Antibacterial Activity and Time-kill Assay of Terminalia catappa l. and Nigella sativa l. against Selected Human Pathogenic Bacteria

The current investigation aims to test the susceptibility of human pathogenic clinical isolates and MTCC strains to leaf and seed extracts of Terminalia catappa and Nigella sativa. Disc diffusion assay, micro dilution assay and minimum Bactericidal Concentration investigated the susceptibility of bacteria to the test extracts. The active extract was subjected to phytochemical screening, separation of the phytochemicals by Thin Layer Chromatography, bioactivity guided assay and Timekill assay. Acetone and methanol extracts of T.catappa revealed, significant inhibition of clinical origin Staphylococcus aureus followed by Proteus vulgaris and the MTCC strains Staphylococcus aureus, Salmonella typhi, Pseudomonas aeroginosa and Bacillus subtilis. Nigella sativa inhibited the growth of clinical origin Staph.aureus and MTCC strain of Staph.aureus, Salmonella typhi and B.subtilis. Minimum inhibitory concentration for all the test bacteria was reported in the range of 5000μg/ml to 9 μg/ml in T. catappa extract. Most sensitive being the clinical isolate Staph. aureus and Proteus vulgaris. The bactericidal concentration for the test bacteria was found to be between 5000μg/ml and 625μg/ml. Phyto-chemical analysis of leaf extracts of T. catappa found to have dominated by polyphenols (Terpenoids, steroids, flavonoids, flavones, saponins and tannins) and N.sativa extracts recorded the presence of alkaloids, proteins and oils and fats. TLC profiling of the acetone extract revealed many antibacterial active bands. Bands having Retention factor 0.47 and 0.52 were active against the test bacteria. Time kill assay of the acetone extract of T. catappa were carried out for the first time. The extract exhibited dose dependent bactericidal and bacteriostatic activity against the clinical isolates.


iNtROduCtiON
Recently there has been increase in the spread of untreatable microbial infections and bacteria cause 90% of the infections 1. World Health Organization reports that infectious diseases cause 50% deaths. The non-selective use of synthetically originated antibiotics, microorganisms have led to the development of multidrug resistance, which poses a serious health concern 2 . The resistance to drug may be caused mostly by the unregulated use of antibiotics and poor hygienic conditions and affects severely in every aspect of life 3 . The escalating resistance in microorganisms is due to the phenomenon of genetic mutations or acquired antibiotic resistant genes influenced by ill-suited use of antibiotics 1 . Additionally antibiotics are associated with unfavorable consequences on the host including hypersensitivity, allergic reactions and immune-suppression 4 . The multiple drug resistance has enforced researchers in search of new drugs with antimicrobial property from different sources like medicinal plants, which serve as acceptable source of novel antimicrobial agents 5 . Rational localization of biologically active components from folk medicines and systemic evaluation will result in finding of novel efficacious drugs, which are potentially active against pathogens 6. Antimicrobial drugs with efficacious mode of action should be developed in order to surpass the downside of current antimicrobial drugs 7 . A large number of medicinal plants are reported to exhibit antimicrobial activity 2 . Reports of WHO states that medicinal plants would be the best root to acquire diverse of drugs 1 . In this context, plants promise a source of natural antimicrobial agents 8 . Antimicrobial activities of plants are ascribed to the presence of phytochemicals like tannins, phenols, alkaloids, terpenoids and flavonoids 9 . The application of plant based drugs in the treatment against pathogens is obtaining great acceptance because of scientific interest and non toxicity properties 10 .
Approximately 119 drugs isolated from plants are used in treating infectious diseases worldwide 11 . Not less than 50% of the drugs which are used in treating clinically infectious diseases accounts to have originated from natural and natural product derivatives 12 . Since phytochemicals have the ability to inhibit the growth of infectious microbes, many plants are still being used for their antimicrobial properties and many other biological activities. Inspite of existence of vigorous antimicrobial drugs, the emergence of resistance microbes has created an immense interest in the exploration and the outcome of efficacious drugs 13 .
Terminalia catappa is a large tropical tree of the family Combretaceae 1 . Different parts of the plant are used in folklore medicine and studies have revealed various medicinal properties 15 . Various research disclose the medicinal uses like microbial inhibition of leaf aqueous and methanolic extract of the plant 16 . Bacterial inhibitory activity of leaf aqueous extract 17 , anti-inflammatory property of leaf ethanolic extract 18 , wound healing activity of bark, antioxidant and radical scavenging activity of leaf aqueous extract, anticancer and antiaging activity of ethanol and aqueous extract of leaves have also been reported 19 . Anti-methicillin potential of phytochemicals of T.catappa have also been evident 20 . Insilico studies of compounds from T. catappa are responsible for hepatoprotective and hepatotoxic properties 21 . Extracts of the leaves of the plant also possess anticancer, anti-HIV reverse transcriptase, hepatoprotective, Anti-inflamatory, Anti-hepatitis and Aphrodisiac effects 22 . Phytochemicals include tannins (Punicalagin, Punicalin, Chebulagic acid, geranin, granitin B), flavonoids (Vitexin, Rutin, Isovitexin), and Terpenoids (Ursolic acid, Asiatic acid) which are responsible for therapeutic activity 23 .
Nigella sativa, belongs to the family Rananculaceae 24 . It is native to Mediterranian regions such as South west Asia, Southern Europe and North Africa 25 . Seeds contain yellowish volatile oil, fixed oil, alkaloids, saponins, minerals and vitamins 26 . The reported studies related to Nigella sativa have illustrated wide range of biological activities such as antioxidant 27 , anti-inflamatory 28 , antidiabetic 29 , anti prostate cancer effects 30 , antibacterial 31 , immunomodulatory effect 32 , and hepatoprotective 33 .

MATeRiALS AnD MeTHoDS Preparation of the plant extract
Mature leaf material was collected from fields in and around Mysuru, Karnataka. The leaves were shade dried and powdered using laboratory blender. N. sativa seeds were collected from the local market, powdered and used for successive solvent extraction by soxhlet extractor using polar and non-polar solvents. After extraction the solvents were evaporated to dryness under reduced pressure and preserved the extracts at 4°C for future analysis.

Microorganisms and growth conditions:
Human Antibacterial potency of the test plant extracts were done by disc diffusion method employing the CLSI M02-A document 36 . Inoculums were prepared and adjusted to McFarland standard with cell density of 1to 1-2 X10 8 CFU/ ml. Standardized inoculum (0.1ml) was swabbed to the previously solidified MH agar plate. Sterile discs were loaded with test plant extract (100mg/ ml) and impregnated onto the plates followed by incubation for 18-24 hours at 35 ±2°C. Standard antibiotics and solvents were employed as positive and negative control. All experiments were carried out in triplicates. Zone of inhibition (ZOI) around the disc was measured in mm.

Microbroth dilution method for determining Minimum inhibitory concentration (MiC) and Minimum Bactericidal Concentration (MBC)
MIC was established by employing 96 well microtiter plate according to the CLSI M07-A9 document 37 . The test plant extract, which showed the inhibitory activity, were selected for determining MIC and MBC. The test plant extract were serially diluted two fold to obtain concentration of 5000-9μg/ml. The experimental set up included reference drug controls and solvent as positive and negative controls respectively. Aliquot of standardized inoculum (1.5 X 10 8 colony forming units/ml) was added to all the wells. The micro dilution plate was covered to avoid drying followed by incubation. Inhibition of bacterial growth was confirmed by addition of 20μl of aqueous solution of 2, 3,5-Triphenyl tertrazolium chloride (TTC) and re-incubated for 4-5h. The colorless well was designated as the MIC, which inhibited the growth of bacteria. Change of colour from colourless to pink indicated the presence of viable cells. MBC was ascertained by sub culturing 10μl of test dilution from the lowest concentration well by streaking on the previously sterilized and solidified MH agar with overnight incubation at optimum temperature. The well, which gave no bacterial colony on the agar medium, was designated as MBC.

Phytochemical composition of the active test extracts
The test extracts were subjected to qualitative phytochemical analysis according to the established procedures to determine the presence of different class of secondary phytochemicals 38,39 .

Thin layer chromatography (TLC) of the active extracts
The active extracts were subjected for separation of phytochemicals. Based on the review of literature and slight modification, EMW (Ethyl acetate: methanol: water) with 40:5.4:4 ratio showed better separation of the compounds and it was best-suited eluent system for the separation of phytocompounds from members of Combretaceae. The retention factor (Rf) of the separated phytochemicals was calculated 40 .

TLC bioautography
Agar overlay technique 41 was adopted with minor modifications to locate the antibacterial bands. The TLC plates (silica gel G f 254 , Merk) were spotted with test plant extract(acetone extract) and developed in the pre saturated developing chamber using EMW as eluent system. After the solvent front reached the optimum distance the chromatogram was removed and visualized under long and short UV to locate the separated compounds. The chromatogram was dried overnight and subjected to overlay bioautography.
One milliliter of standardized test inoculum was mixed with 10ml molten Muller Hinton agar. TLC chromatograms were placed in the sterilized plates and thin layer of media inoculated with the test bacteria was flooded on the chromatogram. Plates were incubated overnight at 37°C. ZOI around the separated phytochemicals can be seen as a clear area without the growth of the test bacteria. As  a confirmatory test the chromatograms were flooded with microbiological agar containing TTC and further incubates for 30 minutes for optimum colour development. The ZOI is seen as white zone against the pink background.

Time kill assay
Protocol of kill time assay was adopted 42 with slight modifications. The active extract of T. catappa was set at a concentration of 4MIC and 8MIC. A control tube was maintained without the test extracts. Each tube was then inoculated with standardized suspension of clinical isolates of S.aureus and P.vulgaris At prefixed time points aliquots were withdrawn from each concentration diluted serially (10 -1 to 10 -4 ) and aliquots were plated on agar plates. Colony counts were performed after overnight incubation at 37°C in ambient air. Colony counts were averaged and expressed as log 10 cfu/ml.

Satatitical analysis
Values are presented as Mean ±SEM. ANOVA was employed to determine the difference in specific means followed by tukey's post hoc test at P<0.05. Graphpad prism (version 8) and Microsoft excel 2007 was used in generating graphs and analyzing the data.

Disc diffusion assay
The test organisms exhibited different sensitivity for the extracts tested which is shown in Fig. 1 to 4. All the test pathogens exhibited significant sensitivity towards acetone and methanol extracts of T.catappa. Acetone extract had good inhibitory activity against the test organisms in contrast with the methanol extract. Significant inhibition was observed against the clinical isolate Staph.aureus (22.5mm) followed

Micro dilution assay
Considering the results obtained from disc diffusion assay, deducing the lowest inhibitory concentration (MIC) of active extracts was considered necessary and the results are shown in Fig. 5 and 6.
Of all the organisms tested, the clinical isolate Staph.aureus and P. vulgaris was the    most sensitive to the acetone extract with lowest concentration of 39μg/ml and 312μg/ml respectively, Kleb.pneumoniae and Salm.typhi were less susceptible with concentration 1250μg/ ml and 625μg/ml respectively. The acetone extract also exhibited inhibitory effect at the lowest concentration against all the MTCC strains tested except E.coli with concentrations between 1250μg/ml and 9μg/ml. Methanol extract showed inhibitory activity towards the test pathogens but slightly less effective compared to the acetone extract having concentration between 5000μg/ml and 625μg/ml for clinical isolated bacteria. The minimum concentration of methanol extracts required against B.subtilis (MTCC 121) Salm.typhi (MTCC 733), Staph.aureus (MTCC 7443), Pseudo. aeruginosa (MTCC 424), and B. cereus (MTCC 1272) was in the range between 1250μg/ml and 78μg/ml.

Minimum Bactericidal Concentration
Acetone extract reported bactericidal effect at 1250μg/ml for clinical isolate Staph. aureus where as the bactericidal effect for Kleb.   Results of phytochemical analysis presented in Table 3. Broad group of phytochemicals were present in the acetone and methanol extract like terpenoids, flavonoids, saponins, cardiac glycosides, phenols and tannins. Hexane extract of Nigella sativa was dominated by alkaloids, oils and fats.

TLC profiling and bioautography
Thin layer chromatography of acetone extract using  (Fig. 9). Clear zone of inhibition around the separated bands was indicative of antibacterial property of the compounds present.
Colorless areas indicate inhibition of the test organisms by the separated phytocompounds.

Kill time assay
The time kill profile for clinical isolates Staph. aureus and P.vulgaris are presented in Table 4 showing the reduction in viable cell counts at particular time intervals. For both bacterial strains a separate time kill profile was produced over a period of 24 hours of incubation following inoculation. Difference in the viable counts for both bacterial strains were more after 4h post inoculation. At concentration corresponding to 8MIC bactericidal activity as observed at time point of 2-4h for Staph.aureus (Fig. 13) after which regrowth of the bacteria was observed indicating concentration dependent killing of the test bacteria. However, 4MIC concentration did not kill the bacteria but slowed down the growth compared to initial control. For P. vulgaris (Fig. 12) maximum reduction of viable cell counts was seen after 24h of incubation at 8 MIC concentration with near bactericidal effect of 3.37 log 10 Cfu/ ml and 4MIC concentration reduced the viable cell counts after 8h of incubation with 3.9log10

disCussiON
The present investigation reports the acetone and methanol extract of T.catappa leaf and hexane extract of N.sativa seeds demonstrated the antibacterial activity against the human   pathogenic bacteria and clinical isolates. The preliminary method for evaluating the sensitivity of the test bacteria towards the plant extract is inhibition zone testing by disc and well diffusion assay. However, to test the efficacy of the extracts, broth dilution assays are also used preferably microbroth dilution assay 41 . Acetone and methanol extract of T.catappa demonstrated varying level of activity with inhibition zones ranging between 11.66 and 23.33 mm for all the test bacteria.
The results are in accordance with the previous reports where in polar solvent (methanol and aqueous) extracts of T. catappa leaves in addition to twenty other plants were evaluated against bacteria (Gram positive and gram negative) and found that majority of the test strains were susceptible to methanol extract having inhibition zone 5-18mm 16 . The differences in the antibacterial activity can be due to the different chemical composition and the distinct mechanism of action of their bioactive constituents 43 . The results can also be attributed with previous reports, which recorded the aqueous extracts of T.catappa exhibited inhibitory activity against Staph. aureus, Kleb.pneumoniae, E.coli and Candida albicans 44 . Several other reports have revealed the antimicrobial activity of leaves and fruits of T.catappa on different gram positive and negative bacteria 45 . The susceptibility results obtained by disc diffusion assay of leaf methanol extract of T. catappa against Staph.aureus, pseudo. aeroginosa, B.subtilis and a clinical isolate Staph.aureus demonstrated that gram-positive organisms were more susceptible 46 . Polyphenols are reported to have antibacterial activity 47 . Various polyphenols isolated from T.catappa like catappanin, geranin, gallantonic, leutolin, apigenin, orientin, isovitexin, catachien, kampferol, genistein, querccetin, ellagic acid, chlorogenic acid, ferulic acid , arjunetin,, ursolic acid, gallic acid, arjunoilc acid, betulinic acid and several compounds from T.catappa have been reported. The above isolated compounds from the plant could be acreditted to the invitro susceptibility of bacteria found in another study 48 . Several other workers [49][50][51] have also reported the antibacterial properties of various other parts of T. catappa against human pathogenic bacteria. Our results exhibit a wide spectrum inhibitory activity towards both gram-positive and negative bacteria. We report strong antibacterial inhibition by polar solvent extracts (acetone and methanol) of T.catappa against clinical isolate Staph.aureus followed by Salm.typhi (MTCC 733) and clinical isolate P.vulgaris.
The Evaluation of essential oil for antibacterial activity, which showed the suppression of test pathogens exhibiting varying zone of inhibition 54 . The results can be related to other reports where the essential oil and oleoresins exhibited the significant inhibition of test organisms 55 . N-hexane extract and seed oil of N.sativa on selected human pathogenic bacteria was evaluated and found that the test microbes were inhibited by both extract and oil 56 . The results obtained in the study are in concordance with the previous studies. The methanol extracts obtained from the seeds were evaluated for antibacterial activity by and found that Staph. aureus and Pseudo.aeroginosa were sensitive to the extracts 57 . N-hexane, methanol and aqueous seed extract of N.sativa was evaluated for their invitro susceptibility activity where methanol extract showed considerable inhibition of Staph. aureus, Escherichia coli and Salm. enterica 58 . The efficacy of N.sativa oil is attributed to its quinone constitutes in the fixed and essential oil, which is, endowed with thymoquinone a significant bioactive constituent making up 30-48% of total constituents. Other functional constituents include p-cymeme, carvacrol, thymohydroquinone, dihydrothymoquinone, thymol, α-thujene,tanthole, β-pinene, α pinene and γ-terpinene 59 .
The MIC values of T.catappa showed, that acetone extract was more potent in inhibiting the test bacteria and methanol extract was slightly less potent than the acetone extract. However, hexane and chloroform extract had no activity against the test strains owing to the fact that polar component of the extract is posing the antibacterial action. Hexane extract of N. sativa revealed the presence of alkaloid, which is the major component of the seeds. The results are in accordance with the earlier work 72 , which reported the occurrence of tannins, alkaloids, flavonoids and sterols.
TLC bioautography helps in locating the antibacterial active spots on the chromatogram. EMW solvent system was capable in eluting the antibacterial compound in acetone extract of with Rf value of 0.59, 0.52, 0.47 and 0.39. Bioautography studies revealed that most of the separated compounds had antibacterial potential, which can be due to the occurrence of polyphenols in the test plant extract, which is also confirmed from the qualitative phytochemical analysis. Time kill studies are important because they provide information about pharmacodynamics of the antibacterial agent 73 . Extracts showed variable kinetics against test bacteria. Time kill findings displayed different levels of time dependent and concentration dependent inhibition of the organisms. Bacteriostatic and bactericidal activity was displayed by the extract mainly against clinical isolates but regrowth of the test bacteria occurred after specific time interval which could be attributed to the use of lesser concentration of the extracts and also the gram positive and negative bacteria differ in cell wall and the membrane composition which regulate their susceptibilities to plant metabolites. At higher concentration bactericidal activity may be achieved invitro. Time kill kinetics of T.catappa against selected human pathogenic bacteria was carried out for the first time, however there are fewer reports related to time kill kinetics of fungi 74 but, reports on bacteria are scarce to the best of our knowledge.

CONClusiON
The results achieved in this study indicate that T.catappa and N.sativa are potential sources of antibacterial agents against the bacterial clinical isolates in particular. The acetone extract of T.catappa exhibited wide spectrum activity against (gram-positive and gram-negative) MTCC and clinical isolates. The extracts also inhibited the test bacteria at a very minimal concentration. Bioautography profile of acetone extract resulted in potent antibacterial bands, which exhibited inhibition both MTCC and clinical isolates was observed. Time kill assays indicated time and concentration dependent activity. Both bacteriostatic and bactericidal activity was observed against clinical isolates. Use of higher concentrations could lead to bactericidal activity against the test bacteria. The extracts can be employed in improving human health in the prevention of treatment of infectious diseases and to Fight the emergence and spread of resistant organisms. Further pure compound isolation, structure elucidation and mechanism of action of the isolated molecule followed by invivo toxicity studies may lead to potential drug in the ongoing search for antimicrobial botanicals.

ACKnoWLeDGMenTS
Authors are thankful to Center for Innovative Studies in Herbal Drug Technology, Department Of Studies in Botany, University of Mysore, Manasagangotri, Mysuru, Karnataka for providing the facilities and to the Department of Microbiology, Mysore Medical College and Research Center for providing the clinical bacterial isolates.