1. health and fitness
2. disease

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PRELIMINARY PHYTOCHEMICAL INVESTIGATION
AND STUDY ON ANTIMICROBIAL ACTIVITY OF
TRIDAX PROCUMBENS LINN
KAMBLE S. I,
DAHAKE P. R,
Department of Botany and Department of Seed
Technology Phulsing Naik Mahavidyalaya Pusad,
District Yavatmal, Maharashtra State, India
Department of Botany and Department of Seed
Technology Phulsing Naik Mahavidyalaya Pusad,
District Yavatmal, Maharashtra State, India
ABSTRACT
Over the past few decades the use of antibiotics is under threat as many commonly used
antibiotics have become less effective against certain illnesses due to emergence of drugresistant bacteria. The continuous search for the source of new antibiotics is needed to face
the problem of increasing resistant strains of bacteria. The disease causing bacteria have
evolved the genetic ability to transmit and acquire resistance to drugs used as therapeutic
agents. The discovery of antibiotics to combat these pathogens marked a resolution in the
20th century. One way to prevent antibiotic resistance is by exploring new bioactive
compounds from traditional medicine which is not based on the existing synthetic
antimicrobial agents. Phytochemicals from medicinal plants showing antimicrobial
activities have the potential of filling this need, because their structures are different from
those of the more studied microbial sources. The present study was conducted to evaluate
the antimicrobial activity from ethanolic extract against different life threatening
pathogenic microorganisms and screening for various phytochemical constituents of
Tridax procumbens Linn. The antibacterial activity of the plant extract of Tridax
procumbens L. was studied against gram positive and gram negative bacteria. The
ethanolic extract displayed broad spectrum activity against all the test organisms. The
antibacterial activity of the extracts was compared to the drug Ampicillin and Penicillin.
Phytochemical screening of the plant revealed the presence of tannins, alkaloids,
glycosides and saponins. The results of this study support the traditional use of Tridax
procumbens Linn. Whole plant as an antibacterial agent.
Keywords: Drug-Resistant Bacteria, Medicinal Plants, Phytochemical Constituents, Tridax
Procumbens Linn., Antimicrobial Activity, Phytochemical Screening,
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INTRODUCTION:
Man always been surrounded by countless microorganisms. The disease producing microbes are playing a very
important role in human life. Pathogenic microorganisms are always trying to develop resistance to the various
antimicrobial agents used for their control. Therefore, the chemotherapy of infectious diseases has proved to be
a continuous struggle. Many efforts have been done to discover new antimicrobial compounds from various
kinds of sources such as soil, micro organisms, animals and plants. One of such resources is folk medicine and
systematic screening of them may result in the discovery of novel effective compounds. Antimicrobials of
plant origin have enormous therapeutic potential and have been used since time immemorial.
They have been proved effective in the treatment of infectious diseases simultaneously mitigating many of
the side effects which are often associated with synthetic antibiotics. Phytochemicals from medicinal plants
showing antimicrobial activities have the potential of filling this need, because their structures are different
from those of the more studied microbial sources, and therefore their mode of action may likely to differ.
There is growing interest in correlating the phytochemical constituents of a medicinal plant with its
pharmacological activity.
Tridax procumbens Linn belongs to family Asteraceae is a green perennial plant and is available in all
seasons in many parts of India. The plant yielded interesting compounds like luteolin, β-amyrin, β amyron,
lupeol, tria contanol, fucosterol, campasterol, stigma sterol, besides arachidic acid, lauric acid, palmatic acid,
flavones and glycosides. The extracts of Tridax procumbens have been reportedn to have various
pharmacological effects,antimicrobial activity against both gram positive and Gram negative bacteria. The
extracts of T.procumbens have been reported to have various pharmacological effects including
antimicrobial activity, wound healing property and immunomodulatory activity on the experimental animals.
METHODS AND MATERIALS:
Collection and processing of plant material:
The fully grown plants of Tridax procumbens were collected from the local area and taken care for its
freshness, healthy and free from any deformation. These plants were dried at room temperature then blended
into powder by mixture blender which then passed from the sieve to get the equal size particles. The powder
should be aseptically kept in air tight container at the moisture free place.
Soxhlet extraction of plant:
For the extraction of Tridax procumbens the selection of solvents is done with care to meet extrability and
regulatory criteria. Depending upon the solubility ethanol was selected for the extraction procedure. 100gm
of powder is accurately weight and is transferred to the cup made up of ‘Whateman filter paper’ and placed
into the extraction thimble. 500ml of ethanol was taken in round bottom flask and heated up to its boiling
point, i.e. 650C. The ethanol gets evaporated and moved in to the condenser where it was converted in to
liquid trickled in to the extraction chamber containing the plant material. The powder was extracted for 48
hrs. At the end of the extraction process, the flask containing the methanolic extract was removed and
extract was condensed at 500C in water bath for overnight. The weight of extract was measured and
percentages of yield of the plant material were calculated. The extract was stored at 40C for further work.
(Sahu and Padhy, 2013)
Phytochemical screening:
The ethanolic extract of Tridax procumbens was screened for the phytochemical content by using different
chemical test for each component. The ethanolic extract of the plant was used for the phytochemical test to
detect the presence of alkaloids, tannins, saponins, flavonoids, cardiac, glycoside, anthraquinones and
steroids according to standard method as follows. (Sawant and Godghate, 2013)
a) Alkaloids: A 5ml quantity of concentrated extract was taken into a test tube and 1 ml HCl was added the
mixture was heated gently for 20 min cooled and filter, the filtrate was used for Hager’s test.
b) Flavonoids: Alkaline reagent test: Extract was treated with 10 % NaOH solution; formation of intense
yellow color indicates presence of Flavonoids.
c) Steroids: 1ml extract was dissolved in 10 ml of chloroform & equal volume of concentrated H2SO4 acid
was added from the side of test tube. The upper layer turns red and H2SO4 layer showed yellow with green
fluorescence .This indicates the presence of steroid.
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d) Tannin: 4ml extract was treated with 4 ml FeCl3 formation of green color indicates that presence of
condensed tannin.
e) Saponins: 5 ml extract was mixed with 20 ml of distilled water then agitated in graduated cylinder for 15
min formation of foam indicates Saponins.
f) Cardial Glycosides: Plant extract treated with 2 ml glacial acetic acid containing a drop ofFeCl3 .A
brown color ring indicates the presence of positive test.
g) Anthraquinones: About 0.5gram of the extract was taken into a dry test tube and 5ml of chloroform was
added and shaken for 5 min. the extract was filtered and the filtrate shaken with equal volume of 100%
ammonia solution. A pink violet or red color in the ammonical layer indicates the presence of free
anthraquinones.
Thin layer chromatography of extract:
The qualitative analysis was made by using TLC. The compounds in the extract have been proceeds for the
separation of by thin layer chromatography. For Tridax procumbens extract Chloroform: Ethyl acetate:
Methanol: Butanol (30:15:30:25) was used. Silica gel plates were prepared and dried in hot air oven at 1100
C for 15min. the test sample was plotted on the TLC plate & plate was run in the solvent system. After
running the solvent front up to one third portions, the plate was taken out and visualize under the U.V.
transilluminator and Rf values for the respective bands were measured.
Isolation of test organisms:
Pure cultures of the test organisms used for antibacterial activity were isolated from the water and soil
sample by using selective media. The characterization of the test organism was done by using IMVIC test.
All the test organisms were cultured on nutrient agar slant. The cultures were maintained by sub-culturing
periodically and preserved at 40C prior to use. (Alzoreky, N.S. and K. Nakahara. 2003)
The gram negative bacteria includes; Escherichia coli, Pseudomonas aeruginosa, Vibrio cholera and
Enterobactor aerogenes. While the gram positive bacteria includes; Bacillus megaterium, Staphyloccus
aureus, Streptococcus pneumoniae and Streptococcus pyogenes.
Screening for antibacterial activity:
All the test organisms were screened for the antibacterial activity against methanolic extract of Tridax
procumbens by agar well diffusion method. With the introduction of variety of antimicrobials it becomes
necessary to perform the antimicrobial susceptibility test. For this the antimicrobial agent was allowed to
diffuse out into the medium and interact in a plate freshly speeded with the test organism. Stock solution of
ethanolic extract of Tridax procumbens was prepared to carry out the antimicrobial activities against
selected cultures for the further process. For the preparation of the stock solution 1 gm of ethanolic extract
was accurately weight and dissolved in 10 ml of DMSO; giving concentration of the stock solution as 100
mg/ml. this solution is then centrifuged and supernatant liquid was collected in a separate test tube, covered
with paraffin wax and stored at 40C for further use. (Sing and Jain, 2012)
Agar well diffusion method:
The suspensions of all the organisms were prepared as per Mac-Farland Nephelometer Standard. A 24 hrs
old culture was used for the preparation of bacterial suspension. Suspensions of organisms were made in
sterile isotonic solution of sodium chloride (0.9% w/v) and the turbidity was adjusted. The Muller-Hinton
agar plates for the bacteria were prepared and 0.1 ml of fresh 18 hours old broth culture was spread on the
respective media. After spreading the culture, wells of 6 mm in diameter was made at the centre of the plate
by using sterile cork borer. The wells were open with the help of sterile forceps. Then 100 µl of stock
solution was added by using micropipette in each well. The final concentration in the well was 10 mg/ml.
The extract was allowed to diffuse; hence the prepared plates were kept in deep refrigerator for 25 minutes.
After this plates were incubated at 370C for 24-48 hours. The zone of inhibition was measured in mm and
recorded. The diameter of the zone of inhibition around each well was taken as measure of antibacterial
activity. Each experiments was carried out in triplicates and mean diameter of the inhibition zone was
recorded. Ampicillin and Penicillin was used as control for gram negative and gram positive bacteria
respectively. (Rajput and Pal, 2011)
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RESULTS AND DISCUSSION:
TABLE NO.1: PHYTOCHEMICAL SCREENING OF ETHANOLIC EXTRACT TRIDAX
PROCUMBENS PLANT
Sr. No. Phytochemical constituents Ethanolic extract
1
Alkaloids
+
2
Saponins
+
3
Tannins
+
4
Steroids
5
Flavonoids
6
Anthraquinones
7
Glycosides
+
Key: (+) present, (-) absent
Table no.1 shows phytochemical screening of ethanolic extract of Tridax procumbens Linn. The presence of
different phytoconstituents was determined by using various tests like Mayer’s test, Killer Killani test,
Salkowski test, Bortrager’s test, Alkaline test and Potassium hydroxide test for Alkaloid, Glycosides,
Steroids, Saponins, Anthraquinones, Flavanoids and Tannins respectively. The ethanolic extract was found
to contain Saponins, Tannins, Alkaloids and Glycosides.
FIGURE NO.1: TLC OF TRIDAX PROCUMBENS ETHANOLIC EXTRACT
Figure no.1 showing the thin layer chromatography of Tridax procumbens ethanolic extract. Active
phytochemical compounds are analyzed by thin layer chromatography by using Chloroform: Ethyl acetate:
Methanol: Butanol (30:15:30:25) and principle pigments were separated. In this case three bands were
separated with Rf values of 0.41 (Green spot), 0.53 (Red spot) and 0.78 (Brown spot).
TABLE NO.2: ANTIMICROBIAL ACTIVITY OF ETHANOLIC EXTRACT OF
TRIDAX PROCUMBENSAGAINST GRAM NEGATIVE BACTERIA
Sr. No.
1
2
3
4
Test Organism
Pseudomonas aerogenosa
Escherichia coli
Enterobacter aerogenes
Vibrio cholerae
Zone of Inhibition (mm in diameter)
19.3 mm
15.6 mm
18.2 mm
20.8 mm
Table no.2 shows agar well diffusion method for demonstration of antimicrobial activity of ethanolic extract
of Tridax procumbens against gram negative bacteria. The zone inhibition around the well observed for
gram negative bacteria varies from 15mm-21mm in diameter with highest for Vibrio cholerae at 20.8mm
and lowest for Escherichia coli at 15.6 mm. (Fig. no.2) Results show that bacteria are sensitive to ethanolic
extract of plant.
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FIGURE NO.2:- ZONE OF INHIBITION AGAINST GRAM NEGATIVE BACTERIA
TABLE NO.3: ANTIMICROBIAL ACTIVITY OF ETHANOLIC EXTRACT
OF LAWSONIA INERMISAGAINST GRAM POSITIVE BACTERIA
Sr. No.
1
2
3
4
Test Organism
Bacillus megaterium
Staphyloccus aureus
Streptococcus pneumoniae
Streptococcus pyogenes
Zone of Inhibition (mm in diameter)
17.7 mm
15.3 mm
16.5 mm
19.4 mm
Table no.3 shows agar well diffusion method for demonstration of antimicrobial activity of ethanolic of
from Tridax procumbens against gram positive bacteria. The zone of inhibition around the well observed for
gram positive bacteria varies from 15mm-20mm in diameter with highest for Streptococcus pyogenes at 19.4
mm and lowest for Staphyloccus aureus at 13.3mm. (Fig. no.3) Result shows that bacteria are less sensitive
to ethanolic extract of plant as compaired with gram negative bacteria.
FIGURE NO.3:- ZONE OF INHIBITION AGAINST GRAM POSITIVE BACTERIA
In current study the result showed that Tridax procumbens sample collected from local region of Pusad city,
demonstrated antimicrobial activity against different bacterial pathogens. The ability of plant extract to
inhibit the growth of all the tested organisms indicates its broad spectrum of activity. (Figure no. 2 and 3)
Gram negative bacteria were found to be more sensitive towards ethanolic extract of plant than that of gram
positive bacteria. Herbal medicines are valuable and readily available resources for primary health care and
complementary health care system. These plants may prove to be antimicrobial activities, but more
pharmacological investigations are necessary. Present time the emergence of multi-drug resistance in human
and animal pathogenic microbes as well as undesirable side effects of certain antibiotics has triggered
immense interest in the search for new antimicrobial drug of plant origin.
In similar investigation Priyadarshini and Iyer in 2013 observed the antimicrobial activity of chloroform extract
against E. coli (14mm), P. aerogenosa (13mm) and S. aureus (16mm). Sharma and Sharma in 2010 found that
aqueous extract of plant inhibit the growth of S. Aureus (26mm), B. subtilis (19mm), E. coli (28mm) K.
pneumoniae (22mm) and M. luteus (27mm). Christudas et al in 2012 revels the inhibition ability of ethanolic
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extract against B. faecalis (16mm), B. subtilis (13mm), E. coli (16mm) and P. aeruginosa (12mm) at
concentration of 800µg/ml. Bama et al in 2012 found the inhibition of V. cholerae (23.9± 0.7 ) and S. aureus
(24.6±0.36 ) at concentration of 20 µg/ml. Bharathi et al in 2012 observed the antimicrobial activity of ethyl
acetate and methanol extract against S. aureus, K. pneumonia, S. typhi, E. coli and B. cereus.
Priyadarshini and Iyer in 2013 observed the presence of tannins, flavonoids and saponins in chloroform
extract of plant. Sharma and Sharma in 2010 found that aqueous extract of plant contains alkaloids, tannins,
flavonoids and saponins. Christudas et al in 2012 demonstrated the presence of steroids, tannins, and
alkaloids from plants extracts. Solanki in 2014 reported the presence Steroid, Tannin, Saponin, Alkaloids,
Phenol and Flavonoids from solvent extracts of plants. Sanghavi et al in 2014 shows the presence of
quercetin by using HPTLC method.
CONCLUSION:
According to current investigation, plant based secondary metabolites have great therapeutic potential as
they can serve the purpose with lesser side effects that are often associated with synthetic antimicrobial
agents. The present results revealed that the extract of Tridax procumbens L. was effective against both
Gram-positive and Gram-negative bacteria. Presence of chemical compounds viz. alkaloids, tannins,
flavonoids and saponins of Tridax procumbens L. may inhibit the bacterial growth. Further studies are going
on this plant in order to isolate, identify, characterize and elucidate the structure of the bioactive compounds.
REFERENCES:
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