Antimicrobial Activity and Phytochemicals Analysis of Vernonia aemulans, Vernonia amygdalina, Lantana camara and Markhamia lutea Leaves as Natural Beer Preservatives

MATERIALS AND METHODS

Collection of plant samples: The fresh leaves of V. aemulans (Idoma), V. amygdalina (Umubirizi), L. camara « Umuhengeri» and M. lutea «Umusave» were collected from Huye and Musanze districts of Rwanda. The plants were authenticated at the Botanic Laboratory, Department of Biology, School of Science, College of Science and Technology, University of Rwanda.

Preparation of extracts: Plant leaves collected were washed thoroughly with distilled water. The leaves were dried under oven at 45°C for 3 days. The dried leaves were well grinded into fine powder using electrical grinder and then stored in air tight containers for further use. Two hundred and fifty grams of the pulverized plant material was extracted for 3 days in absolute ethanol (Sigma-Aldrich) and sterile water²⁴. The separated extracts were then filtered through Whatman’s No. 1 filter paper and the filtrates were then separately condensed to dryness using rotary evaporator. The thick extracted mass was then dried at room temperature. Dried extract was collected in an air tight container and stored at 4°C for further analysis.

Antimicrobial activity essay: The antimicrobial activity of various extracts from L. camara, M. lutea, V. amygdalina and V. aemulans were screened against Escherichia coli (CWBI-WT), Bacillus subtilis (CWBI-FZB42), Saccharomyces cerevisiae (CWBI-F451), Staphylococcus aureus (CHUB) and Salmonella typhimurium (CHUB) obtained from the collection of Centre Wallon de Biologie Industrielle (CWBI) and Butare University Teaching Hospital (CHUB). The bacterial isolates were first sub-cultured in a nutrient broth (Sigma-Aldrich) and incubated at 37°C for 18 h, while the yeast was sub cultured on a Sabouraud broth (Sigma-Aldrich) for 48 h at 28°C.

The antimicrobial assay was carried out by the disc diffusion method as described elsewhere^25,26. The extracts were concentrated and dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich) to a final concentration of 10 mg mL^–1 and sterilized through 0.45 μm millipore filters. One hundred microliters of suspension containing 10³ colony forming unit (CFU mL^–1) of bacteria and 10⁵ CFU mL^–1 of yeast were inoculated on Mueller Hinton agar (Sigma) and Sabouraud agar (Sigma-Aldrich), respectively. Discs of 6 mm diameter were impregnated with 10 μL of extracts and placed on the inoculated agar. The plates containing bacteria were incubated at 37°C for 24 h, while those containing yeast were incubated at 28°C for 48 h. The inhibition zones were measured for determining the antimicrobial activity and the findings were tabulated.

Minimum inhibitory concentration: The determination of Minimum inhibitory concentration for bacterial and yeast strains was performed using agar dilution method²⁷. Varying concentrations of each extract (0.125-20 mg mL^–1) were prepared in 5% DMSO and filter sterilized (0.45 μm). The extract was mixed with sterilized Mueller Hinton agar dispensed into sterile petri dish. Five microliters of standardized inocula (matching 0.5 McFarland turbidity standards) of various test isolates was seeded on Mueller Hinton agar plate. Test strains on solvent free Mueller Hinton agar and DMSO incorporated in Mueller Hinton agar served as growth and solvent controls respectively. Minimum inhibitory concentration was determined after 24-48 h incubation at 37°C.

Qualitative and quantitative analysis of phytochemicals: To detect the presence of tannins, alkaloids, flavonoids, saponins, glycosides, carbohydrates steroids, phenolics, phlobatannins and terpenoids, the phytochemical screening of the extracts was done using the method described elsewhere^28-30.

The phytochemicals which are present in the ethanol extracts of V. aemulans, V. amygdalina, L. camara and M. lutea were determined and quantified by standard procedures as described by Gracelin et al.³¹.

Determination of total phenols: A total phenolic compound was determined spectrophometrically according to Folin-Ciocalteu colorimetric method³². One hundred micrograms of the extract of the sample was weighed accurately and dissolved in 100 mL of Triple Distilled Water (TDW). One milliliter of this solution was transferred to a test tube, then 0.5 mL 2 N of the Folin-Ciocalteu reagent and 1.5 mL 20% of Na₂CO₃ solution was added and ultimately the volume was made up to 8 mL with TDW followed by vigorous shaking and finally allowed to stand for 2 h after which the absorbance was taken at 765 nm. These data were used to estimate the total phenolic content using a standard calibration curve obtained from various diluted concentrations of gallic acid.

Determination of total flavonoids: The method is based on the formation of the flavonoids aluminium complex which has an absorptivity maximum at 415 nm³³. One hundred microliters of plant extracts in methanol (10 mg mL^–1) was mixed with 100 μL of 20 % aluminum trichloride in methanol and a drop of acetic acid and then diluted with methanol to 5 mL. The absorbance at 415 nm was read after 40 min. Blank samples were prepared from 100 mL of plant extracts and a drop of acetic acid and then diluted to 5 mL with methanol. The absorption of standard rutin solution (0.5 mg mL^–1) in methanol was measured under the same conditions. All determinations were carried out in triplicates.

Determination of total alkaloids: Five grams of the sample was weighed into a 250 mL beaker and 200 mL of 10% acetic acid in methanol was added and covered and allowed to stand for 4 h. This was filtered and the extract was concentrated on a water bath to one-quarter of the original volume. Concentrated ammonium hydroxide was added drop wise to the extract until the precipitation was complete. The whole solution was allowed to settle and the precipitated was collected and washed with dilute ammonium hydroxide and then filtered. The residue is the alkaloid, which was dried and weighed²⁸.

Determination of total tannins: Sample (500 mg) was weighed into a 50 mL plastic bottle. Fifty millilitres of distilled water was added and shaken for 1 h in a mechanical shaker. This was filtered into a 50 mL volumetric flask and made up to the mark. Then 5 mL of the filtered was pipetted out into a test tube and mixed with 2 mL of 0.1 M FeCl₃ in 0.1 N HCl and 0.008 M potassium ferrocyanide. The absorbance was measured at 120 nm within 10 min³⁴.

Determination of total saponins: The samples were ground and 20 g of each were put into a conical flask and 100 cm³ of 20% aqueous ethanol were added. The samples were heated over a hot water bath for 4 h with continuous stirring at about 55°C. The mixture was filtered and the residue re-extracted with another 200 mL 20% ethanol. The combined extracts were reduced to 40 mL over water bath at about 90°C. The concentrate was transferred into a 250 mL separatory funnel and 20 mL of diethyl ether was added and shaken vigorously. The aqueous layer was recovered while the ether layer was discarded. The purification process was repeated. Sixty millilitres of n-butanol was added. The combined n-butanol extracts were washed twice with 10 mL of 5% aqueous sodium chloride. The remaining solution was heated in a water bath. After evaporation the samples were dried in the oven to a constant weight; the saponin content was calculated³⁵.

Statistical analysis: The experiments were conducted in triplicate and the results were expressed as mean with standard deviation. Statistical analysis of the data was performed using SPSS package program. Statistical significance was taken at 95% confidence interval when p<0.05. When analysis of variance (ANOVA) revealed a significant effect (p<0.05), the data means were compared by the least significant difference (Duncan’s multiple range test) test.

RESULTS

Antimicrobial evaluation: The results of antimicrobial activities of M. lutea, V. aemulans, L. camara and V. amygdalina leaves are presented in Table 1. These results shown that all extracts tested possess high inhibitory effects against B. subtilis (9.2-26.5 mm of inhibition zone diameter) and S. aureus (5.3-23.1 mm of inhibition zone diameter), but not possess any antimicrobial activity against S. cerevisiae. The extracts of V. aemulans, V. amygdalina and L. camara possess antibacterial activities against E. coli and S. typhimurium with inhibition zone diameter ranging from 3.0-18.6 mm. These Gram-negative bacteria were found to be resistant against the extracts of M. lutea leaves. However, the diameters of inhibition zones of ethanol extracts were larger than those of aqueous extracts (p<0.05) for all bacteria tested.

Ethanol extracts of V. amygdalina, V. aemulans, M. lutea and L. camara leaves showed varying degree of Minimum Inhibitory Concentration (MIC) (Table 2). Growth of B. subtilis was inhibited at a MIC of 2 mg mL^–1 for all extracts analyzed. The MIC values of 2, 8 and 16 mg mL^–1 were recorded for extracts of L. camara, V. amygdalina and V. aemulans respectively against S. typhimurium. The E. coli was inhibited at a MIC of 2 mg mL^–1 for extracts of V. aemulans and V. amygdalina and at a MIC value of 16 mg L^–1 for L. camara. It was observed also that the extracts of M. lutea exerted more activity on S. aureus with MIC value of 1.5 mg mL^–1.

Phytochemical analysis: Phytochemical screening of the extracts was carried out to detect the presence of tannins, alkaloids, flavonoids, saponins, glycosides, phenolics, steroids, phlobatannins and terpenoids in ethanol extract of V. amygdalina, V. aemulans, L. camara and M. lutea from Rwanda and the positive results are showed in Table 3.

Table 1:	Antimicrobial activities of extracts of some Rwandan plants (100 µ mL–1)
R: Resistant at the concentration of 100 µ mL–1 of aqueous and ethanol extracts, a-cMeans in a same column with different letters are significantly different (p<0.05)

Table 2:	Minimum Inhibitory Concentration (MIC) of some Rwandan plants against E. coli, S. aureus, S. typhimurium, B. subtilis and S. cerevisiae
R: Resistant to the concentrations from 0.125-20 mg mL–1 of extracts

Table 3:	Phytochemical screening of ethanol extracts of V. aemulans, V. amygdalina, L. camara and M. lutea leaves
+: Presence, -: Absence (not detected)

On a total of 9 phytochemical tests, 8 were positive in ethanol extract of V. amygdalina, V. aemulans and L. camara leaves. Seven tests were positive in ethanol extracts from M. lutea leaves.

The amount of phytochemicals which were found in ethanol extracts of V. aemulans, V. amygdalina, L. camara and M. lutea were quantitatively determined by standard procedures. Among the five components analyzed tannins content (99±2.6 mg g^–1) was highest in V. amygdalina extracts followed by flavonoids (70.2±1.5 mg g^–1), saponins (64±2.3 mg g^–1), phenolic compounds (35.5±2.2 mg g^–1) and alkaloids (32±0.6 mg g^–1) as shown in Fig. 1. While flavonoids content were high in the extracts of V. aemulans, L. camara and M. lutea with the concentrations of 87.4±1.7, 58.3±1.8 and 62.4±3 mg g^–1, respectively. The V. aemulans extract contained 82.6±3 mg g^–1 of tannins, 44±0.6 mg g^–1 of saponins, 40.7±0.7 mg g^–1 of alkaloids and 22.6±0.3 mg g^–1 of phenolic compounds. In L. camara extract 42.8±1.8 mg g^–1 of phenolic compounds, 37.8±0.1 mg g^–1 of alkaloids, 30.8±1.3 mg g^–1 of saponins and 9.2±0.6 mg g^–1 of tannins were found.

Fig. 1:

Phytochemical components of ethanol extract of V. aemulans, V. amygdalina, L. camara and M. lutea leaves

In M. lutea extract 27±1.7 mg g^–1 of tannins, 17.1±0.8 mg g^–1 of saponins and 8±0.5 mg g^–1 of alkaloids were observed.

DISCUSSION

Many plant extracts have been known to possess antimicrobial activities and are proposed as of food preservatives^26,36,37. In the present study, the aqueous and ethanol extracts of V. amygdalina, V. aemulans, L. camara and M. lutea leaves possessed antibacterial activity against food spoilage bacteria and food-borne pathogens.

However, ethanol extracts of V. amygdalina, L. camara and V. aemulans exhibited pronounced activities against Gram-positive (B. subtilis and S. aureus) than Gram-negative bacteria (E. coli and S. typhimurium). In certain case, e.g., in extracts of M. lutea leaves, the results showed the total absence of antibacterial activity against the Gram-negative bacteria tested. Pelczar et al.³⁸ suggested that the difference in susceptibility of Gram-positive and Gram-negative bacteria to various antimicrobial agents probably depends on structural differences in their cell walls. Cell wall of Gram-negative bacteria is protected by an outer membrane that prevents permeation of the active molecule.

Several similar studies reported the antibacterial activities of V. amygdlina, L. camara and M. lutea against E. coli and S. aureus^39-43. But, the antimicrobial activities of these plants against S. cerevisiae and S. typhimurium were not tested in their studies.

In this study, the results showed that all extracts analyzed did not possess antimicrobial activity against S. cerevisiae, which is reassuring for their use as hops substitute in African brewing beers due to the role played by S. cerevisiae in African beers fermentation. This resistance can be explained by the presence of bitter compounds in these plants as the case in hops. Studies of the antibacterial properties of hop compounds showed that they inhibit growth of Gram-positive bacteria and not Gram-negative bacteria and S. cerevisiae^44-46. Unlike bacteria, yeast cells have a number of unique features that prevent cell damage from bittern compounds. These include modifying the cell wall in response to hop stress; reducing the concentration of alpha acids in the cell vacuole and actively purging alpha acids from the cell itself⁴⁶.

However, according to their microbial properties, V. amygdalina, V. aemulans and L. camara have an advantage over the hops because they are able to inhibit the growth of both Gram-positive bacteria (B. subtilis and S. aureus) and Gram-negative bacteria such us E. coli and S. typhimurium. The antibacterial inhibitory effects of these plants can be attributed to the presence of tannins, flavonoids, saponins, phenolic compounds and alkaloids in extracts of V. amygdalina, V. aemulans and L. camara leaves.

These phytochemical constituents were further reported to be responsible for many antimicrobial activities of different plant species^41,47,48. Their concentration, composition, structure and functional groups serve an important role in determining antimicrobial activity. Phenolic compounds are generally the most effective due to their chemical structures⁴⁹, which may be divided into different categories including simple phenolic compounds, flavonoids, quinones, tannins and coumarins. Phenolic compounds contribute to the sensory properties when added to food and have antioxidant and antimicrobial properties^36,50, characteristics that are useful in extending the shelf-life of food. The antimicrobial effect of phenolic compounds may be due to their ability to alter microbial cell permeability, thereby permitting the loss of macromolecules from the interior. They could also interfere with membrane function (electron transport, nutrient uptake, protein, nuclein acid synthesis and enzyme activity) and interact with membrane proteins, causing deformation in structure and functionality³⁷. Flavonoids have been reported to be synthesized by plants in response to microbial infections and are good antibacterial agent⁵¹. Tannins have been demonstrated antibacterial activities⁵². With proline-rich proteins, tannins form irreversible complexes which may be able to inhibit the cell-wall-protein synthesis of bacteria⁵².

Some alkaloids from plants have also been used as antimicrobials in food⁵³. Recently, saponins have been used as a preservative and/or used as a part of a preservative system to inhibit and/or reduce growth of spoilage microorganisms of beverages and foods⁵⁴.

CONCLUSION

The results of the present study indicate that all extracts of V. aemulans, V. amygdalina, L. camara and M. lutea leaves possess potent antibacterial activity against selected food spoilage bacteria and food-borne pathogens which might be due to the presence of tannins, flavonoids, saponins, glucosides and phenolic compounds in these plants. However, M. lutea extracts did not exhibit antibacterial activity against Gram-negative bacteria such as E. coli and S. typhimurium. All extracts analyzed did not possess antimicrobial activity against S. cerevisiae, which plays an important role in beer fermentation. Therefore, V. aemulans, V. amygdalina and L. camara leaves can be used as natural beer preservatives with considerable market opportunities in African brewing industry due to their strong antimicrobial activity imparting extended shelf-life with less harmful effects.

ACKNOWLEDGMENT

The authors thank the Swedish International Development Agency (Sida) and the "Académie de Recherche et Enseignement Supérieur (ARES, Belgium), as partners of the University of Rwanda for their financial support.