COLUMN CHROMATOGRAPHIC SEPARATION OF BIOACTIVE COMPOUNDS FROM TAGETES ERECTA LINN.

Abstract

Traditional medicines in many developing countries rely on the plant phytochemicals as a source of drug in curing many diseases. Tagetes erecta Linn. is an ornamental plant containing valuable therapeutic phytochemicals. In the current investigation, ethanol extraction of the dried flower sample was subjected to column chromatographic separation. Around 185 fractions were separated and recorded with different Rf values. The fractions were analyzed qualitatively and it was proved that the flower sample was rich in tannins, saponins, carbohydrates, alkaloids, quinines, terpenoids, phenols and coumarins.

Full text

Devika and Koilpillai, IJPSR, 2015; Vol. 6(2): 762-766. E-ISSN: 0975-8232; P-ISSN: 2320-5148
International Journal of Pharmaceutical Sciences and Research 762
IJPSR (2015), Vol. 6, Issue 2 (Research Article)
Received on 18 June, 2014; received in revised form, 28 August, 2014; accepted, 13 November, 2014; published 01 February, 2015
COLUMN CHROMATOGRAPHIC SEPARATION OF BIOACTIVE COMPOUNDS FROM
TAGETES ERECTA LINN.
R. Devika* and Justine Koilpillai
Department of Biotechnology, Aarupadai Veedu Institute of Technology, Paiyanoor, Tamil Nadu, India.
ABSTRACT: Traditional medicines in many developing countries
rely on the plant phytochemicals as a source of drug in curing many
diseases. Tagetes erecta Linn. is an ornamental plant containing
valuable therapeutic phytochemicals. In the current investigation,
ethanol extraction of the dried flower sample was subjected to column
chromatographic separation. Around 185 fractions were separated and
recorded with different Rf values. The fractions were analyzed
qualitatively and it was proved that the flower sample was rich in
tannins, saponins, carbohydrates, alkaloids, quinines, terpenoids,
phenols and coumarins.
INTRODUCTION: Since time immemorial, Man
utilized the potent biochemicals from plants and
converted them into marvelous assortments of
industrial use in various sectors like pharmacy,
chemical, dye, pesticides etc 1. The plant based
secondary metabolites are taxonomically distinct 2
and are highly potential and beneficial in the field
of medicine 3. Indian ayurvedic system (basically
plant origin) has been proved to be predominantly
successful in curing many type of diseases 4 and the
plant extract has been used as anti – inflammatory
agent, astringent, aphrodisiac, liver tonic etc. and
used to treat asthma, ulcers, emaciation, insomnia,
senile dementia and bronchitis 5. Plant
phytochemicals can be derived from any part such
as bark, leaves, flowers, roots, fruits, seeds etc.3 in
combination with secondary metabolites which are
taxonomically distinct for any particular plant 2.
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DOI:
10.13040/IJPSR.0975-8232.6(2).762-66
Article can be accessed online on:
www.ijpsr.com
DOI link: http://dx.doi.org/10.13040/IJPSR.0975-8232.6(2).762-66
The naturally occurring phytochemicals includes
alkaloids, flavonoids, tannins, steroids, phenolics,
terpenes, volatile oils etc., which are used in
medicines, dyes, cosmetics, flavors and foods 6.
Many research studies proved that natural
antioxidants from plant sources can effectively
inhibit oxidation of food and reduce the risk of age
dependent diseases 7, 9. Methanolic extracts of
Polyathia longifolia have yielded 20 known and
two new organic compounds which showed highly
cytotoxic properties9 and the ethanolic extraction
separation possessed a potent nitric oxide radical
scavenging activities 10, 11.
The genus Tagetes consists of 56 species 12 which
was originated in North and South America and
widely cultivated in other Asian countries like
Bhutan, China, Nepal, India etc.13 which has a
strong historic evidence for its religious and
therapeutic value 14, in the treatment of hiccups,
dermatitis, athlete’s foot, colitis, wound burns etc
15. These plants are rapid growing annuals with the
height ranging from 6 inches (dwarf plants) to 3 ft.
Bearing large pompon- like 16, floral heads of 4-6
cm diameter having both ray and disc florets
(Blooms are golden, orange, yellow etc.). Since
Keywords:
Phytochemicals, Therapeutic,
Column Chromatography, Extraction
Correspondence to Author:
R. Devika
Research Scholar, Sathyabama
University, Chennai, Tamil Nadu,
India
E-mail: vineethdevika@gmail.com

Devika and Koilpillai, IJPSR, 2015; Vol. 6(2): 762-766. E-ISSN: 0975-8232; P-ISSN: 2320-5148
International Journal of Pharmaceutical Sciences and Research 763
ancient era, various parts of Tagetes has been used
as skin wash dye 17, food additives 18, fodders 19,
pesticides 20. The flower part of the plant are
efficient therapeutic in the treatment of epileptic
fits (Ayuveda), astringent, carminative, scabies etc
21, 22.
MATERIALS AND METHODS:
The ornamental plant, Tagetes erecta Linn. Belongs
to Asteraceae family. The whole plant has been
proved to have high therapeutic value23. The flower
Samples were segregated separately from the plant.
The dried powered flower sample was subjected for
ethanol extraction process. The extracted sample
used for separation of phytochemicals by standard
column chromatographic method.
Isolation methods with Column
Chromatography:
Ethanol extract (20g) was subjected to column
chromatography on silica gel (100 – 200 mesh –
Merck) packed and eluted with mixture of n-
Hexane, chloroform, ethyl acetate, ethanol,
methanol and water of increasing polarity to obtain
fractions.
Twenty grams of the method extract was
chromatographed over silica gel column (100-200
mesh). The admixture was packed on a silica gel
column (Merck, India) and eluted started with
100% hexane and increased with solvent polarity
chloroform, ethyl acetate, ethanol, methanol, and
water in the ratio of 90:10, 80:20, 70:30, 50:50,
30:70, 20:80, 10:90. Ethanol (70:30) gave a
colorless compound and on further purification
with acetone and methanol the isolated compound
yield was 200mg.
Thin Layer Chromatography:
The Thin Layer Chromatography was developed in
Twin through chamber with silica gel 60 F254 pre
coated aluminuim plate of 0.2 mm thickness using
ethyl acetate: methanol (1:1) as the developing
solvent system and Rf value was calculated.
Distance moved by the molecule the spot)
Distance moved by the molecule
(Location by the spot)
Rf Value =
Distance moved by the mobile phase
(Solvent Front)
Visualization was carried out by dipping the plate
in vanillin sulfuric acid (1%) and heat on 105˚c
when the colour of the spot appeared distinctly.
RESULTS AND DISCUSSION: The dried
powdered flower sample of Tagetes erecta linn.
was subjected to ethanol extraction. About 20g of
the extract was subjected to column
chromatography on silica gel (100-200 mesh -
Merck) pack as shown in Fig 1. The selection of
solvents in a systematic order proves the effect of
polarity on the extraction and the extracted
phytochemicals 24. About 185 fractions with
different Rf values were separated during column
chromatography procedure. (Table 1).
FIG 1: COLUMN CHROMATOGRAPHY OF TAGETES
ERECTA LINN. FLOWER EXTRACT
The column fractions of Tagetes erecta Linn.
flower sample was subjected to various qualitative
phytochemical tests like carbohydrates, tannin,
saponin, alkaloid, quinines, glycosides, terpenoids,
phenols and coumarin as per standard procedure 23.
It was evident that the fractions from 1 -58 showed
positive result for alkaloid and terpenoid (Table 2)
and the fractions from 59 – 80, 81-90 showed
positive result for alkaloid, terpenoid and coumarin.
It was also evident that the fractions form 91-165
showed positive result for alkaloids, terpenoids,
phenols and coumarin but the fractions from 126 to
165 had tannins in addition. The fractions from 166
to 185 registered terpenoids and coumarin in their
fractions (Table 2).

Devika and Koilpillai, IJPSR, 2015; Vol. 6(2): 762-766. E-ISSN: 0975-8232; P-ISSN: 2320-5148
International Journal of Pharmaceutical Sciences and Research 764
TABLE 1: Rf VALUES OF FLOWER EXTRACTION FRACTIONS OF TAGETES ERECTA LINN.
FIG 2: IDENTIFICATION OF CLEAR SPOTS FRACTIONS UNDER UV 254 nm
FIG 3: IDENTIFICATION OF SPOTS FRACTION UNDER UV 366nm
Fractions
Rf values
1-25
-
26-45
0.32, 0.46
46-58
0.30, 0.45
59-68
0.42, 0.56
69-72
0.41, 0.62
73-80
0.22, 0.25, 0.31
81-84
0.18, 0.25, 0.32
85-90
0.44, 0.45,
91-95
0.44, 0.45,
96-102
0.14, 0.33, 0.45,
103-108
0.56, 0.57, 0.58, 0.59, 0.66
109-113
0.57, 0.58, 0.60, 0.71,
114-117
0.32, 0.44, 0.50, 0.54,
118-121
0.19, 0.25, 0.44
122-125
0.25, 0.31, 0.38, 0.42
126-130
0.22, 0.32, 0.40, 0.44
131-134
0.20, 0.25, 0.31, 0.76
135-139
0.21, 0.25, 0.32
140-143
0.25, 0.32, 0.36
144-153
0.20, 0.24, 0.35
154-159
0.46, 0.51
160-165
0.44, 0.56
166-170
0.42, 0.69
171-175
0.40, 0.72
176-186
-

Devika and Koilpillai, IJPSR, 2015; Vol. 6(2): 762-766. E-ISSN: 0975-8232; P-ISSN: 2320-5148
International Journal of Pharmaceutical Sciences and Research 765
FIG 4: IDENTIFICATION OF SPOTS FRACTION UNDER VISIBLE LIGHT AT 512 nm
TABLE 2: PHYTOCHEMICAL ANALYSIS OF COLUMN FRACTIONS OF TAGETES ERECTA LINN.
Phytochemical test
Inference of Column Fractions of Tagetes erecta linn.
1-58
59-80
81-90
91–102
103-125
126-139
140-165
166-225
Carbohydrates
-
-
-
-
-
-
-
-
Tannins test
-
-
-
-
-
+
+
-
Saponin test
-
-
-
-
-
-
-
-
Alkaloid test
+
+
+
+
+
+
+
-
Quinones
-
-
-
-
-
-
-
-
Glycosides test
-
-
-
-
-
-
-
-
Cardiac glycosides test
-
-
-
-
-
-
-
-
Terpenoids test
+
+
+
+
+
+
+
+
Triterpenoids
-
-
-
-
-
-
-
-
Phenols
-
-
-
+
+
+
+
-
Coumarins
-
+
+
+
+
+
+
+
CONCLUSION: From the above investigation it is
evident that Tagetes erecta Linn. Flower sample is
rich in pytochemicals having high therapeutic
value. On futhert isolation and purification of these
bioactive compounds will pave away for discovery
of new effective drug for curing various diseases in
the future.
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How to cite this article:
Devika R and Koilpillai J: Column Chromatographic Separation of Bioactive Compounds from Tagetes Erecta Linn.. Int J Pharm Sci Res
2015; 6(2): 762-66.doi: 10.13040/IJPSR.0975-8232.6 (2).762-66.