Fruit Pulp Extracts of Ficus racemosa and Aegle marmelos: Ethnopharmacological Approach for curing the Diabetic Foot Ulcer

Diabetic Foot Ulcer (DFU) are the most common complication represent a central cause of morbidity among Diabetes mellitus (DM) mostly type–ІІ, also reported amputation of foot region the impact of this complication cause mortality until treated. Biofilms are the sole responsible for over 90% of all chronic wounds in case of DFU. Staphylococcus aureus , Pseudomonas aeruginosa , and Proteus sp, are prime pathogens causing biofilm, whereas higher prevalence occupied by Pseudomonas aeruginosa during chronic condition. The objective of the current investigation is to determine the value of methanolic fruit pulp extracts of Ficus racemosa and Aegle marmelos against isolated key biofilm former Pseudomonas aeruginosa and associated bacterial pathogens. The phytochemical constituents for fruit pulp extracts of F. racemosa and A. marmelos were determined using FT-IR analysis. Minimum Inhibitory Concentration (MIC), Biofilm Inhibitory Concentration (BIC) and antibacterial activity were performed to find out the efficiency of the extract. F. racemosa was shown the better antibiofilm activity than A. marmelos . Remarkably, Sub-MIC level showed increasing inhibitory activity as concentration increases (0.5 mg/mL). Microscopic analysis showed dose dependant reduction in the biofilm architecture as compared to control. The extracts of fruit pulp of F. racemosa and A. marmelos show the anti-biofilm activity and which might be used as a substitute medicine in DFU. But it remains for the further analysis to elucidate the active binding molecule against biofilm former P. aeruginosa.


INTRODUCTION
Diabetic Foot Ulcer (DFU) is referred to a clinical complaint, but simply it is a merely wound on the foot of a patient who is diabetic (Green et al., 2013). A 50%-70% of chance to formation of diabetic foot in persons with diabetes mellitus (Markakis et al., 2016;Kirsner et al., 2010;Boulton et al., 2005). Foot lesions induces the high morbidity and mortality and represent the most common origin of hospitalization in patients with diabetes. It influences a person's quality of life, resulting to a decline to social, physical and psychological functions (Davis et al., 2006) which have high frequency to amputation of the leg (Jeffcoate and Harding, 2003). Diabetics with foot ulcers commonly experience infection with Gram-positive (Staphylococcus aureus and Enterococcus) and Gram-negative (Pseudomonas aeruginosa, Escherichia coli, Klebsiella sp, Proteus sp, etc., and some anaerobes) organisms (Khan et al., 2016) which lined up in the infection site in the form of "Biofilms". Now-a-days, the formation of multidrugresistant bacteria is of great concern due to wideranging use of antibiotics, GMO's and so on. These bacteria have built-in abilities to discovery new ways to be resistant and can pass along genetic materials that allow other bacteria to become drug-resistant as well (CDC, 2017). Multi-drug resistant gene can transfer through the conjugation process between populations or within population in the natural environment (Stickler et al., 1998). Nowadays, plantbased products have found widespread application in the medical and food industries as alternatives to conventional therapy. Generally, essential oils of plants have been widely used to treat several ailments (Ríos and Recio, 2005) and the use of its extracts has also gained popularity as they contain various bioactive compounds whose antimicrobial properties yield wide spread application in the pharmaceutical industry (Shan et al., 2011).
The objective of present investigation is to explore the use of ethno-medicinally properties of Ficus racemosa and Aegle marmelos fruit pulp extracts for the possible remedy of the DFU biofilm former bacterial pathogens.

bacterial strains
Four bacterial pathogens (Pseudomonas aeruginosa, Pseudomonas mendocina, Proteus mirabilis and Shewanella xiamenensis) were found from wound swabs of diabetes foot ulcers patients from Karpagam Medical College Hospital, Coimbatore with proper authentication and directions. Todd Hewitt's Broth (THB) was employed for cultivation of bacterial pathogens and which was maintained at -20°C for future studies.

Fruit pulp extraction and FT-IR analysis
F. racemosa and A. marmelos were obtained from local market in Coimbatore, Tamil Nadu. 50 mL of methanol was used for preparation of extraction from 10 g of fruit pulps. The prepared extract was filtered using the filter paper. The process was carried out by rotary vacuum evaporation to remove the solvents from filtrate. The concentrated extract was dissolved in DMSO and store in -4°C for further analysis (Packiavathy et al.,2012). The functional groups of derived extracts were determined by the Fourier-transform infrared spectroscopy using the protocol of Durrell (1964).

Antibacterial activity assays
The different concentration of prepared extracts (25, 50 and 75 µg/mL) were prepared a n d u s e d f o r A g a r w e l l d i ff u s i o n a s s a y. Streptomycin (0.03 mg/mL) was used as positive control and meanwhile DMSO was employed as negative control. The isolated bacteria (Pseudomonas aeruginosa, Pseudomonas mendocina, Proteus mirabilis and Shewanella xiamenensis) were swabbed on Muller Hinton agar plates. After that well was created and different concentration of extracts was poured. Finally, the plates were incubated at 37°C for 24 hours. The zone of inhibition was measured after the incubation period. The growth curve analysis was done using the 2 mg/mL of F. racemosa and A. marmelos fruit pulp extracts for bacterial pathogens. The bacterial pathogens were grown in LB broth and supplemented with fruit pulp extracts. The cell density was determined according the procedure of Abraham et al.,(2011) and assessed using the UV-Visible spectrophotometer at 600nm. The Minimal Inhibitory Concentration (MIC) assay was performed using the F. racemosa and A. marmelos fruit pulp extracts for inhibition of isolated bacterial growth.

Antibiofilm assay (Microscopic view)
The inhibition of biofilm formation by bacterial pathogens was determined by the antibiofilm assay via protocol of Nithya et al., (2011). Fruit pulp extracts of Ficus racemosa and Aegle marmelos (0.0625-1 mg/mL) were prepared and used for this analysis.

FT-IR analysis of fruit pulp extracts
The functional group of Ficus racemosa possess the active components based on the peak value in the infra-red radiation. The FT-IR spectrum was identified 09 functional groups between 500 to 4000 frequency wavelengths. The presence of intense bands at 817. 28 Table 2).

Antibacterial activity assay
The fruit pulp of methanol extracts from Ficus racemosa showed maximum antibacterial activity against test pathogens of Pseudomonas aeruginosa (20mm) and Proteus mirabilis (17mm) at varying degree of inhibition than A. marmelos. The maximum inhibition was observed in high concentration at 75 µL against the test bacterial pathogen (Table 3).

Growth curve analysis
In growth curve analysis, extracts had good effect on the cell biomass inhibition against bacterial pathogens at 1 mg/mL concentration which was apparent from relatively high to low growth in the cell density. Results reveal that the cell mass inhibition was observed in Ficus racemosa extract possess high bactericidal effects at the frequent incubation periods than A. marmelos (Figure 4).

Minimal Inhibitory Concentration (MIC) Assay
Sub-MIC of 2 mg/mL were taken at vary concentrations (0.062-0.5 mg/mL) and inhibition of all pathogens was drastically observed as the concentration increases. Pseudomonas aeruginosa and Proteus mirabilis highly shown the sensitivity against the fruit pulp extract of Ficus racemosa compared to Aegle marmelos at 0.5 µL concentration ( Figure 5). Inhibition assay (Microscopic View) I n L i g h t m i c r o s c o p i c a n a l y s i s , Pseudomonas aeruginosa and Proteus mirabilis were subjected to the methanol extracts of Ficus racemosa and Aegle marmelos due to the high sensitivity, biofilm susceptibility analysis was assessed using the inhibitory concentrations of 0.0625-1 mg/mL concentration (Fig. 6). Significant reduction in the biofilm formation was observed in the test concentration when compared to their respective controls. This elucidated that the fruit pulp extracts of Ficus aeruginosa inhibited the biofilm former than Aegle marmelos. and Rhl (RhlI and RhlR proteins). The Rhl system showed the prominent characteristics having a role in immune invasion and biofilm formation (Lequette and Greenberg, 2005). In this study, Ethnopharmacology approaches were designed to eradicate the biofilm former because these isolates highly resist in multi-drug combinations and also required high doses of antibiotics to eliminate this biofilm phase. Isolated pathogens were exposed to fruit pulp extracts of F. racemosa and Aegle marmelos which shown an efficient activity against the biofilm formers. FTIR analysis showed the presence of active compounds with varying medicinal properties includes alkyl groups, aryl groups, carbonyl compounds, cyclohexane ring vibrations, Hydroxyl, Isothiocyanate, Methylene, Olefinic group, Phosphate (Aromatic), Alkene and Alcohol group compound possess anti-cancer, anti-oxidant, anti-microbial, wound healing activity. Proteus mirabilis and Pseudomonas aeruginosa divide in one plane in chains of varying lengths in selection agar media and Gram staining (Todar, 2006). They are generally strong fermenters of carbohydrates, resulting in the production of lactic acid, a property used in the dairy industry and hold a catalase, Oxidase and IMViC negative (Kilian and Bulo, 1976). The present study results were show the correlate with the earlier report of Gram staining and Biochemical test.
Fruit pulp extracts of Ficus racemosa showed good antibacterial activity against test pathogens of Pseudomonas aeruginosa (20mm) and Proteus mirabilis (17mm) at the concentration of 75 µL respectively, whereas in A. marmelos, Pseudomonas aeruginosa (15mm) and Proteus mirabilis (10mm) showed its activity at the maximum concentration of 75 µl. Sequential inhibition rate were noted as the concentration increases. Comparatively, fruit extracts of F. racemosa showed maximum activity than A. marmelos.
In Growth Curve analysis, both the fruit extracts minimize the cell density of all pathogens especially Pseudomonas aeruginosa, Proteus mirabilis and Pseudomonas mendocina. As compared with A. marmelos, Ficus racemosa exhibited the maximum inhibitory level, thus biofilm activity against the Pseudomonas aeruginosa and Proteus mirabilis of Ficus racemosa was proceeded for further analysis. Similar results were observed in Minimum Inhibitory Concentration (MIC), extracts of Ficus racemosa showed the inhibition range at 0.5 mg/mL in case of Pseudomonas aeruginosa and Proteus mirabilis, followed by Pseudomonas mendocina and Shewanella xiamenensis and the extracts of Aegle marmelos showed the meager response compared to F. racemosa respectively. The antibiofilm activity at vary concentrations (0.062-1 mg/mL) of fruit pulp extracts showed gradual inhibitory effects from 0.25 concentration, whereas in A. marmelos inhibitory range were steadily low from the range of 0.25. Pseudomonas aeruginosa and Proteus mirabilis were steadily viewed under microscope which was exposed to fruit pulp extracts since these are chief biofilm formers.

CONCLUSION
The present investigation clearly indicated the fruit pulp of Ficus racemosa extracts contain the primary and secondary phytochemicals which are high effective against the prime biofilm former Pseudomonas aeruginosa and Proteus mirabilis in efficient way which was evident and confirmed by different In vitro studies. Studies has also ensured or suggested that this bioburden will be reduced or eradicated through substitute biomaterial in the form of dressing bandage or spray which coated with phytoconstituents from Ficus racemosa could be a possible remedy for DFU patients. Furthermore, studies should involve in understanding of action to exploit as potent of in vivo biofilm mechanism against pathogens.

ACKNOwLEDGMENT
First author thanks the Sri Ramakrishna College of Arts and Science for providing the laboratory facilities to perform the experiments. The corresponding author thank to the Karpagam Academy of Higher Education for providing the laboratory facilities to conduct the research work and also the author thank to DST-FIST, New Delhi for infrastructure facility (SR/FST/LS-1/2018/187).