Pharmacognostical and Preliminary Phytochemical Screening of Erythrina indica Linn.

V.I. Zalavadiya¹*, V.K. Shah², N.R. Sheth³ and Sumit Chakraborty¹

N.R Vekariya Institute of Pharmacy and Research Center, C.L College Campus, Bilkha Road, Junagadh –362001, Gujarat, India.

²A.P.M.C. College of Pharmaceutical Education and Research, Motipura, College Road, Himmatnager, Gujarat, India.

³Department of Pharmaceutical Sciences, Saurashtra University, Rajkot, Gujarat, India.

ABSTRACT:

Erythrina indica Linn (Leguminosae), a genus of trees or shrubs, is widely distributed in tropical and subtropical regions of India .The barks are used traditionally as astringent, febrifuge and in leprosy and fever. Scientifically reported activities of Erythrina indica are Analgesic, Antibacterial activity, Anthelmintic Activity, Hypoglycaemic Activity and Diuretic activity. The present study involves Pharmacognosy and preliminary phytochemical investigations of the stem bark of Erythrina indica .This study consisted of the morphological and microscopical study of the plant; the phytochemical screening and testing for alkaloids, glycosides, tannins, steroids and flavonoids; TLC study and HPTLC fingerprinting. The parameters from the above were recorded with an objective of drawing an attention on the plant as well as a reference for further scientific investigations.

KEYWORDS: Erythrina indica, Flavanoids, Microscopical, Pharmacognosy.

1. INTRODUCTION:

Latin: Erythrina indica, syn, E. variegata

Erythrina Linn (Leguminosae), a genus of trees or shrubs, rarely herbs, is widely distributed in tropical and subtropical regions. About eight indigenous species and ten introduced ones occur in India.¹ The barks are used traditionally as astringent, febrifuge and in leprosy and fever. Leaves are Anthelmintic, Laxative and Diuretic. Paste of leaves is applied externally to cure inflammations and to relieve pain in the joints. Juice is also used to relieve earache and toothache².During the last three decades, over 50 flavonoids have been obtained from 15 Erythrina species ³, with prenylated flavanones, isoflavones and pterocarpans being the major nonalkaloid secondary metabolites isolated ^{4, 5, 6 .}These compounds are of biological importance as they exhibit various pharmacological activities. Formulations available are Paribhadra taila, Paribhadra Avleha, Paribhadradi kshara, Paribhadradi lepa. Reported activities of Erythrina indica Linn. are Sedative, Cytotoxic, Nitrogen extractability and functional properties, Analgesic Activity, Antibacterial activity, Diuretic activity, Hypoglycaemic Activity, Anthelmintic Activity.

The present study was undertaken for Pharmacognostical and Phytochemical study of the Bark extract of this plant and study of TLC with HPTLC fingerprinting for better separation of chemical compounds.

2. MATERIALS AND METHODS:

2.1 Collection and Authentication of plant:

The Bark of Erythrina indica Linn. Were collected from Rajkot during the month of September- October, 2008. The plant was authenticated by Botanical Survey of India, Jodhpur. Further, the plant was identified by comparing it morphologically and microscopically with the description given in different standard texts and floras.^{7, 8}Fresh bark of plant were cleaned, dried at room temperature and powdered.

2.2 Macroscopic Observation:

The bark was subjected to macroscopic studies which comprised of organoleptic characters of the viz., colour, odour, appearance, taste, smell, texture, fracture, etc.

2.3 Microscopical study:⁸

Free hand sections of the Bark of E. indica were taken and the stained section was mounted. The dried powder of the Bark of E. indica was examined for its microscopic characters after passing through sieve No. 60. The sections and powder were then viewed under low power 10 X and 40 X. The microphotographs were taken using Olympus CH20i microscope attached with Magnus MIPS camera.

2.4 Powder analysis:⁹

The dried powder of the bark of Erythrina indica was examined for its microscopic characters. The powder was passed through sieve No. 60 and observed under the microscope for the microscopical characters.

2.5 Physicochemical constants:⁹^-11

Physicochemical parameters like

Foreign organic matter; Determination of moisture content; Ash values e.g.(a) Total ash (b) Acid insoluble ash (c) Water soluble ash; Extractive values e.g.(a) Hot percolation (b) Cold maceration such as Ethanol soluble extractive, Water-soluble extractive, Ether soluble extractive; Foaming index; Determination of swelling index were checked.

2.6 Phytochemical study:

2.6.1 Preparation of Extract:^{10, 12}

The Powdered plant material was repeatedly extracted in a soxhlet apparatus using different solvents according to increase in polarity, starting from Petroleum ether followed by Benzene, Chloroform, Ethyl acetate, Methanol, Aqueous (Chloroform: water-1:99).The extracts were evaporated and concentrated. The concentrate extracts were used for phytochemical analysis of different extracts.

2.6.2 Phytochemical analysis of different extracts of bark of Erythrina indica Linn: ^{10, 12, 13}

The extract obtained from successive solvent extraction were then subjected to various qualitative chemical tests to determine the presence of various phytoconstituents like alkaloids, glycosides, carbohydrates, phenolics and tannins, phytosterols, fixed oils and fats, proteins and amino acids, flavonoids, saponins, gums and mucilage using reported methods.

2.7 Identification by TLC (Thin Layer Chromatography):⁸

Chromatographic conditions:

· Stationary Phase: Glass plate coated with silica gel G and activated at 110°C

· Solvent system: Toluene: Ethyl acetate (8:2)

· Test solution: Ethanolic, Methanolic, Chloroform and Hydro alcoholic extracts of E. indica.

· TLC estimation: The test samples were applied on TLC plate prepared with silica gel-G (activated) having a thickness of about 0.5mm.The chromatogram was developed in Toluene: Ethyl acetate (8:2). The plate was air dried and sprayed with spraying reagent.

· Detection: After spraying with 10 % Methanolic sulfuric acid followed by heating at 110°C for 5-10 min.

2.8 Development of HPTLC (High Performance Thin Layer Chromatography) finger print profile for Hydro alcoholic extract of Erythrina indica:

· Stationary Phase: 10×10 cm Aluminium Precoated Silica gel 60 F₂₅₄ Plate (0.2 mm thickness) of Merck Pvt. Ltd. (India)

· Solvent system: Toluene: Ethyl acetate (8:2)

· Test solution: Dissolve 100 mg dried Hydro alcoholic extract in 10 ml ethanol.

· HPTLC estimation: The test samples were applied in volumes of 4, 6, 8, 10, 11, 12 and 13 ml on TLC aluminum plates pre coated with silica gel. The chromatogram was developed in Toluene: ethyl acetate (8:2). The plate was air dried and scanned at 254 nm and 366 nm in absorbance mode.

3. RESULT AND DISCUSSION:

Morphology of plant: Fig.1 and 2 shows the morphology of plant.

Erythrina indica Linn. Commonly known as ‘Pandervo’ in Gujarati. A medium sized quick growing Tree attaining 18 m in height armed with dark colored, conical prickles. Bark: Mature dried stem Bark about 0.5-2.0 cm thick, smooth, exfoliating in narrow strips; outer surface yellowish to yellowish grey, lenticels found at short intervals longitudinal lines on the outer surface, yellowish to cream colored. Leaves trifoliolate, 15-30 cm long deciduous; leaflets 10-15 cm long and nearly as broad, rhomboid ovate; Petioles 10-15 cm long, unarmed, readily disarticulating; petiolules 8-13 mm long; stipels thick, roundish, gland like, persistent; stipules lanceolate 1 cm long, very caducous. Fruits pods, torulose, 15-30 cm long, are containing up to 12 seeds; seeds red to dark purple or brown, seeds 4-8, subreniform, 2 by 1 cm. Flowers appearing before the leaves, coral red, in dense racemes, 10-23 cm long, arranged in clusters of 1-3 on a puberulous or tomentose rhachis; peduncles stout, woody, reaching 15 cm long; pedicels 6 mm long; bracts small, triangular, tomentose, deciduous; bracteoles 4 mm long, subulate, tomentose; Calyx (before the expansion of the flower) tubular, 5- toothed at the tip, 2.5-3.2 cm long; corolla bright red, papillionaceous, 5-6.3 cm long.

Microscopy of bark:

Transverse section of Mature Bark Fig 3 Showed Stratified and lignified cork (fig.3a) of about 2-9 or more alternating bands of narrow tangentially elongated compressed cells; secondary cortex consists of large, somewhat tangentially elongated to polygonal, parenchymatous cells (fig.3b), a few cells contains prismatic crystals of calcium oxalate (fig.3c), stone cells (fig.3d) occur in singles or in groups which are circular, elongated or rectangular in shape, parenchymatous cells surrounding stone cells groups, contain large crystals of calcium oxalate; crystal fibres numerous, septate and each chamber contains a single prismatic crystals of calcium oxalate.

Powder Characteristics of Erythrina indica Linn:

Organoleptic evaluation:

Colour: Greenish or yellowish grey

Odour: faint characteristic odor

Taste: Bitter, Astringent and disagreeable taste

Microscopic evaluation of powder in Fig.4 showed cork cells (fig.4a), crystal fibres (fig.4b), prismatic calcium oxalate (fig.4c), phloem parenchyma (fig.4d), lignified fibres (fig.4e).

Table 1.Physicochemical analysis

Parameters	Practical values	Std. Values As Per Ayurvedic Pharmacopoeia
Foreign matter	1.2 %	NMT 2%
Moisture content	3.5 %	−
Ash value: Total ash Water soluble ash Acid insoluble ash	12% 3.5% 1 %	NMT 13 % − NMT 1 %
Extractive value: Hot Percolation: Water soluble extractive Cold maceration: Water soluble extractive Alcohol soluble extractive Ether soluble extractive	18% 15% 9 % 2 %	− NLT 7 % NLT2.5 % _
Foaming Index	<100	−
Swelling Index	4.7 ml	−

NMT: Not more than NLT: Not less than

Sr. No.	Solvent		Colour and Consistency	% Yield of Extract (w/w)
1.	Successive extract	Petroleum ether (60-80⁰)	Brownish green (Sticky)	4.5 %
2.		Benzene	Brownish green (Sticky)	3.5 %
3.		Chloroform	Green (Non-Sticky, dry)	5.05 %
4.		Ethyl acetate	Brown (Shiny, Non-Sticky)	1.35 %
5.		Methanol (95 %)	Orange (Shiny, Non-Sticky)	11.65 %
6.		Water: Chloroform (99:1)	Brown (Non-Sticky)	8.6 %
7.	Hydroalcohol (50 %)		Greenish Brown (Non-Sticky)	21 %

Table 2. Successive solvent extractions:

Table 3. Phytochemical analysis of different extracts of bark of Erythrina indica Linn. (Qualitative Chemical Tests of Successive extracts).

Test	Successive Extracts						Hydro alcohol
Test	PE	Benzene	Chloroform	Ethyl acetate	Methanol	Water	Hydro alcohol
Carbohydrates	-	+	+	+	+	+	+
Protein	-	-	-	-	+	+	+
Terpenoid/ steroid	+	+	+	+	-	+	+
Fats andoils	+	+	+	+	-	+	-
Glycoside	-	+	+	+	+	+	+
Alkaloids	-	-	-	-	+	+	+
Tannins/ Phenolic	-	-	-	-	+	-	+
Flavanoids	-	-	-	-	-	-	+

Table 1 show the results of physicochemical analysis which complies with pharmacopoeial data.

Table 2 shows yields of successive solvent extractions. Here higher yield achieved in alcohol and water extract. So 50% hydro alcoholic extract is performed for extraction and compared in phytochemical screening.

Table 4. Reporting of HPTLC fingerprinting of hydroalcoholic extract of E. indica bark

Peak no.	R_f	Peak Area
1	0.04	23797.2
2	0.19	7976.2
3	0.26	9839.1
4	0.34	11885.6
5	0.43	1800.9
6	0.48	18032.2
7	0.65	18032.2
8	0.77	165.0
9	0.85	1250.6

Table 3 shows results of phytochemical analysis of different extracts. Phytochemical Analysis Shows that Maximum Constituents were found in Hydroalcoholic (50%) extract of Erythrina indica bark.

Primary TLC of Erythrina indica (fig. 5) was done to identify and check major compound in Ethanolic, Methanolic, Chloroform and Hydro alcoholic Extract. That showed maximum constituent present in hydroalcoholic extract, so for that extract HPTLC was performed.

HPTLC fingerprinting of Hydro alcoholic Extract of Erythrina indica Bark is shown in Fig.6 and their Rf values are shown in Table 4.

4. SUMMARY AND CONCLUSION:

The Erythrina indica plant was authenticated by Botanical Survey of India, Jodhpur and also identified by macroscopy and microscopy as described in Ayurvedic Pharmacopoeia. In microscopic observation, Physical parameters were found as per Ayurvedic Pharmacopoeial ranges. Preliminary phytochemical screening of the extracts of Erythrina indica reveals that more yield and maximum constituents presents in Hydroalcoholic extract which showed the presence of Carbohydrate, Protein, Steroid, Glycosides, Alkaloids, Tannins and Flavanoids which may be responsible for their individual pharmacological activities. Primary TLC of Hydro alcoholic extract of Bark of Erythrina indica was done to identify and check major compound in Hydro alcoholic Extract. Also HPTLC supports the TLC profile by showing same nine bands for Hydroalcoholic extract which is useful for further study.

5. REFERENCES:

1) The Wealth of India, a Dictionary of Indian Raw Materials and Industrial Products, C.S.I.R., New Delhi (India), 1952, 3, 195.

2) Haque R., Mohammad S., Achinto S., and Alimuzzaman M.D., Analgesic Activity of Methanolic Extract of the Leaf of Erythrina variegata, J. Pharm. Sci., 2006, 5 , 77-79.

3) Kamat V.S., Chuo F.Y., Kubo I., and Nakanishi K., Anti-microbial agents from an East-African medicinal plant, Erythrina abyssinica, Heterocycles, 1981, 15, 1163–1170.

4) Fomum Z.T., and Ayafor J.F., Erythrina studies: Part 2. Structures of the novel prenylated antibacterial flavanones, sigmoidins A–C from Erythrina sigmoide, J. Chem. Soc., Perkin Trans., 1986, 1, 33–37.

5) Fomum Z.T., Ayafor J.F., and Mbafor J.T., Erythrina studies: Part 1. Novel antibacterial flavanones from Erythrina sigmoidea, Tetrahedron Lett., 1983, 24, 4127– 4130.

6) Fomum Z.T., Nkenfack A.E., and Wandji J., Erythrina studies: Part 15. Pharmacological screening of three esters isolated from Erythrina plants. Ann. Fac. Sci. Chim., 1988, 2, 79– 104.

7) Kirtikar K.R., Basu B.D., Indian medicinal plants, International Book Distributors, Dehradun, 1991, 1, 781-784.

8) The Ayurvedic Pharmacopoeia, Govt. of India, Ministry of health and family welfare, New Delhi, 1^st ed, 2, 133.

9) Brain K.R., and Turner T.D., The practical evaluation of phyto pharmaceuticals, Wright Scientechnica, Bristol, 1975, 4-35.

10) Khandelwal K.R., Pawar A.P., Kokate C.K., and Gokhale S.B., Practical Pharmacognosy, Nirali Prakashan, Pune, 2003, 19-153.

11) World Health Organization Expert Committee, Quality Control Methods for Medicinal Plant Materials, WHO, Geneva, 2002, 9, 22-34.

12) Kokate C.K., In. Practical Pharmacognosy, Vallabha Prakshan, New Delhi, 1986, 107-103.

13) Harborne J.B., In. Phytochemical methods- A guide to modern techniques of plant analysis, Chapman and Hall, New York, 1998, 3.

2.6.1 Preparation of Extract: 10, 12

2.6.1 Preparation of Extract:^{10, 12}