International Journal of PharmTech Research CODEN (USA): IJPRIF ISSN : 0974-4304

Vol.6, No.1, pp 244-251, Jan-March 2014

Anatomical Investigation on the leaves of Piper betle (L) var. Sirugamani 1(SGM1) links an Ethnomedical important

Medicinal plant and its Pharmacognostic relevance.

M Mubeen1*, K Periyanayagam1, S Sathik Basha2

1Department of Pharmacognosy, College of Pharmacy, Madurai Medical College, Madurai - 625020, TamilNadu, India.

2Department of Physics, B. S Abdur Rahman University, Vandalur,

Chennai -600048, TamilNadu, India.

*Corres.author: mub_sb@yahoo.com, Phone: +91 9444793802

Abstract: Piper betle L. var. Sirugamani(SGM1) (Piperaceae) is a deep green heart shaped leaves have many uses in Indian and Chinese ethnomedicine. In this study we aim to establish the pharmacognostic profile of betel leaves to assist in the standardization for quality, purity and for the identification of sample. The fresh and dried leaves were evaluated to determine the macro and micromorphological characters using light and Scanning Electron Microscope (SEM) in which usual leaf tissues, cyclocytic stomata in lower epidermis and apostomatic upper epidermis, glandular trichomes (pearl glands), secretory cells were observed. The leaves were also subjected to elemental analysis by an Energy Dispersive X-ray Spectrometer (EDS or EDAX) which was in connection to the SEM. EDS results showed higher potassium content and thus the leaves may be useful in the maintenance of electrical excitability of nerves and muscles. Keywords: Piper betle, betel leaf, Scanning Electron Microscope, Energy Dispersive X-ray Spectrometer.

Introduction

Piper betle (Synonym: Piper peepuloides Wall and Piper chavya Ham; Eng betel leaf Hindi: Pan, Tambuli; Tamil: Vettilai;) Family – Piperaceae is a perennial, dioecious, creeper, probable native of Malaysia, cultivated in India for its leaves used for chewing. There are about 100 varieties of betel vine in the world, of which about 40 are found in India and 30 in West Bengal[1]. In spite of its alienness, the plant is much more popular in India than in any other country of the world since the antiquity. Betel leaves from different regions vary in smell and taste. The leaf was used in Peninsular Malaysia for masticatory and also for relieving constipation in children and poulticing ulcerated noses. Leaves were often heated and applied to the chest to relieve cough and asthma[2]. The leaves were rolled up, covered with oil as a suppository and as purgative in new borns[3]. Orally used as aphrodisiac[4].The leaves contain good amount of B vitamin (particularly nicotinic

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acid), ascorbic acid and carotene. The volatile oil from the leaves was used as gargle or inhalation in diphtheria and respiratory catarah[5]. The scanning electron microscopy on the leaves have shown the inhibitory effect of betel leaves extract against S.mutans[6]. P.betle a green healer from backyard traditionally used in various diseases including brain and heart. It is also used as a stimulant, masticatory, antiseptic and wound healing[7][8].

The commonly available varieties in TamilNadu, India are namely Karpoori, Patchaikodi, Sirugamani (SGM). The widely available and used P.betle L. var. Sirugamani 1(SGM1) was taken in our study to report on the anatomical profile with the aim of exploring the diagnostic value in order to distinguish the taxa and to help commercialization of its medicinal uses. Microscopical prospective is an integral component of pharmacognosy especially while proposing diagnostic protocols for establishing botanical identity and ascertaining the quality control of raw material[9]. The current study was also designed to investigate the elemental contents (C, O, Mg, P, S, Cl, K, Al, Ca, Si and Fe) of P.betle leaf using variable pressure scanning electron microscope microanalysis (EDS). Hence it helps in the standardization of this herb but also guarantee the quality, purity and to prepare a monograph for the proper identification of this plant.

Experimental

All chemicals used were s.d. Fine Chem. Ltd., Mumbai; AR grade.

Fresh betel leaves were collected in the month of July 2010 locally from Solavandan village, Madurai district (South TamilNadu). Piper betle (L) Sirugamani 1(Piperaceae) was authenticated by Dr.R.Arulmozhian Professor-Horticulture, AC&RI, TamilNadu Agricultural University, Tiruchirapalli, TamilNadu, India.Voucher herbarium Specimen (PCG-1/299) was prepared and is preserved at the herbarium of College of Pharmacy, Madurai Medical College, Madurai. Pharmacognostical evaluation including histochemical and macerate were carried out by standard procedure[10][11][12]. Safranin 4% in 50% alcohol was used to stain transverse section of leaf and petiole. Reagents like potassium iodide, ferric chloride, Sudan III, concentrated HCl, ruthenium red and phloroglucinol with dilute HCl were used for histochemical tests. Concentrated nitric acid (50%) with pinch of potassium chlorate crystals was used as the macerating fluid. Photomicrographs were obtained by observing drug under compound binocular microscope (Laboscope model) with built in analogue camera (Canon Power Shot model A430, 4.0 mega pixel).

The dried leaves for SEM analysis of (1x1 mm) were mounted on the specimen stub using fevicol adhesive. Small leaf sample was mounted directly on scotch double adhesive tape. Samples were coated with gold to a thickness of 100 AO using Hitachi vacuum evaporator. Samples were examined at an accelerating voltage of 20 kV at high vacuum mode. Coated sample were analysed by Joel Scanning Electron Microscope model JSM-5610 LV with an EDS microanalysis attachment. When the leaf was bombarded by the electron beam of the SEM, electrons were ejected from the atoms on the specimens surface and microanalysis was carried out. The EDS consist of X-ray detector, a pulse processor and multiple channel analyser. The EDS X-ray detector measures the number of X - rays emitted versus their energy. A spectrum of the energy versus relative counts of the detected X-rays is obtained and evaluated for the determinations of the elements[13][14].

Results and discussion

Macro and microscopic characteristic of the leaf and petiole

Leaves are alternatively arranged and slightly cordate. About 5-7 veins are arising from the bases to the tip. The leaves are 12-18 cm long and 9-11 cm broad. The leaves are light green and glabrous (Figure 1a). It has an entire margin and also with an often undulated margin. The apex of leaf is acuminate with often unequal base. The petioles are stout and are 5.5-6.5 cm long (Figure 1b). The leaves have aromatic odour and pungent taste. The transverse section of the leaf is dorsiventral, mesomorphic, with fairly prominent midrib. The leaf midrib is semicircular in shape with even outline. The adaxial side is flat with circular secretory canal and small secretory cells, while the abaxial side is hemispherical and has small secretory cells with dark contents. The epidermis of midrib is thin and has spindle shaped cells with smooth surface. Inner to the epidermis is a narrow zone of 2 or 3 layers of collenchyma cells. Vascular bundles are single ovate collateral with cluster of xylem elements and thick arc of phloem (Figure 2).

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The lamina of leaf is smooth with even surface (Figure 3a). It has thickness of 160-170µm with adaxial epidermis of 20µm thickness and abaxial epidermis of 12 µm thickness with short cylindrical finger like glandular trichomes (pearl glands). These glandular trichomes are 30µm long and 5µm thick (Figure 3b). The abaxial epidermis is stomateferous while adaxial epidermis is apostomatic. The stomata are cyclocytic type (Figure 4a,4b). They have single row of short, wide vertically oblong palisade cells and spongy mesophyll of 3 or 4 layers with secretory cells of 25-30 µm in diameter (Figure 5). The petiole is roughly triangular and outline has deep furrows and ridges. Inner part of the ridges is collenchymatous (Figure 6). SEM showed pearlglands, cyclocytic stomata in lower epidermis of leaf and subsidiary cells (Figure 7a,7b). The petiole showed numerous covering trichomes (Figure 7c).

Figure 1a: Habit of Piper betle (L) var.SGM1

Figure 1b: Dorsal surface of leaf

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Figure 2: T.S of leaf through midrib.

Figure 3a: Transverse Section of Lamina

Figure 3b: Transverse Section of Lamina showing glandular trichome

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Figure 4a: Abaxial epidermis showing stomata

Figure 4b: Apostomatic adaxial epidermis

Figure 5: Paradermal section showing secretory cells in the mesophyll

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Figure 6: T.S of Petiole- a portion enlarged

Figure 7 a: SEM study- Lower epidermis showing pearl gland

Figure 7 b: SEM study- Lower epidermis showing stomata

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Figure 7 c: SEM study- Petiole showing trichomes

Microelemental analysis by EDS

Apart from carbon and oxygen the plant contains inorganic elements like Mg, Si, Cl, K, Ca, Zn, were present in which the potassium occupies the maximum weight percent (Table 1, Figure 8). Hence the leaves can be used in the correction of imbalance of acid base metabolism in humans and also may be useful in the maintenance of electrical excitability of nerves and muscles[15].

Table 1: Energy dispersive X-ray analysis on the leaf of Piper betle(L) var.SGM1

Figure 8: Elemental spectrum by Energy Dispersive Spectrometer of the leaf of Piper betle (L) var.SGM1

Elements Weight (%)

Mg 7.29 Si 18.55 Cl 8.22 K 48.06 Ca 7.72 Zn 9.56

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Conclusion

The above findings in this account provides first detailed descriptions of anatomy of the leaves useful to supplement existing information with regard to the identification of Piper betle L. var.SGM1even in the powdered form which distinguish from substitutes and adulterants.These pharmacognostic and physicochemical evaluation of P.betle leaves can be used for further research and revalidation so that the vast economic potentiality of this crop can be adequately established by its consumption which can create employment opportunity to an agricultural worker throughout the year.

“Authors: All research done by the authors”;

“Conflict of interest: none”

Acknowledgement

Dr. P. Jayaraman, Director of Plant Anatomy Research Institute, Tambaram, Chennai, TamilNadu, India for the histochemical studies on leaves and The Department of Physics, Annamalai University, Chidambaram, TamilNadu, India for SEM and EDS are gratefully acknowledged.

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