Biochemical profiling of different extracts of Centratherium anthelminticum seeds and its synergistic, antimicrobial activity on clinical, drug resistant and standard strains of some common microorganisms

Extracts of seeds of Centratherium anthelminticum was prepared by Bioassay guided fractionation using different solvents. Different extracts from the plant Centratherium anthelminticumwas biochemically analyzed for different bioactive components and properties. It shows an inhibitory action on different clinical strains within a range of 100 500μg/ ml.


Introduction
Centratherumanthelminticum (L) Kuntze., (Purple flebane) is a common ingredient in different ayurvedic preparations. The biological activities are related to the structure of the compound to an extent. Most important classes of biologically active compound belong to phenolic, flavonoids, alkaloids, terpenoids etc. Centratherum anthelminticum (L) Kuntze., (Purple flebane).
This plant belongs to the family Astearace and other common names are 'Kattujeerakam' 'Vanajeera', 'Aranyageera' and Kalijiri'. It is seen as herb in hilly areas and sub-tropical regions. Medicinal properties are vested in its seeds, roots, leaves etc. It is effective in treating thread worm infections and gastric troubles. Dry seeds were used in the present study [1].

Material and methods
Plant material: The seeds of Centratherum anthelminticum were collected from the plants Wayanad district, Kerala. It was identified by the Department of Botany, University of Calicut. Voucher no.7214 and was deposited in the herbarium.

Determination of minimum inhibitory concentration (MIC)
MICs for each extract against each microbial strains were determined using nutrient broth. 10 ml of the medium was taken in a 50 ml conical flask and sterilized. The extract (stock diluted to 10mg/ ml in DMSO) was then added to 10 ml of medium in a series of conical flasks to get a concentration of 10000, 1000, 100, 10, 0 μg/ ml initially (10 times dilution). Then double dilution was used to determine the range of inhibitory activity (MIC value) of each extract. A loop-full of fresh culture at log phase (1 x 10 8 CFU/ml, turbidity equal to that of 0.5 MacFarland solution) was added to each flask so as to have concentration of 5 x 10 5 CFU/ ml. It was then incubated at 36 0 for 18 to 24 hrs. The OD at 600 nm was monitored at different concentrations of the drug in the culture media. The least concentration at which the growth of the organism was completely inhibited was recorded as MIC of that extract, comparing the OD with that of the blank. It was then confirmed by inoculating to fresh agar plate [9], [10], [11]. The experiment was done in triplicate and average value of MIC was then taken. It was then statistically analyzed and p< 0.05 was considered as significant. The experiment was repeated for different strains and extracts. The values were compared for gram positive and negative bacteria. The difference in inhibitory effect of the extract on the growth of clinical and standard strains was compared.

Synergistic effect
The method was done using a 96-well micro-titer plate. In this 200 µl of the sterile nutrient broth was added to each well. In the first well of series 1, ie A1, antibiotic at a concentration of 100 µg/ ml was added. In the well B1 sample AS1 at a concentration of 500 µg/ml was added. In the well C1 both antibiotic and AS1 were added. The concentration of the antibiotic and AS1 were the respective MICs or above the MIC values. All the wells were then serially diluted to next well.ie 100 µl from first well has transferred to second and 100 µl from second to third and so on so that a double dilution had obtained. All wells were then inoculated with culture at log phase, mixed well, covered and incubated at 36 0 C for 18 hrs. The growth was observed and MICs were noted and Fractional inhibitory concentrations (FICs) were calculated [12], [13]. Fractional inhibitory concentration (FIC) was calculated using the formula FIC= MIC of drug in combination/ MIC of drug alone + MIC of extract in combination /MIC of extract alone.

Statistical analysis
All data were analyzed using SPSS version 16.0.

Results and discussion
The yield of the aqueous extract was yield was 3.5%. Biochemical analysis shows the presence of alkaloids, saponins and glycosides in aqueous extract ( Table 2, 3). HPLC profiling shows two major peaks at RT 1.6 and 7.6 [ Fig. 1]. Results of the in vitro anti-bacterial activity of the aqueous and methanol extract were summarized in Table 4 and 5.      Aqueous extract showed significantly higher activity against microbes test compared with methanol extract. It showed maximum activity against S. aureus and K. pneumonia. The activity against clinical strains was also significant. It showed significant activity against C.albicans also. The extract showed good activity against drug resistant organism like MRSA [ Table 6]. It showed synergistic effect against the microbes when tested along with antibiotics. When treated along with pencillin the concentration of pencillin was significantly reduced to less than half-fold [ Table 7]. Plants are alternative source of medicines [14,15,16]. Present study shows C. anthelminticum is a potential source against microbial diseases.  HPLC chromatogram at 210 nm shows the presence of three major compounds with retention time 3.08 min, 4.51 min and 8.7 min and area of 13500610 mAu 5728857 mAu and 38969965 mAu respectively.

Conclusion
We can conclude that Centratherium anthelminticumhas the potential source to develop into an antimicrobial agent. Both methanol and aqueous and methanol extract showed significant effect against standard and clinical strains respectively. It has synergistic effect with certain antibiotics also against E.coli as well as S.aureus. This has to be further studied as a combating strategy against drug resistance.