Phytochemistry and in vitro Antimicrobial , Antioxydant Activities of Entandrophragma candollei H

1 Institute of Medical Research and Medicinal Plants Studies P.O Box 6163 Yaoundé, Cameroon. 2 Department of Galenic and Legislation, Faculty of Medicine and Biomedical Sciences, University of Yaoundé I, P.O Box: 337Yaoundé, Cameroon. 3 Department of Pharmacy, Faculty of Medicine and Pharmaceutical Sciences, University of Douala, P.O Box: 2701 Douala, Cameroon. 4 Departments of Organic Chemistry, University of Yaoundé I, P.O Box 812, Yaoundé, Cameroon. 5 Departments of Biochemistry, University of Yaoundé I, Cameroon. 6 Department of Chemistry, Faculty of Sciences, University of Maroua, Maroua, Cameroon.


INTRODUCTION
The increasing incidence of infectious diseases in the world particularly in developing country has stimulated the search for new antimicrobial agents.Plants are used as drugs in many countries for the treatment of many diseases (Mahesh and Satish, 2008).A rich heritage of knowledge to preventive and curative medicines was available in ancient scholastic works included in the Atharvaveda, Charaka, Sushruta etc.
Over 50 % of all modern clinical drugs are of natural product origin (Stuffness et al., 1982) and natural products play an important role in drug development program in the pharmaceutical industry (Baker et al., 1995).Plants produce a diverse range of bioactive molecules, making them a rich source of different types of medicines.The most important of these bioactive constituents of plants are alkaloids, tannins, flavonoids, and phenolic compounds (Hill, 1952).
E. candollei a large tropical forest tree belonging to the Meliaceae family (Notizbl, 1896) is used in Cameroon for the treatment of infectious diseases including malaria, bacterial and fungal infections (Dibong et al., 2011;Din et al., 2011).

Collection of plant material
E. candollei was harvested in Yaoundé-Cameroon, (October 2014) and identified at the Cameroon National Herbarium (HNC), where a voucher specimens are deposited (1722/SRFK).Then, leaves stem and root bark were collected, cut into small pieces, dried at room temperature and powdered.

Test bacteria
The microorganisms used in this study were strains and isolates respectively provided by American Type Culture Collection and Centre Pasteur Cameroon.

Stock solutions and disc preparation
For the antimicrobial activity, stock solutions of plant extracts were prepared at 150 mg/mL in DMSO 10 %.Ciprofluxacin and Nyistatin were prepared in the same conditions.For disc preparation, 15 μL of each stock solution was dropped onto sterilized paper disks (6 mm diameter) and dried at room temperature for a final concentration of 1500 μg / disc.

In vitro antimicrobial screening
In vitro antimicrobial activity was screened by disc diffusion method using Mueller Hinton Agar (MHA) obtained from Mast Group Ltd.The MHA plates were prepared by pouring 15 mL of molten media into sterile plates (90 mm).The plates were allowed to solidify for 5 min and 0.1 mL of inoculum suspension was swabbed uniformly and the inoculum allowed drying for 5 minutes.The different extracts and referents drugs loaded at 1.500 μg /discs were placed on the surface of the medium and allowed to diffuse for 5 min.
The plates were incubated at 35 °C for 24 hours for bacteria and for 48 hours for yeast.Negative control was prepared using 10 % DMSO.Ciprofluxacin, and miconazol were used as positive control.At the end of incubation, inhibition zones formed around the disc were measured with a Vernier Calliper in millimeter.

Determination of minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs)
The MIC was determined by microdilution method according to Clinical laboratory Standards Institute (CLSI) M27-A3 for yeast and M38 for bacteria, using (12 x 8 wells) microtitre plates.In the well of the plate, 100 µL of Mueller Hinton Broth for bacteria and Sabouraud Dextrose Broth for yeast were introduced.Later on, 100 µL of stock solution of extracts/fractions at 80mg/mL were added to the first well and 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 0.5 Macfarland for bacteria and at 2.5 × 10 3 Cells/mL for yeast were introduced in the entire well containing the test substances except the column of blank which constitute the sterility control.
The concentrations ranged from 20 mg/ml to 0.15625 mg/mL for extracts and from 5 µg/mL to 0.195 µg/mL for nystatin and Ciprofloxacin.After the incubation period at 37°C for 48 hours for yeast and 24 hours for bacteria, turbidity was observed as indication of growth.Thus, the lowest concentration without turbidity was considered as the MIC.The MFC or MBC were determined by transferring 50μL aliquots of the clear wells into 150 μL of freshly prepared broth medium and incubated at 37°C for 48 and 24 hours.
The MFC and MBC were regarded as the lowest concentration of test sample which did not produce turbidity, indicating no microbial growth.All tests were performed in triplicates.The bactericidal and fungicidal effects were determined by calculating the CMB or MFC / MIC ratio (CLSI, 2008).The classification of extracts of plant material on the basis of CMI is as follows: -strong inhibition: MIC<500 mg/mL; -Moderate inhibition: MIC from 500 mg/mL to 1500 mg / mL; -Weak inhibition: MIC>1500 mg/mL (Aligiannis et al., 2001).

Antioxidant activity Free radical scavenging activity: DPPH test
Antioxidant activity of extracts was studied using 1,1diphenyl-2-picrylhydrazyl free radical (DPPH) as described by (Mensor et al., 2001).Briefly, 100 µL of extract/fraction prepared at 2000µg/mL were serially diluted and mixed with 900 µL of 0.3 mM 2,2-diphenyl-1-picrylhydrazyl (DPPH) , to give five concentrations range from 12.5 -200 µg/mL (12.5, 25, 50, 100 and 200 µg/mL).After an incubation period of 30 min at 25°C, the absorbance at 517 nm (the wavelength of maximum absorbance of DPPH) were recorded as A (sample) .A blank experiment was also carried out applying the same procedure to a solution without thetest material and the absorbance was recorded as A (blank) .The free radical-scavenging activity of each solution was then calculated as percent inhibition according tothe following equation: The radical scavenging percentages were plotted against the logarithmic values of the concentration of test samples and a linear regression curve was established in order to calculate the RSA 50 , which is the amount of sample necessary to inhibit by 50% the free radical DPPH.Antioxidant activity of the extracts was expressed as RSA 50 defined as the concentration of the test material required to cause a 50% decrease in initial DPPHconcentration.Ascorbic acid was used as a standard.All measurements were performed in triplicate.
Then 500 µL of 10% trichloroacetic acid was added to the mixture and centrifuged at 3000 rpm for 10 min.Supernatant (500 µL) was diluted with 500 µL of water and shaken with 100 µL of freshly prepared 0.1% ferric chloride.The absorbance was measured at 700 nm.Vitamin C and BHT were used as a positive control.

Quantitative determination of total phenolic compounds Phenol content
The total phenolic compounds were determined as described by (Ramde-Tiendrebeogo et al., 2012) with slight modification.The reaction mixture consisted of 0.02 mL of extracts and fractions (2 µg/mL), 0.02 ml of 2N FCR (Folin Ciocalteu Reagent) and 0.4 mL of a 20% sodium carbonate solution.After 20min of incubation at room temperature the absorbance was measured at 760 nm.Distilled water was used as control.A standard curve was plotted using Gallic acid (0-0.2µg/mL).All measurements were performed in triplicate.

Statistical Analysis
Data were statistically analyzed using the software SPSS 12.0 for windows and variance analysis by ANOVA coupled with .Waller-Duncan test where P<0.05 was considered as statistically significant.

Phytochemical content of plant extracts
The qualitative phytochemical screening of crude extracts showed the results presented in table 1.From these results, a number of metabolites classes were identified in all the plant extracts among which tannins, flavonoïds, alkaloids, saponins, terpenoïds, steroids and phenols.

Inhibition Zone Diameters of extracts and reference antibiotic
The inhibition zone diameter ranged from 7-17 mm depending to the plants extracts and microorganisms (

Minimum Inhibitory Concentration (MIC) and Minimum Bacterial Concentration (MBC)
The MIC determination of selected extracts showed the results range from 156.25 to 2500µg/mL (Table 3).The most sensible strain was Staphylococcus aureus ATCC BAA 977 where the MIC values were ranging from 156.25 to 312.50µg/mL.This activity of extract was less than that of standard antibiotic e.g.ciprofloxacin and miconazole.

Total phenolic
The quantitative estimation of the phenolic compounds present in extracts of E. candollei are summarize in figure 1 below.
From these results, the quantity of phenol compounds is highly variable depending to extracts and plant part.Hydroethanol extracts of leaves is highly riche in phenol compounds (4.605mgEAG/mg) while the ethyl acetate extract of stem bark have the less concentration of phenol compounds (2.0529 mgEAG/mg).

DPPH radical scavenging activity
The results for DPPH radical scavenging activity are presented in figure 2 and table 2. All the extracts showed scavenging effects at various levels, depending to extracts and plants parts.At the tested concentration, the radical-scavenging activity (RSA) of these extracts is higher than the BHT except ethyl acetate extract of stem bark.From table 4, the IC 50 were range from 9.1026± 0.1635 to 11.8298±0.2077µg/mL.Ethyl acetate extract of stem bark was the most active while ethyl acetate extract of leaves was the less active.

Ferric reducing/antioxidant power (FRAP)
Concerning the Ferric Reducing Antioxidant Power activity, all the plant extracts interfered with the reduction of Fe 3+ to Fe 2+ , suggesting that they possess chelating activity (figure 3).The reducing power was ranging from 12.5 to 200 µg/mL.The highest reducing power was observed with ethyl acetate of leaves, while the ethyl acetate extract of stem bark was lowest.The phytochemical screening reveals the presence many classes of secondary metabolites whose members have already been shown to exhibit antimicrobial activities.These secondary metabolites obtained exert antimicrobial activity through different mechanisms.Tannins exert its antimicrobial activity by binding with proteins and adhesins, inhibiting enzymes, complexation with the cell wall and metal ions, or disruption of the plasmatic membrane (Cowan, 1999).Saponins have ability to cause leakage of proteins and certain enzymes from the cell (Okwu, 2001).Flavonoïds have the ability to complex with proteins and bacterial cells forming irreversible complexes mainly with nucleophilic amino acids.This complex often leads to inactivation of the protein and loss of its function (Burkill, 1988;Shimada, 2006).Some flavonoids have shown several pharmacological activities including antibacterial and antifungal (Tittikpina et al., 2013).These can explain the wide antibacterial spectrum of this plant.The different antibacterial activity of extracts from plant part of E. Candollei could be explained by the quantity of bioactive group of secondary metabolites in each extract and then, highlight the problem of standardization of therapeutic dose of plant extract used in the treatment of various infectious diseases.
The study of antioxidant activity extracts of E. candollei is a starting point to find the new antioxidant agent that could be used to fight against oxidative stress associated to many infectious diseases.The results showed that E. candollei extracts possess high anti-oxidant activities.In this study, the high reducing power of ethyl acetate of leaves and ethanolic and water extracts of root bark suggested that the phytochemical constituents with higher redox potential were more extractable with these solvents.These data suggest that leaves and root barks of E. candollei may contain several antioxidants with different polarities.The high DPPH radical scavenging activities of the various extracts which were comparable to the standard antioxidant, vitamin C and BHT, suggested that the extracts have some compounds with high proton donating ability and could therefore serve as free radical inhibitors.The results of antioxidants activities of these extracts suggest that they are rich sources of phytochemicals with powerful free radical scavenging phytochemical that could be used to fight against free radical upsurge, oxidative stress and consequently might help in the treatment of oxidative stress-associated metabolic disorders.

CONCLUSION
It may be concluded from this study that the extracts from different organs of E. candollei are active against the tested microorganisms and also have antioxidant effects.In addition, the results confirm the use of the plant in traditional medicine.Now our study will be directed to explore the lead compound responsible for aforementioned activity from this plant.

Fig. 1 :
Fig. 1: Total phenolic content of extracts of E. candollei.leaves, stem and root bark.Values with the same letters are not significantly different (P > 0.05).

Fig. 3 :
Fig. 3: Reducing power activities of the crude extracts of E.candollei.leaves, root and bark as well as vitamin C and BHT.

Table 1 :
Phytochemical composition of extracts of E. candollei.

Table 2 :
Inhibition Zone Diameters of extracts and reference antibiotic.