COLUMN CHROMATOGRAPHIC SEPARATION OF BIOACTIVE COMPOUNDS FROM TAGETES ERECTA LINN.

COLUMN CHROMATOGRAPHIC SEPARATION OF BIOACTIVE COMPOUNDS FROM TAGETES ERECTA LINN.

1. 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

Phytochemicals, Therapeutic,

Column Chromatography, Extraction

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

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 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).

TABLE 1:  R_f 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

FIG 4: IDENTIFICATION OF SPOTS FRACTION UNDER VISIBLE LIGHT AT 512 nm

 TABLE 2: PHYTOCHEMICAL ANALYSIS OF COLUMN FRACTIONS OF TAGETES ERECTA LINN.    

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.

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