Screening of some Cameroonian Medicinal Plants against Bacterial and Yeasts involved in Gastrointestinal Disorders

The present study was designated to evaluated the antimicrobial activities of methanol and ethyl acetate extracts of Carcia arereh, Nelsonia canescens, Ficus thonningu, Bryophillum pinnatum, Cyclosorus striatus, Euphorbia cordifolia, Euphorbia hirta, Erygium foetidum and Mimosa pudica. The selected plants species are used as traditional folk medicine in Cameroon for the treatment of various diseases. Thiazolyl blue tetrazolium bromide colorimetric assay was used to evaluate the antimicrobial activity against 16 microbial species. Preliminary phytochemical screening of plant sample was also carried out. Alkaloids and anthraquinones were the most frequent secondary metabolite in the tested plant extracts. The MIC values obtained ranging from 32 to >1024 g/ml and 128 to >1024 g/m respectively for bacterial and yeast strains tested. Methanol extract of Ficus thonningu leaves shows the best antibacterial activity with MIC values of 32 g/ml against Echerichia coli. The lowest MIC values obtained against yeasts strains was 128 μg/ml. This lowest MIC values was obtained with Erynguim foetidum (AE) Euphorbia hirta (EA and MeOH) and Carcia arereh (MeOH) against 12.5 (1/8), 25 (2/8) and 25% of the yeasts tested strains The results might explain the ethnobotanical use of the studied species for the treatment of gastrointestinal infections and afflictions of the skin.


Introduction
Infectious diseases including bacterial infections continue to be a serious health problem worldwide.Therapy with synthetic tropical applications have most side effects and cannot be afforded by the people due to import cost of the drug.Because of the side effects and the resistance that pathogenic microorganisms build against antibiotics, much recent attention has been paid to extracts and biologically active compounds isolated from plant species used in herbaI medicine (Kokoska et al., 2002;Nostro et al., 2000).
Herbal medicines are the synthesis of therapeutic experiences from generations of traditional healers, and in many countries, the plant drugs are still in use in its traditional way and modern health care systems for treatment of different disorder and to improve the health.It has been estimated that between 60-90% of the populations of developing countries use traditional and botanical medicines almost exclusively and consider them to be a normal part of primary healthcare (WHO, 2002).Consumers are increasingly interested in complementary and alternative medicines, including herbal medicine, as they perceive these forms of healing as being both safe and effective (Gangoue et al., 2004).This trend in use of alternative and complementary healthcare has prompted scientists to investigate the various biological activities of medicinal plants, because they contain many bioactive compounds that can be of interest in therapeutic and also because of their low toxicity.Plant based antimicrobials represent a vast untapped source for medicines and, further exploration of plant antirnicrobials needs to occur.
The present study was conducted to investigate antimicrobial properties of 20 extracts from nine medicinal plants against 16 human pathogenic bacteria and yeast implicated in respiratory, gastrointestinal and systemic infection.The selected plants species are use as traditional medicine in Cameroon for the treatment of various diseases such as Treatment of gastrointestinal Disorders, afflictions of the skin and mucous membranes, respiratory system disorders, parasitic infections, gastric ulcer, hypertension, headache, earache, asthma, arthritis, snake bites, scorpion stings and epilepsy.The ethnobotanical data on the use of these plants and the selection of the plant part to be tested where complemented with a biographic review (Adjanohoun et al., 1996).

Plant Material
Nine Cameroonian medicinal plants were used in this study.The selected plants materiel used were collected in different region of Cameroon.The plants were identified at the National herbarium (Yaoundé, Cameroon) where voucher specimens were deposited under the reference numbers (Table 1).

Sample Extraction
Each plant sample was washed, air dried and finely ground into a fine powder.The obtained powder (200 g) was soaked in either methanol (MeOH; 1l) or ethyl acetate (AE; 1l) for 48 h at room temperature and were homogenized regularly.The plant mixture was then filtered through Whatman n 0 1 paper and concentrated under reduced pressure using a rotary evaporator.The extracts were collected and dried in an oven at 40 °C for complete evaporation of the extraction solvent.The performance of each extraction was evaluated in relation to the mass of dry plant starting material.All extracts were then kept at 4 °C until further use.

Preliminary Phytochemical Screening
Preliminary analysis of chemical constituents such as alkaloids, anthocyanines, flavonoids, saponins, tannins, anthraquinones, polyphenols, sterols and/or triterpenes, and steroids was carried out.These constituents were identified by characteristic colour changes using standard procedures previously described (AOAC, 2005).Each test was qualitatively expressed as negative (−) or positive (+) signs.

Test for Alkaloids (Meyer Reagent)
Fifty milligrams of the tested compound was heated in 10 ml of 2% H 2 SO 4 for 2 min and filtered.To 1 ml of the filtrate, a few drops of the Meyer Reagent was added.The presence of alkaloids was indicated by the obtaining of a white precipitation or turbidity.

Test of Flavonoids
Fifty milligrams of the tested compound was introduced in 5 ml of methanol.To it was added some magnesium chip and concentrated hydrochloric acid drop wise.The presence of flavonoids was indicated by the appearance of a violet or brick red coloration with effervescence.

Test for Saponins
Fifty milligrams of the tested compound was added to 5 ml of distilled water.After homogenization, the mixture was boiled in 5 min, cooled and filtrated.5 ml of the filtrate was vigorously agitated for 10 to 30 s.The presence of saponins was indicated by the appearance of foam which persists within 15 min.

Test for Tanins
Fifty milligrams of the tested compound was added in 5 ml of distiller water.After mixing, the mixture was boiled for 5 min, cooled and filtrated.5 ml of 2% NaCl was added to 10 ml of the filtrate and then filtered.The presence of tannins was indicated by the formation of a precipitate after the addition of 1% gelatin to the filtrate.

Test of Anthraquinones
Fifty milligrams of the tested compound was diluted in 2 ml of chloroform, homogenized and filtered.Next, 1 ml of 10% NaOH was added to 1 ml of the filtrate.The presence of anthraquinone was indicated by the appearance of a red coloration.

Test of Phenols and Polyphenols
Fifty milligrams of the tested compound was dissolved in 15 ml distilled water, headed in a water bath for 15 min then cooled and filtered.To 2 ml of the filtrate was added drop wise 1 ml of 1% FeCl 3 and 1 ml of 1% K 3 Fe(CN) 6 .The presence of polyphenols and phenols was indicated by the apparition of a blue and green precipitate respectively.Treatment of parasitic infections, diarrhoea, dysentery, malaria, dermatitis, and skin infections (Kochar et al., 1981;Abo et al., 1998) Anti-trypanosomiasis (Saidu et al., 2013) Antimicrobial properties (De N, et al., 2009) Antioxidant (Kouitcheu et al, 2015) Nelsonia canescens (Acanthaceae) N° 39542/ HNC Leaves,
Other reported properties include antitumour, sedative and muscle relaxant effects (Raymond et al., 2010).
It may also be effective in the treatment of leishmaniasis (Raymond et al., 2010).Antioxidant and anti-Helicobacter pylori (Kouitcheu et al., 2016) Cyclosorus striatus

Whole plant
The herb has been used traditionally for ages, in the treatment of urogenital disorders, piles, dysentery, sinus, and also applied on wounds (Hafsa et al., 2012).

Test of Triterpenes and Sterols (Liebermann-Burchard Test)
Fifty milligrams of the tested compound was dissolved in 2 ml of chloroform and next some drops of acetic anhydride added to the mixture and 1 ml of concentrated H 2 SO 4 .The presence of triterpenes was revealed by the apparition of violet-red coloration and sterols by a blue greenish coloration.

Test of Steroids
Fifty milligrams of the tested compound was dissolved in 3 ml of chloroform, agitated intermittently for 2 hours and filtered.1 ml of the filtrate was deposited on a porcelain plate.After evaporation, a drop of sulfuric acid was added, stirred and the color noted.To another portion of the filtrate was added a drop of acetic anhydride and the color noted.The reaction was positive if the same color was obtained for both sulfuric acid and acetic anhydride.

Test for Anthocyanins
To 50 mg of the tested compound was added 15 ml of a concentrated HCl, the mixture was boiled.The presence of anthocyanins was indicated by the variation of coloration from orange red to orange blue during boiling.

TBTB Colorimetric Assay for MIC Value Determination
Minimum Inhibitory Concentrations (MICs) were determined by broth micro dilution method previously described by Kuete et al., 2008 with slight modifications.The test samples were first of all dissolved in dimethylsulfoxide and/or Tween 80 (1% (v/v).The solution obtained was then added to MHB for bacteria or SDB for yeasts to give a final concentration of 2048 μg/ml.This was serially diluted two fold in 96-wells microplate.A volume of 100 μl of inoculums (2x10 5 CFU/ml for the yeasts and 2x10 6 CFU for bacteria) was added in each well (96-wells microplate) for final concentrations varying from 0.25 to 1024 μg/ml.The negative control well consisted of 100 μl of appropriate medium (MHB or SDB) with DMSO or Tween 80 (1% v/v) and 100 μl of the standard inoculums.The plates were covered with the sterile lid, then agitated to mix the contents of the wells using a plate shaker and incubated at 37°C for 24 h (for bacteria) or at 25 0 C for 48 h (for yeasts).The assay was repeated thrice.The MICs of samples were determined by adding 50μl of a 0.2 mg/ml thiazolyl blue tetrazolium bromide (TBTB, Sigma-Aldrich) solution followed by incubation at 35°C for 30 min.the TBTB is reduced by viable bacterial dehydrogenases into formazan, a violated blue dye (Gomez-Flores et al., 1995;Sankar et al., 2008).MICs were defined as the lowest sample concentrations that prevented this color change, thus indicating a complete inhibition of microbial growth.

Results and Discussion
The results of the qualitative phytochemical analysis showed that each of the tested plant extract contains at least 3 classes of secondary metabolites (Table 2).Alkaloids and anthraquinones were the most frequent secondary metabolites in the tested plant extracts.

Nelsonia canescens
Nelsonia canescens Ficus thonningu (Leaves) The antimicrobial activity of each extract against microorganisms examined in the present study and their potency were quantitatively assessed by the determination of MIC values.The MIC value obtained using the broth microdilution method is given in Table 3 and 4.
Table 3.The MIC values of the plant material extracts against the bacterial strain tested using microdilution assay  It was considered that if the extracts displayed an MIC less than 100 μg/ml, the antimicrobial activity was good ; from 100 to 500 μg/ml the antimicrobial activity was moderate; from 500 to 1000 μg/ml the antimicrobial activity was weak; over 1000 μg/ml, the extract was considered inactive (Fabiola et al., 2002).Our results shown that most of the extract presented a moderate activity against the pathogenic bacteria and yeasts tested.
Methanol extract of Ficus thonningu leaves presented a good activity against Echerichia coli, following by ethyl acetate extract of Nelsonia canescens against Enterococcus feacalis.Salmonella typhi CA and CK were the most resistant strains tested.This differences in susceptibility between the bacterial strains against antimicrobial substances in plants extract may be explained by the differences in cell wall composition and/or inheritance genes on plasmids that can be easily be transferred among bacterial strains (Karaman et al., 2003).
The results of the qualitative phytochemical analysis showed that alkaloids and anthraquinones are the most frequent secondary metabolite in the tested plant extracts.The presence of these chemical compounds in extracts may explain some of their antimicrobial actions since antimicrobial actions of most of these phytochemical substances have been documented (Kouitcheu et al., 2011).However, the antimicrobial activities demonstrated the could be due to the presence of other antimicrobial substances not covered by the screening.

Conclusion
The results in the present work indicate that some of the plant species assayed possess antimicrobial properties and justify the use of these plants in folk medicine for the treatment of various infected diseases.At present, our group is concerned with the fractionation and the isolation of pure compounds and the elucidation of their structures in order to better evaluate their pharmacological activity in vitro and in vivo.

Table 1 .
Information on the studied medicinal plants

Table 4 .
The MIC values of the plant material extracts against the yeast strains tested using microdilution assay Plant species showed different anti-microbial activity each other with MIC values ranging from 32 to >1024 g/ml and 128 to >1024 g/m respectively for bacterial and yeast strains tested.Methanol extract of Ficus thonningu leaves shows the best antibacterial activity with MIC values of 32 g/ml against Echerichia coli , following by ethyl acetate extract of Nelsonia canescens against Enterococcus feacalis with MIC value of 64 g/ml.It appears that extracts from Mimosa pudica (AE), Euphorbia hirta (AE), Cycloorus striatus (MeOH), Bryophyllum pinnatum (AE), and Carcia arereh (MeOH) inhibited the growth of all the eight tested bacterial strains within a concentration range from 128 to 256 μg/mL.The most susceptible bacterium was E. coli following by Enterococcus feacalis with a susceptibility of 65 (13/20) and 60% (12/20) respectively of the tested plant extract.Moreover, the lowest MIC values (32 and 64 μg/mL) was obtained against these bacterial strains.Salmonella typhi was the most resistant bacterium tested since only 35% (7/20) of extract evaluated here had showed an inhibitory activity against it.Plant sample were more active against bacterial stains than fungal one.Indeed, the lowest MIC values obtained against yeasts strains was 128 μg/ml while it was 32 μg/ml against bacteria strains.This lowest MIC values was obtained with Erynguim foetidum (AE) Euphorbia hirta (EA and MeOH) and Carcia arereh (MeOH) against 12.5 (1/8) , 25 (2/8) and 25% of the tested yeasts strains.Moreover, MIC values of 256 g/ml were also recorded with Euphorbia hirta (EA) and Carcia arereh (AE and MeOH) against 12.5 and 25% of the evaluated yeast.So, these two plant extracts; Euphorbia hirta (EA) and Carcia arereh (MeOH) show the best antifungal activity.The most sensitive yeast was Candida guilliermondi, following by Candida tropicalis, Cryptococcus neoformans, Candida parapsilosis, Candida glabrata and Candida lusitaneae with a susceptibility of 10 (2/20) and 5(1/20)% respectively of the tested plant extract.Candida albicans and Candida krusei were the most resistant yeast tested since none of extract evaluated here had showed any inhibitory activity against them.