INTRODUCTION:

Thespesia populnea soland ex Correa (family Malvaceae) is a large tree found in the tropical regions and coastal forests in India and cultivated in the gardens. All the parts of the plant used in traditional system of medicine. The bark, leaves, flower and fruits are useful in cutaneous infection such as scabies, psoriasis, eczema, ringworm, and guinea worm. The decoction of the bark is commonly used for the treatment of skin and liver diseases. A compound oil of bark and capsules is useful in urethritits and gonorrhea (Anon., 1995). The bark, root, fruits were used in dysentery, cholera and hemorrhoids. The fruits of the plant are used in ayurveda for the control of diabetes (Satyanarayana et al., 2004). The barks and flowers posses astringent, hepatoprotective, antioxidant and anti-inflammatory activities in rats (Ilavarasan et al., 2003a,b; Shirwaikar et al., 1995, Mani vasudevan et al., 2006).

The leaves and bark of this tree are still used to produce oil for the treatment of fracture wounds and as an anti-inflammatory poultice applied to ulcers and boils, as a folk medicine. (Mani Vasudevan et al., 2006). Gossypol was found to be the major component of Thespesia populnea (Akhila and Rani, 1993) producing anti-fertility effects in rats (Ghosh and Bhattacharya, 2004; Murthy et al., 1981) as well as in human beings (Qian and Wang, 1984). Four naturally occurring quinones viz thespone, thespesone, mansonone-D, and mansonone-H have been extracted from heart wood of the plant (Johnson et al., 1999).

The phytochemical study of leaf reveals the presence of lupeol, lupenone, β-sistosterol (Rastogi, 1979) and also acacetin, quercetin, vanillic, syringic, melilotic, and ferulic acid. (Daniel., 2006). Lack of proper standard of medicinal plants may lead to usage of substandard drugs which will cause damage to the faith on traditional system of medicine. Therefore scientific method must be developed to identify and maintain quality of plant drugs. With this aim the present investigation was planned to study the pharmacognostical aspects of Thespesia populnea leaves.

MATERIALS AND METHODS:

Plant materials:

The fresh leaves were collected from our Salaiyur University campus in Chennai, India. The plant material was taxonomically identified and authenticated by Director, Plant Anatomy Research Centre, Chennai. A voucher specimen (PARC/236/07) has been deposited in the herbarium of the same department.

Pharmacognostical studies:

Organoleptic or Macroscopical character:

The freshly leaves of the plant were collected and investigated in different organoleptic features by repeated observations. Morphological studies, such as shape, size, apex, surface, base, margin, venation, taste and odour of leaves, were carried out.

Quantitative Microscopy:

The important identifying characteristic of leaf constants like Stomatal Number, Stomatal Index, Vein islet number, Vein termination number were found out and tabulated (Table No: 1) (Khandelwal., 2004).

Table 1: Quantitative microscopy of Thespesia populnea leaf constant

Parameter	Result
Stomatal number (lower epidermis)	21-24 (lower epidermis)
Stomatal index	13-17% (lower epidermis)
Vein inlet number	12
Vein termination number	16

Anatomical or Microscopical evaluation:

Preparation of specimen:

The leaves were cut and removed from the plant and fixed in FAA (Formalin 5ml + Acetic acid 5 ml + 70% Ethanol 90 ml). After 24 hrs of fixing, the specimens were dehydrated with graded series of tertiary butyl alcohol. Infiltration of the specimens was carried by gradual addition of paraffin wax (melting point 58-60⁰C) until tertiary butyl alcohol solution attained super saturation. The specimens were cased into paraffin blocks.

Sectioning:

The paraffin embedded specimen was sectioned with the help of rotary microtome. The thickness of the section was 10-12 µm. After dewaxing the sections were stained with toluidine blue. Since toluidine blue is a polychromatic stain, the staining results were remarkably good and some phytochemical reactions were obtained. The dye rendered pink color to the cellulose walls, blue to the lignified cells, dark green to suberin, violet to the mucilage, blue to the protein bodies etc., wherever necessary sections also stained with safranin and fast green and iodine (For starch). (Khandelwal., 2004., Kokate., 2005). For studying the stomatal morphology, venation pattern and trichomes distribution, paradermal sections as well as clearing of leaf with 5% sodium hydroxide and epidermal peeling by partial maceration employing Jeffrey’s maceration were prepared. Glycerin mounted temporary preparations were made for macerated materials.

Photomicrographs:

Microscopic descriptions of tissues are supplemented with micrographs wherever necessary. Photographs of different magnifications were taken with Nikon Labphot-2 microscope units. For normal observations bright fields was used. For the study of crystals, starch grains and lignified cells polarized light were employed. Since these structures have birefringent property, under polarized light they appear bright against dark back ground.

Physicochemical evaluation:

Physical parameter such as loss on drying, total ash, acid insoluble ash, and water soluble ash was determined (Anon., 1996). All the results were recorded carefully (Table no: 2).

Table 2: Loss on drying and ash values of powdered leaf of Thespesia populnea

Parameters	Values of three replicates (% w/w)
Loss on drying	6.26%
1) Total ash	5.62%
2) Acid insoluble ash	0.76%
3) Water soluble ash	3.20%

Phytochemical screening:

The dried and powdered leaf was subjected to preliminary phytochemical screening for qualitative detection of phytoconstituents. The dried and coarsely powdered leaf (100 g) was extracted successively with petroleum ether (40-60ºC), chloroform (59.5-60ºC), ethyl acetate (76.5-77.5ºC), and ethanol (90%) in a soxhlet extractor by continuous hot percolation. Finally the marc was macerated with chloroform water. Each time before extracting with the next solvent of higher polarity the powdered drug (marc) was dried in a hot air oven below 50ºC for 10 minutes. Each extract was concentrated by distilling off the solvent, which was recovered subsequently. The concentrated extracts were evaporated to dryness and the extracts obtained with each solvent were weighed. Their percentages were calculated in terms of initial air dried plant material. They are tabulated (Table no: 3).

Table 3: Percent extractives of successive extracts of Thespesia populnea leaf

Solvent	Extractive values (% w/w)
Pet. Ether	3.14%
Chloroform	5.32%
Ethyl acetate	2.62%
Ethanol	10.50%
Water	8.54%

The crude extracts were analyzed for the presence of various phytoconstituents by following standard phytochemical tests and the results were reported (Table no: 4). (Kokate., 2005).

Table 4: Results of phytochemical screenings of successive extracts of leaf of Thespesia populnea

Constituent	Pet. Ether extract	Chloroform extract	Ethyl acetate extract	Ethanol extract	Aqueous extract
Alkaloids	-	-	-	-	-
Carbohydrates	-	-	-	+	+
Glycosides	-	-	-	+	+
Steroids	-	-	-	-	-
Flavonoids	-	-	-	+	-
Saponins	-	-	-	-	-
Fixed oils and fats	-	-	-	-	-
Tannins	-	-	-	+	+
Proteins and amino acids	-	-	-	-	+
Mucilage	-	-	-	-	+

RESULT AND DISCUSSION:

The leaf of Thespesia populnea is alternate and glossy green above and paler green below. The leaf blades were heart shaped 10-20 cm long and 6-13 cm broad, leaf stalks were long about 5-10 cm. the leaves were broadly ovate, cordate, apex acuminate and entire smooth margin. Finely reticulate veined with 5-10 prominent nerves. The primary lateral veins were thick and straight. The secondary lateral veins were fairly thick and wavy. The taste and odour of the leaf were characteristic aromatic and agreeable.

Transverse section of the leaf through the midrib showed bowl shaped abaxial parts and slightly raised adaxial side. Both the adaxial and abaxial epidermal layers were single layered thin walled cubical cells. The upper epidermis was aposomatic. The epidermal cells wide, polygonal, thin walled and the walls were straight or slightly wavy. The lower epidermis was stomatiferous. The stomata were anisocytic with three unequal subsidiary cells enclosing the stoma. The guard cells were elliptical with wide stomatal aperture. The lower epidermal cells were smaller as compared to the upper epidermal cells. The epidermal cells followed by four to five layers of angular collenchyma cells on both the sides. Mucilage cavities were present in the collenchymatous hypodermal region. The vascular strand of the midrib occurs in the shallow arc and has two adaxial vascular bundle on the margin of the arc towards the adaxial part of the midrib (Fig.1).

Figure 1: TS of Thespesia populnea (through midrib) leaf.

X: xylem; Ph: pholem; MB: median bundle; MC: mucilage cavity;

GT: ground tissue; AdB: accessory adaxial bundle; Ads: adaxial side;

Abs: abaxial side; Ta: tanniferous cell; Ep: epidermis.

The lamina of the leaf is dorsiventral, mesomorphic, amphistomatic, globroscent and even. Both the epidermal cells were squarish to rectangular, cuticle was thin and smooth. Palisade tissues were single layered. They were cylindrical less compact and occupy one third of the thickness of the lamina. Spongy tissues were three-four layered, spherical and less compact. Lateral veins were smaller with few xylem and phloem element (Fig.2).

Figure 2: TS of Thespesia populnea leaf.

La: lamina; MB: median bundle; GT: ground tissue; AB: accessory adaxial bundle; Ads: adaxial side; Abs: abaxial side.

The abaxial arc has vertical files of xylem elements alternating with xylem fibers. Facing the adaxial side, phloem occurs on the outer part of the xylem arcs. The two accessory adaxial bundles occur above the main arc and consist of xylem elements in groups facing the abaxial side the ground tissue in parenchymatous, thin walled, angular to spherical. Abundant tannin containing cells and calcium oxalate crystals are present in the ground tissues. The calcium oxalate crystals druses or sphaerocrystals appear bright against dark background under polarized light microscope. These crystals were found in mesophyll tissues. The leaf showed abundance of epidermal trichomes. They were flat, thin and membranous structure formed by many radiation narrow cells of unequal length. In the central part of the plate is a small short stalk cell with which they are attached to the epidermis. The outline of the trichomes is coarsely serrate. This type of trichome is called peltate scale (Fig.3).

Figure 3: TS of Thespesia populnea leaf lamina.

Ade:adaxial epidermis; Abe:abaxial epidermis; PM: palisade mesophyll; SM: spongy mesophyll; LV: lateral vein; BSE: bundle sheath extension.

Figure 4: Trichomes (Peltate scale) of TS of Thespesia populnea leaf.

To ensure the quality of plant material, the macroscopic and microscopic description of medicinal plant is the first step towards establishing it identity and purity. For identification and evaluation of plant drugs by pharmacognostical studies is still more reliable accurate and is inexpensive. (Anon., 2002) The macroscopical studies of Thespesia populnea leaf revealed the presence, of characteristic odour, size and shape and taste. In the microscopic studies indicated the presence of special type of pellate trichome, nature of vascular bundle, leaf constants (Vein islet, Vein termination, Stomatal number, Stomatal index) were the important diagnostic characters. (Khandelwal., 2004 ). In physiochemical studies of various parameters established, like various ashes content which showed the presence of inorganic salts by naturally occurring or adhering to it, or deliberately added to it as a form of adulteration. These values are important quantitative standards. The various extractive values obtained by results showed higher yield in ethanolic extract. (Kokate., 2005). The extracts obtained by exhausting plant materials with specific solvents are indicative of approximate measures of their chemical constituents extracted with those solvents from a specific amount of air-dried plant material. This parameter is employed for materials for which as yet no suitable chemical or biological assay exists.The results also showed higher extractive values in hot extraction, indicating the effect of elevated temperature on extraction.In all methods alcohol yielded higher extractives (Mukherjee., 2002).

The plant material was subjected to preliminary phytochemical screening involving successive solvent extraction by different solvents in order of increasing polarity to obtain diverse polar and non polar phytoconstituents possessing different solubility pattern, followed by various chemical tests for qualitative detection of various chemical constituents. Phytoconstituents like glycoside, saponin, tannin, mucilage, and carbohydrates were present in both aqueous and ethanolic extract. Alkaloids and steroids were absent. The percent extractives in different solvents indicate the quantity and nature of constituents in the extract. (Kokate., 2005, 2006).

CONCLUSION:

The present investigation it can be concluded that the pharmacognostical study of Thespesia populnea leaf yielded a qualitative and quantitative parameters or standards that can serve as an important possible sources of information to ascertain the identity and to determine the quality and purity of the plant material. These information will also be helpful to differentiate Thespesia populnea from the closely related other species and varieties of Thespesia.

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Received on 16.12.2015 Modified on 25.12.2015

Res. J. Pharmacognosy & Phytochem. 8(1): Jan.- Mar. 2016; Page 16-20

DOI: 10.5958/0975-4385.2016.00004.2