International Journal of Herbal Medicine 2015; 3(2): 20-23

E-ISSN: 2321-2187
P-ISSN: 2394-0514
IJHM 2015; 3(2): 20-23
Quantitative Estimation of total phenols and
Received: 15-04-2015 antibacterial studies of leaves extracts of Chromolaena
Accepted: 19-05-2015
odorata (L.) King & H.E. Robins
Dr. Manju Madhavan
Asst Professor,
Department of Botany
Vimala College, Thrissur.
Dr. Manju Madhavan

Abstract
Chromolaena odorata (L) King & H.E. Robins, a species of Asteraceae family. They are known as devil
weed, Communist weed. The plant C. odorata is known for its medicinal importance among the tribal
population. It is a common practice to use the leaf extract is to heal wounds.In the present study, an
attempt is made to evaluate the total phenols in leaf extracts of Chromolaena odorata and to find out the
antibacterial effect of the extracts against wound infecting bacteria Staphylococcus aureus and
Escherichiacoli. Total phenolic content were determined using Catechol as standard. Phenol is
determined in the distilled water, ethanol and acetone extracts. Maximum phenol content is found in
acetone extracts. The antibacterial activities of the extracts were determined by the disc method Acetone
extract showed maximum zone of inhibition and water extract showed the least.

Keywords: Chromolaena, antibacterial activity, phenol content, medicinal plant, wound healing.

1. Introduction
Medicinal plants are nature’s priceless gift to humans. Herbs are used traditionally to cure
many diseases both developing and developed countries. Even though development in the
field of modern medicine temporarily subdued the traditional herbal medicine, now it has
staged a comeback and a “herbal renaissance” is blooming all across the world. According to
WHO in 2008, nearly 80% of the world’s population are depending on herbs for their health
care needs. The antibacterial or microbial activities of plants are attributed to the presence of
secondary metabolites in plants. Phenolic acids, one important class of secondary metabolites
widely spread throughout the plant kingdom. Studies have shown that natural phenols exhibit
good antibacterial activity. Phenol also known as carbolic acid is an organic compound with
chemical formula C6H5OH. Phenolic acids are easily absorbed through the walls of our
intestinal tract, and they may be beneficial to our health because they work as antioxidants
that prevent cellular damage due to free-radical oxidation reactions. They may also promote
anti-inflammatory conditions in our body when we eat them regularly. Phenolic compounds
are essential for the growth and reproduction and are produced as a response for defending
injured plants against pathogens in some plants, they are secreted by the root system in the
form of phytoalexins to check the growth of nearby plants [1]. Some phenolics are water
soluble, some others are soluble in organic solvents, and still others are insoluble polymers.
Although pharmacological industries have produced a number of new antibiotics in current
clinical use, in last decades, resistance of microorganisms to these drugs has increased. In
general, bacteria have the genetic ability to transmit and acquire resistance to drugs which are
utilized as therapeutic agents. So more and more plants, crude drugs are analysed for the
antimicrobial properties.
Chromolaena odorata (L) King & H.E. Robins, a species of Asteraceae family and known in
English as Siam weed, is a perennial shrub native of central & South America. From there
they extends its territory to the Asian countries like India, China, Bangladesh, Thailand etc.
They are known as devil weed, Communist weed etc. It expands rapidly at the onset of the
rainy season and forms impenetrable tangles that may ultimately shade out indigenous
vegetation. The plant’s ability to thrive in a wide variety of soil in the tropics and its short
Correspondence: juvenile stage, flowering in dry season, prolific seed production and strong ability to re-
Dr. Manju Madhavan sprout after burning during land preparation all contribute to its invasiveness. It is a much
Asst Professor,
branched perennial shrub that forms dense tangled bushes 1.5 to 3m in height in open
Department of Botany
Vimala College, Thrissur. condition, and occasionally reaching 6-10m by scrambling up other taller vegetation.
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The stems are circular, hairy or almost smooth and much 2.4. Disc diffusion Method
branched. The leaves are opposite triangular shaped, young The antimicrobial activities of the extracts were also
ones slightly reddish purple, have toothed margins, with 3 determined by the disc method [5]. 15ml of the nutrient agar
main veins, and give off a pungent odour when crushed. medium was dispensed into pre sterilised petridishes to yield a
Flower pale blue or white. Seeds are borne in the composite uniform depth for bacterial inoculation. The sterile discs
flower heads. The individual seed is about 5mm long with a (Himedia) were impregnated with various extracts (1mg/ml)
pappus with angled hooks to aid seed dispersal. As the species and were placed on the agar surface with flamed forceps and
name C odorata suggests, the leaves emit a pungent odour gently pressed down to ensure contact with the agar surface.
when crushed. It is a herb or sub shrub with many paired Streptomycin (10µg) was used as positive control. The discs
branches grow after main stem [2]. were spaced far enough to avoid overlapping rings of
The plant C. odorata is known for its medicinal importance inhibition. Finally, the petridishes were incubated for 24 hours
among the tribal population. It is a common practice to use the at 37 °C for bacteria. The diameter of zone of inhibition is
leaf extract is to heal wounds. C. odorata is reported to have indicated by the clear area which was devoid of growth of
antispasmodic, antiprotozoal, antitrypanosomal, antibacterial microbes. The treatments were repeated thrice and the mean is
and antihypersensitive activities. It has also been reported to taken.
possess anti-inflammatory, astringent, diuretic and The bacterial cultures of S. aureus and E.coli were procured
hepatotropic activities. In the southern part of Nigeria, the from the department of Microbiology, St Mary’s College
leaves of C. odorata are used for wound dressing, skin Thrissur. The liquid broth for bacterial culture were prepared
infection and to stop bleeding according to Hill [3]. As the plant by dissolving 13gms of nutrient broth in 1000ml distilled
is a weed it is available in large quantities. Lot of medicinal water.5ml of this medium were dispensed in test tubes and
properties are attributed to this plant. Important property is its autoclaved. This autoclaved medium in test tubes were taken
power of wound healing. In the present study, an attempt is to the microbiology department and inoculated with the
made to evaluate the total phenols in leaf extracts of bacterial strains. These test tubes were incubated in the
Chromolaena odorata and to find out the antibacterial effect of incubator for 2 days in 37 °C.
the extracts against wound infecting bacteria Staphylococcus
aureus and Escherichia coli. 3. Result
In the present work quantification of total phenol and the
2. Materials and Methods antibacterial activity of C. odorata leaves extracts were
2.1. Source of plant material estimated. Antibacterial activity of the leaf extracts were
The plants grown on the road side and uninhabited plots near assessed against the wound infecting bacteria namely E.coli
Vimala College, Thrissur were collected (Fig 2a).The Fresh and S. aureus.
leaves were collected from the plant. Dust and debris were About 20g of the powdered leaves were soaked in 100ml of
removed from the plant parts and shade dried. Shade dried solvents such as ethanol, acetone and water. Water soluble
leaves were grinded to fine powder by a domestic grinder. extractive value was found to be 1.23g and shows light brown
colour. Ethanol extract showed an extractive value of 1.20g
2.2. Preparation of extract with deep greenish black colour, whereas acetone extract
The extract was prepared using distilled water, acetone and showed light green colour and the extractive value was found
ethanol. The extract was prepared by soaking 20gms of dry to be 0.50g (Table 1). The water extract showed high
leaf powder with 100ml of various solvents for 48hours and extractive value, which is slightly greater than that of ethanolic
kept in a magnetic stirrer for 6 hours. The extracts obtained extract.
from various solvents through filtration were kept for
evaporation in hot air oven to remove the excessive solvents. Table 1: Shows the solvent used for extract preparation, colour of
The dried solvent extracts were stored in a cool dry place. extract and weight of extract obtained from C.odorata leaf powder
Solvent Colour of the extract Extractive Value (gms)
2.3. Estimation of total phenol Acetone Light green 0.50
Total phenolic content were determined according to the Ethanol Deep greenish black 1.20
method of Singleton and Rossi 1965, using Catechol as Water Light brown 1.23
standard [4]. One ml(1mg/ml) of the extracted sample from the
respective solvents were mixed with equal volume of Folin Presence of total phenols in the extracts (1mg/ml) was
and Ciocalteu’s phenol reagent and incubated for 3 minutes at characterized by the presence of bluish green colour (Figure
room temperature, to this 1 ml of saturated sodium bicarbonate 2b). Phenol content in the extract was quantified from standard
(3.5%) was added and final volume was made up to 10 ml with graph plotted for catechol (Figure 1). Phenol content in water
distilled water. The reaction mixtures were kept in dark for extract was found to be 52µg, acetone 106µg and ethanol
90minutes and absorbance was read in Spectrophotometer at 100µg. Maximum phenol content observed in acetone extract
650 nm. The standard solution of phenol was prepared with (Table 2). Water extract showed less phenol content.
200 mg of catechol dissolved in distilled water and made upto
100 ml. The working solution (10 µg, 20 µg, 30 µg, ---140µg) Table 2: Quantity of phenol in different extracts of C.odorata
was prepared by diluting the stock solution with distilled water expressed as µg catechol equivalent per mg of extract
in the proportion 1: 10. Standard graph plotted for catechol
Extract OD Value Conc. Of Phenol
(Fig.1) where 'Y' is the concentration of total phenols in µg
Acetone 0.62 106 µg
and 'X' is the optical density. The phenol concentration in the
extract was calculated from the graph and expressed as µg of Ethanol 0.60 100 µg
catechol equivalent per mg of the extract. Water 0.28 52µg

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The water, ethanol and acetone extracts were studied against antibacterial activity showed that the ethanolic extract of C.
wound infecting bacteria S. aureus. and E.coli. 1mg/ml of each odorata exhibited a zone of inhibition of 10.6mm against S.
extract is taken and were tested against these bacteria. The aureus and E.coli. Acetone extract showed only 12.6mm of
ethanol and acetone extracts showed antibacterial activity. inhibition against S. aureus and E.coli. Whereas water extract
This was clear with the zone of inhibition obtained (Fig.2d, e). didn’t show any activity against E.coli (Table 3).
Streptomycin disc was used as positive control (Fig.2 c). The

Table 3: Showing the Diameter of Zone of inhibition (mm) obtained by antibacterial activities of different leaf extracts of C.odorata.
Extracts E.coli S. aureus
Treatment 1 R1 R2 R3 Mean R1 R2 R3 Mean
Cetone 12 mm 14 mm 12 mm 12.6mm 14 mm 12mm 12mm 12.6mm
Ethanol 10mm 12mm 5mm 10.6mm 12mm 10mm 10mm 10.6mm
Water - - - - 1mm 1mm 2mm 3.2mm
Streptomycine Disc 20mm 22mm

competitive and does not let other flora grow. Although it was
used traditionally for its healing properties, it never enjoyed
the status of a medicinal herb [6].Instead, efforts were always
made to eradicate the so called weed. In the present scenario,
microbes are getting more and more resistant, allopathy
medicines are showing side effects so more and more crude
drugs obtained from plants are analysed for antibacterial
properties. So in the present work, this plenty available plant
has been studied for its antibacterial activity.
Natural preparations from plants, crude extracts contain
phenolic compounds and exhibit antibacterial activity [7]. Plant
phenolics, especially dietary flavonoids, are currently of
growing interest owing to their supposed functional properties
Fig 1: Catechol Calibration Curve in promoting human health. It is well-known that phenolic
compounds contribute to quality and nutritional value in terms
of modifying color, taste, aroma, and flavour and also in
providing health beneficial effects. They also serve in plant
defence mechanisms to counteract reactive oxygen species
(ROS) in order to survive and prevent molecular damage and
damage by microorganisms, insects, and herbivores. The
aqueous extract and the decoction from leaves of this plant
have been used throughout Vietnam for the treatment of soft
tissue wounds and burns for decades. A product made from
Chromolaena named eupolin have already been licensed for
use in Vietnam for soft tissue burns and wounds [6].In the
present study ethanol, acetone and water extracts were
qualitatively and quantitatively evaluated for the presence of
phenol. The presence of phenol was detected on all the three
extracts by the bluish green colouration, quantitatively found
maximum in acetone and minimum in water.
The antibacterial activity is a major factor in the wound
healing property expressed by the herbs [8]. In this work,
antibacterial study of the extracts was conducted aganist the
wound infecting bacteria S. aureus and E.coli. Acetone extract
showed maximum zone of inhibition and water extract showed
the least. The studies by Irobi showed that antibacterial
activity of ethanolic extracts of C odorata and a zone of
inhibition obtained varied from 5mm to 24 mm against
different microbial strains [8]. In one of the experiments
conducted by Taleb-Contini et al. [9]. he crude extracts
(dichloromethanic and ethanolic) from Chromolaena have
been evaluated against 22 strains of microorganisms including
bacteria (Gram-positive and Gram-negative) and yeasts. Vital
4. Discussion
and Windell in their works with C odorata, crude extracts have
In the present study, C. odorata, commonly known as
‘communist weed” or ‘Siam weed’, is studied for the phenolic shown activity, mainly against Gram-positive bacteria. It was
found to be particularly active against Staphylococci [10]. In our
content and antibacterial properties. This plant is considered to
studies phenol content in the ethanol and acetone extract is
be a harmful weed due to its highly invasive, allelopathic
nature. It grows in pastures, marginal lands, open areas, dry found to be more or less equal and the zone of inhibiton
obtained also is the same against both the bacteria.
deciduous forests and interior shrub jungles, where it is highly
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This study too confirms the presence of phenol in leaf extracts
obtained by using different solvents. These extracts also
showed antibacterial activity. Efforts should be made to
exploit the medicinal properties of this abundant herb. The
example of this herb indicates the importance to consider and
evaluate the abundantly occurring weed species on this planet
as potential sources of medicines than as invasive flora.

5. References
1. Sunita Maurya, Dhananjay Singh. Quantitative Analysis
of Total Phenolic Content in Adhatoda vasica Nees
Extracts. Int J of Pharm Tech Res. 2010; 2(4):2403-2406.
2. Chopra RN, Nayer SL, Chopra IC. Glossary of Indian
Medicinal Plants. CSIR ed New Delhi, 1956.
3. Hill AF. Economic Botany. A textbook of usefulplants
and plant products. 2 nd edn. McGraw-Hill Book
Company Inc, New York, 1952.
4. Singleton VL, Rossi Joseph A Jr. Colorimetry of Total
Phenolics with phosphomolybdic-Phosphotungstic Acid
Reagents Am. J Enol Vitic. 1965; 16(3):144-158.
5. Davis WW, Stout TR. Disc plate method of
microbiological antibiotic assay. II. Novel procedure
offering improved accuracy. Appl Microbiol 1977;
22(4):666-670.
6. Vaisakh MN, Pandey Anima. Pharmacognostic study of
leaves of Chromolaena odorata Linn. Int J Pharma Sci
Res. 2009; 3(1):80-83
7. Memnune Sengul, Hilal Yildildiz, Neva Gungor, Bulent
Cetin, Zeynep Eser, Sezai Ercisli. Total Phenolic Content,
Antioxidant and Antimicrobial Activities of Some
Medicinal Plants. Pak J Pharm Sci. 2009; 22(1):102-106.
8. Irobi ON. Antibiotic properties of ethanol extract of
Chromolaena odorata. Vol 35 Pharmaceutical biology
1997; 2(1):111-115.
9. Taleb-Contini SH, Salvador MJ, Watanabe E, Ito IY,
Oliveira DCR. Antimicrobial activity of flavonoids and
steroids isolated from two Chromolaena species. Bra J of
Pharma Sci. 2003, 39(4).
10. Vital PG, Windell LR. Antimicrobial activity and
cytotoxicity of Chromolaena odorata (L. f.) King and
Robinson and Uncaria perrottetii (A. Rich) Merr. Extracts.
J Med Plants Res. 2009; 3(7):511-518.

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