Plants have been utilized as medicines for thousands of years (Samuelsson
and Bohlin, 2004). These medicines initially took in the form of crude drugs
such as tinctures, teas, poultices, powders and other herbal formulations (Samuelsson
and Bohlin, 2004). The acceptance of traditional medicine as an alternative
form of health care and the development of microbial resistance to the available
antibiotics has led to investigate the antimicrobial activity of medicinal plants.
The increasing failure of chemotherapeutics and antibiotic resistance exhibited
by pathogenic microbial infectious agents has lead to the screening of several
medicinal plants for their potential antimicrobial activity (Elizabeth,
Antimicrobials of plant origin are not associated with side effects and have
an enormous therapeutic potential to heal many infectious diseases. For example,
vincristine (antitumor drug), digitalis (a heart regulator) and ephedrine (a
bronchodilator used to decrease respiratory congestion) were all originally
discovered through research on plants. The potential for developing antimicrobials
from higher plants appears rewarding as it will lead to the development of a
phytomedicine to act against microbes (Iwu, 1999). Concern
has been expressed about the rising prevalence of pathogenic microorganisms
which are resistant to the newer or modern antibiotics that have been produced
in the last three decades worldwide (Cohen, 1992; Nascimento
et al., 2000). The identification of organic compounds from the extracts
of plants is of great importance, mainly because they can be used as an excellent
source of pharmaceutical products for phytotherapy (Melecchi
et al., 2002; Lang and Wai, 2001). GC-MS technology
appears to be the requirement for chemical derivatization prior to quantitative
analysis (Mueller et al., 2002; Birkemeyer
et al., 2003; Schmelz et al., 2004).
Gymnema is an antidiabetic plant and well known herbal medicine due to the therapeutic
efficacy of its different species.
Among them, the biological activity of G. kollimalayanum is not explored
so far except the taxonomic sketch (Ramachandran and Viswanathan,
2009). Based on the above cited informations, the present and first-time
report focused on the screening of antibacterial activity and isolation of biological
compounds from the pulverized leaf extracts of G. kollimalayanum, a new
record plant from peninsular India.
MATERIALS AND METHODS
Plant collection: The fresh and healthy leaves of Gymnema kollimalayanum A. Ramachandran and M.B. Viswan were collected from the higher altitudes (>1100 in MSL) of Kolli hills at Namakkal district, Tamil Nadu, India. The plant leaves were washed, air dried and powdered.
Extraction procedures: Both polar (water, methanol, ethanol and acetone) and non polar (Di-chloromethane and chloroform) solvents were mixed with the known amount of plant powder (each 10 g in 100 mL). This mixture was kept in a shaker upto 72 h in room temperature and the mixture was filtered through whatmann no. 1 filter study. The filtered solvent was kept in 100 mL of glass beaker. The crude extraction was kept in hot air oven for allow to evaporation until it reach the dried condition. The required amount of extraction could be taken for the antibacterial test.
Screening for antibacterial activity
Preparation of bacterial inoculums: The eight bacterial cultures
(E. coli, P. aeruginosa, P. vulgaris, S. aureus,
S. pneumoniae, K. pneumoniae, S. typhi and B. subtilis)
were collected from the clinical laboratory, Govt. Hospital, Salem, Tamil Nadu.
The stock culture of each bacterium was subculture on nutrient broth at 37°C
for 12-14 h prior to carry out the antibacterial tests.
Agar well diffusion method: The antibacterial activity was performed
by the agar-well diffusion method as described by Natarujan
et al. (2005) with few modifications. A volume of 15 mL of agar medium
was inoculated with 0.1 mL of fresh overnight culture. Three wells of each plate
(5 mm in diameter) were punched in the agar and filled with 70 μL of the
each extract. After holding the plates at room temperature for 2 h to allow
the diffusion of the extract into the agar, the plates were incubated at 37°C
for 24 h and the diameter of the inhibition zones of each well was measured
(13) (Gupta, 1977). The standard antibiotic (streptomycin)
and DMSO are served as positive and negative control.
GC-MS study: Gas chromatography and mass spectroscopy analysis was performed by GC clarus 500 Perkin Elmer using Elite- 5MS column (5% diphenyl/95% dimethyl poly siloxane) 30x0.25 mnx0.25 μm of thickness. Helium was used as carrier gas at a flow of 1 mL min-1. The injection port was maintained at 250°C and the split ratio was 10:1. Oven temperature programming was done from 5-280°C at 10°C min-1 and it was kept at 280°C for 9 min.
Interface temperature was kept at 250°C. The ionization mode was electron impact ionization and the scanning range was from 45-450 (m/z). Mass spectra were obtained at 0-2 min interval. The spectra of the compounds were matched with NIST Version year 2005 library. The structure of selected biologically active compounds were drawn by Chemdraw, Version 8.0.0 Cambridge Soft Corporation, UK.
RESULTS AND DISCUSSION
The results of GC-MS study from the ethanol leaves extracts of G. kollimalayanum showed a total of 10 compounds were identified with 99.99%. The major compounds were identified as 2-penten-1-ol (E) and 2, 6, 10-dodecatrien-1-ol, 3, 7, 11-trimethyl-acetate (E, E) which accounted for 43.74 and 26.33%, respectively and rest of them are minor compounds (Table 1 and Fig. 1) and their structures are shown in Fig. 2.
This study was supported by Sathya et al. (2010)
investigated on the identification of phytochemicals (terpenoids, glycosides
and alkaloids) from the successive leaf extracts of G. sylvestre (Asclepiadaceae)
using similar technique.
The results of antibacterial properties of aqueous and different organic solvents
extracts of G. kollimalayanum were tested against several bacterial strains
(Table 2) by agar well diffusion method. The methanolic extracts
showed moderate to least activity in all the bacteria except P. aeruginosa.
The ethanol extracts of the plant highlighted better antibacterial potentiality
against the organisms tested. Similarly, the chloroform extracts expressed significant
activity against most of the bacteria except P. aeruginosa and K.
||GC-MS study of ethanolic extraction of G. kollimalayanum
|| GC-MS analysis of leaves of G. kollimalayanum
|| Antibacterial activity of leaf extracts of G. kollimalayanum:
a new record plant
||Structural elucidation of some biologically active compounds:
a) dl-Arabinose; b) 2-penten-1-ol (E); c) L-Galactose, 6-deoxy; d) n-Decanoic
acid; e) pentadecanoic acid, 2, 6, 10, 14-tetramethyl-, methyl ester; f)
phytol; g) 9, 12, 15-Octadecatrienoic acid (Z, Z, Z); h) 1, 6, 10, 14-Hexadecatetraen-3-ol,
3, 7, 11, 15-tetramethyl- (E, E); i) 2, 6, 10-Dodecatrien-1-ol, 3, 7, 11-trimethyl-,
acetate (E, E) and j) Diazoprogesterone
The dichloromethane extracts contribute least activity against tested bacterial
strains excluding B. subtilis showed moderate inhibitory effect. Whereas,
the acetone extract showed broad spectrum of antibacterial potentiality against
all the pathogens. The aqueous extract was reported remarkable antibacterial
activity against P. aeruginosa over standard antibiotic and the same
extracts were found to be least to moderate activity in remaining tested organisms.
The overall results indicate that the aqueous and organic solvents extracts
of G.kollimalayanum showed moderate to better antibacterial activity
against most of the tested bacterial strains. Similarly, the alcoholic (Raja
and Devi, 2010), ethanolic (Satdive et al.,
2003), aqueous-methanol (Pasha et al., 2009),
chloroform and ethyl acetate extracts of G. sylvestre and G. montanum
(Ramkumar et al., 2007) showed broad spectrum
of antimicrobial activity against several bacterial strains include the tested
bacterias. The present results were also comparable to other genera of
Asclepiadaceae, i.e., Cryptostegia grandiflora (Mukherjee
et al., 1999), Oxystelma esculentum (Khan
et al., 2008), Tylophora hirsuta (Bashir
et al., 2009), T. indica (Reddy et
al., 2009), Pergularia daemia (Ignacimuthu
et al., 2009), Calotropis gigantea (Kumar
et al., 2010) and Pentatropis microphylla (Prabha
and Vasantha, 2010) have been proved significant antimicrobial activity.
The present study paves the way for further attention to test clinical validation
of the crude drugs.
The results revealed that plant extracts were effective both for controlling gram positive and gram negative human pathogens. This study encourage the use of herbal extracts as therapeutic agents for treat several diseases caused by the pathogenic bacteria.