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

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 multidrug-resistant bacteria is of great concern due to wide-ranging 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, plant-based 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).

In traditional, Ficus racemosa L and Aegle marmelos L, are used as medicine for the treatment of numerous disorders. Root, fruits, latex, leaf and bark are used to treat the diseases such as carminative, leprosy, diarrhea, anti-dysentery, menorrhagia, vermifuge, astringent, circulatory and respiratory disorders (Sharma and Gupta, 2008). Gluanol acetate, tiglic acid, glucanol, taraxasterol, friedelin, and lupeol acetate are the major component of fruits extracts (Joseph and Raj, 2010) and also have various phenolics in the fruit as chlorogenic acid, ellagic acid, ferulic acid, gallic acid, protocatechuic acid and quercetin (Dhan et al., 2011). Charoensiddhi and Anprung, (2008) reported the presence of Dehydro-p-cymene, hexadecanoic acid, α-Cubebene, β-phellandrene, humulene oxide, caryophyllene oxide, β-caryophyllene and dihydro-β-Ionone in the extract of A. marmelos.

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.

Materials and Methods

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 gram 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 and used for Agar well diffusion assay.  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 h. 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 600 nm. 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.

Figure 1: Microscopic observation of isolates. Click here to View figure

Results

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 cm^-1, 948.52 cm^-1, 1049.28 cm^-1, 1311.59 cm^-1, 1419.61 cm^-1, 1674.21cm^-1, 2908.65cm^-1, 2993.52cm^-1 and 3641.60 cm^-1 corresponding to C-H, C-H (S), H – C and C = C bond vibration indicate the presence of Alkyl groups, Aryl groups, Cyclohexane ring vibrations, Alcohol, Hydroxyl, Olefinic group, Alkene and Alcohol respectively (Fig: 2 & Table: 1). In case of A. marmelos, the bands at 948.98cm^-1,1033.85cm^-1, 1311.59cm^-1, 1419.61cm^-1, 1728.22cm^-1, 1990.54cm^-1, 2916.37cm^-1, 3001.24cm^-1 and 3626.17cm^-1 corresponding to (P – O – C stretch), (Si – O – Si), C – H, C – H stretch, (- NCS), C- H asym /sym stretch and OH stretch bond vibration respectively, indicate the presence of Aromatic phosphates, Silicon-oxy compounds, Carbonyl compound, Olefinic (alkene) group, Aldehyde, Isothiocyanate, Methylene and Secondary alcohol respectively (Fig: 3 & Table: 2).

Figure 2: FTIR analysis of Ficus racemosa fruit pulp methanol extract. Click here to View figure

Figure 3: FTIR analysis of A. marmelos fruit pulp methanol extract Click here to View figure

Table 1: Phytoconstituents of Ficus racemosa by FTIR analysis.

S. No	Frequency (500-4000 cm-1)	Bond and type of Vibrations*	Functional groups
1	817.28	C-H	Alkyl groups
2	948.98	C – H	Aryl groups
3	1049.28	C –H	Cyclohexane ring vibrations
4	1311.59	O – H	Alcohol and Hydroxyl compound
5	1419.61	C – H	Olefinic group
6	1674.21	C=C	Alkene
7	2908.65	–	–
8	2993.52	–	–
9	3641.60	O – H	Alcohol group

 

Table 2: Phytoconstituents of A. marmelos by FTIR analysis.

S. No	Frequency (500-4000 cm-1)	Bond and type of Vibrations*	Functional groups
1	948.98	(P – O – C stretch)	Aromatic phosphates
2	1033.85	(Si – O – Si)	Silicon-oxy compounds
3	1311.59	C – H	carbonyl compound
4	1419.61	C – H	Olefinic (alkene) group
5	1728.22	C – H stretch.	Aldehyde
6	1990.54	(- NCS)	Isothiocyanate
7	2916.37	C- H asym. / sym. stretch	Methylene
8	3626.17	OH stretch	Secondary alcohol

 

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 75µl against the test bacterial pathogen (Table: 3).  

Table 3: Antibacterial activities of methanolic fruit pulp extracts.

S. No	Bacterial Pathogens	Ficus racemosa (µg/ml)			A. marmelos (µg/ml)
		25	50	75	25	50	75
		Zone of Inhibition (mm)
1	Pseudomonas aeruginosa	14	17	20	6	10	15
2	Pseudomonas mendocina	–	6	12	–	–	5
3	Proteus mirabilis	11	12	17	6	4	10
4	Shewanella xiamenensis	7	10	14	2	6	10
5	Streptomycin (0.03mg/ml) (Positive control)	17	16	17	15	14	16
6	DMSO (Negative control)	–	–	–	–	–	–

 

Growth curve analysis

In growth curve analysis, extracts had good effect on the cell biomass inhibition against bacterial pathogens at 1mg/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 (Fig:4).

Figure 4: Growth Curve analysis of fruit pulp extracts Click here to View figure

Minimal Inhibitory Concentration (MIC) Assay

Sub – MIC of 2 mg/ml were taken at vary concentrations (0.062-0.5mg/ml) andinhibition 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 0.5µl concentration (Fig: 5).

Figure 5: Minimal Inhibitory Concentration of fruit pulp extracts Click here to View figure

Biofilm Inhibition assay (Microscopic View)

In Light microscopic analysis, 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.

Figure 6: Light microscopic images of Pseudomonas aeruginosa and Proteus mirabilis biofilms Click here to View figure

Discussion

In Diabetic Foot Ulcers (DFU), deep tissues are unprotected to bacterial colonization, once the protective layer of skin is damaged (Vuorisalo et al., 2009). Based on the DFU swabs 96% of S. aureus, 99% for β- haemolytic Streptococcus and 96% of P. aeruginosa pathogens were reported (Gardner et al., 2014). Multicombinations of bacterial pathogens like Staphylococcus aureus, Enterococcus, Pseudomonas aeruginosa, E. coli, Klebsiella sp and Proteus sp form polymicrobial communities within a matrix of EPS (Extracellular Polymeric Substances) (Banu et al., 2015) may possibly communicate via Quorum Sensing (QS) where the systems interact mainly with exopolysaccharide (EPS) production (De Kievit et al., 2001). There are 2 QS systems in Pseudomonas aeruginosa – Las (LasR & LasI synthase proteins) 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). Theyare 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 75µl respectively, whereas in A. marmelos, Pseudomonas aeruginosa (15mm) and Proteus mirabilis (10mm)showed its activity at the maximum concentration of 75µl. 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 (0.062-1mg/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). 

Conflict of Interest

The authors declare no conflict of interest.

Funding Sources

No fund has been received from any funding agencies for this investigation.

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