Evaluation of the antimicrobial activity of crude extracts and chromatographic fractions of Adenanthera pavonina Linn ( Leguminosae ) seeds

Most available reports on the biological activity of Adenanthera pavonina (AP) are on the bark or leaves and very few are on the seeds. In particular, there are no reports on the biological effects of the chromatographic fractions of A. pavonina seeds hence the present study aimed to evaluate the antimicrobial activity of the crude extract and chromatographic fractions of A. pavonina seeds. The methanolic extract was fractionated and all of the column chromatographic fractions as well as the crude extract were evaluated against different strains of Staphylococcus aureus. The methanolic extract (100 mg/ml) produced zones of inhibition on PHM 002 and PHM 005 while the 50 and 100 mg/ml of the hexane extract only produced inhibition on PHM 001. On the other hand, fractions ST 10-12 F exhibited activity PHM 002 at 50 and 100 mg/ml while fractions ST-13-15F exhibited activity at all concentrations (6.25, 12.5 25, 50 and 100 mg/ ml) against S. aureus PHM 002 strain from the skin. The study provides some justifications for the folkloric use of AP seed powder as an antiseptic paste and warrants further studies to determine the structure of the active compound in chromatographic fraction ST 13 -15F.


INTRODUCTION
Plants are valuable as sources of medicine.The use of natural products with therapeutic properties is as ancient as human civilization and, for a long time, mineral, plant and animal products were the main sources of drugs (Rates, 2001).In the recent decades, there has been growing interest in alternative therapies and the therapeutic use of natural products, especially those derived from plants (Vulto and Smet, 1988;Rates, 2001).*Corresponding author.E-mail: aa.adedapo@ui.edu.ng.Tel: +234 816 2746 222.
Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License This may be due to the fact that the conventional medicine can be inefficient (such as having side effects and ineffective therapy), abusive and/or incorrect use of synthetic drugs results in side effects and other problems.Also, a large percentage of the world"s population does not have access to conventional pharmacological treatment.Medicinal plants have served as valuable starting materials for drug development in both developing and developed countries.Today, more than 80% of the people living in Africa depend on medicinal plants and animal based medicines to satisfy their healthcare requirements (Yedjou et al., 2008).It has also been suggested that folk medicine and ecological awareness suggest that "natural" products are harmless.Many pharmaceutical products are from plants; though pharmaceutical industries have produced a number of new antibiotics in recent times, resistance by microorganism remains a challenge.Action must therefore be taken to reduce this problem by controlling the use of antibiotics, developing research to better understand the mechanism of resistance and embarking on studies to develop new drugs either natural or synthetic (Ahmed et al., 1998;Hussain et al., 2011).
Adenanthera pavonina Linn (AP) is a medium to large deciduous tree of the family Leguminosae also called Fabaceae.The tree is common throughout the lowland tropics up to 300 to 400 m (Jayakumari et al., 2012).In terms of medicinal uses, it has been reported that bark and leaves are astringent, vulnerary and aphrodisiac and are used in ulcers, pharyngopathy and even the heart wood is astringent, aphrodisiac, haemostatic and is useful in dysentery, hemorrhages and gout (Vaidyaratnam and Variers, 1994).The root on the other hand is emetic in nature (Jayakumari et al., 2012).It has been reported that the plant have antiseptic and antiinflammatory activities.The red powder is used as an antiseptic paste.Effects of A. pavonina include antiinflammatory and analgesic from methanolic seed extract (Olajide et al., 2004) and ethanolic leaf extract (Mayuren and Ilavarasan, 2009); and antimicrobial from the bark extract (Ara et al., 2010).The blood pressure lowering effect of the seed extract has also been reported (Adedapo et al., 2009).The main important constituents are flavonoid compounds (Rastogi and Mehrotra, 1991).It is used as an antiseptic paste and also used to treat boils and inflammations (Chopra et al., 1956).
Most available reports on the biological activity of A. pavonina are on the bark or leaves and very few are on the seeds.In particular, there are no reports on the biological effects of the chromatographic fractions of A. pavonina seeds.Chromatography is the key to obtaining pure compounds for structure elucidation, for pharmacological testing or for development into therapeuticals.It also plays a fundamental role as an analytical technique for quality control and standardisation of phytotherapeuticals (Marston, 2007).
In this study, the chromatographic fractions of the seeds of A. pavonina are being explored for its antimicrobial properties with a view to determine the fraction responsible for this effect.

Test organisms
Reference strains were used as suggested by Cos et al. (2006).These are different strains of Staphylococcus aureus; from the nose (strain 1) PHM 001, from the skin (strain 2) PHM 002, from the eye area (strain 3) PHM 003, from the armpit (strain 4) PHM 004 and the standard (strain 5) PHM 005.All microorganisms were obtained from the Department of Pharmaceutical Microbiology, University of Ibadan, Nigeria.

Plant material
The seeds of A. pavonina were collected from the Staff School Compound in the University of Ibadan, Oyo State throughout the month of September 2009.They were authenticated at the Forestry Research Institute (FRIN), Ibadan where a voucher specimen was deposited.

Sample preparation
The seeds were air dried and powdered.The powdered seeds were extracted using cold maceration.The powdered seeds sample (1335.09g) was put in a large flask and defatted for 24 h using 1280 ml-hexane.It was stirred occasionally and filtered.The marc was air dried and placed in a flask.The plant material was covered with 2000 ml of methanol and allowed to stand for four days after which it was filtered and the filtrate concentrated to dryness to yield 30.77 g of extract.The residue was again extracted with another 1,300 ml of methanol and the filtrate was again concentrated to dryness to yield a further 20.61 g of crude methanolic extract.

Phytochemical screening
The phytochemical analysis was performed on the ground (powered) leaf of A. pavonina for identification of the constituents.

Thin layer chromatography (TLC)
The TLC plates of dimensions 20 × 20 cm were used.The thickness of the adsorbent layer was 1 mm.Thin layer chromatography was used to monitor fractions obtained from  (Harborne, 1984).

Column chromatographic separation
The methanolic extract was further subjected to silica gel chromatographic separation using standard procedures.The chromatographic column of length 50 cm and internal diameter 3.5 cm was rinsed with acetone and allowed to dry.It was then clamped vertically with a retort stand, and a plug of cotton wool was inserted at the bottom of the column using a clean glass rod.The column was filled with hexane, and the silica gel slurry was mixed with hexane in a beaker.The slurry was poured into the column and tapped intermittently for uniform distribution of the gel and to avoid bubbles in the column.The column was allowed to drain for some minutes to stabilize it.A portion (20.61 g) of the crude methanolic extract was subjected to chromatography in column silica gel, was eluted with solvent mixtures of increasing polarity and dissolved in methanol by warming on a water bath.The cooled methanolic extract was then introduced on top of the silica gel adsorbent in the column.The column was eluted with solvent mixtures of increasing polarity.Fractions were collected and monitored by thin layer chromatography using appropriate solvent systems.Fractions showing similar TLC patterns were pooled together (Li et al., 2012).
Nutrient agar (28 g) was mixed with distilled water to make up 1 L.
The mixture was heated to boiling on a hot water bath.The molten agar was poured into universal bottles and autoclaved for 30 min at 121 psi.Nutrient broth (4 g) was dissolved with distilled in a conical flask and was made up to 500 ml.The mixture was measured in 10 ml aliquots into test tubes and sterilized in an autoclave for 20 min at 121 psi.The methanolic extract and pooled chromatographic fractions were tested for antimicrobial activity using the reference organisms stated earlier.The respective organisms were subcultured into the nutrient broth from the stock.The overnight culture (0.2 ml) was inoculated into 10 ml of sterile distilled water in a test tube.From the diluted organism, 0.2 ml was taken and inoculated into the cooled nutrient agar and shaken for homogeneity.The sterilized agar was poured into Petri dishes and allowed to set.It was dried and bore holed with a cork borer of diameter 8 mm.Six holes were made on each plate.Different concentrations of the extracts and fractions of A. pavonina seeds were then introduced into the holes.The plates were allowed to stand for 30 min to achieve diffusion after which they were incubated for 24 h at 37°C (Gurib-Fakim, 2006;Jimoh et al., 2011).

RESULTS
The phytochemical screening of methanolic extract of the seeds of A. pavonina showed the presence of tannins, saponins, alkaloids, flavonoids and cardiac glycosides while Tables 1 to 6 show the TLC analysis of crude methanolic and hexane extract of A. pavonina seeds; column chromatographic separation of the crude methanolic extract of A pavonina seeds; TLC analysis of column chromatographic fractions of the methanolic extract of A. pavonina seeds using mobile phase SS5 (chloroform: methanol 7:3); TLC analysis of column chromatographic fractions of the methanolic extract of A. pavonina seeds using mobile phase SS7 (toluene: acetone: ethylacetate 80:40:20); TLC analysis of column chromatographic fractions of the methanolic extract of A. pavonina seeds using mobile phase SS6 (toluene: acetone 4:1) and anti-microbial screening of the crude methanolic extract, hexane extract and chromatographic fractions of A. pavonina seeds, respectively.Column chromatographic analysis of the methanolic extract of A. pavonina seeds resulted in 15 fractions on elution with different solvent mixtures.Similar fractions were bulked on the basis of thin layer chromatographic analysis.
Fraction 15 was found to be the one with highest yield providing enough material for anti-microbial evaluations.TLC analysis of the fractions was done using three different solvent systems.Solvent system 6 (toluene: acetone 4:1) was found to be the most appropriate among the solvent systems as it gave the best resolution of the components.The most suitable solvent system for TLC analysis of the crude methanolic and hexane extracts, solvent system 2 (toluene: acetone: EtOAc 8:2:1) which found to be most appropriate gave 3 spots (Rf values: 0.53, 0.67, 0.84) for the methanolic extract and 2 spots (Rf values: 0.43, 0.60) for the hexane extract.The methanolic extract (100 mg/ml) produced zones of inhibition on PHM 002 and PHM 005 while the 50 and 100 mg/ml of the hexane extract only produced inhibition   on PHM 001.On the other hand, only fractions ST 10-12 F and ST 13-15 F produced inhibition.The results of chromatographic analysis and antimicrobial assay are as shown in the Tables 1 to 6.

DISCUSSION
Phytochemical screening of A. pavonina seed showed the presence of tannins, alkaloids, flavonoids and cardiac glycosides.Saponins are of great pharmaceutical importance because of their relationship to compounds such as sex hormones, cortisones, diuretics, steroids, vitamin D, and cardiac glycosides.Flavonoids have influence on arachidonic acid metabolism, thus could have anti-inflammatory, anti-allergic, anti-thrombotic or vasoprotective effects.Flavonoids and tannins are phenolic compounds and plant phenolics are also a major group of compounds that acts as primary antioxidant or free radical scavengers (Ayoola et al., 2008).Tannins and saponins are also found to be effective anti-oxidants, antimicrobial and anti-carcinogenic agents (Lai et al., 2010).Flavonoids are known to target prostaglandins which are observed in the late phase of acute inflammation and pain perception (Chakraborty et al., 2004).Cardiac glycosides are steroidal glycosides which exert a slowing and strengthening effect on a failing heart.In fact cardiac glycosides are being tested for their anticancer properties (Chen et al., 2006;Frese et al., 2006;Newman et al., 2008).Thin layer chromatographic screening of A. pavonina seed crude extract and column chromategraphic fractions showed the presence of several alkaloid substances on spraying with Dragendorff reagent.Alkaloids are generally quite bitter and many are toxic (Takimoto and Calvo, 2008).Some alkaloids such as vinca alkaloids have been used to treat diabetes, high blood pressure, and the drugs have even been used as disinfectants.However, the vinca alkaloids are most famous for being cancer fighters (Takimoto and Calvo, 2008).The implications of all these is that this plant is of great medicinal importance.
Column chromatographic analysis of the methanolic extract of A. pavonina seeds resulted in 15 fractions on elution with different solvent mixtures.Similar fractions were bulked on the basis of thin layer chromatographic analysis.Fraction 15 was found to be the one with highest yield providing enough material for anti-microbial evaluations.Anti-microbial activities of the extract and components from A. pavonina seeds showed that only fractions ST 13 -15 F was able to inhibit the growth of S. aureus strain 2 isolated from the human skin as all concentrations used.The methanolic extract was effective only at the highest concentration (100 mg/ml) against S. aureus strain 2 and S. aureus strain 5.It was ineffective at all other concentrations against other strains.The hexane extract was effective at concentrations 50 and 100 mg/ml against S. aureus strain 1 isolated from the nose alone, while it was inactive against all other strains of S. aureus at all concentrations.Fraction ST 2-7F showed no activity against any of the S. aureus strains, while fraction ST 10-12F was active against S. aureus strain 2 only at 50 and 100 mg/ml.Flavonoids, alkaloids, saponins and tannins have been reported to be active against several bacteria and fungi (Hassan et al., 2004).The presence of these in the crude extract of A. pavonina could be responsible for the antibacterial activity exhibited by the crude extract and components from A. pavonina in this study.The standard reference, gentamicin 500 µg/ml was able to inhibit all strains of S. aureus.Based on the results obtained from this study, there was preliminary evidence that there reside in the extract and components from A. pavonina seed, compounds with antibacterial activity against isolates of S. aureus.There is a need for further studies to determine the ED 50 , as well as to isolate and characterize the constituents from the active fractions of this plant.

Table 1 .
TLC analysis of crude methanolic and hexane extract of Adenanthera pavonina seeds.

Table 2 .
Column chromatographic separation of the crude methanolic extract of Adenanthera pavonina seeds.

Table 6 .
Anti-microbial screening of the crude methanolic extract, hexane extract and chromatographic fractions of Adenanthera pavonina seeds.