Antimicrobial activity of Bauhinia tomentosa and Bauhinia vahlii roots

The hexane, ethylacetate and methanol extracts from Bauhinia tomentosa and Bauhinia vahlii roots were tested for their antimicrobial activity against Gram-positive bacteria (four strains), Gram-negative bacteria (three strains) and three fungi strains using microdilution methods, for the determination of minimal inhibition concentration (MIC) and the minimal microbicidal concentration (MMC). The MIC values of hexane extracts of B. tomentosa and B. vahlii roots were more than 250 µg/ml. The MIC values of ethylacetate and methanol extracts of B. tomentosa roots varied from 7.81 to 31.25 µg/ml and 31.25 to 62.50 µg/ml, respectively. The MIC values of ethylacetate and methanol extracts of B. vahlii roots varied from 15.63 to 62.5 µg/ml and 62.5 to 250 µg/ml, respectively. MMC values obtained are two times greater than the corresponding MIC values. The activities of ethylacetate extracts are attributed to the presence of flavonoids and that of methanol extracts are attributed to the presence of tannins.


INTRODUCTION
The relatively large Bauhinia genus (family: Fabaceae) consisting of trees, climbers and shrubs is distributed in a wide range of geographic locations.Certain Bauhinia species have a long history of traditional medicinal applications. [1]auhinia tomentosa is a scrambling, many-stemmed shrub or small tree, the branches often dropping, with many slender twigs. [2]It is called as adavimandaramu in Telugu and phalgu in Sanskrit.It has been reported to contain amino acids, [3] proteins, [3] fatty acids, [4] minerals, [5] lectins, [6] protocatechuic acid, [7] phytohemagglutinins, [8] rutin, [9] quercetin [9] and isoquercetin. [10]Bauhinia vahlii is the largest creeper in India and is called adattige in Telugu and asamantaka in sanskrit.It has been reported to contain amino acids, [11] proteins, [11] minerals [12] and flavonoids. [13]Despite the very encouraging traditional medicinal applications of some species of Bauhinia, prior investigations to validate the traditional medicinal applications have not appeared in literature.The aim of this investigation was to evaluate the antimicrobial activity of the crude extracts of B. tomentosa and B. vahlii roots.

Plant materials
The roots of B. tomentosa and B. vahlii were collected in July 2007 in Thalakona and Tirumala, respectively.Botanical identification of the plants was done by Dr. K. Madhava Chetty, Department of Botany, S.V. University, Tirupati.Specimens of B. tomentosa (Voucher number: 1821) and B. vahlii (Voucher number:1716) were conserved in S.V. University herbarium.

Extraction of plant materials
The air-dried and powdered roots of B. tomentosa and B. vahlii (2 kg each) were extracted in Soxhlet apparatus sequentially with hexane, ethylacetate and methanol.The extracts were concentrated in a rotary evaporator under vacuum at a temperature not more than 50°C.

Culture media
Nutrient agar (NA) containing bromocresol purple was used for culturing of Bacillus species while NA was used for other bacteria.Sabouraud glucose agar was used for culturing fungi.Nutrient broth containing 0.05% phenol red and supplemented with 10% glucose (NBGP) was used for the determination of minimal inhibition concentration (MIC) and the minimal microbicidal concentration (MMC).The Muller Hinton agar (MHA) was also used for the determination of MMC.

Reference antibiotics
Chloramphenicol (Sigma-Aldrich, USA) and Nystatin (Sigma-Aldrich) were used as reference antibiotics (RA) against bacteria and yeast, respectively.

Minimal inhibition concentration and minimal microbicidal concentration determinations
The MIC of test samples and RA were determined as follows.The test sample was dissolved in dimethylsulfoxide (DMSO).The test and RA solutions obtained were added to NBGP and made to a final concentration 250 µg/ml.These solutions were serially diluted twofold to obtain concentration ranges of 0.49-250 µg/ml.Inoculums were standardized at 1.5 × 10 6 CFU/ml by adjusting the optical density to 0.1 at 600 nm in a spectrophotometer.Each concentration was added in a well of 96-well microplate containing 95 l of NBGP and 5 µl of inoculum. [14]The final concentration of DMSO in the well was less than 1%.Preliminary analysis with 1% (v/v) DMSO/NBGP did not affect either the growth of the test organisms or the change in color due to microbial growth.The negative control well consisted of 195 µl of NBGP and 5 µl of the standard inoculum. [14]The plates were covered with a sterile plate sealer, then agitated to mix the content of the wells using a plate shaker and incubated at 37°C for 24 h.The assay was repeated thrice.Microbial growth was determined by observing the change of color in the wells.The color was red where there was no growth and yellow when there was growth.The lowest concentration showing no color change was considered as MIC.
For the determination of MMC, 5 µl of liquid from each well that showed no change of color was plated on MHA and incubated at 37°C for 24 h.The lowest concentration that yielded no growth after this subculturing was taken as MMC.

RESULTS AND DISCUSSION
The antibacterial and antifungal activities of the crude extracts were evaluated and the results are reported in Tables 1 and 2 1] and MMC [Table 2] showed that the MMC values obtained are two times higher than MIC on the corresponding sensitive microorganisms.
In conclusion, it can be said that the extracts of B. tomentosa roots were more potent than the respective extracts of B. vahlii roots.The relative potencies of extracts are in the order of ethylacetate > methanol > hexane.
Preliminary chemical examination showed the presence of sterols and terpenoids in hexane extracts, flavonoids in ethylacetate extracts, glycosides and tannins in methanol extracts of B. tomentosa and B. vahlii roots.
Only the antimicrobial activity of B. tomentosa leaves has been reported in the literature. [15]To the best of our knowledge, antimicrobial activitiy of B. tomentosa and B. vahlii roots is being reported for the first time.Nevertheless, this study supports the traditional use of B. tomentosa roots as a vermifuge [16,17] and as a mouth gargle. [18]onsidering the medicinal importance of the tested microorganisms, the results of this study are considered to be very promising in the perspective of new drug discovery from plant sources.Ps. aeruginosa has emerged as one of the most problematic Gram-negative pathogens, with the alarmingly high antibiotic resistance rates. [22]Even with the most effective antibiotics against this pathogen, the resistance rates were detected as 15-20.4% amongst 152 Ps. aeruginosa strains.This pathogen was found to be sensitive to the crude extracts of B. tomentosa and B. vahlii roots.Bacillus species and Es. coli are agents of food poisoning. [23,24]. albicans and other Candida species, causing candidiasis, are increasingly important as they are distributed worldwide, and also they are frequent opportunistic pathogens in AIDS patients.[25] Proteus species are widespread in environment and account for 3% of nosocomial infections in the United States.[26] The incidence of enterococcal bacteremia due to En. faecalis is continuously increasing.[27] S. aureus has been associated with persistent and antibiotic-resistant infections.[28] Our investigation showed that the antimicrobial activity of ethylacetate extracts from B. tomentosa and B. vahlii roots might be related to the presence of flavonoids.Flavonoids are known to exhibit antimicrobial activity through formation of a complex with the bacterial cell wall. The ntimicrobial activity of the methanol extracts from B. tomentosa and B. vahlii roots might be attributed to the presence of tannins.The probable mechanism of these phenolic compounds activity includes enzyme inhibition by oxidized compounds, possibly through reaction with sulfhydryl groups or through more nonspecific interactions with proteins.[29] Finally, antimicrobial activity of the extracts from B. tomentosa and B. vahlii roots may be due to the presence of both antifungal and antibacterial compounds in them.The present study provides an important basis for the use of extracts from these plants for the treatment of infections associated with the studied microorganisms. Isoltion and characterization of bioactive compounds from these two species of genus Bauhinia is currently being carried out in our laboratory.
. The MIC values of hexane extracts from B. tomentosa and B. vahlii roots on all microbial strains were greater than 250 µg/ml.The MIC values of ethylacetate and methanol extracts of B. tomentosa and B. vahlii roots were less than 250 µg/ml.For ethylacetate extract of B. tomentosa roots, MIC values on Pr. vulgaris, S. aureus and other strains were 7.81, 31.25 and 15.63, µg/ml respectively.For ethylacetate extract of B. vahlii roots, MIC values on Pr. vulgaris, S. aureus and other strains were 15.63, 62.5 and 31.25 µg/ml, respectively.For methanol extract of B. tomentosa roots, the MIC values on S. aureus and other strains were 62.50 and 31.25 µg/ml, respectively.For methanol extract of B. vahlii roots, MIC values on Pr. vulgaris, S. aureus and other strains were 62.5, 250 and 125 µg/ml, respectively.The results of MIC [Table

Table 1 : MICs (g/ml) of the hexane, ethylacetate and methanol extracts from B. tomentosa and B. vahlii roots and reference antibiotics
Dugasani, et al.: Antimicrobial activity of Bauhinia species

Table 2 : MMCs (g/ml) of the hexane, ethylacetate and methanol extracts from B. tomentosa and B. vahlii roots and reference antibiotics
a Reference antibiotics (chloramphenicol for bacteria; nystatin for fungi); nd: not determined because MIC > 250 µg/ml