Chemical Constituents of Dissotis perkinsiae (Melastomaceae) and Their Antimicrobial Activity

1 Department of Organic Chemistry, Faculty of Science, University of Yaounde I, P.O.Box:812 Yaounde, Cameroon., 2 Department of Chemistry, Faculty of Science, University of Maroua, P.O.Box: 814 Maroua, Cameroon., 3 Department of Organic Chemistry, Higher Teachers Training College, University of Bamenda, P.O.Box: 39 Bambili, Cameroon., 4 Department of Biochemistry, Faculty of Science, University of Yaoundé I, P.O.Box: 812 Yaoundé, Cameroon., 5 Max Plant Institute for Chemical Ecology, Beutenberg Campus, Hans-Knol.8, D-07745 Jena, Germany.


INTRODUCTION
Application of natural products with therapeutic properties is as old as human civilization.These products have been exploited for human use from thousands of years, and plants have been the chief source of compounds used for medicine.In fact, plants represent the largest sources of active substances that can be used in medical therapy due to the large structural diversity that these metabolites exhibit, being perhaps the oldest source of medicines for man (Brandão et al., 2010).Drugs derived from natural products with antibacterial, antifungal, anticoagulant, antiparasitic, immune suppressive and anticancer activity are capable of treating 87 % of categorized human diseases (Newman et al., 2003).Of the 520 new drugs approved between 1983 and 1994, 39 % were natural products or derived from natural products and 60-80 % of antimicrobial were derived from natural products (Cragg et al., 1997).Such data justifies work in the area of natural products, particularly in view of its importance in the search for new drugs against bacterial and fungal infections.The genus Dissotis, member of the Melastomaceae family is one of the most important genus in tropical forests which represent about 140 species in Africa (Loigier, 1994).
They are climbing shrubs, shrubs or small trees of up to 2 m, and are found in countries as Democratic Republic of Congo, Benin, Nigeria, Cote d'Ivoire, Togo and Cameroon (Maluma, 2005).Several species are used in folk medicine, mainly as antidiarrheic, antimicrobial, skin diseases, antioxidant, fever, malaria, cleansing, antitumoral, anti-rheumatic, and anti-inflammatory agents, as well as to lower blood cholesterol.Dissotis perkinsiae, found in tropical areas of Nigeria, Togo and Cameroon is a shrub with the height of 90-150 cm.The leaves are purple or pink with a diameter of about 10.5 cm (Hutchinson and Dalziel, 1954).It is used in traditional medicine for the treatment of the skin diseases and malaria (Haxaire, 1979).In previous studies, some triterpenoïds, flavonoïds and tannins were identified (Ndjateu et al, 2014).Such data have encouraged the investigation of Dissotis perkinsiae with the aim to search for compounds with antibacterial and antifungal activities.

Sample collection
Dissotis perkinsiae leaves were harvested in Bangoua-Cameroon, (May, 2012) and identified by Mr. Victor Nana (Plant taxonomist) of the Cameroon National Herbarium (HNC), where a voucher specimens are deposited (24719/SRF/Cam).Then, leaves were dried at room temperature and powdered.

Extraction and isolation
1700 g of powdered leaves were extracted by maceration in seven liters (7L) of dichloromethane/methanol (DCM/MeOH, 1:1, v/v) mixture at room temperature for 72 h.The filtrate was concentrated in rota vapor  under reduce pressure at 65 °C to yield 240g of extract.Dried extract was dissolved in water and successively extracted with hexane, dichloromethane, ethyl acetate and n-butanol.All these extracts collected were preserved for chemical analysis.

Antimicrobial activity of Extracts/Fractions and Compounds Tested microorganisms
The micro organisms include bacteria (Staphylococcus aureus NR46374, Klepsiella pneumonia NR41916, Salmonella enterica NR13555, Shigella flexineri NR518) and fungal strains (Candida albicans ATCC P37037, Candida krusei ATCC 6258, Candida parapsilosis ATCC 22019) originally obtained from BEI resources and American Type Culture Collection respectively.The organisms were maintained on agar slope at 4°C and sub-cultured for 24h and 48 h respectively for bacteria and yeasts before use.

Preparation of stock solution of plants crude extracts, fractions and compounds
The different stock solution of crude extracts, fractions and compounds were prepared by dissolving 2 mg in 1 mL of DMSO 10 % for a final concentration of 2 mg/mL.Reference antibiotics were prepared in the same condition by dissolving 512 µg of Fluconazole (Sigma Aldrich) and 2mg of Chloramphenicol and Ampicillin (Sigma Aldrich) in 1 mL of DMSO10 % to yield 512µg/mL and 2 mg/mL respectively.After preparation, the different stock solutions were sterilized with 0.20 µM Syringe Filter and store at -20°C before use.

Antifungal activity Determination of minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC)
The minimum inhibitory concentration (MIC) was determined according to Clinical Laboratory Standards Institute M27-A3 microdilution method (CLSI, 2008) using (12 x 8 wells) microtitre plates.In the well of the first line (1-12), 100 µL of RPMI 1640 (Sigma Aldrich) medium were introduced and 100 µL in the other well of the plates.Later on, 100 µL of stock solution of crude extracts, fractions and compounds were added to the first well.The medium and extract/compound in the first well were mixed thoroughly before transferring 100 µL of the resultant mixture to the well of the second line.Serial two-fold dilutions of the test samples were made and 100 µL of inoculum standardized at 2.5 × 10 4 cells/mL were introduced in the entire well containing the test substances except the column of blank which constitute the sterility control.The concentrations ranged from 0.000488 to 0.5 mg/mL and from 1.25µg/mL to 128µg/mL for crude extracts/fractions/compounds and fluconazole respectively.After 48 hours of incubation at 37°C, the turbidity was observed as indication of growth.MIC was defined as the lowest concentration inhibiting the growth of yeasts.The MFC was determined by transferring 50 µL aliquots of the clear wells into 150 µl of freshly prepared broth medium and incubating at 37°C for 48 hours.The MFC was regarded as the lowest concentration of test sample which did not produce turbidity as above, indicating no microbial growth.All tests were performed in triplicates.

Determination of minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC)
The MIC was determined according to Clinical laboratory Standards Institute (CLSI) M38-A microdilution method (CLSI, 2008) using 96 wells microtitre plates.100µL of Muller Hinton Broth (Lab M Limited Topley House) was introduced in the wells of column 1 (1A-1H), and 100µL in the remaining wells.Later on, 100µL of stock solutions of plants extracts/compounds at 2 mg/mL were added to the first well.The medium and sample in the first well were mixed thoroughly and serially diluted by transferring 100 μL from wells of column 1 to wells of column 2, and so on till column 11.
Thereafter, 100µL of the inoculums were introduced in each well containing the test substances.The wells of column 12 are used as blank.The concentration of substances range from 0.5 mg/mL to 0.000488 mg/mL.After an incubation period at 37° C for 24 hour, turbidity was observed as indication of growth.Thus, the lowest concentration inhibiting the growth of bacteria was recorded as the MIC.The MBC was determined by transferring 50 µL aliquots of the clear wells into 150 µl of freshly prepared broth medium and incubating at 37°C for 24 hours.The MBC was regarded as the lowest concentration of test sample which did not produce turbidity as above, indicating no microbial growth.All tests were performed in triplicates.

Antimicrobial results
The results of antibacterial and antifungal activities of extracts, fractions and compounds from Dissotis perkinsiae are presented in tables 1, 2, 3 and 4.The results indicate that the MIC vary from >0.5 to 0.00078 mg/mL on yeasts and from >0.5 to 0.25 mg/mL on bacteria.This inhibition was found to be extracts, compounds and microorganism dependent.The activity of ethyl acetate and hexane fractions were better than crude extract on the three yeast species.Compounds 2 (Sitosterol-β-D-glucoside) was the most active with the broad spectrum.It is the most active on C. albicans and C. parapsilosis while, compounds 6 (Kaempferol-7-O-β-D-glucoside) was the most active on C. krusei.The antiyeats activity of Sitosterol-β-D-glucoside and Kaempferol-7-O-β-Dglucoside were better than fluconazole (0.032mg/mL) on C. parapsilosis (MIC 0.0078 mg/mL) and C. krusei (0.0039mg/mL) respectively.Sitosterol-β-D-glucoside and Kaempferol-7-O-β-Dglucoside exhibited fungicidal activity on the tested microorganisms, high light the ability of these compounds to kill Candida species.On bacteria, the activity of crude extract was better than fractions.Compounds 2 and compounds 7 showed weak inhibitotry activity respectively against S.enterica and S. aureus with MIC value of 0.5mg/mL.This inhibitory effect was less than activity of Chloremphenicol and Ampicillin (0.000488 mg/mL).
The antimicrobial activity of Sitosterol-β-D-glucoside and Kaempferol-7-O-β-D-glucoside can be explained by the substitution of hydroxyl group in the β-Sitosterol and Kaempferol skeletons a glucoside group which enhance their activity.In fact, the glucoside group is known to be responsible for antimicrobial activity due to their ability to form a complex with cell membrane of microorganisms, and thus, inhibiting microbial growth (Cowan, 1999).The difference in activity of the tested compounds on yeasts and bacteria can be explained by genetic distance or specific mechanisms of action on the cell membrane of the microorganisms.In the same way as many antifungal drugs, these compounds could act on ergosterol biosynthesis.Absent in bacteria, ergosterol isone of the key components of the fungal cell membrane and the main sterol of yeasts and other fungi.It is necessary for the growth of cells and normal membrane function.It serves as a bioregulator of membrane fluidity, asymmetry and membrane integrity and contributes to the proper function of membrane-bound enzymes.Thus, the inhibition of ergosterol biosynthesis lead to the death of fungi cell (Lupetti et al., 2002).

CONCLUSION
The results provide justification for the use of Dissotis perkinsiae in folk medicine to treat various diseases.This study might be considered as a prelude to discover new antimicrobial agents to the problematic pathogenic bacteria and fungi.Moreover, the broad spectrum activity of isolated compounds as Sitosterol-β-D-glucoside gives the opportunity for possible discovery of new, effective components for downstream clinical development.Whereas, many studies have to be carry out to obtain insight into its mode of action and antifungal activity.

Table 1 :
MIC of extract, fractions and compounds on yeasts strains (mg/mL).