Antibacterial activity of crude extracts and pure compounds isolated from Vernonia galamensis leaves

The aim of this study was to test the antibacterial property of the extract of the leaves and isolated compounds of Vernonia galamensis that is traditionally claimed to have diverse medicinal use. The disk diffusion method was used to test the successively extracted dried leaves of V. galamensis on Staphylococcus aureus, Escherichia coli, Salmonella typhi and Shigella boydii. Further fractionation of the acetone extract by a combination of column chromatography, gel filtration using Sephadex LH-20 and Prep-TLC afforded two compounds. The results showed that Vernonia Acetone Extract (VAE) of the leaves of V. galamensis showed weak to moderate antibacterial growth inhibition on the test bacteria. Two active compounds; C-I (vernolide) and C-II (vernonioside) were isolated that were not reported from V. galamensis before. C-I (0.6 mg/disc) showed antibacterial activity on all bacteria except E. coli with minimum inhibitory concentration (MIC) value of 2.5 mg/mL and C-II (0.48 mg/disk) showed growth inhibition only against S. boydii and S. typhi with MIC value of 1 mg/mL. In conclusion, V. galamensis leaves have been proved to possess antibacterial chemicals. The plant can possibly be exploited as a source of lead compounds for antibacterial drug development.

Therefore, the main aim of this study was to assess the antibacterial activity of the crude extract and isolated pure compounds from the leaves of V. galamensis on some pathogenic bacteria.

Plant material
Fresh leaves of V. galamensis were collected from Bule-Hora area about 450 km south of Addis Ababa, Ethiopia, in November 2010.The plant was identified by a botanist and a specimen was kept in the National Herbarium of the Addis Ababa University with voucher number GT/005.Leaves were dried under shade to avoid any contamination and then ground with a blender to an appropriate size (about 0.5 mm) for extraction.The ground plant material was kept in closed container until used.

Extraction and isolation of pure compounds
The powdered leaves of V. galamensis (700 g) were macerated in n-hexane, acetone, ethanol and methanol successively in each solvent at a ratio of 1:7 (w/v) with some minor modifications as previously described by Pino-Rodriguez et al. (2003) for 24 h.The filtration was done using a double layer filter paper (Whatmann No.1) giving filtrate and residue in which the latter was subjected to the next maceration stage.Solvent was made to evaporate using Rotavapor R-210 with Vacuum Pump V-700 (Buchi) to collect crude extracts which were then weighed and kept at 4°C until further use.Preliminary antibacterial tests were conducted for each crude extract and the extract with the best activity was selected for further investigation.The extract Vernonia Acetone Extract (VAE) was then subjected to bioactivity-guided fractionation through flash column chromatography using the following solvent systems: pure n- Tafesse et al. 137 hexane: chloroform, chloroform: methanol with increasing polarity.
Different fractions (about 50mL each) were collected which were then combined based on their thin layer chromatography (TLC) profiles.
Further fractionation was done by gel-filtration using Sephadex LH-20 eluting with 1:1 chloroform/methanol.Non UV active compounds developed on TLC plate were visualized using vanillin sulfuric acid spray.Fractions with similar TLC profiles were combined and Prep-TLC was done for each combined fraction being eluted with ethyl acetate: chloroform (7:3).Bands made on Prep-TLC plate were scrapped off independently resulting in solid mixture of compound and silica.The obtained mixture was dissolved in chloroform and filtered out with filter paper (Whatmann No.1,9cm) to separate the target compound from silica.Each compound was then poured in small container and kept in a hood being left open until the solvent completely evaporated.After completely dried, each pure compound was subjected to 1D ( 1 H and 13 C) and 2D (DEPT and HMBC) NMR for identification.

Antibacterial test
Four pathogenic standard bacterial strains namely, S. aureus (ATCC25223), E. coli (ATCC23923), S. typhi (ATCC13311) and S. boydii (ATCC9207), obtained from Ethiopian Health and Nutrition Research Institute (EHNRI) were used for the antibacterial tests.These bacterial strains were first grown on their own selective media prior to the test by incubating them at 37°C for 24 h.Each was incubated in nutrient broth and their turbidity was compared with McFarland (0.5 standard) before spreading them on the plates.Antibacterial tests were performed for the VAE crude extract and two major isolated compounds using the disk diffusion method (Tadeg et al., 2005).The four bacterial strains were streaked on different (independent) plates made of Muller-Hinton agar using sterilized swaps.VAE at 100, 200 mg and 300 mg and compound I (C-I) and compound II (C-II) dissolved in 1 mL of 3% Tween 80 at 20 and 16 mg respectively were used for the test.
Standard drugs were selected on the susceptibility of the bacteria to the drugs.Each drug at a concentration of 2.5 mg/mL in distilled water was prepared.Sterilized paper disks (6 mm each) were impregnated with 30 µL of each of 100, 200, 300, giving 3, 6 and 9 mg per disk of VAE; 0.6 mg/disk of C-I and 0.48 mg/disk of C-II.Similarly, four paper disks were impregnated with the standard drugs (75 µg/disk each): ampicillin, chloramphenicol, ciproflaxin and erythromycin.The same amount of 3% Tween 80 was loaded on different disks and all of them were left to dry.The paper disks were then kept on the allotted place of each plate that was partitioned externally into compartments.All plates were kept in incubator at 37°C for 24 h.After 24 h, zone of inhibitions were measured by ruler and recorded in mm.The antibacterial tests were done in triplicate for the crude extract and each compound.

Minimum inhibitory concentration
Minimum inhibition concentration (MIC) was conducted for C-I and C-II according to the methods in Taiwo et al. (1999) and Adebolu and Oladimeji (2005).C-I at concentrations of 20, 10, 5 mg/mL, 2.5, 1.25 and 0.65 mg/mL dissolved each in 3% Tween 80 and C-II at 16, 8, 4, 2, 1, 0.5 and 0.25 mg/mL dissolved each in 3 % Tween 80 were prepared.The same procedure used for the antibacterial tests was applied.The MIC of each pure compound was performed only on those bacterial strains that gave positive results.

Statistical analysis
The mean (±SEM) value of zones of inhibition of each triplicate test was recorded and one way ANOVA (Turkey) was used to compare the results of the crude extract, the isolated pure compounds with both the negative and the positive controls; 95% confident interval was considered at significance level of P-value < 0.05.

Crude extracts and isolated compounds
Subsequent extraction of the leaf powder of V. galamensis (700 g) resulted in four crude extracts namely Vernonia Hexane Extract (VHE, 13.5 g), Vernonia Acetone Extract (VAE, 25 g), Vernonia Ethanol Extract (VEE 15 g) and Vernonia Methanol Extract (VME, 17.4 g).Among these, VAE showed the best antibacterial activity during the preliminary test.Bioactivity guided fractionation of VAE resulted in identification of five compounds (compounds I -V).
Fractionation of VAE using hexane: ethylacetae (7:3) resulted in 11 fractions (Fractions A -K) among which fraction E and fraction J were active.Fractionation of E using Sephadex with chloroform: methanol (1:1) solvent system gave three fractions (E1 -E3).Fraction E 3 (40 mg) was a pure white crystal and resulted in compound I. Compound II (34 mg), III (7 mg) and V (5 mg) were obtained from fraction E 2 after Prep-TLC as band 2, 1 and 3 respectively.
C-I (Figure 1 A comparison of our data with previously isolated steroids from other Vernonia species showed compound C-II to be an aglycone of a vernonioside previously isolated from V. amygdalina by Jisaka et al. (1993) from V. cinerea (Yao-Haur et al., 2003) and from V. guineensis by Donfack et al. (2012).Though the glycoside of C-II is well-known, this is the first report of the aglycone from V. galamensis.
There was lack of activity against some tested bacteria by the pure compounds in the present work while the crude extract showed activity which might suggest the presence of synergism of compounds in the later.As reported by Erasto et al. (2006), vernolide is also inactive against some Gram negative bacteria, which might be supportive to the lack of activity by C-I and C-II against E. coli in the present study.
The results of the present study showed that V. galamensis contains phytochemicals that can control the growth of some pathogenic bacteria especially those affecting the gastrointestinal tract normal function.Although vernolide was isolated from other Vernonia spp. it is the first report from V. galamensis.It is also the first report for vernonioside with aglycone from this plant.The effect of the crude extract and C-I against S. aureus can support the traditional use of this plant in wound healing treatments.The inhibitory activity of C-II against S. typhi and S. boydii might suggest that this plant can be a potential candidate for antibacterial drug development.

Conclusion
In conclusion, V. galamensis leaves have constituents that have inhibited the growth of pathogenic bacteria.C-II showed significant inhibition on the growth of S. typhi and S.boydi.Further study is recommended to fully exploit the medicinal importance of the plant.
) gave a molecular ion at m/z = 362 corresponding to molecular formula C 19 H 22 O 7 and consistent with nine degrees of unsaturation (double bond equivalents) which accounted for three double bonds 2D NMR data showed C-I to be a sesquiterpene lactone named as vernolide which was previously isolated from V. amygdalina byErasto et al. (2006).C-II (Figure2) gave a molecular ion at m/z 546 corresponding to molecular formula C 31 H 46 O 8 and consistent with nine degrees of unsaturation.These nine degrees of unsaturation can be accounted for two double bonds (four sp 2 -hybridized carbons at  C 143.5, 134.7,C 170.5) and six rings.The 13 C NMR showed a total of 31 carbon atoms, which is consistent with a steroid nucleus.

Table 1 .
Level of inhibition of Vernonia Acetone Extract (VAE), C-I (vernolide), and C-II (vernonioside) on the test bacteria as compared to the controls.

Table 2 .
Inhibitory activity of the Vernonia Acetone Extract (VAE), compound I (vernolide) and compound II (vernonioside) on the test bacteria.

material Zone of Inhibition in mm (Mean + SEM)
Vernonia leaf Acetone Extract; St. Drugs (Standard Drug): chloramphenicol (for S. typhi), ampicillin (for E. coli), erythromycin (for S. aureus), and ciprofloxacin (for S. boydii).P-value represents a comparison of the effect of the extract or pure compounds with the control drugs.
NT= Not Tested; because this compound lacked activity against this bacterium.
NT= Not Tested; because this compound lacked activity against this bacterium.