INTRODUCTION:

Medicinal plants are part and parcel of human society to combat diseases, from the dawn of civilization. Herbal medicines are in great demand in the developed as well as developing countries for primary healthcare because of their wide biological and medicinal activities, higher safety and lesser costs. Proper uses of plants depend upon the correct identification of these plants and appropriate methods of extraction or processing of the plant products. In the field of Indian medicine certain synonyms are used for more than one or two plant drugs. To remove controversies, confusion and selection of genuine drugs we need physicians and pharmaceutical experts. Pharmacognosy deals with all these aspects.

The present investigation aims at the screening of Tephrosia villosa (L.) Pers. (Fabaceae) for pharmacognostic characteristics. The genus Tephrosia is a pantropical taxa with about four hundred species distributed throughout the world (Gillett, 1971). About twenty four species of Tephrosia were recorded in India (Gamble and Fischer, 1918; Saldanha and Singh, 1984). Most of the Tephrosia species are herbs to under shrubs and grow as weeds.

The genus is well known for its richness in prenylated flavonoids and is considered to possess insect repellant, larvicidal, piscicidal, antimicrobial and anticancer properties (Sarin Jagat et al., 1976; Chen Yuh-Lin, 1978; Bentley et al., 1987). Tephrosia villosa (L.) Pers. is an erect white tomentose viscid under shrub distributed in dry lands. The plant is commonly called as Poonai Kolinji in Tamil. Fresh roots are considered as hypoglycemic (Yoganarasimhan, 2000). Leaf juice is used for dropsy (Yoganarasimhan, 2000; Rahmatullah Qureshi et al., 2010) and roots are used in preparation of toothpaste (Jayaweera, 1980 – 1982). Root, leaves and bark are used as anthelmintic and antipyretic and also cure diseases of liver, spleen, heart, blood and leprosy (Varaprasad Bobbarala et al. 2009). Root powders are used to cure stomachache (Giday et al., 2009). Juice of the leaves is given in dropsy and also useful in diabetes (Chopra et al., 1956). The plant has natural distribution in deciduous forests in most forest districts of Deccan, Carnatic and Tamil Nadu. In view of its medicinal importance and the fact that no Pharmacognostical work is available on the species, the present investigations were undertaken. This will help in evaluating or assuring the quality of raw drug.

MATERIALS AND METHODS:

The identified plant of Tephrosia villosa was collected from Sivanthipatti hills near Palayamkottai. It was confirmed with voucher specimen (No: 4303) deposited at the Survey of Medicinal Plants Unit, Govt. Siddha Medical College, Palayamkottai. The taxonomic features of the plant confirmed with the Flora of Presidency of Madras (Gamble, 1915–1921) and The Flora Tamil Nadu Carnatic (Mathew 1983–1988). The plant parts were soaked in 70% alcohol, free hand sections of the leaf, stem and root were taken for detailed microscopic observations and figures (plate 1-4) were drawn by following Johansen (1946). Dry powder of the leaf, stem and root was used for chemical analysis. Physico chemical analysis was carried out as per standard procedure Anonymous (1966). The fluorescence analysis of the powder drug under Ultra Violet was done according to the methods described by Chase and Pratt (1949). The preliminary phytochemical analysis was done by the methods described by Brinda et al., 1981. Biochemical estimation for protein (Lowry et al., 1951), Phenol (Farkes and Kiraly 1962), Starch (Sadasivam and Manickam 1992), Amino acid (Jeyaraman, 1981) and Tannin (Aparna Buzarbarua, 2000) were carried out.

RESULTS AND DISCUSSION:

Macroscopic studies:

The plant is a subshrub and grows in the poor soil of waste land in the plains. The young branches and lower side of the leaflets are villous. The leaves are pinnately compound with 5-7 pairs of leaflets, obovate-oblanceolate, 1.5-2 x 0.4-0.8 cm, characteceous. glabrous above, sericeous below, base cuneate, margin entire, apex obtuse, retuse ; petiole to 1 cm ; petiolule 2 mm ; stipules subulate, 3 mm. Pseudoracemes axillary, to 7 cm ; flowers paired on rachis ; pedicel to 4 mm. Flowers pink, 1 cm across. Calyx – tube 1 mm, sericeous; lobes lanceolate, setaceous, ciliate; upper lobes 4 mm; lower one 5 mm. Corolla pink; standard orbicular, 1 x 1 cm, sericeous; wings oblong – obovate, 1 x 0.4 cm; keels 8 x 4 mm, beaked. Staminal sheath 6 mm; filaments 2 mm. Ovary 5 mm; style 4 mm, glabrous. The fruits 3mm long and about 8 seeded. Pod 3 x 0.4 mm, sericeous, continous within, apex slightly curve not horned.

Synonyms:

Cracca villosa L Tephrosia hirta Buch. Ham, T. incana Graham ex Wight and Arn. T. arngentea Pers.

Regional names:

Telugu: Nagurenpali Tamil Poonai kolinjii Uriya: Setohdothiya

Microscopical characters of Tephrosia villosa:

Leaflet:

The leaflet has fairly prominent midrib and thick lamina (Plate:1). The midrib is flat on the adaxial side and rectangular on the abaxial side. It is 200 µm thick in vertical plane and 250µm wide in horizontal plane. The midrib has a thin epidermal layer of small, thick walled cells. There is a single prominent top-shaped vascular bundle. This is surrounded by a single layer of dilated hyaline parenchymatous bundle sheath cells which extend up to the adaxial epidermis. The vascular bundle has three or four vertical short rows of angular fairly wide xylem elements and a thick horizontal band of phloem elements. The vascular bundle has a thick conical mass of sclerenchyma cells on the adaxial end and an arc of sclerenchyma band on the abaxial side. The lateral vein is also adaxially flat and short and conical on the abaxial side. The vascular strand of the lateral vein also has similar structure as that of the midrib. It has a collateral xylem and phloem with sclerenchyma caps on the adaxial and abaxial ends.

Lamina is 120 µm thick. It has wide rectangular thin walled adaxial epidermis which is 15 µm thick. The abaxial epidermis has circular or squarish thin walled cells. The lamina is amphistomatic; stomata occur on both adaxial and abaxial epidermis. The mesophyll tissue of the lamina consists of a wide adaxial zone of three layers of narrow, cylindrical palisade cells. Similar types of two layers of short cylindrical palisade cells occur on the abaxial part also. Along the median part, these are uniseriate, large, circular hyaline spongy parenchyma cells.

The leaf margin is semicircular measuring 120 µm thick. The epidermal layer along the marginal part is wide with barrel shaped or circular thick walled cells. There is a small circular marginal vascular bundle with collateral vascular elements and parenchymatous bundle sheath.

Venation pattern:

The primary veins are thick and run parallel to each other and the secondary / tertiary vein branch profusely forming reticulate venation. The vein islets are distinct forming polyhedral outline. Vein terminations are seen in some of the vein-islets; they are un-branched, long or short and slender.

Epidermal cells and stomata:

The epidermal cells are small, polygonal in surface view; their anticlinal walls are straight and thin. Some of the epidermal cells are circular and angular, thick walled and lignified. These cells are the basal cells from which the epidermal trichomes originate. Many epidermal cells have dense accumulation of mucilage.

Stomata occur on both sides of the lamina; but they are more in number on the abaxial side. The stomata are either anisocytic type with three unequal subsidiary cells; some of the stomata are anomocytic without distinct subsidiaries. The guard cells are elliptic with wide stomatal pore. They are 15 x 20 µm in size. The stomatal number is about 100/mm².

Rachis (Petiole):

Both proximal and distal parts of the rachis were studied. The proximal part of the rachis is roughly circular in sectional view with two thick, semicircular wings on the adaxial – lateral sides (Plate: 2). The rachis is 550 m in vertical plane and 600 µm in horizontal plane. The rachis has fairly prominent epidermal layer of radially oblong cells, three or four layers of outer parenchyma cells and central portion with wide angular, thin walled parenchyma cells. The xylem tissue occurs in thick continuous cylinder with thin layer of phloem on the outer side. The vascular cylinder is surrounded by a continuous thick cylinder of sclerenchyma cells with gelatinous inner walls. The adaxial wings have prominent collateral vascular bundle bearing a thick arc of bundle cap sclerenchyma.

Distal part of the petiole:

The terminal (distal) part of the petiole is elliptical in sectional view. It measures 550 µm horizontally and more than 700 m vertically. It consists of a prominent epidermal layer, about four layers of outer ground tissue and central part with wide compact angular parenchyma cells. The vascular cylinder is elliptical in sectional view with an adaxial gap and two small circular vascular strands. The vascular cylinder has an outer, three cells thick, gelatinous fibre sheath enclosing wide continuous zone of phloem and dense parallel lines of thick walled angular xylem elements.

Stem:

The stem is unequally four – angled in cross – sectional outline. It is 1.2 mm thick. The surface is densely pubescent (Plate: 3). The epidermal layer is thin, continuous, comprising of small square shaped cells. Dense uniseriate trichomes arise from the epidermal cells. The cortex consists of four or five layers of angular thick walled compact parenchyma cells. The cortical zone is uneven in thickness due to wavy contour of cortical sclerenchyma cylinder. The sclerenchyma cylinder occur inner to the cortical boundary; it is in the form of thick discontinuous arcs and it is 30-50 µm thick.

The vascular cylinder is fairly thick and continuous, enclosing wide pith. The vascular cylinder has outer discrete masses of phloem fibres in a single outer circle followed by narrow secondary phloem. The cells of the secondary phloem are in regular radial short rows.

Xylem cylinder is uneven in thickness. It includes wide, wedge shaped segments and narrow radial files of mostly xylem fibres and less frequency of vessels. The vessels in the wedge shaped segments are wide, angular thick walled and are in short radial multiples. The vessels are 20-25 µm wide. The xylem fibres are angular is cross sectional view. They have thick, lignified walls and wide lumen. The pith has wide, circular or angular parenchyma cells. They are compactly arranged. Calcium oxalate crystals are sparsely seen in the cells around the cortical sclerenchyma cylinder. Crystals are also sparsely seen in the cortical cells.

Root:

Young root:

The thin root has a superficial, smooth layer of periderm which consists of four or five layers of thin walled phellem cells. The wide cortical zone comprises of 8 - 10 layers of large, tangentially oblong thin walled compact parenchyma cells. There are solitary, scattered sclerenchyma cells in the cortex. Xylem occurs in the centre in dense, solid core with wide, circular, thick walled, diffusely distributed vessels mixed with narrow circular vessels. Xylem fibres are thick walled and lignified. The wide vessels are 40-50m in diameter. Phloem occurs in thin continuous sheath around the xylem cylinder (Plate: 4).

Thick root:

The thick root has wide and well developed periderm. It is about 150 µm wide and consists of more than 20 layers of thin walled, tabular, suberised and much compressed phellem cells. Inner to the cortex, it is occupied by thick, irregular cylinder of sclerenchyma cell. Secondary phloem is in thin continuous cylinder ensheathing the xylem. Secondary xylem is in wide, circular dense cylinder which consists of mostly xylem fibres. The vessels are solitary and diffusely distributed. The vessels in the central part are wider than those at the periphery. The wider vessels are 70 µm in diameter; the narrow vessels are 20 µm. Xylem fibres are thick walled and lignified. They are not in regular radial files. Xylem rays are fairly prominent and they are straight, extending from the centre towards the periphery.

Table - 1 Comparative Fluorescence analysis of leaf, stem and root of Tephrosia villosa

S. No.	Treatment	Plant parts
		Leaf		Stem		Root
		Visible light	UV light	Visible light	UV light	Visible light	UV light
1.	Powder + acetone	green	dark green	yellow	greenish yellow	pale yellow	fluorescent yellow
2.	Powder + ethyl alcohol	green	dark green	yellow	greenish yellow	pale yellow	greenish yellow
3.	Powder + 50% H₂SO₄	green	dark green	pale yellow	yellow	yellow	dark green
4.	Powder + 1 N HCl	pale green	green	pale yellow	yellow	pale brown	yellow
5.	Powder + 1N NaOH	yellowish green	green	light yellow	greenish yellow	pale yellow	yellow
6.	Powder + 50% HNO₃	Pale green	green	yellow	green	pale yellow	dark green
7.	Pet – ether extract	yellowish green	green	yellow	greenish yellow	golden yellow	brown
8.	Benzene extract	yellowish green	green	yellow	yellowish green	light yellow	greenish yellow
9.	Chloroform extract	green	dark green	pale yellow	greenish yellow	brownish yellow	greenish yellow
10.	Methanol extract	dark green	green	yellowish brown	greenish yellow	brown	dark yellow
11.	Powder + H₂O	light green	green	colourless	yellow	pale yellow	yellow

Fluorescence analysis:

The leaf, stem and root powder of Tephrosia villosa and the extracts of the powder on various solvents were examined under ordinary light and UV light. These powders were also treated with different reagents and the change in colour was recorded. These results were presented in Table – 1

The methanol extract of root powder shows yellow colour in visible light but appears fluorescent yellow under UV light. Generally the leaf treated with various chemical reagents exhibits green or dark green colour under UV light, but stem powder exhibits yellow or greenish yellow colour under UV light.

Quantitative determination:

The percentage of total ash, water soluble ash, acid insoluble ash, water soluble extractive value, sulphated ash and moisture content value were presented in Table - 2. It is helpful for determining the quality and purity of crude drugs especially in the powdered form.

Table: 2 Comparative analysis of physicochemical characters of leaf, stem and root of Tephrosia villosa (L.) Pers.

S. No	Test	*Leaf*	*Stem*	*Root*
1.	Total ash	11.26	3.8	3.1
2.	Water soluble ash	7.5	2.5	4.7
3.	Acid insoluble ash	7.5	2.54	2.81
4.	Sulphated ash	4.45	2.02	2.5
5.	Moisture content	53.45	46.42	61.14
6.	Water soluble extractive	6.76	2.21	2.89
7.	Alcohol soluble extractive	8.5	4.25	3.75
Extractive value
8.	Petroleum ether extract	6.6	6.1	6.0
9.	Benzene extract	7.9	5.8	6.1
10.	Chloroform extract	7.7	5.9	5.8
11.	Methanol extract	9.6	8.7	8.4

Phytochemical screening

Preliminary phytochemical analysis of the various extracts of stem, leaf and root powder of Tephrosia villosa are performed and the results obtained are presented in Table 3. Alkaloid is reported in all the five extracts of stem powder. Triterpenoid and steroid is absent in the leaf extracts. Catechin is reported only in the chloroform and water extract. The methanol extracts of leaf powder shows positive to saponin and phenol.

Biochemical and Physiological Analysis:

It was found that 1gm of leaf 95.64mg/g of protein, 3.03mg/g of phenol, 2850µg /g of tannin, 34.90mg/g of amino acid and 242.00mg/g carbohydrates. 1gm of stem contains 105.55mg/g of protein, 1.42 mg/g of phenol, 4750µg/g of tannin, 32.00 mg/g of amino acid and 123.64mg/g carbohydrates. 1 gm of root contain 168.51 mg/g of protein, 34.98mg/g of lipids, 1.45 mg/g of phenol, 1530µg/g of tannin, 195.82mg/g of starch and 16.50 mg/g of amino acid.

CONCLUSION:

The macroscopic and microscopic characters, fluorescence analysis, physico-chemical determination and preliminary phytochemical screening can be used as a diagnostic tool in the correct identification of plants. The adulterants if any in the plant material can also easily identified by these studies.

ACKNOWLEDGEMENT:

We thank The Principal, St. Xavier’s College, Palayamkottai and Dr. K. Natarajan, Head, Department of Plant Biology and Biotechnology, St. Xavier’s College, Palayamkottai for providing laboratory facilities and guidance

Table – 3 Phytochemical screening of leaf, stem and root of Tephrosia villosa (L.) Pers.

S. No

Extract

Samples

Saponin

Tannin

Alkaloid

Flavones

Amino acids

Protein

Phenol

Steroid

Triterpernoid

Catachin

Anthroquinone

Sugar

Pet. ether

Leaf

Stem

Root

Benzene

Leaf

Stem

Root

Chloroform

Leaf

Stem

Root

Methanol

Leaf

Stem

Root

Water

Leaf

Stem

Root

NOTE: + denote Present ; - denote Absent

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Received on 24.07.2014 Modified on 16.08.2014

Res. J. Pharmacognosy & Phytochem. 6(4):Oct. - Dec.2014; Page 190-194

Pharmacognostical Studies on Tephrosia villosa (L.) Pers. (Fabaceae)