Antimicrobial activity and phytochemical screening of various parts of Ixora coccinea

Ixora coccinea L. (Rubiaceae) has been used traditionally for a variety of ailments and also cultivated for ornamental purposes. The present study investigated antimicrobial activity of methanolic extracts of various parts of I. coccinea and determined the chemical groups of the active constituents. Antimicrobial activity was assessed using agar disc diffusion, microdilution and thin layer chromatography (TLC) bioautography assays. Methanolic extracts of leaf, flower and stem of I. coccinea displayed good antimicrobial activity, with inhibition zone in the range of 6.7 to 11.3 mm. minimum inhibitory concentration (MIC) values for all three extracts ranged from 0.78 to 3.125 mg/ml. Leaf and stem extracts of I. coccinea showed broad-spectrum antimicrobial activity. Of interest, stem extracts had MIC values against Staphylococcus aureus that were only 62.4 times less potent than the vancomycin. Likewise, leaf and stem extracts displayed good antimicrobial activity of 62.4 and 31.2 times, respectively lesser than gentamycin against Shigella flexneri. Minimum bactericidal/ bacteriostatic concentration (MBC) values for active extracts ranged from 0.78 to 6.25 mg/ml. TLC bioautography and phytochemical screening of the leaf and stem extracts showed that the antimicrobial activity of these extracts may be attributed to compounds belonging to terpenoid, flavonoid, coumarin, alkaloid and phenolic groups.


INTRODUCTION
Plants are the oldest source of pharmacologically active substances and have provided humans with many medically useful compounds (Cordell, 1981).Plants produce a diverse array of secondary metabolites, many of which have antimicrobial activity.Hence, natural products in particular medicinal plants remain as a potential source of new antimicrobial agents (Cowan, 1999).The increasing prevalence of multidrug resistant strains of bacteria and the recent appearance of strains with reduced susceptibility to clinically used antibiotics (Rubiaceae) is a small evergreen flowering shrub found throughout Asia (Latha and Pannikar, 1998).It has been raises the specter of untreatable bacterial infections and adds urgency to the search for new infection-fighting strategies (Sieradzki et al., 1999).Therefore, there is a continuous search for substances from plant sources with proven antimicrobial activity.Ixora coccinea L. used traditionally for a variety of ailments and also cultivated for ornamental purposes.The leaves are used to treat diarrhoea, the roots are used to treat hiccup, fever, sores, chronic ulcers and skin diseases.The flowers have been used in catarrhal bronchitis and dysentery (Sivarajan and Balachanadran, 1941).It is reported that this plant contains anthocyanins in flowers; methyl ester of palmitic, stearic, oleic and linoleic acids in root oil; octadecadienoic acid, saponins and tannins from root bark (Chopra et al., 1956;Grainge and Ahmed, 1988), alkaloids, flavonoids, sapogenins, sterols, terpenes and phenols (Annapurna et al., 2003).The aqueous extract of the I. coccinea demonstrated antino-ciceptive, antiinflammatory and antitumor effects in mice (Rathnasooriya et al., 2005).Annapurna et al. (2003) has reported the antimicrobial activity of I. coccinea leaf extract based on disc diffusion method.However to date, there is no in depth antimicrobial evaluation done on this plant.With this in view, the present study was undertaken to evaluate in detail the antimicrobial activity of methanolic extract of various parts of I. coccinea against series of microorganisms and to identify the chemical groups of the bioactive constituents.

Chemicals and reagents
Chloramphenicol was purchased from Acros Organics (New Jersey, USA).Gentamycin and amoxicillin were purchased from Sigma Aldrich (Steinheim, Germany) while vancomycin, miconazole and para iodonitrotetrazolium (INT) were purchased from Sigma (St. Louis, USA).Amphotericin B was purchased from Himedia (Mumbai, India).Penicillin G, gentamycin, chloramphenicol, amoxicillin and tetracycline impregnated discs were purchased from Oxoid (Hampshire, England).Muller Hinton agar, Muller Hinton broth, Potato Dextrose agar and methanol were purchased from Merck (Darmstadt, Germany) while potato dextrose broth was purchased from Lab Scan Analytical Sciences (Bangkok, Thailand).Dimethyl sulfoxide (DMSO) was purchased from Fisher Scientific (Leicestershire, UK).

Plant material and extraction
Fresh plant materials of I. coccinea (leaves, flowers and stem) were collected from Penang, Malaysia.A voucher specimen (Collection No. 11038) has been deposited at the Herbarium of School of Biological Sciences, Universiti Sains Malaysia.The plant parts were washed with water to remove dirt prior to the drying process at 40°C for a week.The dried plant materials were powdered (100 gm) and extracted repeatedly with methanol (500 ml) by maceration for a week.Fresh methanol was replenished every two days.The extracts were filtered and the filtrates were concentrated in vacuo using rotary evaporator at 45°C.The concentrated extracts were subjected to freeze drying to obtain dry powdered extracts.

Microorganisms and media
Test microorganisms were obtained from the School of Biological Sciences and School of Pharmaceutical Sciences, Universiti Sains Malaysia, Penang, Malaysia.The test microorganisms used in this study were: Bacillus cereus (ATCC 10876), Streptococcus pneumoniae (ATCC 6303), S. aureus (ATCC 25923), Escherichia coli (ATCC 25922), Klebsiella pneumoniae (laboratory strain), S. flexneri (ATCC 12022), Enterobacter aerogenes (laboratory strain) and Saccharomyces cerevisiae (laboratory strain).The strains were maintained and tested on Muller Hinton agar (bacteria) and potato dextrose agar (fungus).For the antimicrobial tests, strains were grown overnight in Muller Hinton agar (bacteria) and Potato Dextrose agar (fungus) in an anaerobic chamber at 37°C under atmosphere consisting 10% CO 2 , 10% H 2 O and 80% N 2 .

Agar disc diffusion assay
The antimicrobial activity of different parts of I. coccinea was initially evaluated by agar disc diffusion assay (Bauer et al., 1966).20 ml Muller-Hinton agar for bacteria or potato dextrose agar for fungi, sterilized in a flask and cooled at 45 to 50°C, were transferred to petri dishes (diameter of 90 mm) and allowed to solidify.Inoculum were prepared by mixing a few microbial colonies with Muller Hinton broth (bacteria) or potato dextrose broth (fungi) and incubated at 37°C for 3 h to get an approximately standard 0.5 Mac Farland solution.Then, inoculum suspensions were streaked over the surface of the media on each plate using sterile cotton swab to ensure the confluent growth of the organism.For antimicrobial testing, a 50 mg/ml stock solution of each extract (leaf, flower and stem) was prepared in methanol.Whatman filter paper No.1 discs of 6 mm diameter were used in this assay.Each sterile disc was impregnated with 20 μl of various extracts of I. coccinea (concentration 1 mg/disc) and then placed onto the agar plates which had previously been inoculated with the test microorganisms with sterile forceps and pressed gently to ensure contact with the inoculated agar surface.Amoxicillin (30 μg/disc), penicillin G (10 μg/disc), vancomycin (10 μg/disc), chloramphenicol (30 μg/disc), gentamycin (10 μg/disc), tetracycline (30 μg/disc) and miconazole (30 μg/disc) were used as positive controls while methanol (20 μl) was used as the negative control.
Finally, the inoculated plates were incubated at 37°C for 18 h and at 28°C for 48 h for the bacteria and fungi, respectively.The diameters of the inhibition zones were measured in milimeters.All measurement was carried out in triplicate.Extracts showing inhibition zones of more than 8 mm were selected for subsequent determination of MIC, MBC and minimum fungicidal concentration (MFC).

Determination of minimum inhibitory concentration (MIC)
The MIC of I. coccinea extracts were determined by serial two-fold dilution method as described by Eloff (1998).The extracts were initially dissolved in 50% DMSO to make up a concentration of 50 mg/ml.Then stock solution was serially diluted two-fold by using Muller Hinton broth (MHB) as diluents.Each well was inoculated with 100 μl of suspension containing 1.5×10 8 CFU/ml (equivalent to McFarland 0.5) of the culture.Final concentration of extracts and positive controls ranged from 25 to 0.006 and 0.4 to 0.00039 mg/ml, respectively.The 96 well plates were incubated at 37°C for 18 h for bacteria and at 28°C for 48 h for fungus.Amoxicillin, chloramphenicol, vancomycin and gentamycin (for bacteria) and miconazole (for fungus) served as positive controls, respectively while 50% DMSO was used as negative control.Microbial growth was evaluated by addition of 50 μl of 0.2 mg/mL of freshly prepared solutions of MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5 diphenyltetrazolium bromide) or INT (para iodonitrotetrazolium) dissolved in water into each of the microplate wells.The covered microplates were incubated further for 30 min.MIC was defined as the lowest drug concentration of extract inhibiting growth of microorganism and there was no colour changes observed in the wells from yellow to purple for MTT and colourless to pink for INT.

Determination of MBC and MFC
MBC or MFC was defined as the lowest concentration of extracts that showed complete inhibition of colonies of microorganisms on agar plates.A loopful of each bacterial or fungal culture in each microplate well was inoculated to the culture medium agar plate and incubated under same conditions as described earlier (Shin et al., 2004).Each assay was carried out in triplicate.

Bioautography
Bioautography was performed using bacterial cultures (S. aureus and S. flexneri) which were found to be the most susceptible to leaf and stem extracts following method described by Masoko and Eloff (2005).The developed TLC plates were carefully dried and inoculated with fine spray of the concentrated suspension of actively growing microorganisms containing 10 9 cfu/ml approximately.The plates were incubated overnight at 37°C and then sprayed with 2 mg/ml solution of INT and further incubated for 2 to 3 h.The presence of white spots indicates presence of compounds that inhibited the growth of tested microorganisms.

Phytochemical screening
The leaf and stem extracts of I. coccinea were found to have antibacterial effect, thus were selected for phytochemical screening by TLC.TLC was performed on aluminium-backed TLC plates (Merck, Silica gel 60 F 254 ).Plates (10 × 5 cm) were loaded with 10 spots (20 mg/ml) of each extract.The prepared plates were developed with chloroform: methanol (9.5: 0.5).The components were visualized under visible and ultra violet light (254 and 365 nm), respectively.The developed plates were sprayed with the following reagents for detection of respective chemical groups: natural products reagent for flavonoid (Wagner et al., 1984), aluminium chloride for phenolic compounds, methanolic potassium hydroxide for coumarins (Harbone, 1973) and Dragendorff's reagent for alkaloids and Liebermann-Burchard reagent for terpenoids (Krebs et al., 1969).

Methanolic extracts of various parts of I. coccinea
were evaluated for antimicrobial activity on nine pathogenic microorganisms by using agar disc diffusion assay and microdilution assay.The growth inhibition zones measured in agar disc diffusion assay are presented in Table 1.The disc diffusion results showed the I. coccinea extracts have potential antimicrobial activity against all tested bacteria strains and fungi S. cerevisiae with zone of inhibition between 6.7 to 11.3 mm.However their antimicrobial effects were found to be less potent than the reference antibiotics.Methanol used as negative control showed no inhibitory effect against the microorganism's growth.In subsequent experiments, the MIC, MBC and MFC values of the I. coccinea extract were determined against selected susceptible microorganisms.The results are summarized in Tables 2 and 3.The MIC, MBC and MFC values of the test extracts of I. coccinea ranged from 0.78 to 6.25 mg/ml.In general, stem extract of I. coccinea was found to be most active extract followed by the leaf extract.Flower extracts was found to be the least active.Interestingly, the stem extract was found to have good antimicrobial activity against S. aureus and S. flexneri with MIC value of only 0.78 mg/ml.By comparing MIC values, the stem extract was found to be only 62.4 times less potent than vancomycin for S.aureus and 31.2 times less potent than gentamycin for S. flexneri.The stem extract of I. coccinea was also found to have good antimicrobial activity against S. cerevisiae.DMSO showed no toxic effect at 25% for E. aerogenes, S. cerevisiae and S. aureus and at 12.5% for the rest of the microorganisms.Based on MIC results, MBC and MFC were determined for the active extracts against selected microorganisms.Antimicrobial substances are con-sidered as bacteriostatic agents when the ratio MBC/MIC>4 and bactericidal agents when the ratio MBC/MIC≤4 (Gatsing et al., 2009).The summary of the microbicide and microbistatic effects were given in Table 4.In the present study, leaf and stem extract showed the ratio MBC/MIC≤4 for most of the test microbes, suggesting that these extracts may act as bactericidal agent on these strains.Stem extract showed MBC/MIC ratio of more than 4 on S. aureus and S. flexneri which may be classified as bacteriostatic agent.
The leaf and stem extract were further subjected to bioautography and phytochemical screening.Bioautography results demonstrated strong inhibition zones of these extracts against the growth of S. aureus and S. flexneri (Figure 1a and b; Table 5).There are few inhibition spots noted suggesting that more than one compound may be responsible for the observed antimicrobial effect.Interestingly, the active compounds (spots label A and D) of leaf extract which inhibited both microorganisms have almost same R f values.The active compounds of leaf extract may belong to terpenoids, flavanoids, coumarins and alkaloids groups of compounds.As for the stem extract, spot B (R f value of 0.39) and C (R f value of 0.18),

DISCUSSION
The antimicrobial activities of medicinal plants have been reported by many researchers (Cowan, 1999;Motamedi et al., 2010;Shariff, 2001;Shin et al., 2004).Most antibacterial medicinal plants are active against grampositive strains while few are active against gramnegative bacteria (Herrera et al., 1996;Meng et al., 2000;Scrinivasan et al., 2001).In the present study, antimicrobial activity of methanolic extracts various parts of I. coccinea was evaluated against series of microorganisms.Among all three extracts of I. coccinea, leaf and stem extracts were found to be more active than flower extract.In particular, the leaf and stem extracts were active against few pathogenic microorganisms such K. pneumonia, S. aureus and S. flexneri.Therefore, these extracts may be potential source leads for antimicrobial agents against these microorganisms.Annapurna et al. (2003) has previously reported on screening of antimicrobial activity of I. coccinea leaves extract using disc diffusion assay.The present findings were similar to the study whereby leaf extract was active against S. aureus with inhibition zone of 18 mm.Studies that used agar disc diffusion to detect antimicrobial activity of plant extract revealed poor accuracy and difficult nature of the method.According to Allen et al. (1991), low levels of antimicrobial activity of plant extracts are not detectable in agar disc diffusion method.MIC of I. coccinea extracts was carried out using microplate assay because this assay is based on correlation of bacterial growth with inhibitory effect of plant extract at certain concentration.This method has been identified as being more accurate than agar disc diffusion and less tedious (Eloff, 1998).S. aureus and S. flexneri was the most susceptible microorganisms against I. coccinea extracts (leaf and stem) with lowest MIC value 0.78 mg/ml.
In addition, these extracts were found to be bacteriostatic S. aureus and S. flexneri.According to Van and Viljoen (2009), as DMSO may exhibit antimicrobial efficacy, MIC values equivalent to or greater than that found for the DMSO control should be omitted from the data and considered not susceptible.In the present study, DMSO was found to have no inhibitory effect at the MIC values of the active extracts.Subsequently, the active constituents were identified by TLC bioautography and characterized by spraying with various reagents.
Bioautography is a useful technique for detecting bioactive compound(s) as well as indicator for separation technique during bioassay-guided isolation of active compounds (Masoko et al., 2007).For each extract, more than one active compound was identified.A group of compounds belonging to the terpenoids, flavanoids, alkaloids, coumarins and phenolics family may be responsible for the antimicrobial activity of stem and leaf extracts.These compounds are producing exciting opportunity for the expansion of modern chemotherapies against wide range of microorganisms (Lutterodt et al., 1999).

Conclusions
I. coccinea extracts have potential to be developed as antimicrobial agents, in particular against S. aureus and S. flexneri.Further studies on isolation and identification of the active principles and evaluation of possible synergism among these constituents for their antimicrobial activity are currently ongoing.

Table 1 .
Zone of inhibition (mm) by various extracts of I. coccinea and reference antibiotics.

Table 2 .
Minimum inhibitory concentration (MIC) of various extracts of I. coccinea and reference antibiotics.
AMX, amoxicilin; VC, vancomycin; CMC, chloramphenicol; GM, gentamycin; MICO, miconazole; Values given in bracket is the number of times the extract is less potent than the reference antibiotics.

Table 3 .
Minimum bactericidal/bacteriostatic concentration (MBC) and minimum fungicidal/fungi static (MFC) concentration of various extracts of I. coccinea GM, gentamycin; MICO, miconazole.Values given indicate bracket is the number of times the extract is less potent than the reference antibiotics.

Table 4 .
Summary of the microbicide and microbiostatic effects of I. coccinea extracts on selected strains.

Table 5 .
Summary of the bioautography of I. coccinea extracts against selected micro-organisms.