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Isolation and characterization of antimicrobial compound from Chromolaena odorata

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Journal of Phytology 2011, 3(10): 26-32
ISSN: 2075-6240
Available Online: http://journal-phytology.com/

Isolation and characterization of antimicrobial compound from

Chromolaena odorata
S. L. Sukanya 1,2, J. Sudisha 1, H. S. Prakash 1 and S. K. Fathima 2*

1Department of Studies in Biotechnology, University of Mysore, Manasagangotri, Karnataka 570006, India

2Department of Microbiology, Maharani's Science College for Women, JLB road, Mysore, Karnataka 570005, India

Abstract
Solvents such as methanol, ethanol, ethyl acetate, hexane and chloroform with extracts of Chromolaena odorata were tested
against clinical bacteria (Escherichia coli and Staphylococcus aureus ) and phytopathogenic bacteria ( Xanthomonas
vesicatoria and Ralstonia solanacearum). Among treatments, maximum In vitro inhibition was scored in methanol extracts of
C. odorata which offered inhibition zone of 8mm, 7mm, 5mm and 7mm against tested bacteria E. coli, S. aureus, X.
vesicatoria and R. solanaccearum, respectively. Further, ethyl-acetate and hexane extracts of C. odorata on TLC produced 9
spots with varied level of inhibition. Partially purified compound from TLC (band 2) showed maximum inhibition against tested
bacteria. The promising anti-microbial compound from ethyl-acetate and hexane extracts (5:5) of C. odorata was further
purified using column chromatography which recorded 11mm, 10mm, 9mm and 7mm against E. coli, S. aureus, X.
vesicatoria and R. solanaccearum, respectively. The minimum inhibitory concentration (MIC) value for clinical bacteria was
ranged between 0.35 to 4.0 mg/ml and 0.25 to 4.0 mg/ml for phytopathogenic bacteria. HPLC chromatogram showed only
one peak, where the retention time was 21.775 min. Structure of the anti-microbial compound was determined by Fourier
Transform Infrared Spectra (FTIR) and 2D nuclear magnetic resonance (NMR) and liquid chromatography mass spectra (LC-
MS). The structure of anti-microbial compound found to be phenolic groups. LC-MS spectrum of methanolic extract of C.
odorata compound exhibiting intense peak at m/z 301.3109 and the molecular weight of the compound was probably m/z
437.4129.
Keywords: Chromolaena odorata; clinical and phytopathogenic bacteria; antimicrobial assay

INTRODUCTION
Chromolaena odorata (L.) R. king and Rabinson 1970 (formely: young plantations, agricultural crops and smothers vegetation as it
Eupatorium odoratum L.), is an herbaceous perennial belongs to the possesses allelopathic potentialities and growth inhibitors [1, 2, 4].
family Asteraceae (=Compositae), is a diffuse, scrambling shrub that Previous investigations of the leaves and stems of C. odorata
is mainly a weed of plantation crops and pastures of southern Asia revealed the presence of essential oils [5-7], steroids, triterpenes [8
and western Africa [1]. It grows to a height of 3m in the open and 9], and flavonoids [10-16]. Flowers of this plant have been
situation and upto 8m when assumed a scrambling habitat in the subjected to investigate for essential oils [17], fats [18] and alkaloids
interior forests [2]. It is native to Mexico, the West Indies, and tropical [19]. It has been reported to have antispasmodic, antiprotozoal,
South America; it was spread widely by early navigators. It is antitrypanosomal, antibacterial and antihypersensitive activities. It
commonly called saim weed, triffid weed, christmas bush, bitter bush has also been reported to possess anti-inflammatory, astringent,
or jack in the bush. It is a weed competes with 13 major crops in 23 diuretic and hepatotropic activities [20-23]. In the southern part of
countries [3]. Nigeria, the leaves of C. odorata are used for wound dressing, skin
C. odorata commonly known as Eupatorium, is an alien, infection and to stop bleeding. Some specific phenolic compounds
obnoxious and aggressive weed. It grows in pastures, marginal have also been isolated from the plant [24]. The medicinal values of
lands, open areas, dry deciduous forests and interior shrub jungles, plants lie in their component phytochemicals such as alkaloids,
where it is highly competitive and does not let other flora grow. It is tannins, flavonoids and other phenolic compounds, which produce a
menace in plantations, agriculture and other ecosystems. It definite physiological action on the human body [25]. A systematic
suppresses search for useful bioactivities from medicinal plants is now
considered to be a rational approach in neutraceutical and drug
research. In this unfolding scenario, it is necessary to develop ways
Received: August 13, 2011; Revised September 18, 2011; Accepted September
of putting C. odorata to beneficial uses. The assessment of nutritive
26, 2011.
value of C. odorata showed that it has good potential for feeding
*Corresponding Author livestock due to its high crude protein (CP), low fibre and low
S. K. Fathima extractable phenolic contents.
Department of Microbiology, Maharani's Science College for Women, JLB road, In India, numerous studies have been carried out to extract
Mysore, Karnataka, India-570005 various plant materials for screening antimicrobial compounds but
Tel: +91-0821-2420503; Res.: +91-0821-2450165 much attention has not been focused on C. odorata. Therefore, by
Journal of Phytology 2011, 3(10): 26-32 27

considering the possibility of having antimicrobial compounds in C. inhibition of all four different bacteria to all the TLC fractions were
odorata, we planned this study to isolate and characterize the compared with negative control as well as positive control
antimicrobial compound which has considerable agrochemical (Chloromphenicol standard antibiotic disc) (30mcg/disc).
properties, environmentally safe, and economically feasible. The partial purified compound obtained from methanolic extract
MATERIALS AND METHODS of C. odorata by TLC (Described in materials and methods) was
loaded into silica gel (60-120 mesh), (SRL, Mumbai) column (35
Plant material
10mm) and successively eluted with a stepwise gradient of ethyl
Fresh leaves of C. odorata were collected from different
acetate: hexane (0, 10, 25, 50, 75 and 100%). Fractions were
locations of Mysore (12.18 N– 76.42 E, 770 m above sea level),
collected at 20-min intervals.
Karnataka, India, during 2007-2008. The plant was identified
taxonomically and authenticated at the Herbarium, in University of Reverse phase high performance liquid chromatography (RP-
Mysore, Mysore. Fresh leaves were washed thoroughly 2-3 times HPLC)
with running tap water and then with sterile water followed by shade- The analytical HPLC system with Waters LC 600 pump and 996
dried, powdered and used for extraction. Photo diode array detectors, C18 column was used at room
Test microorganisms temperature. Gradient elution propiles such as ‘A’ Trifluro acetic acid
(TFA) and ‘B’ was MeOH-H2O, 1:1. The flow rate was 0.5 ml/min.
Human pathogenic bacteria such as Escherichia coli and
Absorbance was measured at A215 and A225 nm 20µl of fraction was
Staphylococcus aureus were collected from JSS medical college
injected for separation. Further, minimum inhibitory concentration
Mysore, India. Plant pathogenic bacteria such as Xanthomonas
was checked for TLC purified methanolic extract instead of crude
vesicatoria and Ralstonia solanacearum collected from the culture
extract of C. odorata.
collection of department of applied botany and biotechnology,
University of Mysore, India. All the test bacterial species were Fourier transform infrared spectra (FTIR)
maintained on nutrient agar media. The infrared spectra were recorded on Shimadzu IR-470 model.
Preparation of solvent extraction The spectra were scanned in the 400 to 4000 cm –1 range. The
spectra were obtained using paraffin oil technique. One hundred µl
Twenty five gram of shade dried, powder of plant materials were
of paraffin oil with 1mg of sample was taken and the spectra were
filled separately in the thimble and extracted successively with 150
plotted as intensity versus wave number.
ml of methanol using a Soxhlet extractor for 48 hours. All the extracts
were concentrated using rotary flash evaporator. After complete Nuclear magnetic resonance (NMR)
evaporation of solvent, each of these solvent extract was weighed The 2D 1H NMR and 13C NMR spectra were recorded with
and preserved at 4oC in airtight bottles until further use. One gram of Brucker AM-500, Switzerland, (500MHz) spectrophotometer with the
methanol solvent residue was dissolved in 10 ml of respective solvent signal as internal reference and chemical shifts were given in
solvent were used as the test extracts for antimicrobial activity assay. ppm. The compound sample was dissolved (7 mg for 1H NMR and 3
Activity guided purification of anti-microbial compound mg for 13C NMR) in 3 ml of CDCl 3 and analysed by nuclear magnetic
resonance (NMR).
Thin layer chromatography (TLC) plates were prepared by
making slurry of 30 gm of silica gel-G with 60 ml of distilled water. Liquid chromatography mass spectra (LCMS)
Spreading was done manually over glass plates (20x20cm) and air LCMS analysis was carried out using API QSTAR pulsar
dried. The plates were activated in an oven for 3h at 110 0C. Germany and data dependent acquisition, during which compound
Methanolic extracts of C. odorata (100 mg sample dissolved in 1 ml precursor ions were detected by scanning from m/z 50 to 2000 with
of respective solvent) were used for TLC spotting. Separation of the column flow rate of 5µl/min solvent A consisted of 90% (water in
TLC spots was done using different solvent system individually and 0.1% Trifluro acetic acid) and solvent B consisted of 10%
in combinations using ethyl acetate: hexane (5: 5) as mobile phase. (acetonitrile in 0.1% Trifluro acetic acid) and 30 µl sample were
Spots developed on TLC plates were observed under UV injected onto the column.
transilluminator. Spots were eluted separately and isolated fractions RESULTS
were tested for its inhibition against four different micro organisms as
On TLC plates, ethyl acetate : hexane (5:5 v/v) with crude
described above. Further, the fractions showing maximum inhibition
extracts of C. odorata developed various spots upon elution and
were rechromatographed on TLC plates using the same mobile
tested against four bacteria and varied level of bacterial inhibition
phase. This process of repeated chromatography on TLC was
was noticed (Fig.1 A-D).
carried out until a single fluorescing spot was obtained (Fig. 2B). The
28 S.L.Sukanya et al.

Fig. 1. Effect of methanolic crude extract of Chromolaena odorata (L) on against Escherichia coli (A), Staphylococcus aureus (B), Xanthomonas vesicatoria (C) and Ralstonia
solanacearum (D).

0.46, 0.59, 0.65, 0.71, 0.76, 0.85, 0.98 (Fig.

Under UV ethyl acetate : hexane (5:5 v/v) yielded nine spots 2).
with variable fluorescing ability consisting Rf values of 0.21, 0.37,

Fig. 2.TLC Fingerprinting of methanol crude extracts (A) and Purified Compound (B) on TLC of C. odorata.

Fig. 3. Inhibition of tested bacteria with purified compund isolated from C. odorata against Escherichia coli (A), Staphylococcus aureus (B), Xanthomonas vesicatoria (C) and
Ralstonia solanacearum (D).
Journal of Phytology 2011, 3(10): 26-32 29

Again the antagonistic activity was rechecked for two bands. Escherichia coli, staphylococcus aureus and Xanthomonas
Band one showed very good activity (Fig. 3 A-D) compared to band vesicatoria have very good inhibitory effect than and Ralstonia
2. The minimum Inhibitory concentration of purified compound was solanacearum (Table 1).
checked against four different bacteria (as mentioned earlier.)
Table 1. Minimum inhibitory concentration (MIC) of Methanolic extract of Chromolaena odorata Purified compound
Concentration (%) E.coli S.aureus X.vesicatoria R.solanacearum
10 1 3 1 1
20 2 4 2 2
30 3 4 3 2
40 4 5 5 3
60 5 6 6.5 4
80 7 7 7.2 5
100 8.5 7.5 8 6
Chloromphenicol 12 12 15 16
(E.coli = Escherichia coli, S.aureus = Staphylococcus aureus, X.vesicatoria = Xanthomonas vesicatoria, R.solanacearum =
Ralstonia solanacearum)

solvent : methanol (MeOH), Trifluro acetic acid (TFA). HPLC

HPLC analysis
chromatogram showed only one peak, where the retention time was
The elutents used in the RP-HPLC analysis of phenolics are
21.775 min and the percentage of concentration was 100% (Fig. 4).
mixtures of aqueous pH modifiers with a polar, water-soluble organic
Detector A - 2 (225nm)
Pk # Retention Time Area Area Percent
1 21.775 32031050 100.00

Totals 32031050 100.00

Fig. 4. High Performance liquid chromatogram showing single peak

Fourier transform infrared spectra (FTIR) exhibits following absorptions (Table 2).
The Infra- Red spectrum (Fig. 5) of the isolated compound
Table 2. IR Spectrum peak number, wave number and functional groups
Peak No. Wave number Functional Group*
1 3423 cm-1 Intermolecular H bonded O-H stretch
2 2956cm-1 Aromatic C-H stretch
3 2924cm-1 C-H stretching due to methylene group
4 2854cm-1 C-H stretching due to methylene group
8 1644cm-1 Overtone or combination bands
9 1462cm-1 C=C ring stretch
10 1376cm-1 In plane O-H bending
12 1154cm-1 C-O stretching vibrations in alcohol and phenol
13 1076cm-1 C-O stretching vibrations in alcohol and phenol
16 723cm-1 Out of plane aromatic C-H bending
*The above results indicate the presence of phenolic groups.
30 S.L.Sukanya et al.

Fig. 5. IR Spectrum of methanolic extract of Chromolaena odorata compound.

and 2). LC-MS spectrum of methanolic extract of Chromolaena

Nuclear magnetic resonance (NMR) and Liquid
odorata compound exhibiting intense peak at m/z 301.3109 and the
chromatography mass spectra (LC-MS)
molecular weight of the compound was probably m/z 437.4129 (Fig.
1H NMR and 13 C NMR (500 MHz) spectrum data shows the
6).
purified compound to be phenolic compound (Supplementary Fig.
1

Fig. 6. LC-MS spectrum of methanolic extract of Chromolaena odorata compound exhibiting intense peak at m/z 301.3109 and 437.4129 molecular mass of compound.

compounds particular to specific solvents. Various investigators have

DISCUSSION
shown that the use of different solvent influences the ability of the
Separation of the active fraction on TLC showed that two bands bioactive molecule in demonstrating different degrees of
exhibited the antibacterial activity in the crude methanolic extract of antimicrobial effect [27, 28]. The phenolic compounds of natural
C. odorata. Further, detailed investigation of these two bands again origin have the positive property of being soluble in polar solvents.
confirmed the presence of inhibitory activity of the extract. Remaining This leads to the possibility of using reversed phase HPLC (RP-
seven bands indicated the loss of antibacterial activity suggesting HPLC) in their analysis, sufficient retention being achieved by using
synergistic activity of the extract. There is a possibility of synergism acidic conditions in order to avoid the presence of ionized forms of
between the compounds in crude extract than in isolated the analytes. The purified compound from methanolic extract of C.
constituents [26]. odoratum (3mg ml-1) consistently showed good inhibitory effect on
C. odorata is a prolific producer of novel phytochemicals [25]. In above mentioned bacteria. These results demonstrated that C.
the search for active compounds from C. odorata, the majority of odoratum has the potential for inhibiting the growth of above
research has been performed on extracts of the leaves which mentioned bacteria at low concentrations, even though, FTIR
inhibited the growth of clinical bacteria such as Escherichia coli, and spectral data of the purified C. odorata compound showed the
Staphylococcus aureus. However, it has not been tested on presence of phenolic groups. However, from our studies we
phytopathogenic bacteria such as Ralstonia solanacearum and observed that the mass weight of our phenolic compound is m/z
Xanthomonas vesicatoria. It is clear that leaf extract with methanolic 437.4129.
solvent showed varied degree of inhibitory effect on both clinical and The need for new, safe and more effective antibacterial is
plant pathogens. This might be due to the solubility of active
a major challenge to the pesticides industry, especially with
the
Journal of Phytology 2011, 3(10): 26-32 31

increase in phytopathogenic strains resistant to bactericides and also in plantations – An allelopath or a growth promoter? “In
fungicides apart from being hazardous to environment and human proceedings of the fifth annual symposium on plantation crops,
health. On this background, the biotechnological potential of C. held at CPCRI, Kasaragod. Dec 15-18.
odoratum in terms of production of phenolic compound inhibiting [5] Inya-Agha, S.I. B.O. Oguntimein, A. Sofowora and T.V. Benjamin.
both clinical and phytopathogenic bacteria is noteworthy. Results
1987. Phytochemical and antibacterial studies on the essential oil
obtained in the present investigation indicated that C. odoratum
produced a stable phenolic compound with active antimicrobial of Eupatorium odoratum. Int. J. Crude Drug Res. 25: 49-52.
activity. [6] Lamaty, G., C. Menut, P.H.A. Zollo, J.R. Kuiate, J.M. Bessiere,
Field existences of antibiotic resistant phytopathogenic J.M. Quamba and Silou, T. 1992. Aromatic plants of tropical
bacteria are increasing in recent years. WHO banned many Central Africa, IV. Essential oil of Eupatorium odoratum L. from
agriculturally important pesticides due to wide range of toxicity Cameroon and Congo.
against non target organisms including humans which are known to [7] Chowdhury, A.R. 2002. Essential oils of the leaves of Eupatorium
cause pollution [29]. Some of the developing countries are still using odoratum L. from Shillong (N. E.). J. Ess. Oil. Res. 5: 14-18.
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naturally available chemicals from plants, which retards the [8] Talapatra, S.K., D.S. Bhar and B. Talapatra. 1974. Flavonoid and
reproduction of undesirable microorganism, would be a more realistic terpenoid constituents of Eupatorium odoratum. Phytochem.13:
and ecologically sound method for plant protection and will have a 284-285.
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[30, 31]. Many reports on antibacterial activity of plants extract and related compounds; Part XIII. Ind. J. Chem. 15B: 806-807.
against human pathogens and their pharmaceutical application are
available [32-35], but not much has been done on the antibacterial [10] Barua, R.N., R.P. Sharma, G. Thyagarajan and W. Hertz. 1978.
activity of plants extract against plant pathogens [35]. This is mainly Flavonoids of Chromolaena odorata. Phytochem. 17: 1807-1808.
due to lack of information on the screening/evaluation of diverse [11] Metwally, A.M. and E.C. Ekejiuba. 1981. Methoxylated flavonols
plants for their antibacterial potential. Thus the present study reveals and flavanones from Eupatorium odoratum. Planta Med. 42: 403-
that C. odorata is a potential and promising plant. Therefore it should 405.
be successfully exploited for the management of the diseases
[12] Hai, M.A., P.K. Biswas, K.C. Shil and M.U. Ahmad. 1991.
caused by different plant pathogenic bacteria which are known to
cause many diseases in wide variety of crops. Chemical constituents of Eupatorium odoratum Linn.
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solancearum and X. vesicatoria has been demonstrated for the first Effect of Eupatorium odoratum on blood coagulation. J. Med.
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date, LC/ (API) MS technique have been widely accepted for the
Chem. 8: 139-142.
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RP-HPLC conditions. The most frequent application of LC/MS in the [15] Wollenweber, E., M. Dörr and R. Muniappan 1995. Exudate
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