Compounds Identification Using HPLC and FTIR In Eclipta alba And Emilia sonchifolia

Compounds Identification Using HPLC and

FTIR In Eclipta alba And Emilia

sonchifolia

M.Thenmozhi*

Department of Biotechnology, Karpagam University, Coimbatore-641021, Tamilnadu, India

P.K.Bhavya

Department of Biotechnology, Karpagam University, Coimbatore-641021, Tamilnadu, India

Rajeshwari Sivaraj

Department of Biotechnology, Karpagam University, Coimbatore-641021, Tamilnadu, India

ABSTRACT

Engineering modern techniques are used in various fields like drug and medicine. Many plants synthesize substances that are useful to the maintenance of health in humans and other animals. The use of plants as medicines predates written human history. Medicinal plants are in all way having its own immense importance in current era. All plants produce chemical compounds as part of their normal metabolic activities. These metabolic activities can be measured using HPLC, FTIR etc., The present study reports the phytochemicals present in methanolic extract of Eclipta alba and Emilia sonchifolia, the identification of these compounds using HPLC and FTIR. The results revealed the presence of a well number of phytochemicals in them responsible for antimicrobial, antioxidant and curing activities against several diseases.

Keywords: HPLC, FTIR, phytochemicals, Eclipta alba, Emilia sonchifolia, compounds

1. INTRODUCTION

Biotechnology is a field of applied biology that involves the use of living things in engineering, technology, medicine, and other useful applications. By comparison to biotechnology, bioengineering is generally thought of as a related field with its emphasis more on higher system approaches (not necessarily altering or using biological materials directly) for interfacing with and utilizing living things. Medicinal plants are nature’s priceless gift to human. The World Health Organization (WHO) estimates that 80 percent of the world's population presently uses herbal medicine for some aspect of primary health care. The development in the field of modern medicine temporarily subdued the traditional herbal medicine. But it has now stayed a comeback and an “herbal renaissance” is blooming across the world. Green medicines are healthier, safer and harmless than synthetic ones.

The acceptance of traditional medicine as an alternative form of health care and the development of microbial resistance to the available antibiotics has lead researchers to investigate the antimicrobial activity of plant extracts[1]. In recent years there has been renewed interest in natural medicines that are obtained from plant parts or plant extracts. On the order of 40% or more of the pharmaceuticals currently used in Western countries are derived or at least partially derived from natural sources. India has a rich tradition of plant-based knowledge on healthcare. Ayurveda, the indigenous system of Indian medicine, describes thousands of plant species in detail. With its varied climatic zones, India has a rich diversity of medicinal herbs. The forests harbour a large number of plant species, but deforestation has been responsible for the rapid loss of medicinal plant wealth, such that many valuable medicinal plants are under the threat of extinction. Research in the area of plant tissue culture technology has resulted in the production of many pharmaceutical substances for new therapeutics. Advances in the area of cell cultures for the production of medicinal compounds has made possible the production of a wide variety of pharmaceuticals like alkaloids, terpenoids, steroids, saponins, phenolics, flavanoids, and amino acids[2].

those of the more studied microbial sources, and therefore their mode of action may too very likely different. There is growing interest in correlating the phytochemical constituents of a medicinal plant with its pharmacological activity. Screening active compounds from plants has lead to the discovery of new medicinal drugs which have efficient protection and treatment roles against various diseases, including cancer and Alzheimer’s disease [5].

Eclipta alba (L.) Hassk. (Asteraceae), a small, branched annual herb with white flower heads, is native to the tropical and subtropical regions of the world. It is used as a tonic and diuretic in hepatic and spleen enlargement. It is also used in catarrhal jaundice and for skin diseases [6]. Decoction of the plant is used to invigorate the liver, to prevent premature graying of the hair and to staunch bleeding, especially from the uterus. The plant contains specified compounds like ecliptin and wedelolactone. Emilia sonchifolia (Asteraceae), is a herbaceous plant found in India and in other Asian countries. The plant is used in treatment of tumor, inflammation, cough, rheumatism, cut and wound. In China the plant is used for fever and dysentery. The plant exhibits different properties like antimicrobial, antifungal etc. The samoolam or whole plant is taken fresh and crushed well, juice is extracted and given in the dose of 5 – 10 ml daily for three days to cure intestinal worms. The karkam or the paste of samoolam of the plant is given in the dose of 2 – 5 grams preferably with butter milk for bleeding piles. The decoction of the whole plant is effective for fever [7].

2. MATERIALS AND METHODOLOGY 2.1 Collection of plant materials:

The plants of Eclipta alba and Emilia sonchifolia were collected from different places of Calicut and Malappuram Districts, Kerala. Both the plants were further confirmed at Botanical Survey of India, South circle, Coimbatore.

2.2 Preparation of Plant samples:

The live plants collected were washed thoroughly under running tap water and then were rinsed in distilled water, they were allowed to dry for some time. Then these plants were shade dried without any contamination for about 3 to 4 weeks. The dried plant sample was powdered in blender and was stored in airtight containers.

2.2.1 Preparation of Plant extract (Methanolic extract):

50 g of dried powdered samples were soaked in 125 ml of methanol for 16 hours in a rotatory shaker. Whatman No.1 filter paper was used to separate the extract of both plants. The filtrates were used for further phytochemical analysis.

2.3 Preliminary Phytochemical Screening of plant extracts:

Phytochemical screening was performed for the analysis of different phytochemicals like carbohydrates, saponins, oils, fats, flavanoids, terpenoids, alkaloids etc., in methanolic extracts of plant samples. The screening was performed with some modifications from the method of Harborne[8].

2.3.1 Test for Carbohydrates (Fehling’s test):

The extracts were treated with 5.0 ml of Fehling’s solution and kept in boiling water bath. The formation of yellow or red colour precipitate indicates the presence of reducing sugars.

2.3.2 Test for Cardio Glycosides:

A volume of 5 ml of the plant extract was treated with 2 ml of glacial acetic acid containing a drop of ferric chloride solution. Then it was underplayed with 1 ml concentrated sulphuric acid. A brown ring of the interface indicates a deoxy sugar characteristic of cardio glycosides. A violet ring may appear below the ring, while in the acetic acid layer, a greenish ring may form just gradually throughout thin layer.

2.3.3 Test for Saponins:

About 2 g of the powdered sample was boiled in 20 ml of distilled water bath and filtered. The 10 ml of the filtrate was mixed with 5 ml of distilled water and shaken vigorously for a suitable persistent froth. The frothing was mixed with 3 drops of olive oil and shaken vigorously, and then the formation of emulsion was observed.

2.3.4 Test for Fixed oils and fats (Spot test):

2.3.5 Test for Terpenoids:

A volume of 5 ml of the plant extract was mixed in 2 ml of chloroform and concentrated H2SO4 was added to form a layer. A reddish brown coloration of the interface was formed to show the presence of terpenoids.

2.3.6 Test for Alkaloids:

The plant extract was mixed with a few drops of acetic acid followed by Dragendroff’s reagent and mixed well. An orange red precipitate formed indicated the presence of alkaloid.

2.3.7 Test for Steroids and Sterols:

Two ml of acetic anhydride was added to 0.5 g of the plant extract of each sample with 2 ml of H2SO4. The colour change from violet to blue green in the sample indicates the presence of steroids and sterols.

2.3.8 Test for Flavonoids:

Five ml of dilute ammonia solution was added to the aqueous filtrate of the plant extract followed by the addition of concentrated H2SO4. A yellow coloration observed in the extract indicated the presence of flavonoids. The yellow colour disappeared on standing.

2.3.9 Test for Tannins and Phenolic compound:

About 0.5 g of the dried powdered sample was boiled in 20 ml of water in a test tube and then filtered. A few drops of 0.1% ferric chloride was added and observed for brownish green or a blue-black coloration. A few drops of alcohol and ferric chloride solution was mixed with the plant extract. A blue green or red colour indicates the presence of phenol.

2.3.10 Test for Amino Acids and Proteins:

To 1ml extract, 2 drops of freshly prepared 0.2% ninhydrin reagent was added and heated. Blue colour develops indicating the presence of proteins and amino acids.

2.3.11 Test for Quinones:

A few drops of sodium hydroxide was mixed with the plant extract and shaken vigorously. A blue green or red colour indicates the presence of quinones.

2.4 Thin Layer Chromatography (TLC):

The 60 % silica gel (Spectrum) was prepared and was spread evenly in the TLC plate having a 15 mm thickness. Toluene: Acetone: Formic acid (11:6:1 v/v) was used as solvent. Then the plate was marked 1 cm from the bottom and spots were made with the standard and samples. Then the plate was soaked lightly in the bottom with the solvent and was allowed to run until it reaches a 3/4th position. Ninhydrin (3 % ninhydrin in 100 ml butanol containing 3 ml of Acetic acid) was used as the spraying agent and it was sprayed all over the plate and was allowed to dry. The spots were noted and the Rf value was calculated by measuring the distance traveled by the solute and the solvent.

2.5 High Performance Liquid Chromatography (HPLC):

The components present in the whole tissues of Eclipta alba and Emilia sonchifolia were extracted in methanol and analyzed using HPLC, Water’s (USA), Tiger- LC System was used for the analysis. The column used was C 18 Silica. Wave length was 270 nm and the flow rate was 1.0 ml / min. 0.5% Formic acid : Acetonitrile (70 : 30) was used as mobile phase.

2.6 Fourier Transform Infrared Spectrophotometer (FTIR):

The powdered sample of the two plant specimens were treated for FTIR spectroscopy (Shimadzu, IR Affinity 1, Japan). The samples were run at infrared region between 400 nm and 4000 nm. The detector used in the system was Standard DLATGS with a mirror speed of 2.8 mm/sec.

3. RESULTS AND DISCUSSION

3.1 Preliminary Phytochemical Screening:

lead to drug discovery and development. Similar observations for phytochemicals like alkaloids, phenol, Tannin, Fixed oil and fat, flavanoids, saponins, steroids, quinones, cellulose are reported in Acalypha indica, Vitex negundo and Coriandrum sativum [10] .

3.2 Thin Layer Chromatography (TLC):

Thin layer chromatography was performed to confirm the presence of flavanoids in both plants. Purple to violet colour spots were identified when viewed under UV light. And the compound was confirmed by calculating the Rf Value. Similar studies also show that the developed plates observed under UV lamp at long wavelength for wedelolactone (flavanoid) (Rf = 0.56) [11]. In the present study the compounds gave a Rf value of 0.56 and 0.557 which may be due to the presence of flavanoids.

Table 1.Preliminary Phytochemical Screening of Eclipta alba and Emilia sonchifolia

S.NO Compound

Eclipta alba

Emilia sonchifolia

1

Carbohydrates

+ +

2

Cardio glycosides

+ +

3

Saponins

+ +

4

Oils and Fats

+ +

5

Terpenoids

_ _

6

Alkaloids

+ +

7

Steroids and Sterols

_ _

8

Flavanoids

+ +

9

Tannins and

Phenolic compounds

+ +

10

Amino acids

+ +

11

Quinones

+ +

+ = Presence, - = Absence

The HPLC results revealed the presence of a good percentage of flavanoid content. The flavanoid content of Eclipta alba was 0.280% (Fig 1) and for Emilia sonchifolia it was 0.147% (Fig 2). This may be responsible for the antimicrobial activity. The qualitative HPLC profile of S. potatorum seeds were detected at a wavelength of 270 nm due to sharpness of the peaks and proper baseline and recorded its retention time (Rt

Fig 1 HPLC analysis of Eclipta alba

Fig 2 HPLC analysis of Emilia sonchifolia

free ligand and the structurally related 1,3-bis(2-benzimidazyl)-2-thiapropane also exhibit infrared bands round 500 cm-1[12]

Fig 3: FTIR spectroscopy for Eclipta alba

Fig 4: FTIR spectroscopy for Emilia sonchifolia

4. Conclusion

comprehensive analytical techniques are needed to acquire a better understanding of the pharmacological basis of the herb and to enhance the product quality control.

5. REFERENCES

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[3] Krisharaju, A, V., Rao T V N, and Sundararaju. 2005. Assessment of bioactivity of Indian medicinal plants using Brine shrimp (Altenaria salania) lethality assay. Int. J. Appl. Sci Engg. 2: 125-134.

[4] Prakash, D., Suri, S., Upadhyay, G and Singh, B.N. 2007. Total phenols, Antioxidant and Free radical scavenging activities of some medicinal plants. International Journal of Food Science and Nutrition 58(1): 18-28.

[5] Soma, R., Kiranmayee, R., Bhuvaneswari., Archana, G and Lakshmi, N.M. 2010. Phytochemical analysis of Andrographis paniculata extract and its antimicrobial activity. World J Microbiol Biotechnology, 26: 85–91.

[6] Anonymous. 1952. The Wealth of India: Raw Materials. CSIR, New Delhi, India.Vol. III:D-E, pp 127.

[7] Chevallier and Andrew. 1996. The Encyclopedia of Medicinal Plants, 1st Ed. (DK Publishing Inc., New York, USA), pp 55-281. [8] Harborne, J.B. 1984. Phytochemical Methods: A Guide to Modern Techniques of Plant Analysis. Chapman and Hall, London, UK. [9] Mallikharjuna, P.B., Rajanna, L.N., Seetharam, Y.N and Sharanabasappa, G.K.2007. phytochemical Studies of Strychnos

potatorum L.f.- A Medicinal Plant. E-Journal of Chemistry, Vol. 4, No. 4: 510-518.

[10] Vijayakumari, B., Yadav, R.H and Nithya, S.V. 2008. Pharmacognostic aspect of Acalypha indica, vitex negundo and Coriandrum sativum. Biosciences, Biotechnology Research Asia, v. 5(1): 269-276

[11] Thorat, R. M., Jadhav, V. M., Kadam, V. J., Kamble, S. S., and Salaskar, K. P.2009. Development of HPTLC method for estimation of Wedelolactone, Quercetin and Jatamansone in Polyherbal Formulation, International Journal of ChemTech Research, Vol.1: No.4: 1079-1086.