Indian Journal of Pharmaceutical and Biological Research (IJPBR)

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1 Indian J. Pharm. Biol. Res. 2016; 4(1):7-18 CODEN (USA): IJPB07 ISSN: Indian Journal of Pharmaceutical and Biological Research (IJPBR) Original Research Article Microscopical evaluation of leaf of Maytenus emarginata Journal homepage: Bhupendra K. Kumawat 1*, Devendra S. Rathore 1, Mahesh K. Gupta 2, Tarachand 3, 1 NIMS Institute of Pharmacy, NIMS University, jaipur , Rajasthan, India. 2 Oriental College of Pharmacy and Research, Oriental University, Indore, M.P., India. 3 Regional College of Pharmacy, Jaipur , Rajasthan, India. ARTICLE INFO: Article history: Received: 30 December 2015 Received in revised form: 3 March 2016 Accepted: 15 March 2016 Available online: 30 March 2016 Keywords: Maytenus emarginata (Willd.) Ding Hou, Microscopical, Pharmacognostic, Powder Microscopy. ABSTRACT Microscopical investigation was carried out on leaves of Maytenus emarginata (Willd.) Ding Hou belongs to family celastraceae, is an evergreen tree that tolerates various types of stresses of the desert, locally known as Kankero in Hindi, Thorny staff tree in English. Literature survey revealed that not much work has been done on this plant, especially on leaves. So we have taken its detailed microscopical studies to prove its appropriate identification. Microscopical study provide information that the lamina is dorsiventaral and 380µm thick. The Lamina is amphistomatic, the stomata being distributed both on the adaxial and abaxial sides. Calcium oxalate crystals of druses are fairly abundant in the mesophyll tissue. The stomata are cyclocytic type. The venation is densely reticulate. The terminations are either simple or more commonly forked into deuse clusters. Powder Microscopy show that the sclereids are fiber like in shape and size. The sclereids are 500 µm long and 30 µm thick. Long or short, cylindrical mesophyll cells are common with powder. They have dense tannin contents. The cells have thin wall and the cells are up to 200 µm long and 60 µm wide. Calciumoxlate druses are quite abundant in the power. They are spherical bodies with spiny surface. The druses are 30 µm in diameters. The above studies provide useful information in regard to its correct identity, evaluation and help to differentiate from the closely related other species of Maytenus Emarginata (Willd.)Ding Hou. Introduction Maytenus emarginata (Willd.) Ding Hou belongs to family celastraceae, is an evergreen tree that tolerates various types of stresses of the desert, locally known as Kankero in Hindi, Thorny staff tree in English[1, 2]. It is found throught in India (Madhya Pradesh, Uttar Pradesh, Punjab, Maharashtra, Gujarat, Delhi, Bihar, Tamilnadu, and Rajasthan) [3]. The crude plant extracts of the celastraceae in traditional medicine and agriculture is astonishing, and includes stimulant, restorative, male contraceptive, anti-tumor, anti-leukemic, anti-bacterial, insecticidal and insect repellent activities[4]. Various parts of this plant contain immense medicinal properties[5]. Literature survey revealed that not much work has been done on this plant, especially on leaves. So we have taken its detailed microscopical studies to prove its appropriate identification. Pharmacognostical studies mainly include study of morphological characters, microscopical characters and powder microscopy. Microscopy method is an important tool in the evaluation of crude drugs which is applicable at various levels such as the authentification of the crude drugs, study of powdered drugs visualizing calcium oxalate crystals [6]. Microscopy method allows more detailed examination of a drug it can be used to identifying the organized drugs by their known histological characters. It is mostly used for qualitative evaluation of organized crude drugs in entire and powdered form [6]. Materials and methods Plant collection and identification Plant material The proposed study of the plant leaves of Maytenus emarginata (Willd.) Ding Hou were collected from uncultivated fields in and around the Village Maroth of Nagaur District, Rajasthan, India during july-august The Plant was identified from Department of Botany, University of Rajasthan, Jaipur and confirmed by compared with the help of herbarium maintained at the Department of Botany, * Corresponding Author: Bhupendra kumar Kumawat, NIMS Institute of Pharmacy, NIMS University, Jaipur, Rajasthan, India. bhupsa137@yahoo.com 7

University of Rajasthan, Jaipur. The voucher specimen (RVBL21072) was deposited and preserved in Herbarium Department of Botany, University of Rajasthan, Jaipur for further reference.

2 University of Rajasthan, Jaipur. The voucher specimen (RVBL21072) was deposited and preserved in Herbarium Department of Botany, University of Rajasthan, Jaipur for further reference. Taxonomical Identification The species for the proposed study was identified and authentified as Maytenus emarginata (Willd.)Ding Hou(Certificate no. PARC/2013/2065.) by Dr. P. Jayaraman, Botanist, Plant Anatomy Research Centre (PARC), West Tambaram, Chennai. Microscopical studies Collection of specimen Most care was taken to select the fresh leaf of healthy plant Maytenus emarginata (Willd.) Ding Hou. The required sample of fresh leaf with petiole was collected from the plant and fixed in FAA (Farmalin-5ml + Acetic acid -5ml + 70% Ethyl alcohol-90ml). 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 58-60ºC) until TBA solution attained super saturation. The specimens were cast into paraffin blocks[7]. Sectioning. The paraffin embedded specimens were section with the help of rotary Microtone. The thickness of the sections was μm. De-waxing of the sections was done and the sections were stained with Toluidine blue [8]. Since Toluidine blue is a polychromatic stain, the staining results were remarkably good; and some phytochemical reactions were also 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 were also stained with safranin and Fast-green and I 2 -KI (for Starch). Powdered materials of different parts were cleared with NaOH and mounted in glycerine medium after staining. Different cell component were studied and measured [9]. Photomicrographs Microscopic descriptions of tissues are supplemented with micrographs wherever necessary. Photographs of different magnifications were taken with Nikon Labphot 2 Microscopic Unit. For normal observations bright field 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 background. Magnifications of the figures are indicated by the scale-bars [10]. Powder microscopy First of all the leaves were washed with water and dried it in sunlight for one hour and then it was dried in shade. By the help of wood grinder the dried leaves were powdered and were passed through the sieve no. 60 for powder microscopy. The leaf powder is boiled with chloral hydrate for 5-10 minutes, and then stained with phloroglucinol, Toludiene and observed for the microscopic features under high power (40 x) [7]. Results Fig. No. 1: An exomorphic feature of Maytenus Emarginata (Willd.) fresh leaf Original Research Article 8

3 Fig. no. 2: T.S. of lamina (leaf of Maytenus Emarginata) Fig. no. 3: T.S. of lamina showing smooth even midrib ( Abs- Abaxial side, Ads- Adaxial side, Hd- Hypodermis, La- Lamina, Lv- Lateral vein, MR- Midrib, Ph- Phloem, PM- Palisade Mesophyll, SM- Spongy Mesophyll, St- Stomata, VB- Vascular Bundle, X- Xylem ) Original Research Article 9

4 Fig. no. 4: vascular strand of the midrib enlarged ( Abs- Abaxial side, Ads- Adaxial side, Hd- Hypodermis, La- Lamina, Lv- Lateral vein, MR- Midrib, Ph- Phloem, PM- Palisade Mesophyll, SM- Spongy Mesophyll, St- Stomata, VB- Vascular Bundle, X- Xylem ) Fig. no. 5: T.S. of Lamina enlarged Original Research Article 10

( AbE- Abaxial Epidermis, Abs- Abaxial side, AdE- Adaxial Epidermis, Cr- Crystal, LM-

5 Fig. no. 6: Crystal distribution in the lamina as seen under Polarized light. Fig. no. 7: T.S. of marginal part of the lamina. ( AbE- Abaxial Epidermis, Abs- Abaxial side, AdE- Adaxial Epidermis, Cr- Crystal, LM- Leaf Margin, Lv- Lateral vein, MT- Mesophyll Tissue, PM- Palisade Mesophyll, SM- Spongy Mesophyll, St- Stomata ) Original Research Article 11

Mesophyll, Sc- Subsidiary Cell, St- Stomata ) Original Research

6 Fig. no. 8: Druses in the mesophyll cell of the lamina. Fig. no. 9: Paradermal section of the lamina showing the stomata. (Cr- Crystal, Ec- Epidermal cell, Ep- Epidermis, PM- Palisade Mesophyll, Sc- Subsidiary Cell, St- Stomata ) Original Research Article 12

7 Fig. no. 10: Single stoma enlarged (Stained with safranin). Fig. no. 11: A stoma with cyclocytic subsidiary cells (Stained with toludine blue). (GC- Guard Cell, SC- Subsidiary Cell) Original Research Article 13

8 Fig. no. 12: Venation skeleton of the lamina. Fig. no. 13: Vein islet and Vein terminations enlarged. (VI- Vein Islet, VT- Vein Termination) Original Research Article 14

9 Powder Microscopy Fig. no. 14: A fibre sclereid found in the powder. Fig. no. 15: A fibre sclereid as seen under polarized light showing lignified walls and narrow lumen. Original Research Article 15

10 Fig. no. 16: A portion of the sclereid enlarged. (Scl- Sclereids, Sw- Secondary wall) Fig. no. 17: Paranchyma cells filled with tannin. (Cr- Crystals, Pa- Parenchyma) Original Research Article 16

The region where the midrib vascular bundle is located is slightly bent (Fig. no. 2,3). The lamina is dorsiventaral; bent the dorsiventaral symmetry is not well expressed.

11 Fig. no. 18: Calcium oxlate druses seen in the mesophyll tissues (Under polarized light). Fig. no. 19: Crystals enlarged. (Under polarized light) (Cr- Crystals, Pa- Parenchyma) Discussion Anatomy of the leaf The leaf is uniformly smooth and even and midrib is not distinct externally. The region where the midrib vascular bundle is located is slightly bent (Fig. no. 2,3). The lamina is dorsiventaral; bent the dorsiventaral symmetry is not well expressed. The leaf is 400mm thick both in the midrib region and in the lateral laminar region. Both the adaxial and abaxial epidermal layer are thin the epidermal cells are small, spindle shaped and thick walled. There is a subepidermal layer of cells both in the adaxial and abaxial sides; the subepidermal cells are similar to the epidermal cells (Fig. no. 3). The Lamina is amphistomatic, the stomata being distributed both on the adaxial and abaxial sides (Fig. no. 3) The vascular bundle of the midrib is bowl shaped and collateral. It consists of about 10 parallel lines of short xylem elements which are circular, narrow and thick walled along the lower end of the xylem strand occurs a thin arc of phloem elements (Fig. no. 4). on the upper and lower ends of the vascular bundle occurs vertical segment, thin walled, hyaline parenchyma cells. The mesophyll tissue consists of about 8 layers vertically oblong, cylindrical cells. All the cells appear to the palisade cells, the spongy parenchyma being lacking (Fig. no. 3). Most of the mesophyll cells are filled with tannin (Fig. no. 5). Calcium oxalate crystals of druses are fairly abundant in the mesophyll tissue. They diffusely distributed in tissue. The cells containing the druses are not modified in shape and size (6, 8) Leaf margin (Fig. no. 7) The marginal part of the lamina is blunt and semi circular. It is 380µm thick. The hypodermal layer is not evident in Original Research Article 17

12 themarginal part. The epidermis and the mesophyll tissues are not changed in the margin. Stomata (Fig. no. 9, 10, 11) The stomata occur both on the upper and lower epidermal layers. They are sinken below the epidermis. In surface view the epidermal cells appear small, polygonal in outline with straight and thick anticlinal walls. The stomata are cyclocytic type each stomata is surrounded by 4-6 rectangular subsidiary cells (Fig. no. 10, 11) the guard cells are µm in size. Venation (Fig. no. 12, 13) The venation is densely reticulate. The veins are thick and straight. The vein-islets are narrow, variable shape, size and in orientation. The vein boundaries are thick and straight. The vein-terminations are present in most of the vein-islets. The terminations are either simple or more commonly forked into deuse clusters (Fig. no. 13). Powder Microscopy Sclereids (Fig. no. 14, 15, 16) Foliar Sclereids are abundant in the leaf powder. The sclereids are fiber like in shape and size. They are located in all the veins and are parallel to the veins. The sclereids are long, narrow with pointed ends. They have very thick lignified secondary walls and narrow lumen. The sclereids are 500 µm long and 30 µm thick. Mesophyll cells (Fig. no. 17) Long or short, cylindrical mesophyll cells are common with powder. They have dense tannin contents. The cells have thin wall and the cells are up to 200 µm long and 60 µm wide Druses (Fig. no. 18, 19) Calciumoxlate druses are quite abundant in the power. They are spherical bodies with spiny surface. The druses are 30 µm in diameters. Conclusion The leaves of Maytenus emarginata (Willd.)Ding Hou belonging to family Celastraceae was selected for Microscopical study, on the basis of Literature survey which revealed that not much work has been done on this plant, especially on leaves. So we have taken its detailed microscopical studies to prove its appropriate identification. The above studies provide useful information in regard to its correct identity, evaluation and help to differentiate from the closely related other species of Maytenus Emarginata (Willd.)Ding Hou. The above studies also provide proper future identification of the plant, and serves as a standard monograph for identification and evaluation of plant. The determination of these characters will help future researchers in phytochemical as well as a pharmacological analysis of this species. Conflict of interest: We declare that we have no conflict of interest. Acknowledgement The author sincerely thanks to help render by Prof. P. Jayaraman, Director, PARC, Chennai for providing all the facilities and help during Microscopical study. References 1. Kirtikar K.R., Basu B.D., Indian medicinal plants. Lalit Mohan Basu Publications Allahabad; Second edition, Vol. 1: Nair, N.C & Henry A.N., Flora of the Tamilnadu, India, Vol. I, 1983: Sagwan S., Rao D.V., Sharma R.A., Maytenus Emarginata (willd.); A promising drug for cancer therapy, Asian J Pharm Clin Res, 2011; 4(3): 9:12 4. Pullaiah T., Encyclopedia of world medicinal plants, Sal. Paratyphi, Regency Publication, New Delhi, Ed 1 st Sher mohammed, kasera P. K. and shukla J. K., Unexploited plants of potential medicinal value from the Indian Thar desert, Natural Product Radiance, 2004: 3(2): Kokate C.K., Practical Pharmacognosy, 4 th edition, 2001, Vallabh Prakashan, Delhi;107: , , Sass, J.E., Elements of Botanical Microtechnique, Mc Graw Hill Book Co; New Yark, 1940: O Brien T.P., Feder N. and Mc Cull M.E., Polychromatic Staining of Plant cell walls by Toluidine Blue-O; Protoplasma; 59: Johansen D.A., Plant Microtechnique; Mc Graw Hill Book Co. Newyork : Easu, K., Plant Anatomy; John Wiley and Sons; New York: 767. Cite this article as: Bhupendra K. Kumawat, Devendra S. Rathore, Mahesh K. Gupta, Tarachand. Microscopical Evaluation of Leaf of Maytenus emarginata. Indian J. Pharm. Biol. Res.2016; 4(1):7-18. All 2016 are reserved by Indian Journal of Pharmaceutical and Biological Research This Journal is licensed under a Creative Commons Attribution-Non Commercial -Share Alike 3.0 Unported License. This article can be downloaded to ANDROID OS based mobile. Original Research Article 18

CHAPTER Introduction

CHAPTER Introduction 27 CHAPTER 2 2.1 Introduction Pharmacognosy as a subject of pharmaceutical curriculum focused on those natural products employed in the allopathic system of medicine. Coincident with the increasing attractiveness