CHEMICAL COMPOSITION AND ANTIBACTERIAL ACTIVITY OF PAJANELIA LONGIFOLIA (WILLD.) K.SCHUM AGAINST MULTI DRUG RESISTANT CLINICAL ISOLATES FROM DIABETIC FOOT ULCER

Katherin Steffy, G. Shanthi and V. Natarajan. Division of Microbiology, Rajah Muthiah Medical College, Annamalai University, Tamilnadu, India. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

In India, various traditional health care systems such as Ayurveda, Siddha, Unani etc solely depends on the plants and their products for the recovery of various ailments. Medicinal plants used traditionally found to contain several phytochemical compounds that aid in recovery of infections (Patwardhan et al., 2004). Thus drugs developed from the traditionally used medicinal plants, could provide higher routes to the invention, development and delivery of latest medication with increased performance in terms of value, safety and effectiveness (Kong et al., 2009). In order to elucidate the various properties of medicinal plants systematic research has to be done, so that the plant resources won't be destroyed due to improper use.
Plants which belongs to family Bignoniaceae are said to have significant medicinal values such as antioxidant activity, antiviral activity (Kernan et al., 1998), antiplasmodial activity (Onegi et al., 2002), anti-diarroheal activity (Longanga Otshudi et al., 2000), anti-microbial activity (Akunyili et al., 1991). Pajnelia Longifolia (Wild) Schum of family Bignoniaceae is an evergreen deciduous trees widely distributed in India, Srilanka, Thailand, North Sumatra, Natuna islands and Myanmar (Roy Choudhury et al., 2015). In India Pajnelia longifolia have been distributed in Western Peninsular India, North East India and Nicobar islands (Chander et al., 2015). Traditional healers in the tribal communities of Tripura have been using bark of Pajnelia longifolia for the treatment of Jaundice (Kumar Anil., 2007). Tribal communities of Assam have been using leaves of Pajnelia longifolia for the treating skin and nail infections (Chin et al., 2007). Ethnomedical practioners of Dakshina kannada district of Karnataka state uses pajnelia leaves for treating eczema (Padayana, 2011). Thus the aim of the present study is to identify various secondary metabolites present in petroleum ether, ethyl acetate, ethanol and aqueous extract of Pajnelia longifolia leaf through phytochemical and GC-MS analysis and evaluation of its antibacterial potential against standard bacterial strains and antibiotic resistant bacterial isolates from Diabetic Foot Ulcer. Collection of Plant material:-Plant leaves were collected from the forest areas of Western Ghats, South India from the month of June 2016. The plants were identified and authenticated by Kerala Forest Research Department (KFRI/SILVA/GEN16). The plant material was thoroughly washed with clean water allowed to shade dry. The dried leaves were pulverized to coarse powder and kept in an airtight container.

Preparation of Plant extract:-
Dried pulverized plant material was successively extracted with series of selected solvents from high to low polarity such as Petroleum ether, Ethyl acetate, Ethanol and Aqueous. 350gm of plant material was exhaustively extracted with 2 liters of respective solvents successively for twelve hours in a soxhlet apparatus. At the end of each extraction, the plant material was dried and then repacked for the next course of extraction. The crude extract obtained at every step was filtered with whatmann No.1 filter paper, was concentrated using rotary evaporator under reduced pressure and stored at 4⁰C. Preliminary phytochemical screening:-All the extracts were analyzed for preliminary phytochemical qualitative screening to identify the presence of various secondary metabolites as per methods given by Harborne. (1998). Antibacterial Assays:-Bacterial isolates:-Antibiotic Susceptibility pattern of Clinical bacterial isolates from Diabetic foot ulcer was determined by disc diffusion method according to the standard guidelines prescribed by Clinical And Laboratory Standards Institute (CLSI., 2016) .Clinical isolates that exhibited Multi drug resistance were confirmed tested with VITEK 2® Compact automated system (bioMérieux, Marcy l' Etoile, France) using GN Test Kit VTK2/GP Test Kit VTK2 were included in the study. Reference type strains of Staphylococcus aureus (ATCC 29213), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), Enterococcus faecalis (ATCC 29212) obtained from CSIR-National Chemical Laboratory, Pune were also included in antibacterial study.
Preliminary evaluation of antibacterial activity by disc diffusion Method:-Disc diffusion assay of plant extract was done by Kirby-bauer method (Bauer et al., 1966) as per CLSI guidelines (CLSI, 2016). Sterile Whatmann No.1 filter paper discs of 6 mm diameter of each were impregnated with 24 mg/ml, 48 mg/ml of plant extract dissolved in 20% Dimethyl Sulphoxide (DMSO) (Fischer-Scientific). Bacterial suspensions were prepared in columbia based blood agar (Himedia Laboratories) from overnight growth and the turbidity was adjusted to 0.5 McFarland standards that gives a colony count of approximately 1x10 8 cfu/ml. Using a sterile swab lawn culture of organisms was prepared on Muller Hinton Agar (Himedia Laboratories) and disc impregnated with plant extract was placed along with positive control antibiotic Vancomycin (30 µg) for Gram Positive organisms, Colistin (10 µg) for Gram Negative organisms and negative control (20% DMSO). The plates were incubated at 37ºC for 24 hours, the zone of inhibition of each well was measured and values were noted.

Determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC):-
MIC was determined by Macro dilution method (Wiegand et al., 2008). Plant extracts were serially diluted with Muller Hinton broth (Himedia laboratories, India) and bacterial suspensions were prepared according to manner described in disc diffusion were added to tubes containing plant extracts to obtain final desired concentrations of 0.19 mg/ml to 48 mg/ml. The inoculated tubes were incubated for 37°C for 24 hours under aerobic conditions. Vancomycin for Gram Positive organisms and Colistin for Gram Negative organisms, in a concentration ranging from 0.125 µg/ml to 512 µg/ml. Turbidity was checked after 24 hours of incubation. The lowest concentration of extract that produced no visible growth was considered as MIC.
For the determination of the MBC, tubes that produced no visible growth were gently mixed 100 µl were pipette onto a sterile blood agar plates and allowed to dry. The growth control and sterility control tubes were subcultured in same manner. All the test plates checked for the colony count after overnight incubation. Concentration of the extract that inhibited the growth of bacterial colony are considered as MBC and was calculated by comparing final inoculums volume with number of colonies as rejection values as per CLSI guidelines (CLSI, 1999).

Statistical Analyses:-
All the experiments were performed in triplicates, with the results being expressed as Mean ± SEM of three Independent experiments. The means were statistically compared using One-way ANOVA followed by post hoc Dunnett's Multiple Comparison's test by using GraphPad Prism version 5. P˂0.05 was considered as statistically significant.    Table.2. GC-MS profile of the ethanol extract of P. longifolia showed 6 major peaks ( Figure.1b) also described in Table.3.   Based on MIC/MBC ranges summarized in Table.5, ethyl acetate and ethanol extracts of P. longifolia leaf were only significant against MDR clinical isolates from DFU as well as against standard bacterial strains. The minimum inhibitory concentration (MIC) of Ethyl acetate extract and ethanol extracts of P. longifolia leaf against all bacterial strains ranged between 12 mg/ml to 48 mg/ml.  Our present study identified the presence of various phytochemical constituents in ethyl acetate and ethanol extracts of P. longifolia leaf and also major chemical compounds were analyzed through GC-MS. Antibacterial studies of plant extracts against MDR bacterial isolates from DFU and ATCC bacterial strains displayed moderate range of bactericidal properties. However, further studies should be done on isolation and identification of the pharmacologically active compounds to understand the exact molecular mechanism of action. The purified constituents contributing to antibacterial activity would be then used for novel cost-effective drug leads.