Journal of Herbal Medicine and Toxicology 3 (2) 127-132 (2009) ISSN : 0973-4643 Original Article PHARMACOGNOSTICAL AND PRELIMINARY PHYTOCHEMICAL STUDIES ON THE LEAF OF ALSTONIA MACROPHYLLA M. S. Khyade and N. P. Vaikos Department of Botany, Dr. Babasaheb Ambedkar Marathwada University, Aurangabad - 431 004, [M.S.] Corresponding author e mail- mskhyade@rediffmail.com, drvaikos@gmail.com Received- 10th March, 2009, Revised- 30th April, 2009, accepted- 10th May, 2009 ABSTRACT : Alstonia macrophylla Wall. ex G.Don (Apocynaceae), which is used in the traditional systems of medicine. The leaf is considered one of the important plant organs for the treatment of various disorders such as stomachic, skin diseases urinary infections, sprains, bruises and febrifuge. It is also known for its anticholeric and high vulnery effects. The present investigation has therefore carried out to provide requisite pharmacognostic details. Morphological and anatomical aspects as well as differential microchemical response have been worked out to identify the diagnostic features of the leaf. Physical constant values involving moisture content, ash and extractives as well as qualitative and quantitative estimation of various phytochemicals have been studied. The presence of lipid, saponin, tannin, alkaloid, phenol, steroid, flavonoid, and some other chemical constituents are recorded. Key words: Alstonia macrophylla, leaf drug, pharmacognosy, phytochemistry, INTRODUCTION MATERIALS AND METHODS Alstonia macrophylla Wall.ex G.Don (Apocynaceae) is an evergreen tree grows in the low hill rain forest of south East Asia. Medicinally it is used for the same purpose as that of Alstonia scholaris [1]. The decoction of leaves and stem bark is widely used in stomachic, skin diseases and urinary infections [2].The leaves are known to have anticholeric and high vulnery effects and are greased with hot coconut oil for sprains, bruises and dislocated joints as poultice and useful as febrifuge [3].Moreover the leaf vapours are also inhaled in fever by the tribal community- Shompen in Nicobar Islands [4, 5]. Various studies have been carried out with A. macrophylla on the antibacterial, antifungal and anti-inflammatory activities [6, 7], alkaloids [8-12] and presence of tannin, flavonoids, sterols, triterpenes and reducing sugars [12]. Since there is no pharmacognostic work on the leaves of A. macrophylla, the present investigation is undertaken. Samples of Alstonia macrophylla were collected from the Botanical Garden of Dr. Babasaheb Ambedkar Marathwada University Aurangabad (MS), India. It was identified, confirmed and authenticated by comparison with an authentic specimen. A voucher specimen of the plant deposited in the Departmental herbarium for the future reference. Collected fresh leaves were washed and used for the present study. The fresh leaf was used for the study of macroscopic and microscopic characters. For microscopic studies, the epidermal peels and cross sections of leaves were prepared. The sections were stained in safranin (1 %), light green (1 %) and mounted in DPX after the customary dehydration. Some hand sections were also examined in glycerin. Microphotographs of leaf were taken by using Jenaval and Mirax Laborec Cameras affixed to microscope. 127 Journal of Herbal Medicine & Toxicology The quantitative leaf microscopy like palisade ratio, stomatal index, vein islet and vein termination number were carried out as described methods [13]. are confined to lower surface. They are paracytic and anomocytic. The trichomes are unicellular, thick walled and unbranched [Figures A, D and E]. The histochemical colour reactions were performed by the standard methods [14, 15]. The physicochemical values such as ash, acid insoluble ash, water soluble ash and extractive values were done by reported methods [16]. Phytochemical studies such as qualitative and quantitative were done from the shade dried drugs. For qualitative phytochemicals, standard prescribed methods were followed [1719].The quantitative phytochemicals such as fats [20], alkaloids [21], tannins and phenolics [22] and saponins [23] were estimated. Quantitative microscope The leaf microscopic characters like stomatal frequency, stomatal index, vein islet number and vein termination number were determined (Table.1).The quantitative determination of some pharmacognostic parameters are useful for setting standards for crude drugs. The vein islet and vein termination numbers and other parameters determined in the quantitative microscopy, are relatively constant for plants and can be used to differentiate closely related species. Histochemical colour reactions RESULTS AND DISCUSSION The leaves of A. macrophylla are observed to be in whorls of 3-4, crowded at the ends of branches. They are oblanceolate to oblong, narrowed at the base, entire, acute or subacute. The leaves are glabrous and shining above where as paler and softly pubescent on the nerves beneath. T.S. of Leaf The leaf is dorsiventral and hypostomatic. The upper epidermis has barrel shaped closely arranged thick walled cells. The cuticle is thick. The lower epidermis is of small cells and thin walled. The stomata are restricted to lower surface. The trichomes are one celled and thick walled. The hypoderm consists of single layer. At certain places it has 2-layers. The mesophyll is differentiated into palisade and spongy tissue. The palisade is two layered and spongy tissue is of loosely arranged cells [Figures A, B, and C]. The vascular tissue has bicollateral vascular bundles which extend through the mesophyll. The midrib has typical an arc shaped strand. Two end of an arc develop more phloem strands which meet to each other forming a ring like structure. The ground tissue is of parenchymatous cells, which contain calcium oxalate crystals. Epidermal features The upper epidermis is of large, usually tri to polygonal cells, the cell walls thick and more or less straight walled. The lower epidermis is made up of small cells with cell walls thin and slightly wavy. The stomata The histochemical colour reactions were carried out on transverse section of the fresh leaf (Table.2). The results indicated that presence of lignin, starch, fats, alkaloids, saponins, tannins, flavonoids and calcium oxalate crystals. Histochemical localization of certain important compounds enables to get a preliminary idea of type of compounds and their accumulation in the plant tissues. Based on this study, one can chose the organ or tissue where the required compounds are located. Physico-chemical characters The physico-chemical characters such as moisture content, total ash, acid insoluble ash, acid soluble ash and extractive values in chloroform, alcohol and water of the dried leaf powder were calculated in terms of air dried sample as shown in (Fig. 2). The physical constants evaluation of the drug is an important parameter for in detecting adulteration or improper handling of the drugs. The moisture content of the drug is not high (5.8%), thus it discourages bacterial, fungi or yeast growth, as the general requirement for moisture content in crude drug is not more than 14 %. Equally important in the evaluation of crude drugs, is the ash value and acid insoluble ash value determination. The total ash is particularly important in the evaluation of purity of drugs, i.e. the presence of or absence of foreign inorganic matter such as metallic salts or silica. Thus it appears that the plant A. macrophylla is useful in the traditional medicine. 128 M. S. Khyade et al. Table 1 : Quantitative microscopy Range Mean Stomatal frequency 30-49 35.3 Stomatal index 15-22 18.0 Vein islet number 21-30 26.0 Vein termination 16-22 19.0 Table 2 : Histological colour reactions Reagents Constituents Colour Histological Zone Degree of Intensity Aniline SO4 + H2SO4 Lignin Yellow Xy. ++ Weak Iodine solution Starch Blue Mesophyll + Sudan III / IV Fats Pink / Red Mesophyll + Dragendroffs reagent Alkaloids Turbidly Brown Mesophyll, M. cor. +++ Ba (OH)2 + K2Cr2O7 + CaCl2 Saponins Yellow Mesophyll, M. cor. ++ Fecl3 Tannins Blue green M. cor. + Vanillin + HCl Flavonoids Yellow M. col., Mesophyll, M. cor. +++ AgNO3 + H2O2 Crystals Black Mesophyll, M. col. ++ Note= M. cor. - Midrib cortex; Xy. - Xylem; Scl. - Sclerenchyma; M.col- Midrib collenchyma. Table 3 : Qualitative chemical analysis Phytochemicals Acubins / Iridoids ++ Alkaloids a) Dragendorff’s reagent +++ b) Mayer’s reagent +++ c) Wagner’s reagent +++ Anthraquinone -- Cardiac glycoside -- Coumarins -- Flavonoids +++ Leucoanthocyanins -- Phlobatannin ++ Reducing sugars + Simple phenolics ++ Steroids ++ Saponins + Tannins Test – a true tannin +++ Test – b pseudotannin + Terpenoid +++ 129 Journal of Herbal Medicine & Toxicology 130 M. S. Khyade et al. Phytochemical evaluation The results of qualitative phytochemicals are presented (Table III) and the values of quantitative phytochemicals obtained are presented in Fig 3. Different chemical compound such as iridoids, alkaloids, flavonoids, phlobatannin, phnenolics, steroids, saponins, tannins and terpenoids are detected in A. macrophylla, which could make the plant useful in treating different ailments and having potential for providing useful drug for human use. This is because the pharmacological activity of any plant is usually traced to particular compound. The presence of tannins and other phenolic compounds, which have antiseptic properties, could explain the use of this plant for the treatment of various 131 Journal of Herbal Medicine & Toxicology diseases. It has also been mentioned that the antioxidant activity of plants might be due to their phenolic compounds [24]. Flavonoids are a group of polyphenolic compounds with known properties which include free radical scavenging, inhibition of hydrolytic enzymes and anti-inflammatory action [25]. It should be noted that steroidal compounds are of important and interests in pharmacy due to their relationship with such compounds as sex hormones [26]. The saponin reduces the body cholesterol by preventing its reabsorption, increasing its excretion, thereby reducing blood pressure [27]. Standardization of herbal drugs is a topic of great concern. 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