presented epithelial damage and disorganization of the mucosa.
| Table 1: | Weights (g) of animals exposed to ethanol toxicity and treated with different extracts of Celosia trigyna |
Group 1: Normal control, group 2: Negative control, group 3: Treated with methanol extract, group 4: Treated with dichloromethane extract, group 5: Treated with ethyl acetate extract, group 6: Treated with hexane extract, means with same superscript letters within the column indicate no significant difference while means with the different letters indicate significant difference (p<0.05, Duncan Post hoc multiple range test comparisons) | |
| Fig. 1(a-f): | Photomicrographs of the Liver in (a) group 1-normal control (intact histoarchitecture), (b) Group 2-negative control (disorientation of the hepatocytes (Black circle) characterized with distortion of the sinusoids (Green arrow), (c) Group 3-treated with methanol extract (note the partly disorganized histoarchitecture), (d) Group 4-treated with dichloromethane extract (note the similarity in the histoarchitecture compared with group 1), (e) Group 5-treated with ethyl acetate extract (note the partly disorganized histoarchitecture) and (f) Group 6-treated with hexane extract (note the similarity in the histoarchitecture compared with group 1 and 4) |
| CV: Central vein, Red arrow: Hepatocyte, H and E 100× | |
| Fig. 2(a-f): | Photomicrographs of the Ileum in (a) Group 1-normal control (arrow showing intact epithelium), (b) Group 2-negative control (disruption of the epithelium-Black circle), (c) Group 3-treated with methanol extract (note the pattern of orientation and sloughed villi) (Red circle), (d) Group 4-treated with dichloromethane extract (intact epithelium) (arrow), (e) Group 5- treated with ethyl acetate extract (note the pattern of orientation and sloughed villi) (Red circle) and (f) Group 6-treated with hexane extract (note the intact epithelium) |
| H and E 400× | |
| Fig. 3(a-f): | Photomicrographs of the Ileum in (a) Group 1-normal control, (b) Group 2-negative control, (c) Group 3-treated with methanol extract, (d) Group 4-treated with dichloromethane extract, (e) Group 5-treated with ethyl acetate extract and (f) Group 5-treated with hexane extract |
| Elastic fiber: Black VVG 1000× | |
| Fig. 4(a-f): | Photomicrographs of the liver in (a) Group 1-normal control, (b) Group 2-negative control, (c) Group 3-treated with methanol extract, note the scanty reticular fibers, (d) Group 4-treated with dichloromethane extract, (e) Group 5-treated with ethyl acetate extract, Note the scanty reticular fibers and (f) Group 6-treated with hexane extract, note the reticular fibres within the perisinusoidal space and around the wall of the central vein in groups 1, 4 and 6 |
| Reticular fibres: Black Goddon and Sweets 100× | |
| Fig. 5: | Mean reticular fibre density 1600 mm–2 |
Values are given as Mean±SEM, means with different letters differs significantly at p<0.05 while means with the same letters does not differ significantly at p<0.05, Group 1: Normal control, group 2: Negative control), group 3: Treated with methanol extract, group 4: Treated with dichloromethane extract, group 5 Treated with ethyl acetate extract, group 6: Treated with hexane extract |
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Also, the villi were compromised when compared with group 1 (Fig. 2). There were various degrees of remediation in the groups treated with the leave extracts of Celosia trigyna. Remarkable improvements were noticed in the groups treated with DCM and hexane extracts.
Elastic fibers were appreciably deposited in the mucosa and muscularis externa of the ileum in groups 4 and 6 which is comparable with group 1. There were only mild traces of elastic fibers in group 2, 3 and 5 when compared with group 1 (Fig. 3).
The reticular fibers were sparsely stained in group 2 animals when compared with normal control (Fig. 4). There were improvement in the deposition of reticular fibers in groups 4 and 6 when compared with groups 3, 5 (Fig. 4). The reticular fibers in the untreated group was significantly lower (p<0.05) when compared with the normal control. The groups treated with methanol and ethyl acetate extracts were significantly different when compared with the normal control but the group treated with DCM extract was not significantly different from the normal control. The untreated group and the groups treated with methanol and ethyl acetate were not significantly different from each other as shown in Fig. 5.
DISCUSSION
The liver and ileum are very vital organs in the body. While the liver sub serve so many functions among which is: Metabolism, detoxification and production of bile, the ileum on the other hand, is involved in the absorption of vitamin B12, bile salt and every product of digestion that escapes the jejunum. The abuse of ethanol has been on the increase worldwide thus leading to alcoholic liver disease5. Alcoholic liver disease is the leading cause of morbidity and mortality as a result of liver failure, liver cancer and cirrhosis5. Ethanol can induced oxidative stress and by so doing, impair the antioxidant defense mechanism16. When the antioxidant defense mechanism is compromised, this can lead to cellular damage10. Cellular damage to the mucosal affect digestion and assimilation of nutrients leading to weight loss due to malnutrition7 as observed in group 2 of this study. In a similar fashion, Olaibi et al.17 in their work on the histomorphometric study of stomach and duodenum of aspirin treated Wistar rats concluded that weight loss could be due to decline digestive functions as a result of severe injuries by aspirin administration to the gastrointestinal mucosa. There was an improvement in the weight difference of the extract treated groups, this may be due to phytochemicals and antioxidant potentials of Celosia trigyna as reported by Ofusori et al.18. Celosia trigyna contains proteins, amino acids, minerals and vitamins19. In addition, Celosia trigyna is highly rich in magnesium, zinc, calcium, iron and vitamins A, B, C, E, K20,21. Herbal therapies that contain high level of antioxidants are capable of safeguarding body viscera from oxidative damage by reducing free radicals produced by toxicants such as ethanol. Antioxidants are found in majority of herbs consumed by man and animals. In most cases, the antioxidant component of a particular plant may have to work in concert in order to achieve a medicinal potential.
The hepatocellular damage characterized by steatosis, neutrophils infiltration and fat droplets in the liver of the untreated group were gradually been restored to near normal in the extracts treated groups when compared with the normal control. The extracts of Celosia trigyna ameliorated the epithelial damage and disorganization of ileum mucosa when compared with group 1. Better remediation was achieved in the groups treated with DCM and hexane extracts than those treated with methanol and ethyl acetate extracts. This observation is similar to the pattern of events in the study documented by Ofusori et al.13, where they observed that the DCM and hexane extracts ameliorated the gastric lesion induced by ethanol better than the methanol and ethyl acetate extracts. The reason for the effectiveness of DCM and hexane extracts may be due to the rich antioxidant properties. Ofusori et al.13 isolated the following compounds: Lutein, chondrillasterol acetate, chondrillasterol and pheophytin a from the combined hexane and DCM extracts of Celosia trigyna. Lutein which is the most abundant constituent of Celosia trigyna13 has a wide range of beneficial activities which ranges from antioxidant, antihypertensive, antiulcer, antidiabetic and anti-inflammatory and antiatherogenic22-25. According to Serpeloni et al.26, lutein scavenge reactive species and cause gene expression that is needed for improved antioxidant response, thereby enhancing oxygen transport. The positive influence of lutein in the redox state of cells could be due to its antigenotoxic and antioxidant effects.
The elastic fibres are very important in the normal functions of the gastrointestinal tract. They help in the resilient rebound of the stretched wall of the gastrointestinal tract. The gradual deposition of the elastic fibres in the muscularis externa of the ileum of the extract treated groups as against the scanty deposit in the untreated group is suggestive of enhanced mechanical movement necessary to propel the chyme in corroboration with Ofusori et al.27.
Reticular fibres are mesh-like in nature and predominant in viscera such as liver. They provide supportive mechanism for the liver28. Some medical conditions or toxicants can elicit hepatic fibrosis. These conditions, when present, leads to the depletion of reticular fibres (a pathological feature) common in hepatic fibrosis29. A significant improvement observed in the deposition of reticular fibres within the perisinusoidal space and around the wall of the central vein in the extract treated groups is an indication that Celosia trigyna has the capability of halting the advancement of hepatic fibrosis. The mechanism for achieving this may be through the amelioration of hepatocellular degeneration caused by ethanol administration. Ofusori et al.13 reported a significant cellular turn over after the administration of Celosia trigyna in ethanol induced gastric lesion. The synergic effects of these isolated compounds (chondrillasterol, lutein, chondrillasterol acetate and pheophytin a) may have as well been responsible for these observations as reported by Ofusori et al13. Alcohol liver disease is the cause of most death in adult consumers and it is characterized by inconstant degrees of cirrhosis, inflammation and progressive fibrosis30. Cubero et al.31 confirmed that hepatic stellate cells, the cell type implicated in liver fibrosis are always triggered by toxins such as alcohol due to the elicitation of reactive oxygen species-induced damage as observed in this study. Before the development of alcoholic cirrhosis (the end-stage liver disease), abnormalities such as alcoholic hepatitis, steatosis and hepatic fibrosis are the observable features32 characterized by decrease in reticular fibres as noticed in this study. The administration of graded doses of aqueous extract of Camellia sinensis by Lodhi et al.33 in ethanol-induced liver damage led to regeneration of the hepatocytes with little or no evidence of microvesicular steatosis in a similar fashion displayed in this study. Stickel et al.5 and Stickel34 called for great concern in the growing incidence of hepatocellular carcinoma which occurs in about 1-2% of alcoholic cirrhotics per year. Ethanol consumption is deleterious to the liver and ileum as seen in this study and should be avoided or taken with caution, herbal products such as Celosia trigyna is capable of restoring the compromised viscera to near normal and thus recommended for further studies aimed at corroborating our findings.
CONCLUSION
It can be concluded that there were improvements in the histoarchitectural outline of the liver and ileum in the extract treated groups when compared with the untreated group. These improvements were more pronounced in the groups treated with DCM and hexane extracts than those treated with methanol and ethyl acetate extracts. The reason for this observation may be due to the rich phytochemical compounds present in the DCM and hexane extracts. The extract of Celosia trigyna has demonstrated a very good potential in ameliorating the degenerative changes in the liver and ileum of ethanol-induced toxicity in adult Wistar rats.
SIGNIFICANCE STATEMENT
This study discovered that the leave extracts of Celosia trigyna is capable of ameliorating ethanol induced toxicity in the liver and ileum of rats, with the DCM and hexane extracts demonstrating better remediation. Therefore, Celosia trigyna can be beneficial in the management of ethanol-induced complications. This study will assist researchers in the development of alternative therapeutic approach in the management of alcohol related disorders.
ACKNOWLEDGMENT
The authors would like to thank the Research Journal of Medicinal Plants for publishing this article free of cost and to Karim Foundation for bearing the cost of article production, hosting as well as liaison with abstracting and indexing services, and customer services.
The authors also appreciate Mr. M.S Ige of the Department of Anatomy and Cell Biology, Obafemi Awolowo University, Nigeria for his assistance during the histopathological procedures.