Phytochemical and Antimicrobial Screening of Alugbati (Basella Rubra Linn.) Leaves and Stems Extracts Against Pathogenic Microoganisms

BURCE, J.M., MARCELLANA, W.B, & TUANDO, C.M. Department of Biology, College of Arts and Sciences, Rizal Technological University, Boni Avenue, Mandaluyong city ABSTRACT Qualitative analysis of the compounds of medicinal importance in Basella rubra Linn was performed on its ethanol, methanol, and aqueous extracts from leaves and stems. Phytochemical investigation of this plant yielded more than seven (7) different compounds; mainly cardiac glycosides, saponins, tannins, flavonoids, terpenoids, carbohydrates, and reducing sugars. Highest yield of cardiac glycosides in ethanol extracts leaves and stems, tannins in methanol and ethanol extracts of leaves and stems, respectively, terpenoids in aqueous and ethanol extracts of stems, carbohydrates in aqueous extracts of stems and reducing sugars were found in aqueous and ethanol extracts of leaves. These secondary metabolites showed inhibitory effects in all test organisms. The antimicrobial properties of alugbati leaves and stems extracts were tested against Gram-positive bacteria (Bacillus subtilis, Staphylococcus aureaus) and Gram-negative bacteria (Proteus vulgaris Escherichia coli, Pseudomonas aeruginosa, and Salmonella typhii) and pure cultures of fungi such as Aspergillus niger, Candida albicans, and Rhizopus stolonifers. In this study, the results of the various extracts revealed that the maximum activities were exhibited by methanol and aqueous extracts in stems against Salmonella typhii and Proteus vulgaris whereas, aqueous extracts in leaves showed no inhibitory activity against Escherichia coli, and Pseudomonas aeruginosa. Both leaves and stems of ethanol extracts possessed the highest inhibition for Bacillus subtilis and Salmonella typhii. Alugbati leaves and stems extracts showed antifungal activity against Aspergillus niger, Candida albicans and Rhizopus stolonifers. A. niger has the highest mean inhibition in ethanol extract of stems, and least inhibition in ethanol extract of leaves. No activity was seen in aqueous, and methanol extracts in leaves except Candida albicans and Rhizopus stolonifers in ethanol extracts of leaves. INTRODUCTION

Basella rubra (Malabar nightshade leaves or alugbati), a perennial herb is abundant and can be found in the backyards in the Philippines. It has spread throughout the tropical countries and is one of the best tropical spinach widely adapted to a variety of soils (Omar,2004). Alugbati is a succulent, branched, smooth, twinning, herbaceous vine, several meters in length. Leaves are fleshy, ovate or heart-shaped, 5-12cm long, stalked tapering to a pointed tip with a cordate base. Basella rubra had been studied to exhibit antimicrobial property against many different test organisms, with an exception to Pseudomonas aeruginosa (Sen et.al, 2010). It has an emollient and astringent properties which soften, soothe, and correct dryness and sealing of skin. It also constricts body tissues and effective in stopping the flow of blood and other secretion. A phytochemical analysis of alugbati is necessary in determining the essential components which makes the Alugbati a very important plant in pharmaceutical industry. The objectives of the study are:to qualitatively screen the phytochemical properties of Basella rubra, to determine the effect of using different solvent in the Phytochemical properties of Basella rubra and to determine potential antimicrobial activity from the leaves and stems of Basella rubra.

MATERIALS AND METHODS

Table 1. Phytochemical analysis of Basella rubra Linn. (Alugbati). Phytochemical Screening

Aqueous Extract Stem Leaves

Ethanol Extract Stem Leaves

Methanol Extract Stem Leaves

1. CardiacGlycosides

-

+

+++

+++

++

++

2. Saponins

++

++

++

++

+++

+++

+

++

+

+++

+++

+++

4. Flavonoids

++

++

++

++

++

++

5. Terpenoids

+

+++

+++

++

++

++

+++

+

++

+

++

+

+

+++

++

+++

++

++

3. Tannins

6. Carbohydrates 7. Reducing sugars Legend: (-) Negative

(+) Slight

(++) Moderate

(+++) Frequent

Source: Table 1. Ashutosh, M., Kumar, P., Ranjan, M., Susil, K., Ashutosh, M 2009).Phytochemical Screening of Ichnocarpus Frutescens Plant Parts, International Journal of Pharmacognosy and Phytochemical Research,1(1): 5-7.

In this study, table 2 shown the amount of aqueous crude extracts yielded from the various solvents. This result shown that the different solvent used in the extraction method affects changes in the content of the extracts. It was also observed that the effects were higher in methanol and aqueous extracts and less in ethanol extracts. This indicates that the active constituents of the plant parts have more ability to dissolve in methanol and aqueous solvent than ethanol solvents used in this study. Though ethanol solvent produced lower amount of extract they exhibited relatively higher effect than aqueous and methanol solvent which was obtained in lower quantity. It indicates that the amount of yield does not always influence in inhibiting the growth of bacteria but the active compounds found in the extract play a major role (Jeyaseelan et.al, 2011). A single solvent extraction may not be enough to exhaustively extract certain compounds responsible for the activity (Hebsibah & DhanaRajan, 2010).

All of the three extracts of Basella rubra Linn. showed varying degrees of antimicrobial activities against the test organisms, and their effectiveness were assessed by the diameter of inhibition zones. In this study, the results of the various extracts revealed that the maximum activities were exhibited by methanol and aqueous extracts in stems against Salmonella typhii, (29.67mm in stem and in leaves 14.33) and Proteus vulgaris (22.67mm) whereas, aqueous extracts in leaves showed no inhibitory activity against Escherichia coli, and Pseudomonas aeruginosa. Both leaves and stems of ethanol extracts possessed the highest inhibition for Bacillus subtilis (19.67 mm) and Salmonella typhii (17.67mm). The results indicated that the different extracts of alugbati have inhibitory effect on the growth of the test organisms due to the active compounds present (Table 3).The immense inhibition of aqueous extract in stem may link to the highest presence of carbohydrates in as recorded in Table 1 and the highest presence of Cardiac glycosides of both leaves and stems of ethanol extracts. The highest occurrence of tannins and saponins may constitute to the inhibition of methanol extracts of both leaves and stems. In Table 4, Aspergillus niger has the highest mean inhibition of 10.33 mm in ethanol extract of stems, and least inhibition in ethanol extract of leaves. No activity was seen in aqueous, and methanol extracts in leaves except Candida albicans (3 mm) and Rhizopus stolonifers (3.67 mm) in ethanol extracts of leaves. There are several reasons for the lack of antifungal activity in the extract, either the plant part used or the type of extraction might have resulted in the lack of activity in this study or the time of collection of herbal material and climate, which might, in turn, affect the amount of active constituents in the plant material (Obasi et. al, 2010). Figure 1. Comparison of different solvent system extracted from the leaves and stems of Basella rubra Linn. on the inhibitory activities of Different microorganisms

Table 2. Average recovery of different solvent extracts from 100g of alugbati leaves and stems

Different solvent extract Aqueous

Leaves Stems Amount Amount (mL) (mL) 320mL 290mL

Ethanol

120mL

70mL

Methanol

150mL

180mL

Figure 2. Effects of Inhibitory activities different extracts -Aqueous, Ethanol, and Methanol of leaf and stem in Different microorganisms

CONCLUSION

RESULTS AND DISCUSSIONS

Qualitative analysis of the compounds of Basella rubra Linn were performed on ethanol, methanol and aqueous extract of leaves and stems. The medicinal value of these plants lies in some chemical substances that have a definite physiological action on the human body. The most important of these bioactive constituents of plants are alkaloids, tannins, flavonoids and phenolic compounds (Hill, 1952). Phytochemical investigation of this plant has yielded more than 7 different compounds; mainly cardiac glycosides, saponins, tannins, flavonoids, carbohydrates, and terpenoids, as shown in Table 1.

Alugbati demonstrate different active compounds such as cardiac glycosides, saponins, tannins, flavonoids, terpenoids, carbohydrates and reducing sugars. However, the presence of these compounds varies significantly depending on the solvents used, some solvents showed extreme occurrence of specific compounds and some solvents had minor presence. Furthermore, the antimicrobial properties of Alugbati have shown substantially high in all test organisms however depending on the solvents being used. Aqueous extract inhibited some test organism but does not demonstrate consistency since aqueous leaves is the only extracts that showed inhibition. These results might be because different solvents extracted different bioactive compounds, and we used polar solvents so that, in effect it only extracted polar compounds. In addition to that these bioactive compounds may represent the distinction between the inhibition of bacterial and fungal strains especially the inhibition it demonstrate in a very resistant Pseudomonas aeruginosa. Also the experiment provides some scientific justification for the utilization of the stems and leaves extracts from alugbati to treat infectious diseases. However, it is important to be further purified through antibacterial activity guided fractionation to isolate, characterize and elucidate the compound responsible for antimicrobial activity.

RECOMMENDATION

The phytochemical substances present in alugbati leaf and stem should be isolated and purified to obtain their maximum therapeutic potentials. The sensitivity to other bacterial strains in Alugbati extract may also be used to further analyze the potential of the extract as antimicrobial agent. Other solvents are also recommended for the extraction process because we only used polar solvents and other non polar solvents extract non polar compounds. It is important to further study the Basella rubra efficacy as an antimicrobial agent by isolating specific compounds.