Vol. 8(10), pp. 423-429, 10 March, 2014

DOI: 10.5897/JMPR11.1281
ISSN 1996-0875
Copyright © 2014 Journal of Medicinal Plant Research
Author(s) retain the copyright of this article
http://www.academicjournals.org/JMPR

Full Length Research Paper

Antimicrobial activity and phytochemical screening of

various parts of Ixora coccinea
Mani Maran Marimuthu1, Claira Arul Aruldass2, Uma Mageswary Sandrasagaran2, Suriyati
Mohamad3, Surash Ramanathan1, Sharif Mahsufi Mansor1 and Vikneswaran Murugaiyah2*
1
Centre for Drug Research, Universiti Sains Malaysia, Penang, Malaysia.
2
School of Pharmaceutical Sciences, Universiti Sains Malaysia, Penang, Malaysia.
3
School of Biological Sciences, Universiti Sains Malaysia, Penang, Malaysia.
Accepted 10 November, 2011

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.

Key words: Antimicrobial, Ixora coccinea, minimum inhibitory concentration, minimum

bacteriostatic/bacteriocidal concentration, bioautography.

INTRODUCTION

Plants are the oldest source of pharmacologically active bacteria and the recent appearance of strains with
substances and have provided humans with many reduced susceptibility to clinically used antibiotics
medically useful compounds (Cordell, 1981). Plants pro- (Rubiaceae) is a small evergreen flowering shrub found
duce a diverse array of secondary metabolites, many of throughout Asia (Latha and Pannikar, 1998). It has been
which have antimicrobial activity. Hence, natural products raises the specter of untreatable bacterial infections and
in particular medicinal plants remain as a potential source adds urgency to the search for new infection-fighting stra-
of new antimicrobial agents (Cowan, 1999). The in- tegies (Sieradzki et al., 1999). Therefore, there is a conti-
creasing prevalence of multidrug resistant strains of nuous search for substances from plant sources with

*Corresponding author. E-mail: vicky@usm.my. Tel: 046534583.

Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution
License 4.0 International License
424 J. Med. Plants Res.

proven antimicrobial activity. Ixora coccinea L. used Malaysia, Penang, Malaysia. The test microorganisms used in this
traditionally for a variety of ailments and also cultivated study were: Bacillus cereus (ATCC 10876), Streptococcus
pneumoniae (ATCC 6303), S. aureus (ATCC 25923), Escherichia
for ornamental purposes. The leaves are used to treat coli (ATCC 25922), Klebsiella pneumoniae (laboratory strain), S.
diarrhoea, the roots are used to treat hiccup, fever, sores, flexneri (ATCC 12022), Enterobacter aerogenes (laboratory strain)
chronic ulcers and skin diseases. The flowers have been and Saccharomyces cerevisiae (laboratory strain). The strains were
used in catarrhal bronchitis and dysentery (Sivarajan and maintained and tested on Muller Hinton agar (bacteria) and potato
Balachanadran, 1941). It is reported that this plant dextrose agar (fungus). For the antimicrobial tests, strains were
contains anthocyanins in flowers; methyl ester of palmitic, grown overnight in Muller Hinton agar (bacteria) and Potato
Dextrose agar (fungus) in an anaerobic chamber at 37°C under
stearic, oleic and linoleic acids in root oil; atmosphere consisting 10% CO2, 10% H2O and 80% N2.
octadecadienoic acid, saponins and tannins from root
bark (Chopra et al., 1956; Grainge and Ahmed, 1988),
alkaloids, flavonoids, sapogenins, sterols, terpenes and Antimicrobial evaluation
phenols (Annapurna et al., 2003). The aqueous extract of
Agar disc diffusion assay
the I. coccinea demonstrated antino-ciceptive, anti-
inflammatory and antitumor effects in mice The antimicrobial activity of different parts of I. coccinea was initially
(Rathnasooriya et al., 2005). Annapurna et al. (2003) has evaluated by agar disc diffusion assay (Bauer et al., 1966). 20 ml
reported the antimicrobial activity of I. coccinea leaf Muller-Hinton agar for bacteria or potato dextrose agar for fungi,
extract based on disc diffusion method. However to date, sterilized in a flask and cooled at 45 to 50°C, were transferred to
there is no in depth antimicrobial evaluation done on this petri dishes (diameter of 90 mm) and allowed to solidify. Inoculum
were prepared by mixing a few microbial colonies with Muller Hinton
plant. With this in view, the present study was undertaken broth (bacteria) or potato dextrose broth (fungi) and incubated at
to evaluate in detail the antimicrobial activity of metha- 37°C for 3 h to get an approximately standard 0.5 Mac Farland
nolic extract of various parts of I. coccinea against series solution. Then, inoculum suspensions were streaked over the
of microorganisms and to identify the chemical groups of surface of the media on each plate using sterile cotton swab to
the bioactive constituents. 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
MATERIALS AND METHODS of 6 mm diameter were used in this assay. Each sterile disc was
impregnated with 20 μl of various extracts of I. coccinea
Chemicals and reagents (concentration 1 mg/disc) and then placed onto the agar plates
which had previously been inoculated with the test microorganisms
Chloramphenicol was purchased from Acros Organics (New Jersey, with sterile forceps and pressed gently to ensure contact with the
USA). Gentamycin and amoxicillin were purchased from Sigma inoculated agar surface. Amoxicillin (30 μg/disc), penicillin G (10
Aldrich (Steinheim, Germany) while vancomycin, miconazole and μg/disc), vancomycin (10 μg/disc), chloramphenicol (30 μg/disc),
para iodonitrotetrazolium (INT) were purchased from Sigma (St. gentamycin (10 μg/disc), tetracycline (30 μg/disc) and miconazole
Louis, USA). Amphotericin B was purchased from Himedia (30 μg/disc) were used as positive controls while methanol (20 μl)
(Mumbai, India). Penicillin G, gentamycin, chloramphenicol, was used as the negative control.
amoxicillin and tetracycline impregnated discs were purchased from Finally, the inoculated plates were incubated at 37°C for 18 h and
Oxoid (Hampshire, England). Muller Hinton agar, Muller Hinton at 28°C for 48 h for the bacteria and fungi, respectively. The
broth, Potato Dextrose agar and methanol were purchased from diameters of the inhibition zones were measured in milimeters. All
Merck (Darmstadt, Germany) while potato dextrose broth was measurement was carried out in triplicate. Extracts showing
purchased from Lab Scan Analytical Sciences (Bangkok, Thailand). inhibition zones of more than 8 mm were selected for subsequent
Dimethyl sulfoxide (DMSO) was purchased from Fisher Scientific determination of MIC, MBC and minimum fungicidal concentration
(Leicestershire, UK). (MFC).

Plant material and extraction Determination of minimum inhibitory concentration (MIC)

Fresh plant materials of I. coccinea (leaves, flowers and stem) were The MIC of I. coccinea extracts were determined by serial two-fold
collected from Penang, Malaysia. A voucher specimen (Collection dilution method as described by Eloff (1998). The extracts were
No. 11038) has been deposited at the Herbarium of School of initially dissolved in 50% DMSO to make up a concentration of 50
Biological Sciences, Universiti Sains Malaysia. The plant parts were mg/ml. Then stock solution was serially diluted two-fold by using
washed with water to remove dirt prior to the drying process at Muller Hinton broth (MHB) as diluents. Each well was inoculated
40°C for a week. The dried plant materials were powdered (100 with 100 μl of suspension containing 1.5×108 CFU/ml (equivalent to
gm) and extracted repeatedly with methanol (500 ml) by maceration McFarland 0.5) of the culture. Final concentration of extracts and
for a week. Fresh methanol was replenished every two days. The positive controls ranged from 25 to 0.006 and 0.4 to 0.00039 mg/ml,
extracts were filtered and the filtrates were concentrated in vacuo respectively. The 96 well plates were incubated at 37°C for 18 h for
using rotary evaporator at 45°C. The concentrated extracts were bacteria and at 28°C for 48 h for fungus. Amoxicillin,
subjected to freeze drying to obtain dry powdered extracts. 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
Microorganisms and media 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 diphenyl-
Test microorganisms were obtained from the School of Biological tetrazolium bromide) or INT (para iodonitrotetrazolium) dissolved in
Sciences and School of Pharmaceutical Sciences, Universiti Sains water into each of the microplate wells. The covered microplates
 Marimuthu et al. 425

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

a
Mean zone of growth inhibition (mm) (n=3)
Strain I. coccinea extracts (1mg/disc) Reference antibiotic (μg/disc)
Leaf Flower Stem AMX 30 PEN G 10 VC 10 CMC 30 GM 10 MICO 30 M
Gram positive bacteria
Bacillus cereus 7.0±1.0 7.0±0 6.0 ±0 12.7 ±0.6 -
Streptococcus pneumoniae 9.0 ±0 8.3 ±0.6 8.3±0.6 28.7±0.6 -
Staphylococcus aureus 8.0±0 9.0 ± 0 9.0±0 13.0±0 -

Gram negative bacteria -

Escherichia coli 6.0±0 6.7±0.6 8.7±0.6 19.7±0.6
Klebsiella pneumonia 8.7±0.6 6.0±0 8.7±0.6 19.0±0 -
Shigella flexneri 8.8±0.6 8.0±0 9.0±0 20.3±0 -
Pseudomonas aeruginosa 8.0±0 9.0±0 9.0±0 20.0±0 -
Enterobacter aerogenes 10.0±0.5 8.0±0 9.0±0 29±0
-
Fungi
Saccharomyces cerevisiae 11.0±0 10.0±0 11.3±0.6 12.7±0.6 -
a
Diameter of inhibition zone including disc diameter of 6.0 mm. Values for growth inhibition are presented as mean±SD; ‘-’ no inhibition zone observed; AMX, amoxicilin; PEN G,
penicillin G; VC, vancomycin; CMC, chloramphenicol; GM, gentamycin; MICO, miconazole; M, methanol.

were incubated further for 30 min. MIC was defined as the aureus and S. flexneri) which were found to be the most chloroform: methanol (9.5: 0.5). The components were
lowest drug concentration of extract inhibiting growth of susceptible to leaf and stem extracts following method visualized under visible and ultra violet light (254 and 365
microorganism and there was no colour changes observed described by Masoko and Eloff (2005). The developed TLC nm), respectively. The developed plates were sprayed with
in the wells from yellow to purple for MTT and colourless to plates were carefully dried and inoculated with fine spray of the following reagents for detection of respective chemical
pink for INT. the concentrated suspension of actively growing micro- groups: natural products reagent for flavonoid (Wagner et
organisms containing 109 cfu/ml approximately. The plates al., 1984), aluminium chloride for phenolic compounds,
were incubated overnight at 37°C and then sprayed with 2 methanolic potassium hydroxide for coumarins (Harbone,
Determination of MBC and MFC mg/ml solution of INT and further incubated for 2 to 3 h. 1973) and Dragendorff’s reagent for alkaloids and
The presence of white spots indicates presence of Liebermann-Burchard reagent for terpenoids (Krebs et al.,
MBC or MFC was defined as the lowest concentration of compounds that inhibited the growth of tested 1969).
extracts that showed complete inhibition of colonies of microorganisms.
microorganisms on agar plates. A loopful of each bacterial
or fungal culture in each microplate well was inoculated to RESULTS
the culture medium agar plate and incubated under same Phytochemical screening
conditions as described earlier (Shin et al., 2004). Each Methanolic extracts of various parts of I. coccinea
assay was carried out in triplicate. The leaf and stem extracts of I. coccinea were found to were evaluated for antimicrobial activity on nine
have antibacterial effect, thus were selected for phyto-
chemical screening by TLC. TLC was performed on
pathogenic microorganisms by using agar disc
Bioautography aluminium-backed TLC plates (Merck, Silica gel 60 F254). diffusion assay and microdilution assay. The
Plates (10 × 5 cm) were loaded with 10 spots (20 mg/ml) of growth inhibition zones measured in agar disc
Bioautography was performed using bacterial cultures (S. each extract. The prepared plates were developed with diffusion assay are presented in Table 1. The disc
426 J. Med. Plants Res.

Table 2. Minimum inhibitory concentration (MIC) of various extracts of I. coccinea and reference antibiotics.

Minimum inhibitory concentration (mg/ml), n=3 % DMSO not

Strain I. coccinea extracts Reference antibiotic inhibiting
Leaf Flower Stem AMX VC GM MICO microorganisms
Gram positive bacteria
Streptococcus pneumoniae 1.56 (500×) 3.125 (1000×) 0.78 (250×) 0.003125 12.5
Staphylococcus aureus 1.56 (125×) 12.5 (1000×) 0.78 (62.4×) 0.0125 25.0

Gram negative bacteria

Klebsiella pneumoniae 1.56 (2000×) 3.125 (4000×) 1.56 (2000×) 0.00078 12.5
Shigella flexneri 1.56 (62.4×) 1.56 (62.4×) 0.78 (31.2×) 0.025 12.5
Entrobacter aerogenes 1.56 (125×) 3.125 (250×) 1.56 (125×) 0.0125 25.0

Fungi
Saccharomyces cerevisiae 1.56 (250×) 1.56 (250×) 0.78 (125×) 0.00625 25
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.

diffusion results showed the I. coccinea extracts S. aureus and S. flexneri with MIC value of only suggesting that these extracts may act as
have potential antimicrobial activity against all 0.78 mg/ml. By comparing MIC values, the stem bactericidal agent on these strains. Stem extract
tested bacteria strains and fungi S. cerevisiae with extract was found to be only 62.4 times less showed MBC/MIC ratio of more than 4 on S.
zone of inhibition between 6.7 to 11.3 mm. potent than vancomycin for S.aureus and 31.2 aureus and S. flexneri which may be classified as
However their antimicrobial effects were found to times less potent than gentamycin for S. flexneri. bacteriostatic agent.
be less potent than the reference antibiotics. The stem extract of I. coccinea was also found to The leaf and stem extract were further
Methanol used as negative control showed no have good antimicrobial activity against S. subjected to bioautography and phytochemical
inhibitory effect against the microorganism’s cerevisiae. DMSO showed no toxic effect at 25% screening. Bioautography results demonstrated
growth. In subsequent experiments, the MIC, for E. aerogenes, S. cerevisiae and S. aureus and strong inhibition zones of these extracts against
MBC and MFC values of the I. coccinea extract at 12.5% for the rest of the microorganisms. the growth of S. aureus and S. flexneri (Figure 1a
were determined against selected susceptible Based on MIC results, MBC and MFC were and b; Table 5). There are few inhibition spots
microorganisms. The results are summarized in determined for the active extracts against selected noted suggesting that more than one compound
Tables 2 and 3. The MIC, MBC and MFC values microorganisms. Antimicrobial substances are may be responsible for the observed antimicrobial
of the test extracts of I. coccinea ranged from 0.78 con-sidered as bacteriostatic agents when the effect. Interestingly, the active compounds (spots
to 6.25 mg/ml. ratio MBC/MIC>4 and bactericidal agents when label A and D) of leaf extract which inhibited both
In general, stem extract of I. coccinea was the ratio MBC/MIC≤4 (Gatsing et al., 2009). The microorganisms have almost same Rf values. The
found to be most active extract followed by the summary of the microbicide and microbistatic active compounds of leaf extract may belong to
leaf extract. Flower extracts was found to be the effects were given in Table 4. In the present terpenoids, flavanoids, coumarins and alkaloids
least active. Interestingly, the stem extract was study, leaf and stem extract showed the ratio groups of compounds. As for the stem extract,
found to have good antimicrobial activity against MBC/MIC≤4 for most of the test microbes, spot B (Rf value of 0.39) and C (Rf value of 0.18),
 Marimuthu et al. 427

Table 3. Minimum bactericidal/bacteriostatic concentration (MBC) and minimum fungicidal/fungi static (MFC) concentration of various
extracts of I. coccinea

MBC and MFC (mg/ml), n=3

Strain I. coccinea extracts Reference antibiotic
Leaf Flower Stem VC GM MICO
Gram positive bacteria
Staphylococcus aureus 6.25 (250×) 6.25 (250×) 0.025

Gram negative bacteria

Klebsiella pneumoniae 6.25 (500×) 6.25 (500×) 0.0125
Shigella flexneri 6.25 (62.5×) 0.1
Entrobacter aerogenes 3.125 (16×) 3.125 (16×) 0.2

Fungi
Saccharomyces cerevisiae 0.78 (62.4×) 0.0125
VC, vancomycin; 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.

Extracts of various part of I.

Microbicide effect on specific strains Microbiostatic effect on specific strains
coccinea
Entrobacter aerogenes, Staphylococcus -
Leaf
aureus, Klebsiella pneumoniae

Saccharomyces cerevisiae, Entrobacter Staphylococcus aureus, , Shigella flexneri

Stem
aerogenes, Klebsiella pneumonia

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

Microorganism A B C D E F
Extract Inhibition zone
Terpenoid + + + + + +
Flavonoid + + + + + +
Coumarin + + + + + +
Alkaloid + - - + - -
Phenol - - + - - +
Rf value 0.37 0.39 0.18 0.33 0.38 0.16
Growth inhibition of microorganisms: ‘+’ present; ‘-’ absent.

which inhibited S. aureus were similar to the spot E (Rf et al., 2010; Shariff, 2001; Shin et al., 2004). Most
value of 0.38) and F (Rf value of 0.16) which inhibited S. antibacterial medicinal plants are active against gram-
flexneri. These spots may consist of terpenoids, positive strains while few are active against gram-
flavanoids, alkaloids, coumarins and phenolic negative bacteria (Herrera et al., 1996; Meng et al., 2000;
compounds. Scrinivasan et al., 2001). In the present study,
antimicrobial activity of methanolic extracts various parts
of I. coccinea was evaluated against series of micro-
DISCUSSION organisms. Among all three extracts of I. coccinea, leaf
and stem extracts were found to be more active than
The antimicrobial activities of medicinal plants have been flower extract. In particular, the leaf and stem extracts
reported by many researchers (Cowan, 1999; Motamedi were active against few pathogenic microorganisms such
428 J. Med. Plants Res.

  
Figure 1. Bioautography of I. coccinea extracts against selected microorganisms.
(Labelled spots indicate growth inhibition of microorganisms).

K. pneumonia, S. aureus and S. flexneri. Therefore, Bioautography is a useful technique for detecting
these extracts may be potential source leads for bioactive compound(s) as well as indicator for separation
antimicrobial agents against these microorganisms. technique during bioassay-guided isolation of active
Annapurna et al. (2003) has previously reported on compounds (Masoko et al., 2007). For each extract, more
screening of antimicrobial activity of I. coccinea leaves than one active compound was identified. A group of
extract using disc diffusion assay. The present findings compounds belonging to the terpenoids, flavanoids,
were similar to the study whereby leaf extract was active alkaloids, coumarins and phenolics family may be
against S. aureus with inhibition zone of 18 mm. Studies responsible for the antimicrobial activity of stem and leaf
that used agar disc diffusion to detect antimicrobial extracts. These compounds are producing exciting
activity of plant extract revealed poor accuracy and opportunity for the expansion of modern chemotherapies
difficult nature of the method. According to Allen et al. against wide range of microorganisms (Lutterodt et al.,
(1991), low levels of antimicrobial activity of plant extracts 1999).
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 Conclusions
growth with inhibitory effect of plant extract at certain
concentration. This method has been identified as being I. coccinea extracts have potential to be developed as
more accurate than agar disc diffusion and less tedious antimicrobial agents, in particular against S. aureus and
(Eloff, 1998). S. aureus and S. flexneri was the most S. flexneri. Further studies on isolation and identification
susceptible microorganisms against I. coccinea extracts of the active principles and evaluation of possible syner-
(leaf and stem) with lowest MIC value 0.78 mg/ml. gism among these constituents for their antimicrobial
In addition, these extracts were found to be activity are currently ongoing.
bacteriostatic S. aureus and S. flexneri. According to Van
and Viljoen (2009), as DMSO may exhibit antimicrobial Conflict of Interests
efficacy, MIC values equivalent to or greater than that
found for the DMSO control should be omitted from the The author(s) have not declared any conflict of interests.
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 ACKNOWLEDGEMENTS
active constituents were identified by TLC bioautography
and characterized by spraying with various reagents. Mani Maran Marimuthu is supported by the USM
 Marimuthu et al. 425

Fellowship Scheme from the Institute for Postgraduate Lutterodt GD, Ismail A, Basheer F, Mohd HB (1999). Antimicrobial
effects of Psidium guajava extract as one mechanism of its
Studies (IPS) of Universiti Sains Malaysia. We would like
antidiarrhoeal action. Malaysian J. Med. Sci. 6:17-20.
to thank Ms Geethaa Sahgal, Ms Suhanya Parthasarathy Masoko P, Picard J, Eloff JN (2007). The antifungal activity of twenty-
and Ms Kamilla Lingam (Centre for Drug Research, four Southern African Combretum species (Combretaceae). S. Afr. J.
USM) for their technical assistance throughout this study. Bot. 73:173-183.
Motamedi HE, Darabpour M, Gholipour, Seyyed Nejad SM (2010).
Antibacterial effect of ethanolic and methanolic extracts of Plantago
ovata and Oliveria decumbens endemic in Iran against some
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