INTRODUCTION:

Medicinal values of plants and herbs are immense and they are recommended for various ailiments. The medicinal plant Eclipta alba (L.) Hassk commonly known as False Daisy, yerba de tago, and bhringraj, is a plant belonging to the family Asteraceae.¹ Root well developed, cylindrical, greyish. It is also named 'kehraj' in Assamese and karisalankanni in Tamil. The whole plant and seeds have great medicinal value. Bhrngaraja is equally useful both, internally as well as externally. In glandular swellings and filariasis, it is used to mitigate swelling and pain, by applying the paste. The chronic and infected wounds get cleansed and heal better with application of its paste². It also alleviates the dispigmentation of the skin and renders normal colour to the skin. The fresh fuice of the plant is instilled in the ears and eyes in otitis and conjunctivitis to ameliorate pain. The massage with its fresh juice is effective to alleviate headache. The nasal drops of its juice, mixed with milk, are beneficial in migraine. The medicated oils of bhrngaraja are widely used as hair tonic and to prevent hairfall and prematre graying of the hair. Bhrngaraja is the most common ingredient incorporated in numerous market preparations of various hair oils³. Internaly, bhrngaraja is useful in many diseases. It is keen stimulant to digestive system. It augments the appetite and improves digestion. It is an effective cholegouge, hence benevolent in hepatosplenomegaly as well as hepatitis⁴.

The fresh juice of the plant (15-30ml) given along with castor oil curbs the tendency for getteing repeated worm infestations. Bhrngarajaas an adjunct relieves mucous and facilitates free breathing in asthma and cough. It is one of the best blood purifiers, stimulates the liver and alleviates the general oedema all over the body. It also augments the haemoglobin percentage in anaemia. The fresh juice of plant with marica (Pipernigrum) fruit powder is recommended in the treatment of anasarca and anaemia. Bhrngaraja augments the acuity of vision and is salutary in night blindness. The seeds are commonly used as aphrodisiac in sexual debility⁵.

EXPERIMENTAL:

Plant material

Plant sample of Eclipta alba was collected from peshawar. The plant was dried under shade and powdered. The plant powder and the extracts of the powder in various solvents were examined under ordinary light and in UV-light (365 nm). The fluorescence characters were determined according to the methods of Chase and Pratt.⁶ The percentage of loss of weight on drying, total ash, water-soluble ash, acid-insoluble ash, and residue on ignition were determined by employing standard methods of analysis as described in pharmacopoeia of India (1966)⁷. The percentage of extractive values of the leaf powder in various solvent systems was also determined.

Preliminary phytochemical analysis of the extracts of the plant powder in various solvents has been performed^8,9. The various extracts of the powder of Eclipta alba were subjected to thin-layer chromatographic studies using “Silica gel-G for TLC”. The silica gel coated plates were dried and activated at 110ºC. UV-fluorescence lamp (365 nm) and iodine chamber was used to find out the spots and the Rf values were calculated. The water extract of the plant powder was subjected to thin layer chromatography studies using using n-butanol: acetic acid: water solvent system (4.0: 1.1:4.9).

Phytochemical screening

Phytochemical screening was performed using standard procedures.

Test for reducing sugars (Fehling’s test)

The aqueous ethanol extract (0.5ml in 5 ml of water) was added to boiling Fehling’s solution (A and B) in a test tube. The solution was observed for a colour reaction.

Test for anthraquinones

0.5ml of the extract was boiled with 10 ml of sulphuric acid (H₂SO₄) and filtered while hot. The filtrate was shaken with 5 ml of chloroform. The chloroform layer was pipette into another test tube and 1 ml of dilute ammonia was added. The resulting solution was observed for colour changes.

Test for terpenoids (Salkowski test)

To 0.5ml each of the extract was added 2 ml of chloroform. Concentrated H2S04 (3 ml) was carefully added to form a layer. A reddish brown colouration of the interface indicates the presence of terpenoids.

Test for flavonoids

Three methods were used to test for flavonoids. First, dilute ammonia (5 ml) was added to a portion of an aqueous filtrate of the extract. Concentrated sulphuric acid (1 ml) was added. A yellow colouration that disappear on standing indicates the presence of flavonoids. Second, a few drops of 1% aluminium solution were added to a portion of the filtrate. A yellow colouration indicates the presence of flavonoids. Third, a portion of the extract was heated with 10 ml of ethyl acetateover a steam bath for 3 min. The mixture was filtered and 4 ml of the filtrate was shaken with 1 ml of dilute ammonia solution. A yellow colouration indicates the presence of flavonoids.

Test for cardiac glycosides (Keller-Killianitest)

To 0.5ml of extract diluted to 5 ml in water was added 2 ml of glacial acetic acid containing one drop of ferric chloride solution. This was underlayed with 1 ml of concentrated sulphuric acid. A brown ring at the interface indicated the presence of a deoxysugar characteristic of cardenolides. A violet ring may appear below the brown ring, while in the acetic acid layer a greenish ring may form just above the brown ring and gradually spread throughout this layer.

Test for saponins

To 0.5ml of extract was added 5 ml of distilled water in a test tube. The solution was shaken vigorously and observed for a stable persistent froth. The frothing was mixed with 3 drops of olive oil and shaken vigorously after which it was observed for the formation of an emulsion.

Test for tannins

About 0.5 ml of the extract was boiled in 10 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 colouration

Test for alkaloids

0.5 ml of extract was diluted to 10 ml with acid alcohol, boiled and filtered. To 5 ml of the filtrate was added 2 ml of dilute ammonia. 5 ml of chloroform was added and shaken gently to extract the alkaloidal base. The chloroform layer was extracted with 10 ml of acetic acid. This was divided into two portions.

Mayer’s reagent was added to one portion and Draggendorff’s reagent to theother. The formation of a cream (with Mayer’s reagent) or reddish brown precipitate (with Draggendorff’s reagent) was regarded as positive for the presence of alkaloids.

RESULTS AND DISCUSSION:

Fluorescence Analysis

The plant powder of Eclipta alba and the extracts of the powder in various solvents were examined under ordinary light and UV light (365 nm). The powder was also treated with various chemical reagents and the changes in colour were recorded. These results are presented in Table 1.

Table.1 Fluorescence characters of Eclipta alba powder and their extracts in different solvents.

S.No	Particulars of treatment	Under ordinary light	Under UV light
1	Powder as such	Dark green	Green
2	Powder + 1N NaOH (aqueous)	Dark brown	Dark green
3	Powder + 1N NaOH (ethanol)	Yellow green	Dark green
4	Powder + 1N HCI	White	Light green
5	Powder + H2 SO4 (1:1)	Black	Black
6	Powder + HNO3 (1:1)	Light brown	Light green
7	Extracts
a)	Petroleum ether	Light green	Green
b)	Benzene	Light green	Green
c)	Chloroform	Light green	Dark green
d)	Ethanol	Dark green	Green
e)	Water	Light yellow	Light green

Quantitative determination

The percentage of loss of weight on drying, total ash, water-soluble ash, acid insoluble-ash and residue on ignition were obtained by employing standard methods of analysis as described in pharmacopoeia of India⁷. The percentage of extractive values in petroleum ether, benzene, chloroform, ethanol and water were also determined. The results are presented in Table 2.

Table 2.Physico –chemical characters of the Eclipta alba.

S.No	Particulars	Eclipta alba %
1	Loss of weight on drying	2.4
2	Total ash	16.65
3	Acid – insoluble ash	1.00
4	Water-soluble ash	35.00
5	Residue on ignition	42.63
6	Extractive values
(a)	Petroleum ether	2.80
(b)	Benzene	13.00
(c)	Chloroform	10.80
(d)	Ethanol	9.40
(e)	Water	26.00

Phytochemical screening

5g of the plant powder of Eclipta alba was extracted with petroleum ether, benzene, chloroform, ethanol and water in soxhlet apparatus. The different extracts were tested for the presence of steroids, reducing sugars, cardiac glycosides, terpenoids, alkaloids, anthraquinones, tannins and flavonoids. The phytochemical tests performed and the results obtained are presented in Table 3.

Thin layer chromatography

Thin layer chromatographic studies have been performed for the petroleum ether, benzene, chloroform, ethanol and water extracts of the plant powder of Eclipta alba. The plates were first viewed through UV-fluorescence viewing cabinet (365 nm) before keeping in an Iodine chamber and the Rf values of the fluorescing spots were noted.

Then the plates were developed in the Iodine chamber and Rf values were noted of the various solvent systems tried for thin layer chromatographic studies, there was no common solvent system for all the four different extracts. Different solvent systems have been found to be effective to get the maximum number of spots for the various extracts. The results are presented in Table 4.

Table 3.Preliminary phytochemicalscreening of powder of Eclipta alba

S. No	Name of extract	Test	Result
1	Ethanol extract	Fehling test (for reducing sugars)	Fehling -A = + Fehling -B = +
		Anthraquinones	-
		Terpenoids (salkowski test)	+
		Flavonoids	+
		Saponins	+
		Alkaloids	+
		Tannins	+
		Cardiac glycosides	+
2	Chloroform	Fehling test (for reducing sugars)	Fehling (A) = - Fehling (B) = +
		Anthraquinones	-
		Terpenoids (salkowski test)	+
		Flavonoids	+
		Saponins	-
		Alkaloids	-
		Tannins	-
		Cardiac glycodides	+
3	Benzene	Fehling test (for reducing sugars)	Fehling (A) = - Fehling (B) = +
		Anthraquinones	-
		Terpenoids (salkowski test)	+
		Flavonoids	-
		Saponins	+
		Alkaloids	-
		Tannins	-
		Cardiac glycodides	-
4	Petroleum ether	Fehling test (for reducing sugars)	Fehling (A) = + Fehling (B) = +
		Anthraquinones	-
		Terpenoids (salkowski test)	+
		Flavonoids	-
		Saponins	+
		Alkaloids	-
		Tannins	+
		Cardiac glycodides	-
5	Water	Fehling test (for reducing sugars)	Fehling (A) = + Fehling (B) = +
		Anthraquinones	-
		Terpenoids (salkowski test)	+
		Flavonoids	+
		Saponins	+
		Alkaloids	+
		Tannins	-
		Cardiac glycodides	-

“+” = presence of the compound; “-“= Absence of the compound

Table 4.Thin – layer chromatographic behaviour of the extracts of Eclipta alba

S.No	Name of the extract	Solvent system used	Rf values of the spots
S.No	Name of the extract	Solvent system used	Ordinary light	Under UV light	In iodine chamber
1	Petroleum ether	Benzene :Chloroform (1:1)	0.02	--	--
2	Benzene	Chloroform : Ethanol (9.5:0.5)	0.8, 0.53	--	0.42
3	Chloroform	Chloroform : Ethanol (9.5:0.5)	0.82, 0.53	--	0.4
4	Ethanol	Chloroform : Ethanol (8:2)	--	--	--
5	Water	1 – Butanol : Acetic acid : Water (4.0:1.1:4.9)	--	--	--