MATERIALS AND METHODS

Plant material: The rhizomes of Ampelocissus grantii were collected in the area of Sapone at about 45 km from Ouagadougou (Burkina Faso), in March 2009. The plant was taxonomically authenticated at the Department of Plant Biology and Ecology of the University of Ouagadougou.

The rhizomes were washed, cut to small fragments and carefully dried during 2 weeks at ambient temperature in the laboratory under continuous ventilation, away from sunlight and dust. The samples were then crushed to fine powder with a mechanical crusher and the powder was hermetically sealed in polythene bags and stored away from light and moisture until the time of extraction.

Preparation of plant extracts: Different extracts were made from powdered rhizomes using acetone (70%v/v) and water. Aliquots of 25 g were made and each aliquot was soaked with the appropriate solvent. Acetone extract was left under shaking conditions at room temperature for 24 h. Water extract was boiled for 1 h. At the end of the extraction, each extract was filtered using Whatman No. 1 filter paper. Acetone was completely removed at low pressure with a rotary evaporator (BÜCHI, Labortechnik, Switzerland). The acetone extract and the water extract were concentrated, lyophilized and stored at 4°C until analysis.

Phytochemical screening: Extracts were screened for phytochemical constituents using standard procedures of analysis (Harborne, 1998; Sofowora, 1993; Trease and Evans, 2002). Dragendorff’s test, FeCl₃ test, Frothing test, Libarman-Burchard’s test, Killer-killani’s test were performed for respectively alkaloids, flavonoids, saponins, terpenes and steroids, cardiac glycosides detection. All chemicals were from Sigma-Aldrich, Germany, obtained with local suppliers.

Determination of total phenolic contents: Total Phenolic Contents (TPC) from extracts were quantified using Folin-Ciocalteu’s method adapted to a 96 well-plate (Dicko et al., 2002) with minor modifications. One hundred microlitters of Folin-Ciocalteu’s reagent (10%, v/v) was added to 20 μL of each lyophilized plant extract dissolved in water and adjusted to 100 μg mL^-1. After 5 min incubation at room temperature, 80 μL of sodium carbonate (75 g L^-1) were added to each well containing the previous mixtures. The final volume per well was 200 μL. The 96 well-plate (Greiner Labortechnik, Germany) was slightly shaken and incubated for 30 min at room temperature in the darkness. The absorbance was measured at 735 nm using a multiwell plate reader (μQuant Bio-Tek Instrument, Inc., USA) on line interfaced with a computer. Absorbencies were automatically recorded using KC junior software version 1.31.5 (Bio-Tek instrument, Inc., USA). Gallic acid was used as standard and the calibration equation was Y = 0.061X+ 0.003 (R² = 0.998), where X is the gallic acid concentration in μg mL^-1 and Y is the absorbance read at 735 nm. All the analyses were done in triplicate and results (average of triplicate analysis) were expressed as gallic acid equivalent per gram of lyophilized sample.

Flavonoid content determination: Total flavonoid contents (TFC) were determined by the aluminium chloride (AlCl₃) colorimetric assay method (Quettier-Deleu et al., 2000) adapted to 96 well-plate, using quercetin as a standard. The extracts were dissolved in ethanol 75% and 100 μL of this solution were mixed with 100 μL of 2% AlCl₃. After 15 min of incubation at ambient temperature, the absorbance was measured at 435 nm using the multiwell plate reader. Three replicates were made for each test sample. For each sample, a blank was prepared by substituting AlCl₃ by the solution used to dissolve extracts. Quercetin was used as a standard for the calibration and a calibration curve (ranging from 0 to 50 μg mL^-1) was generated, with the regression equation of Y = 0.035X+0.026 (R² = 0.999), where X is the concentration of Quercetin in μg mL^-1 and Y is the absorbance reading at 435 nm. All experiments were performed in triplicate. The total flavonoid contents (average of the triplicate analysis) were expressed as Quercetin equivalent in milligrams per gram of lyophilized sample.

Antioxidant activity index determination: The Antioxidant Activity Index (AAI) was determinate according to the method described by Scherer and Godoy (2009). This method is based on the 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radical test. Briefly, the scavenging activity of the extracts against DPPH was evaluated spectrophotometrically by a slightly modified method of Mensor et al. (2001) with adaptation on the 96-well plate. Graded concentrations of extracts ranging from 0.781 to 100 μg mL^-1 obtained by two-fold dilutions were prepared and 100 μL of each dilution were mixed with 100 μL of DPPH (100 μg mL^-1 in ethanol). Samples were kept for 15 min at room temperature in the dark and then the absorbance was measured at 517 nm. Ascorbic acid (Vitamin C) and Butylated Hydroxyanisole (BHA) were used as references. The ability to scavenge DPPH radical was calculated by the following equation:

where, RSA is the percentage of free radical scavenging activity, Abs(control) the absorbance of DPPH radical + ethanol and Abs(sample) the absorbance of DPPH radical +sample extract or standard. The IC₅₀ (concentration providing 50% inhibition) of extracts and standards was determinate using regression curves in the linear range of concentrations. The antioxidant activity index (AAI) was then calculated as follows:

where, [DPPH] is the final concentration of DPPH.

Antimicrobial assays
Disc diffusion method: The in vitro antibacterial activity of the extract was studied by the paper disc diffusion method of Bauer et al. (1966) as described previously (Zongo et al., 2009). Bacteria grown on Mueller-Hinton Broth (MHB) (Liofilchem, Italy) at 37°C for 18 to 24 h were suspended in saline solution (0.9% NaCl) and adjusted to a turbidity of 0.5 Mac Farland standard corresponding to 10⁸ colony forming units (cfu) mL^-1 (McFarland, 1907; Sittiwet and Puangpronpitag, 2009). Bacterial suspensions were used to inundate sterile Petri dishes (90 mm diameter) containing 15 mL Mueller-Hinton Agar (MHA) (Liofilchem, Italy). Plant extracts were dissolved in DMSO at 100 mg mL^-1 and filtered with a Millipore filter (0.22 μm) for sterilization. Ten microlitters were pipetted to impregnate sterile paper discs (6 mm diameter). The discs were then placed onto the surface of inoculated Petri dishes. Gentamicin (10 μg) and Ciprofloxacin (5 μg) (Liofilchem, Italy) were used as the positive control. Paper discs soaked in DMSO without extract were used as negative control and DMSO didn’t show inhibition effects to the growth microorganism. Petri dishes were incubated aerobically at 37°C for 18 to 24 h. The results were recorded by measuring the zones of growth inhibition surrounding the disc indicating the presence of antimicrobial activity. All tests were performed in duplicate and the antibacterial activity was expressed as the mean of Diameters of Inhibition Zone (DIZ) produced.

Broth microdilution assay: A broth microdilution method was performed to determine the Minimum Inhibitory Concentration (MIC) and the Minimum Bactericidal Concentration (MBC) as recommended by NCCLS (2008). The inocula of the bacterial strains were prepared from 18 to 24 h broth cultures and suspensions were adjusted to 0.5 Mc Farland standard turbidity. The extracts were first dissolved in 10% DMSO and then sterilized as described previously. One hundred microliter of this stock extract solution were transferred into the first well of a 96-well sterile plate (Cellstar, Greiner Labortechnik, Germany) previously filed with 100 μL of nutrient broth. Serial twofold dilutions were made in to 11 consecutive wells. Into each well, 95 μL of nutrient broth and 5 μL of the inocula were added to achieve concentrations of extracts ranging from 5 to 0.0049 mg mL^-1. The final volume in each well was 200 μL. On the same plate, some wells with specific medium and microorganism were used as control of the growth and other inoculated wells containing only 200 μL of the media were used for sterility control. Each plate was mixed on a plate shaker at 300 rpm for 20 sec and then incubated at 37°C for 24 h. Bacterial growth was indicated by the presence of turbidity and a pellet on the well bottom. The MIC is the lowest extract concentration demonstrating no visible growth in the broth. The MBC which is the lowest concentration of the extract at which 99.99% or more of the initial inoculum was killed was determinate as described previously (Zongo et al., 2009; Akomo et al., 2009). One hundred microlitter from each well demonstrating no visible growth were removed to spread onto Petri dishes filed with sterilized Plate Count Agar (PCA) (Liofilchem, Italy) medium. Petri dishes were incubated at 37°C for a total period of 48 h.

Antifungal screening: The antifungal assay was performed by disc diffusion method and microdilution technique (NCCLS, 2008; Singh et al., 2008) as described above with minor modifications. A strain of Candida albicans was used for this test. Potato Dextrose Broth (PDB) and Potato Dextrose Agar (PDA) were used. The plates were incubated at 28°C for 48 to 96 h. Standard disc of Nystatin (100 IU) (Liofilchem, Italy) was used as positive control.

RESULTS

The result of the phytochemical screening is presented in Table 1. This reveals presence of flavonoids and saponins and absence of alkaloids in both extracts. Lieberman-Burchard’s test was negative with water extracts and weakly positive with acetone extract indicating that terpenes and steroids are slightly present in hydro-acetone extract and absent in water extract. Likewise, cardiac glycosides have been detected in water extract but not in acetone extract.

Results of TPC and TFC determination by Folin-Ciocalteu method and aluminium chloride colorimetric assay method are summarised in Table 2. These results show that the TPC and TFC levels were higher in the acetone extract than in the water extract. The TPC and TFC amount in acetone extracts were, respectively 124.26 mgQE/100 g and 12.41 mgQE/100 g of lyophilized extract while in the water extracts, TPC amount was 74.25 mgQE/100 g and TFC 6.32 mgQE/100 g.

The free radical scavenging activity of the extracts by DPPH method shows that acetone extract is more efficient to scavenge DPPH free radicals than the water extract (Fig. 1). However, both extracts showed lower activity than BHA and vitamin C. The AAI of the extracts are shown in Table 3, these results show that acetone extract has strong antioxidant activity (AAI = 4.6) but this activity remain lower than activity of BHA and vitamin C (AAI = 7.30 and 10.66 respectively in this study). Water extract has a weak antioxidant activity (AAI = 0.21).

The results in Table 4 show the antimicrobial activity of the extracts of A. grantii against bacteria and fungal strains recorded by disc diffusion method. Our results show that both extracts exhibited weak to moderate activity against all microorganisms used in this study excepted Pseudomonas aeruginosa witch gave no inhibition with the water extract.

Table 1:	Phytochemical screening of acetone extract (AGae) and water extract (AGwe) of A. grantii
-: Negative (not detected), +: Positive (detected)

Table 2:	Total phenolic compounds (TPC) by Folin Ciocalteu’s method and total flavonoids content (TFC) by AlCl3 method

Table 3:	Antioxidant activity of A. grantii extracts by DPPH free radical scavenging method

The largest DIZ (15 mm) has been recorded with Enterococcus faecalis 10907 CIP followed by that obtained with Bacillus subtilus ATCC 21332 (13 mm). The MICs and MBCs values of extracts of A. grantii obtained with microdilution assay are shown in Table 5. The lowest MIC value (0.625 mg mL^-1) was recorded with E. faecalis and B. subtilus confirming their susceptibility in disc diffusion method. MIC values ranged from 0.625 to 5 mg mL^-1.

Fig. 1:	DPPH radical scavenging activity of acetone extract (AGae) and water extract (AGwe) of A. Grantii compared with BHA and Vitamin C. percentage Radical Scavenging Activity = [abs(control)-abs(sample)]/ abs(control)] x 100

Table 4:	Diameters (mm) of inhibition zone by disc diffusion assay
Nd: not determined, Cipro: Ciprofloxacin, Genta: Gentamicin, Nyst: Nystatin

Table 5:	Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) or fungicidal concentration (MFC) in mg mL-1 obtained by microdilution method

DISCUSSION

The indiscriminate use of commercial antimicrobial drugs has lead to multiple drug resistant strains (Motamedi et al., 2010). Beyond the increasing prevalence of drug resistance, some synthetic antibiotics are demonstrating undesirable side effects. This has highlighted the urgent need for new drugs particularly affordable for poorest countries (Raja et al., 2010). In many parts of the world, the extracts of medicinal plants are used for their antibacterial, antifungal and antiviral properties (Hassawi and Kharma, 2006). A. grantii is a plant well known in traditional medicine for its properties. It has been reported that the family Vitaceae include 14 genera and 800 species described but only a few number of them contain no significant alkaloids. Like in this study, previous studies did not detect alkaloids with Ampelocissus extracts (Raffauf, 1996). Our results showed that polyphenolic compounds amount is higher in acetone extract than water extract. It have been reported that 70% aqueous acetone was more efficient to extract polyphenolic compounds compared to water and other solvent such as alcohol (Lamien-Meda et al., 2008; Wang et al., 2009). Phenolic compounds are generally more soluble in polar organic solvents than in water. However, there are contradictory reports in the literature regarding the best solvent for polyphenols extraction. Some studies have demonstrated that ethanol is the best solvent (Mohsen and Ammar, 2009). The presence of these compounds in water extract is interesting because water is a universal solvent and is generally used in traditional settings to prepare the plant decoctions for health remedies. It was observed that the extracts containing high levels of TPC were also potent DPPH radical scavenger. The acetone extract showed a strong antioxidant activity. An extract is considered to show poor antioxidant activity when AAI<0.5, moderate antioxidant activity when AAI between 0.5 and 1.0, strong antioxidant activity when AAI between 1.0 and 2.0 and very strong when AAI>2.0 (Scherer and Godoy, 2009). The relationship between phenols content and antioxidant activity has been underlined in literature (Sthishkumar et al., 2009; Hussein et al., 2010). Apart from the antioxidant activity exhibited, acetone extract showed the better antimicrobial effect.

An antimicrobial activity can be considerate when the diameter of inhibition zone (DIZ) observed is 9 mm or more around the paper disc (Kitzberger et al., 2006). Generally, DIZ obtained with gram-positive bacteria are larger than those obtained with gram-negative bacteria. This is in phase with several previous studies which demonstrated that gram-positive bacteria are more sensitive to plant extracts and their components than gram-negative bacteria (Kelmanson et al., 2000; Masika and Afolayane, 2000; Sahin et al., 2002; Karaman et al., 2003; Karou et al., 2005; Masoodi et al., 2008; Zongo et al., 2009; Khanahmadi et al., 2010). Indeed, the Gram-negative bacteria which are responsible for a large number of infectious diseases have a unique outer membrane that contains lipopolysaccharides which render them impermeable to certain antibacterial substances including antibiotics (Tortora et al., 2001; Clements et al., 2002). In some cases the MIC was equivalent to the MBC, indicating a bactericidal action of the extracts. Lower MIC and MBC value and higher zones of inhibition for acetone extracts connote higher solubility of phytoconstituents in the acetone extract compared to the water extract. Obtained results are in agreement with what was reported in literature concerning the biological activities of polyphenols. Indeed, there are several data in literature demonstrating the antibacterial, antifungal and antioxidant activity of these compounds (Haslam, 1996; Rice-Evans et al., 1996; Yoshida e t al., 2000; Karou et al., 2006; Fattouch et al., 2007; Jimoh et al., 2008; Mohsen and Ammar, 2009). However it is important to notice that over compounds such as alkaloids have strong antimicrobial activities (Karou et al., 2005; Zongo et al., 2009). What is important with plant extract is the fact that antibiotic resistance does not interfere with the antibacterial action of plant extracts. Plant extracts with antimicrobial activities are generally active on multi-drug resistant human pathogens (Oskay et al., 2009).

CONCLUSION

This study justifies the uses of this plant to treat several infectious diseases and cancers in folk medicine. Investigations may continue in order to know the limit of toxicity of this plant and its in vivo potencies. The studies can lead to news active structures or lead to Enhanced Traditional Medicines (ETM) which are cheaper and disposable for population in developing countries.

ACKNOWLEDGMENTS

The authors are grateful to the International Programme in the Chemical Sciences (ISP/IPICS) for the partial financial support during this study and to Mr. Dieudonné KINDA, an herbalist from Ouagadougou for his help during plant material collection.