Phytochemical Evaluation of Extracts of Stem of Eclipta alba (Bhringaraja)

Agarwal A*¹, Gupta V², Gupta Amita² and Jayalakshmi S¹

¹Department of Chemistry, College of Pharmacy, IFTM, Moradabad, (U.P.), India

²School of Pharmaceutical Sciences, Jaipur National University, Jagatpura, Jaipur, (Rajasthan), India- 302025

ABSTRACT

This paper deals with the detailed phytochemical evaluation of the stem of Eclipta alba (Bhringaraja) family: (Compositae). The preliminary phytochemical screening shows that different extracts of plant have different constituents like carbohydrate, alkaloids, phenolics, steroids, glycosides, fat and oils. TLC of the hexane extract after column extraction was shown R_f value 0.78 in Petroleum ether: Hexane: Chloroform: Methanol, used as mobile phase in 2:4:2:1 ratio and TLC of pet. ether extract also shown R_f value is 0.72 in Petroleum ether: Hexane: Chloroform, used as mobile phase in 4:3:1 ratio after column extraction. This is evaluated by the instrumental analysis (UV, IR, and NMR) of hexane and pet. Ether extract. They shows consequently that pet. ether extract contains alkane (2940 Cm^-1, d 0.827), alkene (1630 Cm^-1, d 1.253- 1.351), monomer of aliphatic acid (1760 Cm^-1 , d 2.169), aromatic amine (1570 Cm^-1, d 2.593) etc. and hexane extract contains alkane (2940 cm^-1,d 0.801-0.873) , alkene (1625 Cm^-1, d 0.988-1.075 ) aldehyde (1740 Cm^-1, d 2.154 – 2.288) and aromatic alcohol (1110Cm^-1, d 5.324). These data suggests that the extracts (pet. ether and hexane) may have the glycoside, aliphatic acid, steroids, fats and oils. They are pharmacologically important.

KEY WORDS: Eclipta alba, flavanoids, glycosides, thin layer chromatography.

INTRODUCTION

The Sanskrit word Bhringaraja literally means that which bestows hair the splendid colour like that of a humming bee. It has numerous synonyms describing its properties of promoting the growth of hair and making them black and lustrous. The botanical name of bhringaraja is Eclipta alba. The yellow bhringaraja is Wedelia calendulaceae. Bhringaraja belongs to family Compositae ^(1-2). It grows commonly in moist places as a shrub in the tropical and sub tropical regions of all over the world. Eclipta alba is an erect and prostate, much branched, strigosely hirsute, annual, often rooting at the nodes. Leave opposite; sessile; oblong-lanceolate; shallow serrate, acuminate. Stem single from base but with many spreading branches, herbaceous, erect or ascending. Flower white, pappus of 2.5 minute teeth. Root cylindrical, grayish, a number of secondary branches arise from main root. Seed 0.1 cm wide, dark brown, hairy and non-endospermic ⁽³⁾.

The plant is bitter, acrid, thermogenic, alterative, anti-inflammatory, anthelmintic, anodyne, vulnerary, ophthalmic, digestive, carminative, emetic, haematinic, diuretic, aphrodisiac, anodyne, hair tonic, deobstruent, absorbent, depurative, tonic and febrifuge. It in useful in hepato-splenomegaly and its associated disorders, elephantiasis, inflammations, gastric disorder, anorexia, worm infestation, skin disease, wounds, ulcers, ophthalmic disorders, headache, hypertension, strangury, leprosy, pruritus, fever, jaundice, toothache, earache. It is good for blacking and strengthening hairs and stopping hemorrhages and fluxes and for strengthening gums. Seeds are used as aphrodisiac ⁽⁴⁾.

Fig. 1: UV/Visible absorption spectra of compound separated from pet.ether extract of stem of Eclipta alba by column chromatography, which show many absorptin peak at particular wave length

Fig.2:IRspectra of compound separated from pet.ether extract of stem of Eclipta alba by column chromatography

The herb Eclipta alba contains mainly coumestans i.e. wedelolactone (I) and demethylwedelolactone (II) ⁽⁵⁾, polypeptides, thiophene derivative, amino acids ⁽⁴⁾, steroids, glycoside ⁽⁶⁾, triterpene saponins ⁽⁷⁾, and flavanoids ⁽⁸⁾.

In spite of this plant have numerous chemical constituents which have many pharmacological activity and medicinal uses but phytochemical evaluation of stem of this plant has not been published. Hence the present investigation is an attempt in this direction and includes preliminary phytochemical screening of different extract, column chromatography and TLC of pet ether and hexane extract , and analytical evaluation of chemical constituents of pet.ether and hexane extract of stem of Eclipta alba.

Fig. 3a: ¹H NMR spectra of compound separated from pet.ether extract of stem of Eclipta alba by column chromatography

Fig. 3b: ¹H NMR spectra of compound separated from pet.ether extract of stem of Eclipta alba by column chromatography

MATERIALS AND METHOD:

Experimental site:

The study was conducted in the College of Pharmacy, IFTM (Institute of Forign Trade and Management), Moradabad, Uttar Pradesh Technical University, Lakhnow, Uttar Pradesh, India. NMR was done in Department of Chemistry, Punjab Technical University, Chandigarh.

Plant material:

The plant Eclipta alba was collected from local area of Lodhipur, Moradabad, Uttar Pradesh on August 2006 and was authenticated by the Dr. V.K. Lal, Deputy Director (Ministry of Health and Family Walfare , Dept. of Ayush , Govt. of India ), New Delhi and specimen has been deposited in the Herbarium of College of Pharmacy, IFTM, Moradabad (U.P.), India. The stem of plant Eclipta alba cut in to pieces and shade dried and were subjected to size reduction to a course powder by using dry grinder.

Table 1: Preliminary phytochemical screening of stem extracts of Eclipta alba

Test	Petroleum ether	Hexane	Chloroform	Methanol	Water
Carbohydrate Steroid Glycosides Alkaloids Phenolics Fat and oils	- + - - - +	- + + - - +	+ + + + + -	+ + - + + -	- + - + + -

+ Denotes the presence of respective class of compounds.

Table 2: Interpretation of different wave no. of IR Spectra of compound separated from pet. ether extract by column chromatography

Wave no. (cm^-1)	Vibration Type	Interpretation
2940 2850 1460	C-H Stretching C-H Stretching C-H Bending	Alkane
1630 780	C=C Streching C-H Bending	Conjugated Alkene
1570 1335 780	N-H Bending C-H Streching OOP N-H Bending	Primary Aromatic Amine
2850 1760 1420	C-H Streching C=O Streching C-O Bending	Monomer of Aliphatic Acid
780 425	OOP Bending OOP Ring Bending	Substituted Aromatic Ring

Fig. 4: UV/Visible absorption spectra of compound separated from hexane extract of stem of Eclipta alba by column chromatography, which show many absorptin peak at particular wave length

Chemical & instrument:

Test tube, beaker, soxhlet apparatus, water bath, glass plate of 10 cm long and 2 cm wide, column and other common glassware were used. For the phytochemical evaluation double beam UV-VIS Spectrophotometer of Ecil UV 5704 SS, IR instrument of Shimadzu and NMR instrument of Bruker AVANCE II 400 NMR Spectrometer were used. Solvents viz. pet ether, hexane, chloroform, ethanol, methanol and n-butanol were obtained from local company of India and they were of high purity and analytical grade. Some reagent / chemical viz. silica gel. G (120) and sodium hydroxide were procured from Ranbaxy Fine Chemicals Ltd. Mumbai. India.

Fig. 5: IR spectra of compound separated from hexane extract of stem of Eclipta alba by column chromatography

Preparation of Extract:

The preparation of various extract was done by using different solvent of increasing polarity. About 500 gm of dried powder was extracted with petroleum ether at 25-30⁰C by continuous percolation by using soxhlet apparatus. The extraction was continuing for 72 hrs. The petroleum ether extract was filtered and concentrated to get a dry mass. A dark green waxy residue was obtained. The mark left after petroleum ether extract was taken and it was again extracted with hexane up to 72 hrs in soxhlet apparatus. The hexane extract was filtered and concentrated to get dry mass. The mark left after hexane extract was taken and it again extracted with chloroform up to 72 hr in soxhlet apparatus. The chloroform extract was filtered and concentrated to get a dry mass. The mark left after chloroform extraction was taken and it was again extracted with methanol at 30⁰C up to 72 hr. in soxhlet apparatus. The methanol extract was filtered and concentrated to get a dry mass.

Table 3: Interpretation of chemical shift (d) of ¹H NMR Spectra of compound separated from pet. ether extract by column chromatography

Chemical Shift (d)	Interpretation
d 0.827 d 1.253- 1.351 d 2.033 d 2.593 d 2.169 d 2.253 d 7.55	CH3CH2R Alkane -CH2-C-C=C Alkene -CH2-C=C Alkene CH3-NH2 Amine CH3-CO-OH Aliphatic Acid CH3-Ar Substituted Aromatic Ring Aromatic Proton

Table 4: Interpretation of different wave no. of IR Spectra of compound separated from hexane extract by column chromatography

Wave no.(cm^-1)	Vibration Type	Interpretation
2940 Cm^-1 2840 Cm^-1 1455 Cm^-1	C-H Stretching C-H Stretching C-H Bending	Alkane
1625 Cm^-1 840 Cm^-1 660 Cm^-1	C=C Stretching C-H Bending C-H Bending	Conjugated Alkene
2840 Cm^-1 1740 Cm^-1	C-H Stretching C=O Stretching	Aldehyde
1410 Cm^-1 1330 Cm^-1 1110 Cm^-1	O-H In plane Bending O-H In plane Bending C-O Stretching	Aromatic Alcohol
840 Cm^-1 660 Cm^-1 510 Cm^-1	oop Bending oop Ring Bending oop Ring Bending	Substituted Aromatic Ring

The mark left after the methanol extraction was taken and extracted with distilled water in the same manner and aqueous extract as filtered and concentrate to get dark brown residue was obtained.

Preliminary phytochemical screening of extracts of the stem of Eclipta alba:

Preliminary phytochemical screening of pet. ether, hexane , chloroform , aqueous and methanol extract of stem were carried out by using standard procedures described by Kokate (1986b) ⁽⁹⁾ Harborne (1998) ⁽¹⁰⁾, Agrawal O.P (2000) ⁽¹¹⁾.

Chromatographic studies:

Pet. ether extract:

A solid mass of petroleum ether extract was taken and dissolve in petroleum ether and adsorb it on silica gel 120 and dry it in oven below 40⁰C. This material was fitted in column for isolation of pure compound. Silica gel 120 was used as stationary phase. Petroleum ether and hexane in 2:1 ratio was taken as mobile phase.

TLC study was performed of the column extract for identification and authentication of pure compound. Dry column extract was dissolved in the methanol and apply on TLC plate where silica gel G used as stationary phase and petroleum ether: Hexane: chloroform used as mobile phase in 4:3:1 ratio.

Hexane Extract:

Solid mass of hexane extract was taken and dissolved in hexane and adsorb it on silica gel 120 and dry it in oven below 40⁰C. This material was fitted in column for isolation of pure compounds. Silica gel G 120 in use as stationary phase. Hexane and chloroform in 3:1 ratio was taken as mobile phase.

Fig. 6a: ¹H NMR spectra of compound separated from hexane extract of stem of Eclipta alba by column chromatography

Then solid mass of column extract was dissolved in methanol and apply on TLC plate where silica gel G used as stationary phase and Petroleum ether: Hexane: Chloroform: Methanol used as mobile phase in 2:4:2:1 ratio. And check in the UV range fluorescence.

Phytochemical evaluation by instrumentation:

Phytochemical analysis of pet. ether and hexane extract were performed. Solid mass of the pet. Ether and hexane extract were dissolved as sequence in pet. ether and hexane. Different dilutions were prepared separately for recording the wavelength and absorbance of compounds by the UV spectrophotometry. Then IR was performed by using dried KBr (analytical grade) plates for both extracts. NMR was performed by dissolving the extracts in the DMSO (Di-Methyl Sulphoxide) separately.

RESULT AND DISCUSSION:

Preliminary phytochemical screening:

The result of phytochemical analysis of different extracts of stem of Eclipta alba were shown that pet. ether extract contains only steroids, fat and oils. In addition Hexane extract contains glycosides. And aqueous extract contains steroids, alkaloids and phenolics. Ethanol extract contains carbohydrate, phenolics, steroids and alkaloids. In addition Chloroform extract contains glycosides (Table 1). Other extracts (leaf, root and whole plant) of different parts of Eclipta alba also have this type of constituent so due to these constituents many pharmacological activity are reported. They are, hepatoprotective ^{(12, 13)}, antiviral ⁽¹⁴⁾, anti-bacterial ⁽¹⁵⁾, anti-inflammatory ⁽⁷⁾, neuropharmacological activity ⁽¹⁶⁾, analgesic or anti-noceceptive ^(7,17), anti-hyperglycemic ⁽¹⁸⁾, antifatigue ⁽¹⁹⁾, antibiotic activity ⁽²⁰⁾, antifungal ^(21,22), bronchodialatory ⁽⁷⁾ promoter of blackning and growth of hair ⁽²³⁾, Diuretic, hypotensive, and hypocholesterolemic ⁽²⁴⁾, immunostimulatory ^(25,26).

Fig. 6b: ¹H NMR spectra of compound separated from hexane extract of stem of Eclipta alba by column chromatography

Chromatographic studies:

The pet.ether extract dissolves in pet. ether and after repeated column chromatography over silica gel afforded one pure brown coloured compound which was crystal out. Then for authentication pure substance, was applied on the silica gel G plate then one spot only obtained which have R_f value 0.78 and M.P. was 210⁰C.

The hexane extract dissolves in hexane and after repeated column chromatography over silica gel afforded one pure greenish brown coloured compound which was crystal out. Then for authentication pure substance was applied on the silica gel G plate then one spot only obtained which have R_f value 0.72 and M.P. was 180⁰C.

Phytochemical evaluation by instrumentation:

The UV of brown coloured compound (separated from pet. ether extract) was performed which have highest peak at 573.5 nm and absorbance 0.205 and lowest peak at 481.5 nm and absorbance was 0.027(Fig. 1). Then IR of this compound was performed which showed different absorption band for Alkane (C-H stretching 2940 cm^-1, C-H stretching 2850 cm^-1, C-H bending 1460 cm), Conjugate alkene (C=C stretching1630 cm^-1, C-H bending 780 cm^-1), Primary aromatic amine (N-H bending 1570 cm^-1, C-N stretching 1335 cm^-1, OOP bending 780 cm^-1), Monomer of aliphatic acid (C-H stretching 2850 cm^-1, C=O stretching 1760 cm^-1, C-O bending 1420cm^-1), Substituted aromatic ring (bending 780 cm^-1, ring bending 425 cm^-1) (Table 2) (Fig. 2). NMR also proves that the compound have Alkane CH_3-CH_2-R (d 0.827), Alkene -CH₂-C-C=C (d 1.253- 1.351), Alkene -CH₂-C=C (d 2.033), Amine CH₃-NH₂ (d 2.593), Aliphatic Acid CH₃-CO-OH (d 2.169), Substituted Aromatic Ring CH₃-Ar (d 2.253), Aromatic Proton (d 7.55) (Table 3) (Fig. 3a & 3b).

The UV of greenish brown coloured compound (separated from hexane extract) was performed which have highest peak at 574 nm and absorbance 0.340 and lowest peak at 453 nm and absorbance was 0.044 (Fig. 4). Then IR of this compound was performed which showed different absorption band for Alkane (C-H stretching 2940 cm^-1,C-H stretching 2840 cm^-1, C-H bending 1455 cm), conjugate Alkene (C=C stretching 1625 cm^-1, C-H bending 840 cm^-1, C-H bending 660 cm^-1), Aldehyde (C-H stretching 2840 cm^-1, C=O stretching 1740 cm^-1), Aromatic alcohol (O-H in plane bending 1410 cm^-1, O-H in plane bending 1330 cm^-1, C-O stretching 1110 cm^-1), Substituted aromatic ring (oop bending 840 cm^-1, oop ring bending 660 cm^-1, oop ring bending 510 cm^-1 ) (Table 4) (Fig. 5). NMR also proves that the compound have Alkane CH₃-CH₂-R (d 0.801-0.873), Alkene -CH₂-C-C=C (d 0.988-1.075), Aldehyde -C-CHO (d 2.154 – 2.288), Phenol (Aromatic Alcohol) HO-Ar (d 5.324), Aromatic Proton (d 7.79) (Table 5) (Fig. 6a & 6b).

Table 5: Interpretation of chemical shift (d) of ¹H NMR Spectra of compound separated from hexane extract by column chromatography

Chemical Shift (d)	Interpretation
d 0.801-0.873 d 0.988-1.075 d 2.154 – 2.288 d 5.324 d 7.79	CH3CH2R Alkane -CH2-C-C=C Alkene -C-CHO Aldehyde HO-Ar Phenol (Aromatic Alcohol) Aromatic Proton

The interest in medicinal and aromatic plants has been shown all over the world because of safe and effective constituents of plants products and in particularly the presence of active principle of medicinal plants. The result of present study indicate the presence of many pharmaceutical constituents in various extracts which may have the various pharmacological activities and evaluation of the some pharmaceutical active constituents of some extracts like pet. ether and hexane extracts by instrumentation (TLC, Column Chromatography ,UV ,IR and NMR).

CONCLUSION:

The result of present study has provided supportive scientific evidence that the stem extract of Eclipta alba possess some chemical principles that may be pharmacologically important and evaluation of constituents of some extracts by instrumentation analysis.

ACKNOWLEDGEMENT:

The authors are grateful to Dr. A.K Wahi (Dean), College of Pharmacy, IFTM, Moradabad, Uttar Pradesh.

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Received on 07.08.2009

Accepted on 10.10.2009

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Research J. Pharmacology and Pharmacodynamics 1(3) Nov - Dec. 2009; 134-139