Research Article S.Thenmozhi et al. / Journal of Pharmacy Research 2011,4(4),1259-1262 ISSN: 0974-6943 Available online through http://jprsolutions.info Pharmacognostical and phytochemical investigation on leaves of trifolia linn. S.Thenmozhi*, R.Shanmuga Sundaram, Jena Prabhat Kumar and Choudhury Golak Bihari *Department of Pharmacognosy, Faculty of Pharmacy, Jeypore College of Pharmacy, Rondapalli, Jeypore - 764002, Koraput, Odisha, . S.Thenmozhi, *Department of Pharmacognosy, Faculty of Pharmacy, vinayaka mission’s college of pharmacy, vinayaka missions university, salem, tamil nadu .Received on: 05-12-2010; Revised on: 14-01-2011; Accepted on:09-03-2011 ABSTRACT Vitex trifolia Linn (Verbanaceae) is commonly known as chaste and is reported to have good medicinal values in traditional system of medicines. The present study deals with pharmacognostical examination includes the morphological, microscopical characters and physical constants of vitex trifolia leaves including determination of loss on drying, ash values and extractive values. The preliminary phytochemical screening of various leaf extracts was also carried out and it is revealed the presence of various phytoconstituents like carbohydrates, flavonoids, protein and amino acids, tannins, phytosterols and saponins. The results of the standardization may throw immense light on the botanical identity of the Vitex trifolia which may furnish a basis of judging the authenticity of the plant and also to differentiate the drug from its allied species and detect adulterants.

Key words: Vitex trifolia, Pharmacognostic evalution, Physicochemical analysis.

INTRODUCTION Standardization of natural products is a complex task due to their heterogeneous composition, 4. Microscopic Evaluation: which is in the form of whole plant, plant parts or extracts obtained thereof. To ensure reproducible quality of herbal products, proper control of starting material is utmost essential. Preparation of specimen: The first step towards ensuring quality of staring material is authentication. Thus, in recent The healthy leaves were selected carefully for the study of microscopical characters. The years there has been a rapid increase in the standardization of selected medicinal of specimen leaves of Vitex trifolia were fixed in FAA (Formalin-5ml + Acetic acid-5ml + 70% potential therapeutic significance (Wallis, 1985)15. Despite the modern techniques, identifi- Ethyl alcohol-90ml). After 24 hours of fixing, the specimens were dehydrated with graded cation of plant drugs by pharmacognostic studies is more reliable. According to the World series of TBA (tertiary- Butyl alcohol). Infiltration of the specimens was carried out by gradual Health Organization, the macroscopic and microscopic description of a medicinal plant is the addition of paraffin wax (melting point 58 - 60° c) until TBA solution attained super saturation. first step towards establishing the identity and the degree of purity of such materials and should The specimens were cast into paraffin blocks. be carried out before any tests are undertaken (WHO Library, 1998)16. Sectioning of specimen: Vitex trifolia Linn. belonging to the family of Verbenaceae is commonly known as chaste tree The paraffin embedded specimens were sectioned with the help of Rotary Microtome. The (English), Nirnochi (Tamil) and jalanirgundi (). It is distributed throughout India in thickness of the sections was 10-12µm. De-waxing of the section was done by customary tropical and subtropical regions. Height of the plant 1-3.5 meter. Flowers are appearing in procedure (Johansen, 1940)9. The sections were stained with toludine blue (0.25 % having a summer or late summer, and 6-12 inch long. Leaves are commonly used as poultice for pH of 4.7) as per the specified method (O’Brien et al., 1964). Since Toludine blue is a rheumatic pains, inflammations, sprains, fever, anthelmintic, improve memory, favour the polychromatic stain, the staining results were remarkably good and some cytochemical growth of hair, good for the eyes, leucoderma, bad taste in mouth and bronchitis3-4. Roots are reactions were also observed. The dye rendered pink colour to the cellulose walls, blue to the used to treat febrifuge, painful inflammations, cough and fever. Flowers are used in treating lignified cells, dark green to suberin, violet to the mucilage and blue to the protein bodies. fever and fruits in amenorrhoea (Chopra et al., 1956)2. This plant is known to possess various Wherever necessary, sections were also stained with safranin, fast-green and N/10 Iodine to active constituents viz, essential oils (Pan et al., 1989), halimane type diterpenes, vitetrifolins identify the presence of lignified cells and starch grains. (Ono et al., 2001)12 and several pharmacological properties have been studied viz, antipyretic (Ikran et al., 1987), antibacterial (Hossain et al., 2001), against asthma and allergic diseases To study the histology of stomata, venation pattern and trichome distribution, paradermal (Ikwati et al., 2001). In view of its diverse medicinal applications and in order to ensure sections (parallel to the surface of leaf) were taken. The clearing of leaf was done with 5% complete quality of its supply in terms of adulteration and substitution prevailing on the crude sodium hydroxide solution or epidermal peeling by partial maceration, employing Jeffrey’s drug markets of India, the present communication deals with a complete pharmacognostical maceration fluid (Sass, 1940). The mounting of macerated/cleared materials was performed evaluation of the leaves of vitex trifolia. The study include macroscopic and microscopic with glycerine. Powdered materials of different parts were cleared with sodium hydroxide evaluation, fluorescence analysis of powder, physico-chemical values, preliminary phytochemical solution and mounted in glycerine medium after staining. Different cell components were screening of different extractives1. studied and measured.

MATERIALS AND METHOD: Photomicrographs: Microscopic descriptions of tissues can be described with micrographs wherever necessary. 1. Collection and Authentication of Plant Material: Photographs of different magnifications were taken with Nikon labphoto-2 microscopic unit. The plant specimens for the proposed study were collected from shevoroyan hills, Salem, For normal observations bright field was used. For the study of crystals, starch grains and Tamilnadu, India during the month of Jan. 2010. The plant material was taxonomically lignified cells, polarized light was employed. Since, these structures have birefringent property identified and authenticated by Dr. A. Balasubramanian, Consultant, Central siddha research, under polarized light; they appear bright against dark background. Magnifications of the figures Salem, Tamilnadu. A voucher specimen (letter number: CSR08/DBT/115, dated: 12-4- are indicated by the scale-bars. Descriptive terms of the anatomical features are as given in the 2010).has been deposited in the Herbarium of the Department of Pharmacognosy, Vinayaka standard anatomy books (Easu, 1964a & 1964b)3-4. mission’s college of pharmacy, Tamilnadu, India, for future reference. 5. Determination of Physical Constants: 2. Chemicals: (i) Ash Values All the solvents and chemicals used were purchased from Merck chemical (LR grade), India. The ash values are useful to determine the quality and purity of the crude drug. Ash contains inorganic radicals like phosphate, carbonates and silicates of sodium, potassium, magnesium, 3. Macroscopic Examination: calcium, etc. Such variable are then removed by treating with acid. Different ash values like The leaves of Vitex trifolia was studied individually for its morphological characters such as total ash, water soluble ash, acid insoluble ash and sulphated ash were determined as per colour, odour, taste, shape, size, etc. standard procedure mentioned in Indian pharmacopoeia 1996.

(ii) Extractive Values *Corresponding author. Extractive values are useful for evaluation of crude drugs. It gives an idea about the nature of the Dr.R.Shanmuga Sundaram chemical constituents present in the crude drug. Different extractive values like water soluble Department of Pharmacology, extractive and alcohol soluble extractive value were determined as per standard procedure mentioned in Indian pharmacopoeia 1996. Jeypore College of Pharmacy, Rondapalli, Jeypore-764002, Koraput, Odisha, India.

Journal of Pharmacy Research Vol.4.Issue 4. April 2011 1259-1262 S.Thenmozhi et al. / Journal of Pharmacy Research 2011,4(4),1259-1262 (iii) Foreign Organic Matter and Moisture Content has a median, wide arc-shaped strand and a group of adaxial accessory strands (Fig3.1). The Foreign organic matter was determined from the weight of the drug taken and moisture content median arc of vascular strand consists of several parallel rows of xylem elements with narrow was determined by loss on drying method in terms of percent w/w as per standard procedure parenchymatous space in between the xylem rows. In each row of xylem, these are about six cells which are angular and thick walled (Fig 3.2). The adaxial accessory bundles are circular mentioned in Indian Pharmacopoeia 1996. and collateral (Fig 3.2). The strand has a small group of xylem elements and wide zone of phloem. 6) Reaction of Powdered Drug with Different Reagents Powdered drug was treated with different reagents and colored shown by that treatment is noted down (Ansari et al., 2006).

7) Fluorescence Analysis Fluorescence characteristic of powdered drug with different reagent were observed under day light and U.V. light after drug treatment with different reagents (Ansari et al., 2006).

8) Preliminary Phytochemical Screening Freshly prepared various extracts of leaves were tested for the presence of phytochemical constituents by using reported methods (Harborne, 1992, Kokate, 1994, Khandelwal, 2005)5,9- 10.

RESULTS AND DISCUSSION: 1: MACROSCOPIC EVALUATION: Leaves are variable, some simple and some three foliolate; leaflets elliptic or oblong- obovate, usually obtuse, the terminal leaflet sessile, 5-6.3 by 2.5-3.8 cm the lateral smaller, sessile, all glabrous above, very densely white-tomentose beneath, base ta- pering; common petioles 1.3-1.6 cm long. The leaves are dark green in colour and 3.1 T.S of Leaf through Midrib with Lamina: bitter taste. Fig. 1. The Leaves of Vitex trifolia 2. MICROSCOPIC EVALUATION:

Leaflet: [Fig 2.1, 2.2]: The leaflet has smooth and even adaxial surface and densely pubescent uneven abaxial side. It is 140 µm thick along the ridged part of the leaflet and 120 µm thick in between the ridges. The adaxial epidermis is thick and prominent; the epidermal cells are rectangular and tangen- tially oblong, fairly thick walled and have thin cuticle. These are a subepidermal layer of dilated and hyaline squarish or cylindrical cells. The epidermal layer is 10 µm thick; the hypodermal layer is 15µm thick. The abaxial epidermis is narrow with thin walled cylindrical cells. They bear dense nonglandular trichomes. The mesophyll consists of four layers of vertically elongated narrow cylindrical cells. The height of these cells is more along the first row and the height is reduced gradually towards the lower part. All these cells are palisade; and spongy parenchyma cells are not evident.

3.2 T.S of Midrib – Vascular bundle Enlarged: [AbB – Abaxial Bundle; AbS – Abaxial Side; AdB – Adaxial Bundle; AdH – Adaxial Hump; Ep-Epidermis; GT – Ground Tissue; Ph – Phloem; X – Xylem]. Fig. 3: Anatomy of the Midrib: Venation [Fig4.1, 4.2., 5.1, 5.2]: The venation pattern of the lamina shows uniformly thin and straight lateral veins. The veins 2.1 T.S of Lamina through Lateral Vein under Low Magnification: are covered by hyaline parenchymatous bundle sheath cells (Fig4.1). The vein-islets are not well defined; they are open and not distinct. The vein-terminations are distinct; they are long, slender, straight or slightly bend and un branched. Stomata are located in the furrows of the abaxial epidermis. They are circular and are anomocytic type (Fig 4.2).

2.2 T.S of Lamina through Lateral vein under Low magnification Enlarged: Fig. 2: Anatomy of the Lamina: Midrib [Fig 3.1, 3.2]: The midrib is thick, projecting prominantly on the abaxial side with a short and wide adaxial hump. The epidermal layer is a narrow with small cells. The ground tissue of the midrib consists of circular thin walled compact parenchyma cells. The vascular system of the midrib Fig. 4.1: Paradermal Section Showing Vein- Islets and Vein-termination:

Journal of Pharmacy Research Vol.4.Issue 4. April 2011 1259-1262 S.Thenmozhi et al. / Journal of Pharmacy Research 2011,4(4),1259-1262 Reaction of Powdered Drug with Different Reagents: The behavior of powder with various reagents under ordinary light by naked eye is given in Table 2

Table.2. Behavior of the Powder of Vitex trifolia with Different Chemical Reagents Treatment Colour

Powder as such Pale green Powder + Conc.sulphuric acid Greenish black Powder + Conc. nitric acid Brownish yellow Powder + Conc. Hydrochloric acid Pale yellow

Powder + 5% I2 Brownish yellow Powder + 5M NaOH Yellowish green Powder + glacial Acetic acid Pale green

Powder + 80% H2SO4 Black

Fluorescence Analysis: The fluorescence analysis of powder with various reagents is observed under day light and U.V. light is given in Table 3. [Bs - Bundle sheath cells; St - Stoma; VI - Vein-islet; VT - Vein- termination Table 3.Fluorescence Characteristics of Powdered Leaves of Vitex trifolia. Fig: 4.2:Abaxial Epidermis with Stomata: Reagents Day light UV Light Drug Powder Pale green Green Drug Powder +1M NaOH Greenish yellow Yellowish green Drug Powder + alcoholic 1M NaOH Yellowish green Pale green Drug powder + 1M HCl Yellowish green Greenish yellow

Drug Powder + 50 % HNO3 Yellowish brown Pale green Drug powder + 5% FeCl3 Greenish black Dark green Drug powder +80% H2SO4 Black Greenish brown Drug powder + Water Greenish yellow Green

Drug powder+Conc.H2SO4 Brownish green Yellowish brown

Preliminary Phytochemical Screening: The preliminary phytochemical screening with the various qualitative chemical tests revealed the presence of various phytoconstituents like carbohydrate, phenolic compounds, flavonoids, protein and amino acids, tannins, phytosterols and saponins. The results were shown in Table 4.

Table 4. Results of Phytochemical screening

Constituents Tests Pet.Ether Ethanol Aqueous 5.1: Cleared Leaf showing Vein- islets and Vein termination: Alkaloids a) Mayer’s reagent - - - b) Dragendorff’s reagent - - - c) Hager’s reagent - - - d) Wagner’s reagent - - - Sugars and a) Molish’s reagent - + + Carbohydrates b) Barfoed’s test - + + c) Fehling’s solution test - + + d) Benedict’s test - + + Phenolic a) Ferric chloride solution - + + compounds b) Lead acetate solution - + + c)Pot.dichromate test - + + Flavanoids a) Shinoda test - + +

b) Conc.H2SO4 - + + c) NaOH test - + + Protein and amino acids a) Biuret test - - + b) Ninhydrin test - - + c) Million’s test - - + Tannins a) Ferric chloride solution + + + b) Potassium dichromate - + + Phytosterols Salkowski reaction - + - Saponins Foam test - + - Fixed oil andFats a) Spot test - - - b) Saponification test - - - + = Present and - = Absent CONCLUSION: The present study is related to pharmacognostical, physical constants and preliminary phy- 5.2: Cleared Leaf showing Vein - islets and Vein Termination Enlarged: tochemical screening of Vitex trifolia Linn. leaves provided useful information about its correct [VI – Vein - islets; VT – Vein - termination]. identity and evaluation. It helps to differentiate from the closely related other species of Vitex Fig. 5: Venation Pattern: trifolia Linn. Phytochemical study is also useful to isolate the pharmacologically active principles present in the drug. The other parameters observed are also useful for the future 3. Determination of Physical Constants: identification of the plant and serves as a standard monograph for identification and evaluation The results were shown in Table no.1 of plant. Table 1. Determination of physical constants: (Ash values, Extractive values, Foreign organic matter and Moisture content) ACKNOWLEDGEMENT Evaluation parameters Yield (% w/w) Authors wish to thank to local people of salem, tamil nadu and Central siddha research, Salem, Tamilnadu, India, for providing valuable information about the plant and its identifi- Total ash 3.64 cation. The author wish to express their gratitude Jeypore College of Pharmacy, Rondapalli, Water soluble ash 1.54 Jeypore, Koraput, Odisha. Acid insoluble ash 2.46 Sulphated ash 1.89 Alcohol soluble extractive 8.62 REFERENCES Water soluble extractive 4.35 1. Ansari M.M., Ahmad J., Ahmad A. and Ansari S.H., (2006) Pharmacognostic characterization Foreign organic matter 0.1 and standardization of Morus alba stem bark. Journal of Medicinal and Aromatic Plant Sciences, Moisture content 4.0 28, 31-36.

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