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International Journal of Universal Pharmacy and Bio Sciences 5(6): November-December 2016
INTERNATIONAL JOURNAL OF UNIVERSAL
PHARMACY AND BIO SCIENCES IMPACT FACTOR 2.96***
ICV 6.16***
Pharmaceutical Sciences RESEARCH ARTICLE …………!!!
PHARMACOGNOSTIC STANDARDIZATION, PHYTOCHEMICAL
EVALUATION AND ANTIMICROBIAL ACTIVITY OF LEAF
EXTRACTS OF TRIDAX PROCUMBENS
Ms. Gharge V.G.*, Ms. Shelar P.A., Dr. Pawar P.K, Dr. Yadav A.V, Mrs. Mohite M.S,
Ms. Patil C. D, Mr. Thorat M. B, Mr. Pawar B. R.
Gourishankar Institute of Pharmaceutical Education & Research, Limb, Satara, India-41501.
KEYWORDS:
Tridax procumbens,
Physical constants,
Microscopy,
Antimicrobial activity.
For Correspondence:
Ms. Gharge Varsha G.*
Address:
Gourishankar Institute of
Pharmaceutical
Education & Research,
Limb, Satara, India-
41501.
ABSTRACT
Purpose: Tridax procumbens is a plant possess medicinal values like
analgesic, anti-inflammatory, antioxidant, antihepatotoxic,
immunomodulatory, antiseptic, etc. To evaluate the scientific basis for
the use of the plant, the antimicrobial activities of extracts of the leaves
were evaluated against some common gram negative and gram positive
bacteria. The study also investigated the chemical constituents of the
plant. Methods: Pharmacognostic characterization: In the present study
pulverized dried Tridax procumbens leaves were investigated for its
physical constant (LOD, ash value, fluorescence analysis). Microscopic
study: The fresh leaf of Tridax procumbens was studied for
microscopical characterization which shows different parts like upper
and lower epidermis, palisade cells, spongy parenchyma in lamina region
while Collenchyma and vascular bundles were observed in midrib region.
The pulverized dried Tridax procumbens leaves were extracted with
methanol using soxhlet apparatus. The powder of Tridax procumbens
leaves were also macerated with chloroform water. The antimicrobial
activity of the concentrated extracts were evaluated by determination of
the diameter of zone of inhibition against both gram negative and gram
positive bacteria using agar disc diffusion and minimum inhibitory
concentration (MIC). Results: Results of the phytochemical studies
revealed the presence of alkaloids, glycosides, steroids, flavonoids,
tannins and the extracts were active against both gram positive and gram
negative bacteria. Conclusions: The methanolic and aqueous extracts of
Tridax procumbens leaves were studied for antimicrobial activity, of
which methanolic extract shows significant results.
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INTRODUCTION:
Tridax procumbens Linn. belongs to the family Compositae. It is commonly known as „Common
button‟ or „Coat button‟ and it is a weed found throughout India.1 It is a semi prostate, annual,
creeper herb. Stem is ascending 30-50 cm height, branched, sparsely hairy, rooting at nodes. Leaves
are simple, opposite, exstipulate, lanceolate or ovate. 3-7 cm long irregularly toothed margin, base
wedge shaped, shortly petioled, hairy on both surfaces. Flowers are tubular, yellow with hairs,
inflorescence capitulum. Tridax procumbens has two types of flower: ray florets and disc florets
with basal palcentation. Flowering-Fruiting occurs throughout the year. Fruit is a hard achene
covered with stiff hairs and having a feathery, plume like white pappus at one end. It shows the
presence of fumaric acid, luteolin and glucoluteolin and ß-sitosterol-3-O-ß-D-xylopyranoside.2 The
plant also shows various pharmacological activities i.e. immunomodulatory3, anti-diabetic
4, anti-
hepatotoxic, anti-oxidant5, anti-inflammatory
6, analgesic
7 and marked depressant action on
respiration. A reputed ayurvedic medicine “Bringraj” is also prepared by the same plant; which is
used for various types of liver disorders8. Its flowers and leaves possess antiseptic, insecticidal and
parasiticidal properties.9
The nature has provided abundant plant wealth for all the living creatures, which possess medicinal
virtues. The essential values of some plants have long been published, but a large number of them
have remained unexplored to date. Therefore, there is a necessity to explore their uses and to
conduct pharmacognostic and pharmacological studies to ascertain their therapeutic properties.
Medicinal plants are of great importance to the health of individuals and communities. Hence an
attempt has been made to investigate antimicrobial activity of Tridax procumbens.
Figure No:-1- Whole Plant of Tridax procumbens.
MATERIAL AND METHOD:-
Collection & authentication
The plant material Tridax procumbens Linn (Compositae) were collected from the Satara district,
Maharashtra, during the month of July in the year 2014 and authenticated by Dept. of Botany,
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Y.C.I.S, Satara, Maharashtra, India. Specimen voucher was deposited in the college hebarium for
further reference. Fresh drug obtained were shade dried and coarsely powdered and passed through
sieve 100 mesh sizes and stored in air-tight containers for further use. The fresh leaves were used
for study of macroscopic and anatomical characters. Collected plant material was shade dried and
coarsely powdered. This coarse powder was used for determination of extractive values, ash value,
LOD and preliminary phytochemical investigation.
Preparation of Extract10
:-
The pulverized dried Tridax procumbens leaves were extracted with methanol using soxhlet
apparatus. The powder of Tridax procumbens leaves were also macerated with chloroform water.
Methanol and water extracts were filtered & evaporated to dryness.
Macroscopic Characteristic11, 12
:-
The macroscopy of fresh leaves were studied according to standard methods.
Microscopic characteristics13
:-
For microscopy hand section of leaf was taken, stained & mounted following usual micro
techniques.
Physical Evaluation:-
The ash values, extractive values and loss on drying were performed according to the officinal
methods prescribed in Indian Pharmacopeia.14
Fluorescence Analysis
Many drugs shows fluorescence when their powder is exposed to ultraviolet radiation. It is
important to observe all materials on reaction with different chemical reagents under U.V. light.
The fluorescence characteristics of powdered drug were studied under U.V. light after treating with
different chemical reagents is reported.
Fluorescence analysis was carried out according to the method of Chase and Pratt15
and Kokoski.16
Phytochemical Screening
The dried leaves were extracted with methanol and water. The behavior of powder with various
chemical reagent and preliminary chemical tests for various extracts were also carried out according
to the standard procedures described by Kokate17
and Horborne.18
ANTIMICROBEAL STUDY19
:-
Collection of microbes
Bacterial strains such as Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and
Salmonella typhi were used for the study. The collected microbes were maintained in Nutrient agar
broth and cultured in Nutrient Agar media. (Hi Media (P) Ltd Mumbai).
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Preparation of the medium
Nutrient agar medium was prepared by dissolving 2.8 g of nutrient agar in 100 ml of distilled water.
The solution was sterilized in an autoclave at 121°C for 15 min. It was cooled and poured into
sterile Petri dishes to solidify. The agar depth of the medium was measured (4 cm).
Determination of antimicrobial activity
Agar well-diffusion method was followed to determine the antimicrobial activity. Nutrient agar
(NA) plates were swabbed (sterile cotton plug) with 8 hour old-broth culture of respective bacteria.
Three wells (10mm diameter) were made in each of the plates using sterile cork borer. About 0.3 ml
of different concentration of plant solvent extract were added using sterilized dropping pipettes in
to the wells and allowed to diffuse at room temperature for two hours. The plates were incubated at
370C for 18-24 hr for bacterial pathogen. Respective proper control of solvent plant extracts were
also maintained. Diameter of the inhibition zones and the values were recorded.
Chromatographic Studies20, 21, 22
Thin Layer Chromatography studies were carried out for extracts to confirm the presence of
different phytoconstituents in these extracts. TLC is a mode of liquid chromatography, in which the
extract is applied as a small spot or band at the origin of thin sorbent layer supported on a
glass/plastic/metal plate. The mobile phase migrates through the stationary phase by capillary
action. The separation of solutes takes place due to their differential absorption/ partition coefficient
with respect to both mobile and stationary phases. Each separated component has same migration
time but different migration distance. The mobile phase consists of a single solvent or a mixture of
solvents. Although, a number of sorbent like silica gel, cellulose, polyamide, alumina, chemically
modified silica gel etc. are used, silica gel (type 60) is most commonly used sorbent. Handmade
plates are prepared by using techniques like pouring, dipping or spraying. The retardation factor
(Rf) is calculated using following formula,
Distance traveled by sample from base line
Rf = ----------------------------------------------------------
Distance traveled by solvent from base line
Thin Layer Chromatography23
The extracts were subjected to thin layer chromatography for the presence of phytoconstituents. In
this technique, the Silica gel-GF254 (for TLC) was used as an adsorbent and plates were prepared
by spreading technique, then air dried for an over-night and activated for one hour at 110°C and
used.
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Preparative Thin Layer Chromatography
A thick layer of silica gel GF-254 was coated on the square shaped plate and activated at 1100C for
one hour. The broad band (2 mm width) of extracted sample was applied on the plate.
Characterization of Isolated Compound
From the separated bands, the substance of interest was scrapped from the plate and it dissolved in
methanol. The mixture was filtered and the filtrate was evaporated to dryness. The isolated
compound was then subjected for further studies. (1mg/ml concentration of the extracts were used).
IR of isolated compound
IR spectrum was recorded in IR- spectrometer in 400-4000 frequency in cm-1
for isolated moiety.
IR spectrum of compound was carried in KBR pellet and reproduced on fig.No.6 and fig.No.7. The
important absorption can be correlated.
RESULT:-
Macroscopic Characteristic of Leaves and stems of Tridax procumbens
The plant is annual or biennial somewhat patently hispid herb. Steam branched, creeping at base,
sub erect or trailing above. Leaves were simple, opposite, serrate or dentate, acute, fleshly and
pubescent, 3 to 7 cm long, 1 to 4 cm wide, with irregularly toothed margins; base wedge- shaped,
shortly- petiole, more hairy on lower surface than upper surface.
Microscopic characteristics of leaves of Tridax procumbens
The Transverse Section of leaf is dorsiventral consists of Midrib and Lamina.
Midrib
It consist of single layered epidermis, on either side, upper epidermis composed of single layer
closely arranged elongated cells externally covered with striated cuticle. Leaf surface contains
simple, multicellular covering trichomes and anomocytic type of stomata. Below the upper
epidermis 3-4 layers of well developed more or less isodiametric collenchymatous tissue were
observed.
Midrib contains centrally located vascular bundle which is collateral surrounded by some
parenchymatous cells filled with dark content. Xylem is well developed and the phloem consists of
strands of sieve tubes and small celled parenchyma.
Lower epidermis consisted of single layer elongated cells with cuticle and just above the lower
epidermis 2-3 layers of parenchymatous cells followed by the layers of collenchymatous cells were
present. Calcium-oxalate crystals were found in spongy parenchyma. Lower epidermis contains
more number of covering trichomes as compared to upper epidermis.
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Lamina
Dorsi-ventral structure with single layered upper and lower epidermis with a layer of elongated
closely arranged cells externally covered with cuticle. Epidermal cells show anomocytic stomata in
surface view; below upper epidermis single layered palisade cells followed by 5-7 layers of
masophyll parenchyma which are rounded in shape and are devoid of intracellular spaces.
Figure No 2:- Microscopy of leaf of Tridax procumbens
Physical evaluation
The Loss on Drying, Ash Values likes (Total Ash, Acid insoluble ash, Water soluble ash),
Methanol soluble extractive, Water soluble extractive of leaf powder are given in table-1.
Table No.1:- Result of physical evaluation of leaves of Tridax procumbens
Sr. No. Physical Constants Result
1. Ash Value (% w/w)
Total Ash
Acid Insoluble Ash
Water Soluble Ash
11.67
4.24
3.50
2. Loss on Drying (% w/w) 84.8
3. Extractive Values (% w/w)
Methanol soluble extractive.
Aqueous soluble extractive
0.200
0.249
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Fluorescence Analysis
The fluorescence characteristics of powdered drug were studied under U.V. light after treating with
different chemical reagents is reported in table no. 2..
Table No.2:- Result of Fluorescence Analysis of leaves of Tridax procumbens
Reagents Fluorescence Observed
Leaf Leaf Powder Stem Powder
At 254 At 366 At 254 At 366 At 254 At 366
Powder + 1N NaOH
In Methanol
Green Green Light Green Light
Green
Dark
Brown
Dark
Brown
Powder + 1N NaOH
In Water
Green Green Dark Brown Light
Green
Dark
Brown
Light
Brown
Powder + 50% HCl Blue Yellow Yellowish
green
Green Dark
Brown
Yellowish
Green
Powder + 50%
H2SO4
Light Green Light
Green
Dark Green Light
Green
Light
Brown
Light
Brown
Powder + 50%
HNO3
Dark
Yellow
Greenish
Yellow
Dark
Yellowish
Dark
Yellowish
Dark
Brown
Light
Yellowish
Powder + Petroleum
Ether
Faint
Yellowish
Green
Faint
Yellowish
Green
Dark
Yellow
Faint
Green
Dark
Brown
Faint
Brown
Powder
+Chloroform
Faint Green Faint
green
Dark Green Faint
Green
Faint
green
Faint Green
Powder +Picric
Acid
Faint Green Faint
green
Yellow Dark
Green
Dark
Brown
Faint green
Powder +5% FeCl3 Yellowish
Green
Yellowish
Green
Dark Brown Faint
Green
Yellowish Yellowish
Green
Powder +5% Iodine Green Faint
Green
Dark Brown Dark
Green
Dark
Brown
Black
Green
Powder +Methanol Black Dark
Green
Black Dark
Green
Light
Brown
Dark Green
Powder +
(HNO3+NH3)
Faint Green Yellowish
Green
Faint
Brown
Light
Green
Dark
Brown
Yellowish
Brown
Table No. 3:- Phytochemical investigation of Methanolic and Aqueous extracts
Sr
No. Name of the test
Leaf
Methanolic extract Aqueous extract
1. Test for sterols + +
2. Test for Triterpenoids + -
3. Test for glycosides + +
4. Test for carbohydrates + +
5. Test for alkaloids + +
6. Test for flavonoids + +
7. Test for tannins + +
8. Tests for proteins - -
9. Test for amino acids - -
10. Test for fats + -
11 Test for Volatile oils - -
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Table No.4:- Antimicrobial activity of Methanolic extracts of Tridax procumbens
Organism
Diameter of inhibition zone in cm
Methanolic extract Streptomycin
100(µ/ml) 100(µ/ml) 200(µ/ml)
Staphylococcus aureus 2 2.9 5
Escherichia coli 1.8 3.1 4
Salmonella typhi 2.1 3 5
Pseudomonas aeruginosa 2.5 3.3 3.5
Figure No 3:- Antimicrobial activity of Methanolic extracts
Table No.5:- Antimicrobial activity of Aqueous extracts of Tridax procumbens
Organism
Diameter of inhibition zone in cm
Water Extract Streptomycin
100(µ/ml) 100(µ/ml) 200(µ/ml)
Staphylococcus aureus 2.1 2.5 4.2
Escherichia coli 2 3 3
Salmonella typhi 2.3 2.4 4
Pseudomonas
aeruginosa
2.4 3.4 4.2
Figure No 4:- Antimicrobial activity of Aqueous extracts
Staphylococcus aureus Escherichia coli Salmonella typhi Pseudomonas aeruginosa
aeruginosa
Staphylococcus
aureus
Escherichia coli Pseudomonas aeruginosa Salmonella typhi
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Thin layer chromatography of Methanolic extract
Stationary phase: Silica gel GF-254
Mobile Phase: Toulene: ethyl acetate: Formic Acid (8.5:1:0.5).
Detection: UV-366
Solvent front: 5
Spot detection: 2.2
Thin layer chromatography of Aqueous extract
Stationary phase: Silica gel GF-254
Mobile Phase: Toulene: ethyl acetate: Formic Acid (8.5:1:0.5).
Detection: UV-366
Solvent front: 5
Spot detection: 2
TLC of Methanolic extract TLC of Aqueous extract
Figure No 5:- Thin layer chromatography of Methanolic and Aqueous extracts
Table No.5:- TLC Profile of steroids
Extract Observation Rf values
No. of spots Colour of spots
Methanolic 1 Yellow 0.44
Aqueous 1 Yellow 0.34
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Table No.6:- IR Spectral peaks of Aqueous extract of Tridax procumbens
Peak Observed Assignment Absorption Expected (cm-1
)
763 -Cl 785-540
1051.2 Aryl or Vinyl Ether 1300-1000
1409.96 Nitro Compound 1330-1540
1276.88 -F 1400-1000
1317.38 -S=O 1375-1300
1564.27 -C=C- 1400-1600
3116.97 -CH 3150-3050
3375.43 -OH 3400-3200
1276.88 Amine 1180-1360
Figure No. 6 – IR Spectra of Aqueous extract of Tridax procumbens
Table No.7:- IR Spectral peaks of Methanolic extract of Tridax procumbens
Peak Observed Assignment Absorption Expected (cm-1
)
599 Br/I Less Than 667
1047 Aryl or Vinyl ether 1300-1000
1219.011575 -F 1400-1000
1575 -C=C- 1400-1600
3126 -CH 3150-3050
3360 -OH 3400-3200
2686.84 Aldehydes 2650-2880
686.66 Alkenes 600-1500
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Figure No. 7 - IR Spectra of Methanolic extract of Tridax procumbens
DISCUSSION:-
1. The herbs were collected from Satara district, Maharashtra region and authenticated. The
herbs were subjected for Pharmacognostic investigation which includes determination of
physical constants such as ash value, extractive value determination and fluorescence
analysis. The powder of herbs shows fluorescence at 254 and 366 nm.
2. Macroscopic and microscopic characteristics of the leaf were studied. The microscopic
study shows that it contains midrib and lamina portion. The lamina shows upper and lower
epidermis, spongy parenchyma, palisade cell layer while midrib portion shows upper and
lower epidermis, collenchyma, vascular bundles, etc., Powder characteristics shows
presence of anomocytic stomata and covering trichomes.
3. The leaves of herbs were subjected to successive extraction by using methanol and water
and these extracts were subjected to phytochemical investigation.
4. Phytochemical investigation of extracts of Tridax procumbens, shows that Aqueous extract
contains sterols, glycosides, carbohydrates, alkaloids. While Methanolic extract shows
presence of sterols, flavonoids, glycosides, carbohydrate, alkaloids and tannins.
5. Chromatographic study of the extracts was carried out. Where Thin layer chromatography
were carried out by using mobile phase Toulene: Ethyl acetate: Formic Acid (8.5:1:0.5)
which shows Rf value 0.44 and 0.34 for steroids for methanolic and aqueous extract
respectively.
6. For these isolated compounds of methanolic extract infra red spectroscopy was carried out
which shows that Tridax procumbens contains hydroxyl group, fluorine, alkenes, aldehydes,
bromine, etc. While aqueous extract shows alkenes, amines, nitro-group, Chlorine, etc.
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CONCLUSION:
Tridax procumbens is widely found in India during rainy season. As there is less information
available on pharmacognostical work on leaves hence the morphological study, microscopical
studies, physico-chemical parameters, fluorescence analysis and chemical tests performed will
guide in the proper identification of the plant species as well as help in authentication of the purity
of the plant. All these parameters also help to build up a suitable plant profile.
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