Intestinal ameliorative effects of traditional Ogi-tutu, Vernonia amygdalina and Psidium guajava in mice infected with Vibrio cholera.

Background: Cholera, a severe acute watery diarrhea caused by Vibrio cholerae is endemic in Nigeria with most cases occurring in the rural areas. In South West Nigeria, some individuals resort to alternative treatments such as Ogi-tutu, Psidium guajava and Vernonia amygdalina during infections. The effectiveness of these alternatives in the prevention and treatment of V. cholerae infection requires experimental investigation. Objective: This study was designed to investigate the ameliorative effects of Ogi-tutu, Vernonia amygdalina and Psidium guajava on intestinal histopathology of experimental mice infected with V. cholerae . Methods: Preliminary investigation of in vitro vibriocidal activities of these alternatives were carried out using agar cup diffusion assay. For ameliorative effects, adult mice were inoculated with 100 μl (106 cells) of Vibrio cholerae and dosed at 0 h (immedi ate prevention) and 4 h (treatment of infection) and their intestines were histopathologically evaluated. Results: The histopathological changes were the same irrespective of the treated groups, but the lesions varied in extent and severity. The ameliorative effects in decreasing order were V. amygdalina > P. guajava > Ogi-tutu . Conclusion: V. amygdalina gave the best ameliorative effects in the prevention and treatment of V. cholerae infection.


Introduction
Vibrio cholerae is a human pathogen that causes a severe acute watery diarrhea called cholera, a waterborne disease characterized by severe dehydration 1,2 .Vibrio cholerae is a major public health problem because of its epidemic potential 3 .Though, there are approximately 200 serotypes, only V. choleare O1 and O139 have been found in epidemic and pandemic cholera 4 .Without treatment, the case fatality rate for severe cholera can be as high as 50% 5 .
Nigeria is in one of the three major cholera foci in the world, with frequent outbreaks and epidemics from different parts of the country 6 .In SouthWest Nigeria, majority of the people living in rural areas use traditional medicines to treat all types of diarrhea 7 .This is in line with the World Health Organization (WHO) estimates of approximately 80% of the world's inhabitants that rely on traditional or herbal medicines for their primary health care 8 .
Regarded as traditional remedy for diarrhea and specifically for the treatment of cholera, is the use of "ogi-tutu" and this is acclaimed to stop the characteristic cholera symptoms.Ogi-tutu is the wet cake mixed with omidun (sour-water) [9][10] .Ogi is a porridge prepared from fermented maize, sorghum or millet in West Africa 11 .Ogi may have potentials for treatment of diarrhea, due to its high level of lactic acid bacteria (LAB) 10 .Also, several plants are used locally in the treatment of diarrhoea [11][12][13][14] .Psidium guajava L. and Vernonia amygdalina [15][16] are two of such plants that are used in SouthWest Nigeria.Vernonia amygdalina is a dark green vegetable known as bitter leaf in English, and ewuro in Yoruba land 17 .The roots and leaves are used in phytomedicine to treat fever, hiccups, kidney disease and stomach discomfort, and as antihelminthic and antimalarial 13 .Psidum guajava L. commonly known as guava of the family Myrtaceae is a plant used in the indigenous system of medicine for the treatment of various human ailments such as wounds, ulcers, bowels and cholera 18 .The plant is also known for its antidiabetics, antispamodic, antidiarrheal and antidysentary properties [19][20] .Empirical proof of these traditional treatments in ameliorating histopathological effects of Vibrio cholerae in the intestinal mucosa need to be investigated.

Study design
A randomized controlled experimental design was used in this study.Ethical approval for the study was obtained from the Research and Ethical Committee, Department of Microbiology, College of Biosciences, Federal University of Agriculture, Abeokuta, Nigeria through Research Proposal Presentation and Approval (Approval number: 2005/06/01/01) .

Vibrio cholerae strains
Toxigenic strain of V. cholerae isolated from an epidemic case in Nigeria was used for the study.A loopful of the working stock was transferred into 3 ml Tryptone Soy Broth (TSB, Oxoid) containing NaCl at final concentration of 3% (Na-TSB broth) and incubated at 37 o C for 18 h.Before inoculation, V. cholerae was grown in Luria Bertani (LB) broth containing ampicillin (50µg) and Streptomycin (50µg) at 37 o C on a roller shaker for 16 to 18 hrs.

Preparation of Ogi-tutu
Corn-Ogi, was prepared by spontaneous traditional fermentation.Ogi slurry was prepared by adding 100 ml of sour water (omidun) to 50 g of ogi cake and then, mixed thoroughly.A hundred milliliters (100 ml) slurry of ogi was measured into sterile 250 ml Erlenmeyer flask.Before inoculation, the ogi slurry samples were maintained at experimental temperature (37 o C) for 1 to 2 hrs to allow for equilibration 21 .

Source of Vernonia amygdalina extract and Psidium guajava decoction
Fresh leaves of each plant were purchased from herb sellers at Itoku market in Abeokuta, Ogun State, Nigeria.Voucher specimens were taken to the Herbarium Unit of the Department of Forestry and Wildlife Management, Federal University of Agriculture, Abeokuta.Vernonia amygdalina was classified as Asteraceae Grp 1 and Psidum guajava L. as Myrtaceae Grp 1. Herbarium sample specimens were deposited with voucher numbers FUNAAB/ FWM/HU/Asteraceae 1/005 and FUNAAB/FWM/ HU/Myrtaceae 1/030.

Traditional Preparation of Vernonia amygdalina extract and Psidium guajava decoction
Raw extract of Vernonia amygdalina was obtained by crushing 100 g using sterile mortar and pestle.Decoction of Psidium guajava was prepared by boiling 100 g of the shade dried leaves in 100 ml distilled water till the volume was reduced to 25ml.The resulted extracts were centrifuged and filtered through a membrane of 0.22 μm pore size before use for investigation.

Procedure for ethanolic extraction of Vernonia amygdalina and Psidium guajava
The leaves were dried at 40 o C in the hot-air oven, powdered and stored in an airtight container for use.The powders were then soaked in 50% redistilled (aqueous) ethanol in a glass container for 48 h.This was followed by filtration.The filtrate was concentrated to dryness using water bath at 40 o C and finally in a dessicator unit.

Preliminary vibriocidal activity by agar diffusion cup plate assay
Agar diffusion method was used to evaluate the vibriocidal activity of the extracts using 100 µl of extracts.Diameters of zones of inhibition were measured to the nearest millimeter.

Oral inoculation of mice
Adult mice (7 to 10 weeks old) were purchased from the Department of Veterinary Physiology, University of Ibadan, Nigeria and transported to the experimental unit of the Department of Pure and Applied Zoology, College of Biosciences, Federal University of Agriculture, Abeokuta, Nigeria where they were left to acclimatize for 72 hrs.During the period of acclimatization, animals were treated by following strictly, the principles of laboratory animal care" (NIH Publication No. 85-23) and mice were fed with pelleted feed and water ad libitum.
Prior to V. cholerae inoculation, the animals were fasted for 24 hrs prior to the experiment and were randomly divided into ten groups, with three animals per group.Eight groups were inoculated orally with 100μl (106 cells) of toxigenic V. cholerae via syringe while the ninth and tenth group served as uninoculated control group.Out of the eight inoculated groups, only six were dosed with ogi-tutu (500 mg/ml), ethanolic extracts of P. guajava (250 mg/kg) and V. amygdalina (250 mg/kg), three groups at 0 hrs (immediate prevention) and three groups at 4 hours (treatment of infection).The remaining two untreated groups were given only distilled water and they served as infected but not treated positive control.Each treated group of animal was monitored for 24 h after which they were sacrificed.However, the uninfected control group (negative control) was sacrificed at 0 hrs and 4 hrs for histopathological comparison.Also, the infected but untreated group (positive control) at 0 hrs was also sacrificed immediately while the delayed group was sacrificed after 24 hrs.
The summary of the experimental treatment groups: Group 1: Inoculated orally with V. cholerae; dosed with Ogi-tutu at 0 hrs; sacrificed at 24 hrs Group 2: Infected with V. cholerae; dosed with Ogi-tutu at 4 hrs; sacrificed at 24 hrs Group 3: Infected with V. cholerae; dosed with V. amygdalina at 0 hrs; sacrificed at 24 hrs Group 4: Infected with V. cholerae; dosed with V. amygdalina at 4 hrs; sacrificed at 24 hrs Group 5: Infected with V. cholerae; dosed with P. guajava at 0 hrs; sacrificed at 24 hrs Group 6: Infected with V. cholerae; dosed with P. guajava at 4 hrs; sacrificed at 24 hrs Group 7: Infected with V. cholerae; untreated (positive control); sacrificed at 0 hrs Group 8: Infected with V. cholerae; untreated (positive control); sacrificed at 24 hrs Group 9: Not infected (Normal negative control); sacrificed at 0 hrs Group 10: Not infected (Normal negative control); sacrificed at 4 hrs representing 4 hrs

Routine histopathology
Mice intestinal samples were eviscerated and harvested after 24 hour.These were immediately washed briefly in phosphate buffered saline (PBS) and then fixed in buffered 10% formalin.After 48 h, the intestinal tissues were trimmed and dehydrated in graded levels of alcohol (50%, 70%, 80% and 100%) and cleared in two changes of xylene.Tissues were embedded in paraffin wax, sectioned at 5 µm and stained with haematoxylin and eosin stains.The stained specimens were further dehydrated in graded levels of ethanol and cleared in xylene.The photomicrographs of the tissues were viewed and taken under light microscope (Leica, UK).
The degree of intestinal histopathological changes were semi-quantified depending on the severity and distribution of the intestinal lesions.Histopathological changes such as degeneration and necrosis of enterocytes, inflammatory cellular infiltration in the lamina propria, regeneration of enterocytes, stumpy and club-shaped villi and goblet cell hyperplasia were scored or assigned as follows: (-) = absent, (+) = 1 to 4 foci/section examined, (++) = 5 to 8 foci/section examined, (+++) = > 9 foci/ section examined.

Preliminary investigation of vibriocidal activities
The three traditional treatment proved effective at preliminary investigation as displayed in clear halos of growth inhibition of Vibrio cholerae.Also, both the traditional and 50% ethanolic extracts of V. amygdalina and P. guajava exhibited similar inhibitory activities.

Histopathology of Ogi-tutu, Psidium guajava and V. amygdalina
The histopathological changes were the same irrespective of the treated groups, but the lesions varied in extent and severity.Table 1 shows that at zero and fourth hour p.i., animals treated with ogi tutu showed moderate diffuse degenerative and inflammatory changes with most of the exudate made up of neutrophils and few lymphocytes.
The villi were stumpy and club-shaped in appearance with mild goblet cells hyperplasia (Plate 1).Tissues treated with Psidium guajava (Plate 2) and V. amygdalina (Plate 3) demonstrated marked anti-inflammatory and anti secretory properties at both zero and 4 th hour p.i, evidenced by the very mild degenerative and inflam-matory changes with mild goblet cells proliferation in the intestinal mucosa of the groups treated with these plants.
The anti-inflammatory and anti-secretory activities were more prominent in V. amygdalina and P. guajava compared with Ogi-tutu.
The infected but untreated group (Plate 4) showed severe infiltration of the lamina propria with neutrophils and lymphocytes, proliferation and regeneration of enterocytes with stumpy and club-shaped villi in this study.
Moderate goblet cells proliferation was also observed, but there was no significant change in the negative control group (Plate 5).

Discussion
Several traditional treatments of diarrhea and specifically, cholera are in practice in SouthWestern Nigeria.These traditional claims require experimental investigation to ascertain their effectiveness.Preliminary investigation of vibriocidal activities of Ogi tutu, V. amygdalina and P. guajava showed in vitro vibriocidal activities.These agreed with findings on antibacterial activities documented in the literature 16,19,[22][23][24] .
However, investigation on histopathological effects gave varied ameliorative effects.Ogi-tutu (500 mg/ml) was less effective in treating V. cholerae infection as evidenced by the moderate diffuse degenerative and inflammatory changes observed at histopathology.Ogi-tutu contain lactic acid bacteria (LAB) which have been shown to possess or have antimicrobial potentials and it is significant in the management of human clinical infections [22][23] .
Vernonia amygdalina showed very high preventive and effective treatment measures in both the immediate and delayed groups of mice demonstrating excellent ameliorative effect and very mild degenerative changes in vivo.
Similar antidiarrheal activity was demonstrated in castor oil induced diarrhea 24 .The varied biological activity of V. amygdalina is said to be likely due to the combination of its active compounds such as saponins and alkaloids, terpenes, steroids, coumarines, flavonoids, phenolic acids, lignans, xanthones and anthraquinones edotides, tannins and sesquiterpene lactone 25 .
Psidium guajava displayed very high activity as both prevention and treatment of V. cholerae infections when compared with positive and negative control groups.The ameliorative effects of this plant were attested to by the very mild degenerative and inflammatory changes observed in this study.Antidiarrheal activity of the leaves has been proven 26 and also, of the fruit in castor oil induced diarrhea 27 .The decoction of the leaves is capable of neutralizing Vibrio cholerae 28 .Pharamacological investigation indicated that its bark, fruit and leaves posses antibacterial, hypoglycemic, anti-inflammatory, analgesic, antipyretic, spasmolytic and CNS depressant properties 19 .

Conclusion
Ogi-tutu displayed less ameliorative effects in the prevention and treatment of V. cholerae infection.However, V.
amygdalina showed better ameliorative effects than P. guajava in infected mice.The ameliorative effects in decreasing order were V. amygdalina > P. guajava > Ogi-tutu.Vernonia amygdalina could be used for preventive and treatment of Vibrio cholerae infection.Further investigations on the active principles of V. amygdalina with its mechanism of action are warranted.This might channel a new pathway and better alternative therapeutic agents in the management of diarrhea associated with V. cholerae.