133| p a g e international standard serial number (issn): 2319 …ijupbs.com/uploads/12....

133| P a g e International Standard Serial Number (ISSN): 2319-8141

Full Text Available On www.ijupbs.com

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.

134 | P a g e International Standard Serial Number (ISSN): 2319-8141


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,



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).



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.



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.



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



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 - -



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



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



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



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.



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.

REFERENCES:

1. Ganju K, Pathak A.K, Pharmacognostic and Phytochemical Evaluation Of

Tridaxprocumbens Linn, Volume 1 Issue 5, 2013; 42-46.

2. Sutar M., Malvankar K., and Singh S., Pharmacognostical And Phytochemical Investigation

Of Leaves Of A Weed Tridax Procumbens Linn. Vol 5, Issue 1, 2013; 29-33.

3. Vyas P Suresh, Tiwari Umesh, Rastogi Bhawna, and Singh Paramjit, Immunomodulatory

effects of aqueous extract of Tridax procumbens in experimental animals. Journal of

Ethnopharmacologoly, 2004; 92: 113-119.

4. Bhagwat Durgacharan A, Killedar, Suresh G, Adnaik, Rahul S, Anti-diabetic activity of leaf

extract of Tridax procumbens. International Journal of Green Pharmacy, 2008; 2(2): 126-

128.

5. Reddipalli Hemalatha, Anti-hepatotoxic and anti-oxidant defense potential of Tridax

procumbens. Int. J. of Green Pharmacy, 2008; l.2 (3): 164-169.

6. Diwan Prakash V, Iravati Karwande, I. Margaret and Sattur PB, Pharmacology and

Biochemical Evaluation of Tridax procumbens on inflammation. Indian J of Pharmacology,

1989; 21(2): 1-7.

7. Jain Hitesh, Formulation and evaluation of analgesic activity of Tridax procumbens Gel.

Indian J of Nat. Prod, 2006; 23(1): 31-33.

8. Ravikumar V, Kanchi Subramanian Shivashangari, Devaki T, Effect of Tridax procumbens

on liver anti-oxidant defense system during lipopolysaccharide-induced hepatitis in

Dgalactosamine sensitised rats. Molecular and Cellular Biochemistry,2005; 269(1-2): 131-

136.

9. Pathak et al., Fitoterapia, 1991; 62: 307.

10. Chatwal GR, Anand SK. Instrumental methods of chemical analysis, 7th ed., Himalaya

Publishing House. 1992; 588-598.

11. Mukherjee PK, Quality control of herbal drugs, Bussiness Horizon‟s, Pharmaceutical

publisher, New Delhi, 2002, 138-141.



12. Evans WC, Trease and evans, Pharmacognosy, W.B. Saunders, Edinburgh London, New

York Philadelphia, 15th Edition, Pg. No.519-520, 545-547.

13. Johansen DA, Plant microtechniques, McGraw-hill Book Company, New York & London,

1st Ed

n 1940, 182-203.

14. Indian Pharmacopoeia, Government of India, Ministry of Health and Family Welfare,

Controller of Publication, 4th ed, New Delhi, 1996. 4(II), A53-A54.

15. Chase, C.R and Pratt, R.S., Fluorescence of Powdered Vegetable drugs with particular

reference to Development of a System of Identification. J. Am. Pharmacol. Assoc. 1949,Pg.

No.38, 32.

16. Kokoshi, C.J., Kokoshi, R.J and Sharma, F.T., Fluorescence of powdered vegetable

drugsunder Ultraviolet radiation. J. Pharm. Asses.1958; 47: 715-717.

17. Kokate, C.K., Practical Pharmacognosy, 1st Ed, Vallabh Prakashan, New Delhi 1986,1,15-

30.

18. Harborne, J.B., Methods of extraction and isolation. In: Phytochemical methods, 3rd ed,

Chapman and Hall, London 1998, 60-66.

19. Perez C,Paul M, Bazerque P, Antibiotic assay by Agar well diffusion method, Acta Boil

Med Ex, 1990, 15, Pg No. 113-115.

20. William Kemp. Organic Spectroscopy, 3rd ed. 1991; 58-269.

21. Chatwal GR, Anand SK. Spectroscopy (Atomic ad Molecular), Himalaya Publishing House,

5th ed. 2001; 2.29-2.234.

22. Sharma BK. Instrumental method of chemical analysis, 22nd ed., Goel Publishing House,

Merrut. 2002; 180-224.

23. Ergon Stahl. Thin Layer Chromatography, A Laboratory Hand Book, 2nd ed., New York

1990; 52-59.

133| p a g e international standard serial number (issn): 2319 …ijupbs.com/uploads/12....

Documents