Evaluation of the antibacterial and antifungal potential of spider saxifrage plant ( Saxifraga flagellaris Willd . )

The current research work was performed to find antifungal and antibacterial potential of spider saxifrage plant. The aim was to find a new source against the selected pathogenic bacterial and fungal strains. Saxifraga flagellaris extract was fractionated. Five fractions were prepared and tested for antimicrobial potential against the selected fungal and bacterial strains. The highest antifungal activity (35-65%) was displayed by the crude methanolic extract against all the selected fungal stains and the lowest antifungal trend was exhibited by the aqueous fraction. However in antibacterial activity the n-hexane fraction showed maximum antibacterial activity (46-48%) against the selected pathogens and more or less same trend was followed by crude methanolic extract, while the least inhibition was shown by aqueous fraction. The other fractions though remained with moderate zone of inhibition but were significant.


Introduction
Plants' primary or secondary metabolites are natural products.Primary metabolites are necessary for the growth and development wile secondary metabolites govern the biochemical pathways [1].Secondary metabolites are formed from the primary metabolites via biosynthetic pathways and may or may not be linked to the growth of the plant but have significant ecological functions.The commercial applications of these extracts include different flavorings, fragrances and drugs synthesis etc. [2].There is enormous diversity in the nature of the plants derived secondary metabolites.Plants have medicinal properties due to the presence of certain secondary metabolites [3].
According to [4] these compounds are specific at different taxonomic ranks like subspecies, species, genera and families.About 50% of the medicines introduced into the market in the last two decades have been obtained from medicinal plants [5].Modern screening methods have given medicinal plants amazing importance because of the enhanced biological impact, structural confirmation and isolation of active metabolites.Currently three generations of medicinal plants are identified [6].Based on the enormous availability of the biochemical variety; medicinal plants provide a limitless resource of the new drugs discovery [7,8].About 25% of the medications and 66% of the anti-inflammatory and anticancer drugs in market and laboratory trials are of the medicinal plants origin [9].Based on the fact that microbes become resistant over time, the antibiotics; which play a fundamental role in the treatment of infectious diseases; become less effective which results in the increasing failures of the therapies.These diseases caused about 50% of the deaths globally and became the 3 rd major reason of deaths

Antibacterial activity Media preparation and agar well diffusion method
Following [23] agar well diffusion Method was used for antibacterial activity.1 liter distilled of water was used to liquefy 25g of Luria Broth, Miller powder at pH adjusted at 7.0.100ml of this media were autoclaved in 250ml flask.The selected bacterial strains were introduced into the flasks and kept overnight at 150RPM at 37 0 C. The agar was allowed to solidify in the petri-dishes and after that 5 holes were tunneled through a sterilized borer.The tunnels were then introduced with the inoculums.The bacterial and fungal strains were selected based on their frequent occurrence in the local hospitals and their resistance to common drugs.

Tested bacterial strains
The selected fungal strains included three gram positive strains i.e.Staphylococcus aureus, Streptococcus mutans and Methicillin-resistant Staphylococcus aureus (MRSA) and a single gram negative strain i.e.Serratia marcescens.

Extract preparation and measurement of zones of inhibition
The crude extract of Saxifraga flagellaris were dissolved in 20mg/ml of Dimethyl sulfoxide (DMSO).Standard antibiotic; Cefotaxime (2mg/ml) was used as positive control while pure DMSO as negative control.75µl of the plant fraction was introduced into the wells in petri-dishes.The samples were incubated for 24 hours at 37 0 C.After incubation diameter of the transparent zone, around each well was measured.The experiment was repeated thrice and standard deviation was calculated from the data obtained.

Antifungal activity Media preparation and agar well diffusion method
Potato dextrose agar (39g) was taken in liter distilled water (1 liter).The mixture was sterilized at 15PSI for 15 minutes in the autoclave.It was cooled at room temperature and poured into the petri-dishes to solidify.The method of [24] i.e. agar well diffusion method was followed.100µl of different strains of the selected fungal strains were placed on the surface of agar plate through a micropipette.The culture was spread by a sterilized inoculation loop.Holes were made in the culture (Plate 1) via a sterile cork borer.Crude extract (75ml) of the Saxifraga flagellaris were added to the petri-dishes and incubated at 37 0 C for 24 hours.Results were calculated by measuring the clear areas around each hole which indicated the extent of antifungal activity of each extract against the tested fungal strain.Each test was repeated thrice and the data was analyzed statistically.

Tested fungal strains
The selected fungal strains included Alternaria alternate, Aspergillus flavus, Polysphondylium pallidum and Fusarium oxysporum.

Results and discussion
The current research work was carried out to evaluate the anti-fungal and anti-bacterial potential of the spider saxifrage plant (Saxifraga flagellaris WILLD.)plant.The results revealed that this plant is a very effective source for the inhibition of the growth of the tested species of both fungi and bacteria.The plant can be investigated for active metabolites to evolve the anti-bacterial and anti-fungal drugs through biochemical and biophysical essays.

Antibacterial activities of Saxifraga flagellaris
In the current research work five fractions of Saxifraga flagellaris extracts were used to know their potential use against the selected bacterial strains that are Staphylococcus aureus, Streptococcus mutans and Methicillin-resistant Staphylococcus aureus (MRSA) and Serratia marcescens.These species were selected for the current experiments on the basis of their frequent pathological reports from hospitals of Khyber Pakhtunkhwa.Results of the anti-fungal activity as shown in ( respectively.Globally several hundred genera of medicinal plants are used as the main sources of anti-bacterial drugs [25,26].According to [27,28] in the previous century most of the faculties in the medicine sector switched their concern from natural to synthetic drugs.But for the last few decades this trend is shifting in the reverse direction [29].In a survey 25% of the drug prescriptions in 35

Conclusion
It is evident from the results of anti-bacterial and anti-fungal activities that the selected plant i.e.Saxifraga flagellaris possessed very significant properties.The crude methanolic and ethanolic extracts were comparatively more active which clarified that polar solvents dissolved more properly in the polar solvents.

21]. Similarly
[22]growth of Mycosphaerella eumusae can be inhibited by different extracts obtained from Allium sativum[22].In this regard the current research work was designed with the aim to evaluate the antibacterial and antifungal activities of Saxifraga flagellaris WILLD.