BJP Antimicrobial and cytotoxic activities of Barleria prionitis and Barleria grandiflora : A comparative study

Leaf juices as well as leaves of Barleria prionitis and Barleria grandiflora are being used by rural people across various regions of India in the treatment of oral ailments such as dental troubles, gum ailments, pyorrhoea, dental carries and mouth ulcers. Zone of inhibition and MIC values obtained for all the extracts suggest ethanolic extract of the herbs were more antimicrobial when compared to the aqueous extract. Results of biofilm suppression were found statistically significant (p<0.05) when compared to control. Cytotoxicity was evaluated by MTT assay on human gingival fibroblast and human dermal fibroblast cell lines for ethanolic extract of the herbs. CTC 50 value was found to be more than 1,000 µg/mL for ethanolic extracts of both herbs. Chlorhexidine was found to be more cytotoxic with CTC 50 value of 12.5–25 µg/mL. Ethanolic extract of B. prionitis and B. grandiflora found significantly cytotoxic (p<0.05) in comparison with control. Video Clip of Methodology: Cytotoxicity assay: 18 min 45 sec Full Screen Alternative


Introduction
Alternative system of medicine largely comprises of the use of plants and plant derived products (Banerjee et al., 2012;Singh and Dubey, 2012;Rawat et al., 2010).Genus Barleria L. belonging to family acanthaceae largely comprises of more than 300 species of shrubs and herbs.These species are mainly found in Asia and Africa (Balkwill and Balkwill, 1998).India is represented by 26 to 32 species, one subspecies and one variety (Shendage and Yadav, 2010;Karthikeyan et al., 2009;Balkwill and Balkwill, 1997).
B. grandiflora Dalz is an another species of this genus, leaves of which are being chewed by rural population across central India for the treatment of mouth ulcer, stomatitis and gingivitis (Sawarkar et al., 2009;Salunkhe et al., 2013;Jayanthi et al., 2014).Literature survey reveals that although these plants and their extracts evaluated for a number of pharmacological activities, not much of the work has been reported towards their usefulness in oral ailment.
The present study aimed to compare the usefulness of leaf extracts of B. prionitis and B. grandiflora in treatment of oral ailment like aphthous ulcers.

Plant material
The herbs had been collected in October, 2014 (flowering stage) from a rural region of Amravati district of Maharashtra (India).The herbs had been identified and authenticated by Prof. Ranjana Mishra, Department of Botany, Durg Science College, Durg (Chhattisgarh).Latter, leaves of B. prionitis and B. grandiflora were collected in February, 2015 (fruiting stage), dried in shade, powdered and used for extraction.

Extraction of plant material
The powdered plant materials had been treated with petroleum ether for defatting.Plant materials, thus, obtained further subjected to hot continuous extraction and cold maceration to get ethanolic extracts and aqueous extracts respectively.All the four extracts, then, treated with dichloromethane and ethyl acetate subsequently; in order achieve complete removal of fatty material from the plant material and designated as ethanolic extract of B. grandiflora, ethanolic extract of B. prionitis, aqueous extract of B. grandiflora and aqueous extract of B. prionitis.

Microorganism and growth conditions
Bacterial cultures Staphylococcus aureus MTCC 3160, Streptococcus mutans MTCC 890 and fungal culture Candida albicans MTCC 3017 were procured from Imtech, India.Lactobacillus sporogens [Sporlac powder, Sanzyme (P) Ltd.] purchased from local chemist.Bacterial strain had been cultured in nutrient blood agar media and fungal strain in Sabouraud dextrose

Principle
Biofilm suppression determined by microtiter biofilm formation assay is a qualitative and quantitative method to study the biofilms.Biofilms are large colonies of micro-organisms surrounded by fluid-filled channels and regarded as the one of the major factors responsible for antibiotic tolerance and resistance.Staining agent crystal violet, will impart violet coloration to the biofilms formed during the assay.In presence of antimicrobial component, the violet coloration will be lesser when compared to control, which can be quantified by reading absorbance values of the test solutions and standards using microcuvetes.

Procedure
Step 1: The overnight cultures bacteria and the fungus had been diluted to 1:100 respectively in Mueller Hinton broth and Sabouraud dextrose broth.Each well, seeded with 100 μL of bacterial and fungal cultures.The microtitre plate had been incubated at 37°C for 24 hours.
Step 2: Two gentle washings were given by sterile distilled water to remove bacterial and fungal cells on the surface or unbound cells.
Step 3: All the test extracts were reconstituted in dimethyl sulfoxide and diluted in respective broth to obtain concentration of 5 mg/mL.100 µL of each of the extracts was then added in to the wells and incubated at 37°C for 4 hours.Similarly, 100 µL of chlorhexidine (50 µg/mL) was added to the wells and incubated at 37°C for 4 hours.100 µL of broths were added to the control wells and incubated at 37°C for 4 hours.
Step 4: Contents of the wells were discarded after incubation.
Step 5: 100 µL of staining agent (0.1% crystal violet) was added to each well and incubation had been carried out at room temperature for 15 min.
Step 6: Sterile distilled water was used to remove excess of staining.Three washings were given with sterile distilled water and plates were then air dried.
Step 7: 125 µL of acetic acid (30%) was used for the destaining of microbial cells.The destaining was carried out for 15 min.
Step 8: Quantification of biofilm was done by transferring the contents of the wells to another microtiter plate and by taking the absorbance readings at 600 nm using spectrophotometer.
Step 9: Percentage inhibition of the biofilm was determined by using the formula, Percentage inhibition = 100 -{( Sample Absorbance/ Control Absorbance) × 100} Where, Sample Absorbance means absorbance observed with EBP, EBG and chlorhexidine separately.Control Absorbance means absorbance observed with 30% acetic acid in water.agar medium.The subcultures of bacterial strains had been prepared in nutrient broth for agar well diffusion assay and for biofilm suppression assay, the subcultures prepared in Mueller Hinton broth.The subculture of C. albicans had been prepared in Sabouraud dextrose broth.The bacterial and fungal subcultures were incubated at 37°C, for 24 hours and used for antimicrobial and biofilm suppression activities.

Cell lines
Human gingival fibroblast and human dermal fibroblast cell lines had been used for cytotoxicity study.DMEM supplemented with 15% FBS, streptomycin (100 µg/mL), penicillin (100 U/mL) and amphotericin B (0.25 mg/mL) was used for maintaining the cell lines.Both the cell lines maintained at 37.1°C with 2.1% CO2 and relative humidity of 44.4% in an incubator.

Antimicrobial activity
The antimicrobial activity against bacterial and fungal strains was performed for comparing the effectiveness of the extracts of both the herbs.The antimicrobial activity of the extracts had been evaluated against oral infectious microbial strains, S. aureus, S. mutans, L. sporogens and C. albicans by agar well diffusion method (Ahmad et al., 1998;Perumalsamy and Ignacimuthu, 2000;Werner et al., 1999).The bacterial cultures and fungal culture had been prepared in nutrient broth agar and Sabouraud dextrose agar respectively by pouring sufficient amount of strains aseptically.The sterile borer (8 mm) was used to make wells in petri plates.All the extracts were mixed in DMSO for obtaining final concentration of 5 mg/mL.The wells had been filled with 1 mL of extracts and compared with chlorhexidine (50 µg/mL) for antimicrobial potential.All the plates were incubated for 24 hours at 37°C.After 24 hours of incubation the zone of inhibition measured in mm.DMSO had been used as control.

Determination of minimum inhibitory concentration (MIC)
Microdilution method with slight modifications (Sarker et al., 2007) was used for determining the MIC for the extracts, chlorhexidine and control.DMSO was used for diluting the extracts and chlorhexidine.All the extracts were diluted further with sterile normal saline for obtaining serial dilutions as 5000, 2500, 1250, 625, 312.5, 106.2, 53.1 and 26.5 µg/mL.The positive standard chlorhexidine was diluted for obtaining serial dilutions as 50, 25, 12.5, 6.2, 3.1, 1.0, 0.5 and 0.2 µg/mL.The control was prepared using DMSO and sterile normal Box 2: Cytotoxicity assay Principle MTT assay is an in vitro method to study cytotoxicity.The method evaluates the cellular metabolic activity resulting into formation of dark purple colored formazan.MTT is converted into formazan due cellular mitochondrial dehydrogenase enzyme.
Step 2: The initial stock solution of test extracts (10 mg/mL) was prepared by dissolving in DMSO and subsequent dilutions had been made in serum free medium.
Step 4: The dilutions (100 µL/ well) added to cells.The plates had been further incubated at 37.1°C with 2.1% CO2 and relative humidity of 44.4%.Cell control (prepared by mixing equal volumes of DMSO and serum free media) had also been maintained.
Step 5: After 24 hours incubation, the morphological changes of the cells were observed.The cytotoxicity was determined by MTT assay.At this stage, the content of the wells was removed gently and 100 µL of MTT solution was added to each well.
Step 6: The plate was incubated for 4 hours at 37.1°C with 2.1% of CO2 and relative humidity of 44.4%.
Step 7: After incubation, the cell supernatant was removed and cell monolayer was washed with PBS twice.
Step 8: Finally, 100 µL of DMSO was added to each well to extract out the cell bound dye and incubation was carried out for 15 min.The absorbance was measured using spectrophotometer at 540 nm.
Step 9: The % cytotoxicity was calculated from absorbance values of treated and control groups.The CTC50 values for the sample were calculated from the dose response curves by linear regression analysis.
The percent cytotoxicity was calculated by using formula:

Percent cytotoxicity = [(C1-T1)/ C1] × 100
Where, C1 = Absorbance of cell control group and T1 = Absorbance of the test saline.The row of wells designated for extracts and standard had been pipetted with 240 µL of the extracts and chlorhexidine.Similarly, 240 µL of control had also been pipetted in to designated wells.These wells had been added with 10 µL of resazurin indicator solution.Preparation of resazurin indicator solution was done by dissolving 270 mg of resazurin in 40 mL of distilled water.Further, 30 µL of nutrient broth and Sabouraud dextrose broth had been pipetted into the wells with respect to the microbial culture to be inoculated.Finally, 10 µL of bacterial or fungal culture had been added to each well.The plates then incubated for 24 hours at 37°C.The MIC taken as the minimum concentration at which color changes from purple to pink or colorless.

Statistical analysis
All the results had been expressed as mean ± standard deviation.Results of biofilm suppression and cytotoxicity were further analyzed using one-way ANOVA followed by Dunnett multiple comparison test.

Antimicrobial activity and determination of MIC
The result of antimicrobial activity suggested the effectiveness of ethanolic extracts when compared to aqueous extracts of both herbs.All the extracts except ethanolic extract of B. prionitis (same MIC for bacterial and fungal strains) found to be more antibacterial (lower MIC) than antifungal.The antimicrobial activity of the extracts found to be less than the chlorhexidine (Table I).The antimicrobial activity of all the extracts was statistically significant (p<0.05) in comparison to chlorhexidine.Among the extracts, ethanolic extract of B. prionitis observed to be most effective against test microbes followed by the extract of B. grandiflora.B. prionitis shown same antibacterial and antifungal potential (MIC value in between 53.1-106.2µg/mL for all test microbes.Extract of B. prionitis showed significant antimicrobial potential with MIC value in between 106.2-312.5 µg/mL for bacterial strain and MIC value of 312.5-625 µg/mL for fungal strain.

Biofilm suppression
Ethanolic extract of B. prionitis and B. grandiflora were significantly suppressing the biofilms of test organisms (p<0.05) when compared to control (DMSO) at tested concentrations (Table II).Particularly ethanolic extract of B. prionitis proved to be more effective than ethanolic extract of B. grandiflora in biofilm suppression but results were not statistically significant (p>0.05) when compared to chlorhexidine.There was no significant difference observed between the ethanolic extract of B. prionitis and chlorhexidine at tested concentrations.Biofilm suppression with ethanolic extract of B. grandiflora observed statistically significant (p<0.05) when compared to standard particularly against all test bacterial strains.Ethanolic extract of B. prionitis and B. grandiflora both at tested concentration found no significant (p>0.05) in biofilm suppression against C. albicans.

Cytotoxicity assay by MTT
Cytotoxic effect of the ethanolic extract of B. prionitis and B. grandiflora on human gingival fibroblast cells were assessed using MTT assay.Based on the results of antimicrobial effects only ethanolic extracts were subjected for evaluation of cytotoxicity.The results had shown ethanolic extract of B. prionitis and B. Grandiflora at >1000 μg/mL caused 50% cytotoxicity (CTC50) to human gingival fibroblast cell lines, human dermal fibroblast cell lines.CTC50 value for chlorhexidine was found to be 12.5-25 µg/mL with human gingival fibroblast cell line.CTC50 value for chlorhexidine found to be 25 µg/mL with human dermal fibroblast cell line (Figure 1).Statistically both B. prionitis and B. grandiflora were found significant when compared to control (p<0.05).

Discussion
The result of the study showed the in vitro effectiveness of ethanolic extracts of both the herbs.Ethanolic extract of B. prionitis was most effective among all the extracts with MIC of 53.1-106.2µg/mL against all tested microbes.MIC for B. grandiflora was found 106.2-312.5 µg/ mL (bacterial strains) and 312.5-625 µg/ mL (fungal strain).Hence, for further study ethanolic extracts had been taken.The extracts evaluated for biofilm suppression and results found at par with chlorhexidine having no significant difference.Ethanolic extract of B. prionitis was found to be showing higher antifungal potential than standard at tested concentrations.Cytotoxicity assessed by MTT assay and CTC50 values for ethanolic extract of B. prionitis and ethanolic extract of B. grandiflora observed to be more than 1000 µg/mL.Kosmulalage et al. (2007) reported strong antibacterial activity of ethanolic extract and some phytochemicals like balarenone, pipataline, 13, 14-stigmasta-5-14-dione -3-ol, isolated from ethanolic extract of B. prionitis.Aneja et al. (2010) showed the potent antifungal and antibacterial activity of methanolic extract of B. prionitis bark against bacteria and fungi involved in oral diseases of human.Kumar et al. (2013)
B. grandiflora has been reported to have phytochemicals like glycosides, anthraquonone, saponins, flavanoids and phenolic compounds (Sawarkar et al., 2009).B. grandiflora yet not been investigated for the isolates.It is a well established fact that biological activities of the crude drugs are due to phytochemicals present in it.Thus it would be a possibility that the antimicrobial activity, biofilm suppression and cytotoxic potential of these herbs may be due to one or more phytochemicals present in them.

Conclusion
The usefulness of B. prionitis and B. grandiflora in treatment of oral ailments may be due to their antimicrobial and cytotoxic potential.Ethanolic extracts of both the herbs were found to be having more antimicrobial potential than to that of aqueous extracts and found less cytotoxic than chlorhexidine.

Figure 1 :
Figure 1: Cytotoxicity activity of the ethanolic extract of B. prionitis (A), B. grandiflora (B) and chlorhexidine (C) on the human gingival fibroblast and human dermal fibroblast cell lines by MTT assay

Table I Antimicrobial activity of leaf extracts
*Indicates statistically significant (p<0.05)antimicrobial activity of extracts in comparison to chlorhexidine; Data are mean; SD are within parenthesis