Plant material
The plant Symplocos racemosa bark was procured from a local market of Amritsar, Punjab, India; which has been identified and deposited in the Department of Botanical and Environmental Sciences, Guru Nanak Dev University, Amritsar wide accession no. 766-768 Bot.& Env. Sc. dated 02/07/15. The plant material was sterilized as described earlier in Sood et al. [15].
Test microorganisms and Inoculum preparation
Reference strains: Enterococcus faecalis (MTCC 439), Staphylococcus aureus (MTCC 740) and Staphylococcus epidermidis (MTCC 435) as Gram positive bacteria; Escherichia coli (MTCC 119), Klebsiella pneumoniae 1 (MTCC 109), Klebsiella pneumoniae 2 (MTCC 530), Pseudomonas aeruginosa (MTCC 741), Salmonella typhimurium 1 (MTCC 98), Salmonella typhimurium 2 (MTCC 1251) and Shigella flexneri (MTCC 1457) as Gram negative bacteria and yeast strains such as Candida albicans (MTCC 227) and Candida tropicalis (MTCC 230) were obtained from Microbial Type Culture Collection (MTCC), Institute of Microbial Technology (IMTECH), Chandigarh, India. A clinical isolate (MRSA) obtained from PGIMER, Chandigarh, India was also used in the study. Their glycerol stocks were preserved at -80°C.
Drug resistant strains- The drug resistant strains used in the study were clinical isolates: MRSA (DSECI 1-11), Enterococcus sp. (DSECI 12) (procured from Shri Guru Ram Das medical College & Hospital, Amritsar (Pb.), India; MDR Escherichia coli (CRIRS 1-12) and MDR Salmonella spp. (CRIRS 13-22) obtained from Central Research Institute, Kasauli (H.P.), India.
The inoculum of these organisms was prepared by 4h activation in a suitable broth followed by its standardization upto 0.5 McFarland standards [15].
Antimicrobial Screening of organic extracts of Symplocos racemosa bark
Different organic solvents were screened to work out the best extractant. The organic extracts were prepared by using solvent-solvent extraction of the aqueous extract with an equal volume of the given organic solvent for three times independently. The pooled organic layers were concentrated; the residue was reconstituted in 30% DMSO and then screened for antimicrobial activity against the test microorganisms by Agar Diffusion Assay (ADA), where 30% DMSO served as a control.
Qualitative and Quantitative phytochemical profiling of Symplocos racemosa
Qualitative analysis for the major groups of phytoconstituents in the powdered plant material was carried out by standard methods as (Additional file 1). The qualitatively detected phytoconstituents were then quantified as described previously [19, 20] and screened for their antimicrobial potential, which was then compared with standard antibiotics (Additional file 1).
Minimum Inhibitory Concentration (MIC)
The ethyl acetate extract and the active phytoconstituents were tested for their MIC against test organisms as per protocol followed in Arora and Sood [21]. The values were then compared with that of the standard antibiotics.
Viable Cell Count (VCC) studies and Post Antibiotic effect (PAE)
Time kill assay of the test extracts was done so as to ascertain the microbicidal/static nature of the compounds. A stock solution was prepared and the study was performed according to Arora and Onsare [22]. A comparison was done with standard antibiotics and the experiment was performed in duplicate.
The PAE of the ethyl acetate extract, flavonoids and cardiac glycosides was performed according to [23]. The diluted suspension of the test organism was exposed to the test compounds for 2 h at 37ºC under shaking. The drug activity was stopped by diluting the suspension in a suitable double strength broth. Hundred microlitre, i.e., 0.1 ml aliquot from each set was plated at an interval of two hours upto 24 h. The PAE was calculated as follows: PAE=T – C (T= time required for the colony count in the test to increase by 1 log₁₀CFU/ml; C= the time required in case of untreated control).
Antibiofilm potential of the phytoconstituents of Symplocos racemosa bark
The antibiofilm potential was tested against four organisms, i.e., Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae 1 and a yeast Candida albicans using protocol detailed in Additional File 2.
Screening for biofilm formation
The biofilm formation by test pathogens was screened out by the microtitre plate according to Stepanovic et al. [24] and Costa et al. [25], where the organisms were grown in suitable broth for 24 h. The 200 µl aliquot of the activated cultures were dispensed into microtitre plates and further incubated at for 24 h, following which the wells were decanted off and stained with 0.1% crystal violet solution for 15 min. The wells were destained with 150 µl of 95% ethanol and the intensity of biofilm formation was affirmed in terms of Optical density (OD) of stained adherent biofilm by using an ELISA reader (Biorad 680-XR, Japan) at 590 nm. The results are interpreted by calculating the cut-off value (ODc), which separates biofilm producers from the non-biofilm producing strains, as follows:
Optical density cut-off value (ODc) = Average OD of the negative control + [3×standard deviation (SD) of negative control].
Upon confirmation the following assays were carried out to establish the antibiofilm potential of most active phytoconstituents.
Inhibition of initial cell attachment
The inhibitory potential of the partially purified phytoconstituents was carried out according to Jadhav et al. [26] and Onsare and Arora [27]. One hundred microlitres of the flavonoids and cardiac glycosides was added to the 96-well microtitre plates, to which an equal volume of the cultures was then added. The plates were incubated at suitable temperature for 24 h. Gentamicin and amphotericin B were used as positive control. Following incubation, the inhibition potential was established using crystal violet assay (described later).
Screening of phytoconstituents for their disruptive potential of preformed biofilms
It was performed as described earlier [25, 27] with slight modifications. One hundred microlitre aliquot of each of the 4h activated cultures were dispensed into a 96-well microtitre plate and were incubated at 37°C/24 h to allow biofilm formation and was treated with equal volume of the phytoconstituents. The plates were further incubated at suitable temperature and biomass content was estimated after 24 h by the crystal violet assay.
Estimation of biofilm biomass by Crystal Violet (CV) assay
The estimation of biofilm biomass was assessed by Crystal Violet (CV) assay [27-29]. Following the treatment of the test organisms, the culture medium from each well was discarded and plates were then washed with sterile distilled water and then dried, which were then stained with 100 µl of 0.1% crystal violet for 15 min. The plates were then repeatedly washed several times with sterile distilled water to remove unabsorbed stain. Absolute ethanol (125 µl) was then added to destain the wells and the absorbance was determined at 590 nm to quantitatively estimate the biofilm density and the percentage inhibition was calculated using the formulae:
Percentage inhibition= 100- [{OD590nm test well/ OD590nm negative control well without antimicrobial agent} × 100].
Estimation of metabolic activity by XTT assay
Post exposure to the flavonoids, cardiac glycosides and standard antibiotics, the metabolic activity of the treated biofilms was assessed using the modified {2, 3-bis [2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide} (XTT) reduction assay [27, 30]. After incubation of the biofilm with test extracts for 24 h, 200 µl of menadione–XTT working solution was added in respective well and further incubated in dark for 2 h at the suitable temperature. Upon incubation, the quantification of color was done using microtitre plate reader (Bio-Rad 680-XR, Tokyo, Japan) at 490 nm. The mean absorbance of test wells was determined in comparison to that of negative control.
Antimicrobial potential of the phytoconstituents against clinical isolates of MRSA, some drug- resistant strains of Escherichia coli and Salmonella spp.
The Flavonoids and cardiac glycosides were tested for their antimicrobial efficacy by ADA against different strains of MRSA, drug- resistant Escherichia coli, Salmonella spp. and Enterococcus sp. The MIC of the most sensitive organisms was also worked out by broth dilution method [31]. The lowest concentration with no visible growth was defined as the minimum inhibitory concentration (MIC). To determine the MBC, the aliquots from the wells showing no visible growth were swabbed onto the nutrient agar plates and incubated at 37°C for 24 h. The concentration corresponding to no growth on the plates was taken as MBC.
In vitro cytotoxicity against cancerous cell lines RD (Human Rhabdomyosarcoma), L20B (Diploid mouse lung cell line) and Hep 2 (Human epithelioma of larynx) by MTT assay
The cytotoxic effect of the most active phytoconstituent, i.e., flavonoids was studied against these three cell lines obtained from Central Research Institute (C.R.I), Kasauli, Himachal Pradesh, India by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay as described previously [32-34] with slight modifications. The experiment was performed in triplicates as per protocol detailed in Additional file 3. A stock solution (10 mg/ml) was prepared and subjected to two fold serial dilutions ranging from 10 mg/ml to 0.039 mg/ml for the experimentation. The percent growth inhibition was calculated with respect to untreated control. The IC50 of the flavonoids was calculated from the dose-response curve generated for each cell line.
Biosafety evaluation of Symplocos racemosa bark
Ames mutagenicity test
The test extracts were subjected to Ames test [21]. The overnight activated inoculum of Salmonella typhimurium (MTCC 1251, IMTECH, Chandigarh) was serially diluted and eventually mixed with equal volume of the extract (MIC concentration) and was then added to the top agar containing a histidine–biotin mixture (1:1 ratio). Here, sodium azide was used as a positive control.
MTT toxicity assay
In order to ascertain the cellular toxicity of the test extracts, MTT [3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay was performed as described in Eren and Ozata [35] with slight modifications. The wells with untreated cells served as control and percent viability was thus calculated.
Acute Oral Toxicity study of Symplocos racemosa flavonoids in Swiss albino mice
In order to validate the non-toxicity of the compound in animal models, acute oral toxicity was studied as described previously [12, 16] with slight modifications. The experiment was conducted at Central Research Institute, Kasauli, Himachal Pradesh, India as per the protocol detailed in Additional file 4. Healthy Swiss albino mice (males and females) weighing between 25 g to 35 g and aged 8 to 10 weeks were obtained from the Animal house, Central Research Institute. The mice were randomly divided into 2 test groups (6 male; 6 female) and 2 control groups (6 male; 6 female), where the test groups were exposed to a single dose (5000mg/kg) of the flavonoids by oral route. This study was carried out in accordance with the principles of the Basel Declaration and recommendations of Organization of Economic Co- operation and Development (OECD) guideline 420 for testing of chemicals. The protocol was approved by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), New Delhi, India (No. CPCSEA/IAEC/CRI/14-114-2016). On the 15th day, the final weight of mice was noted and was anaesthetized in the laboratory using xylaxine and ketamine (5mg/kg and 2.5mg/kg b.wt. respectively). The blood samples were collected via cardiac puncture in non-heparinized tubes. The serum samples, thus collected, were analyzed for determination of alanine aminotransferase (ALT), alkaline phosphatase (ALP), aspartate aminotransferase (AST), total bilirubin (TBIL), urea and creatinine levels. Following blood collection, all the animals were sacrificed by overdose of anesthesia. The vital organs mainly liver, kidney and heart were removed, cleaned with saline and subjected to histopathological analysis.
Identification of the bioactive components of the flavonoids
In order to find out the component responsible for antimicrobial activity of the most active phytoconstituent, i.e., flavonoids, various techniques such as TLC, Preparative-TLC and Gas Chromatography-Mass Spectrometry (GC-MS) analysis were performed.
Thin Layer Chromatography (TLC) analysis
To work out the best solvent system, the quantitatively isolated flavonoids were subjected to TLC on the pre-coated Silica gel F254 plates using different solvent systems. The developed chromatograms were allowed to air dry and were visualized using natural light, UV light (254nm, 365nm) and iodine vapors so as to assess the degree of separation of bands and the Retention factor (Rf) were calculated for the bands resolved in the most suitable solvent system. The best worked out solvent system was taken up for quantitatively separating the bands using Preparative TLC (P-TLC) method.
Quantitative separation of the bands using Preparative TLC (P-TLC) method and their antimicrobial screening
The bands were resolved and were separated in the selected solvent system using powdered Silica gel F254. Once developed, the separated bands were scrapped off carefully into separate vials, which were eluted overnight in methanol. The solvent containing the eluted band was decanted off carefully, which was evaporated to dryness to obtain a constant weight. The separated bands were dissolved in a minimum known volume of methanol and were screened for their antimicrobial activity against Staphylococcus aureus, Staphylococcus epidermidis (Gram positive) and Klebsiella pneumoniae 1, Shigella flexneri (Gram negative) and a yeast Candida albicans using Disc diffusion method, where the filter paper discs were impregnated with 20 µl of this suspension, while methanol alone acted as a negative control. The band/s showing the most significant antimicrobial activity was then subjected to GC-MS analysis.
Gas Chromatography-Mass Spectroscopy (GC-MS) analysis
The GC-MS analysis of the most active band/s was carried out using Thermo Trace 1300GC coupled with Thermo TSQ 800 Triple Quadrupole MS with column BP 5MS (30m X 0.25mm, 0.25µm). The instrument was set to an initial temperature of 60ºC, and maintained at this temperature for 3 min. At the end of this period the oven temperature was raised to 280 ºC, at an increase rate of 15 ºC/ min and maintained for 19 min. Injection port temperature was ensured as 260 ºC and Helium flow rate as 1 ml /min. The ionization voltage was 70eV. The samples were injected in split mode as 10:1. Mass spectral scan range was set at 50-650 (m/z). Using computer searches on a NIST Ver.2.1 MS data library and by comparing the spectrum obtained through GC-MS, compounds present in the flavonoids were identified.
Data analysis
The experiments were performed in duplicate and repeated thrice. The statistical analysis was done at 5% level of significance by IBM SPSS Statistics Data editor Version 20.