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Pharmacognosy Journal 5 (2013) 176e183

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Pharmacognosy Journal

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Original article

Pharmacognostic specifications of eight cultivars of Piper betlefrom eastern region of India

Atiya Akhtar Khan, Satyendra Prasad Bhatnagar, Barij Nayan Sinha, Uma Ranjan Lal*

Department of Pharmaceutical Sciences, Birla Institute of Technology, Mesra, Ranchi 835215, Jharkhand, India

a r t i c l e i n f o

Article history:Received 22 May 2013Accepted 17 July 2013Available online 29 August 2013

Keywords:Piper betle cultivarsPharmacognosyMicroscopicMacroscopicPhysiochemical

* Corresponding author.E-mail addresses: uma@bitmesra.ac.in, urlalvv@ya

0975-3575/$ e see front matter Copyright � 2013, Phhttp://dx.doi.org/10.1016/j.phcgj.2013.07.002

a b s t r a c t

Background: Piper betle L. is a well-known and widely used herb especially in tropical Asiatic countries,which contains several bio-active constituents and possesses health promoting properties. The leaves ofPiper betle L. have been traditionally known for its various therapeutic uses. The pharmacognostical andphysicochemical investigations have not been carried out for eastern Piper betle cultivars.Objective: The present investigation was therefore undertaken to determine the requisite pharmacog-nostic standards for their evaluation to ensure their safety and efficacy.Materials and methods: Different Piper betle L. cultivars (Desi, Bangla, Kali, Haldia, Sanci, Meetha, Birkoliand Magahi) were obtained from Indian States of West Bengal, Odisha, and Bihar (Table 1).Fresh leaves were washed with water, sliced with a microtome and the transverse sections were stainedwith safranin, phloroglucinol, concentrated hydrochloric acid and mounted in glycerine medium. Leavesdried in the shade were finely powdered and passed through a sieve no.180 and a sieve no.125, sepa-rately, to obtain fine powder respectively and then subjected to microscopic examination.Mesophyll tissue is divided up by slendest vein, which is called vein islet. The numbers of vein islet inevery epidermal cell is called Vein Islet Numbers (VIN); Vein termination number is the number ofveinlet termination per square mm of the leaf surface midway between the midrib and the margin (VTN).Since vein islet and vein termination number is consistent and different from plant to plant, thisparameter can be used in authentication. Stomatal Index (SI) is a basilic parameter in authentication,which can be obtained from the following formula: Stomatal Index ¼ (number of stomata/every milli-meter-square) � 100/(number of stomata/every millimeter-square þ number of epidermal cell/everymillimeter - square), which also can be used as a reliable parameter in authentication since it isconsistent in a species of plant. The results were studied using light microscope according to the usualmicroscopic techniques. Different physicochemical parameters such as ash value, extractive value andloss on drying were carried out as per WHO guidelines.Results: In present study eight cultivars of Piper betle were compared with their distinguishing morpho-anatomic details of leaf, dissociation and powders. The microscopic features were systematicallydescribed and illustrated. The physiochemical properties such as loss on drying, total ash value, acidinsoluble ash value, water soluble ash value and extractive values were also carried out. HPTLC analysis ofpetroleum ether extract and essential oil showed confirming the presence of compound in Piper betlecultivars leaf. And detailed key authentication parameters based on these anatomic characteristics werepresented. The various morphological, microscopical, physicochemical standards can be unambiguouslyused to authenticate standardization and distinguish eight cultivars of Piper betle.

Copyright � 2013, Phcog.Net, Published by Reed Elsevier India Pvt. Ltd. All rights reserved.

1. Introduction

Piper betle Linn., (Piperaceae) is an aromatic, shade lovingclimber cultivated mainly in India and other tropical Asiatic

hoo.com (U.R. Lal).

cog.Net, Published by Reed Elsevi

countries (Bangladesh, Pakistan, Malaysia and Indonesia etc).1 Thebetel leaf is known as Paan in India.2 There are more than 700species of plants belonging to the genus Piper, out of which 30species are found in India.3 It is consumed as betle quid with arecanut (Areca catechu), katha (Acacia catechu L.), slaked lime [Ca(OH)2]cardamom (Elettaria cardamomum), clove (Eugenia caryophyllus),etc. which act as a breath freshener with or without tobacco.4,5 Thebetle vine cultivars vary, besides in morphological characters, in

er India Pvt. Ltd. All rights reserved.

Table 1Collection data for samples and morphological characters of Piper betle cultivars.

Cultivars District State Colour Length(cm)

Width(cm)

Haldia Ram Nagar West Bengal Light green 12e14 8.0e10Bangla Midnapur West Bengal Dark green 16e19 14e16Desi Midnapur West Bengal Green 12e14 5.0e7.0Sanci Parganas,

HalisaharWest Bengal Dark green 20e23 15e16

Meetha Midnapur West Bengal Dark green 14e16 10e11Kali Machida West Bengal Dark green 17e20 14e17Magahi Nevada Bihar Green to light green 7.0e9.5 5.5e6.5Birkoli Nayagarh Orissa Greenish yellow 9.0e11 5.0e7.0

A.A. Khan et al. / Pharmacognosy Journal 5 (2013) 176e183 177

flavour and in the aroma of the leaves and the flavour and taste ofthe leaves are due to the essential oil composition.6 Investigationson the Piper betle showed a number of pharmacological propertieslike anti-oxidant,7 antifungal,8 anti-microbial,9 anti-inflamma-tory10 and radio protective11 etc. Phytochemical investigation onleaves revealed the presence of tannins and steroidal compo-nents.12 The terpenoids and phenylpropanoids reported in Piperbetle are 1,8-cineole, cadinene, camphene, caryophyllene, limo-nene, pinene, chavicol, 4-allyl-pyrocatechol, carvacrol, safrole,eugenol, and chavibetol.13,14 Phytochemical screening studies onPiper betle leaves have revealed the presence of flavonoids, sapo-nins, mucilage, reducing sugars, carbohydrates, glycosides, proteinand amino acids and absence of alkaloids and fixed oil.15 There is agood level of traditional and experimental evidences to supportvarious claims and advantages of this widely available plant.Despite its importance, only a few comparative morpho-anatomicaland physicochemical studies have been done on the leaves of Piperbetle.15e19 But our selected eastern cultivars have not beenexplored. Hence the present study was undertaken to explore themfor standardization purpose. In eastern India, Piper betle L. cultivarslike Bangla, Kali, Desi, Haldia, Meetha, Sanci, Magahi and Birkoli arewidely cultivated. In present work we report macroscopic evalua-tion, microscopic evaluation, physicochemical parameters andpreliminary phytochemical screening for standardization andquality assurance purposes of these cultivars from eastern region ofIndia.

2. Materials and methods

2.1. Collection and authentication of plant materials

Fresh leaves of eight cultivars of Piper betle were collected fromeastern region of India (West Bengal, Bihar, and Odisha) inNovember, 2010 (Table 1). Plant leaves were authenticated by theCentral National Herbarium, Botanic Garden Howrah Kolkata, India.A voucher specimen has been deposited (CNH/10B/2011/Tech.II/502). The plants were also authenticated by Dr. (Mrs.) M. N.Naganandini, Department of Pharmacognosy, JSS College of Phar-macy, Mysore, India.

2.2. Macroscopic analysis

Details macroscopic and taxonomic features of the leaves viz.size, shape, colour, surface, venation, apex, margin, base, laminaand texture were studied according to the standard proceduresmentioned in standard textbooks.20,21

2.3. Leaf constants

Fresh leaves were washed and small fragments of leaves weretaken from the middle region of the lamina of mature leaves.

Cleaning of leaf was done to study the venation pattern. Washedleaf fragments were first boiled in 90% alcohol for about 3e5min toremove chlorophyll, and then washed 2e3 times with water, thenboiled again with 10% KOH solution for 2e3 min and washed 4e5times with water. The epidermal layer was peeled off using the helpof pointed needle and forceps and was washed in water. The mar-gins of the cover slip were sealed with DPX (Hi-Media laboratoriesPvt Ltd, Bombay), and the slides were observed under the micro-scope. Stomatal index, palisade ratio, vein termination number andvein islet number were then calculated.22

2.4. Anatomy

For microscopic studies cross section was prepared as per thestandard procedures.23 Wherever necessary, sections were stainedwith safranin, phloroglucinol, concentrated hydrochloric acid andmounted in glycerine medium.24,25 Microphotographs were takenusing Leica DME, DSC Power shot S70 DE [PICT Bridge], 7.1 mega-pixels, wide angle 3.6� optical zoom lens.

2.5. Powder studies

Leaves dried in the shade were finely powdered and passedthrough a sieve no.180 and a sieve no.125, separately, to obtain finepowder respectively and then subjected to microscopic examina-tion. The sample was treated with following reagents and studiedfor their components of diagnostic value, glycerine (50%) as tem-porary mountant phloroglucinol (2% w/v) in ethanol (90%), conc.HCl (50%) for lignin; alcoholic ferric chloride (5% w/v) for phenoliccompounds; Iodine (I-/KI) solution (2%) for starch grains; andruthenium red (0.08%) in lead acetate (10%) for mucilage andobservation was recorded.26,27

2.6. Physicochemical parameters

Physicochemical values such as the percentage of ash values,loss on drying (moisture content) and extractive values were per-formed according to official methods prescribed (Indian Pharma-copoeia, 1996)28 and the WHO guidelines on quality controlmethods for medicinal plant materials (WHO guidelines, 1992).29

2.7. Preliminary phytochemical screening

The shade dried and coarsely powdered leaves were extractedsuccessively with different solvent by using soxhlet apparatus andanalysed using simple chemical tests for preliminary phytochem-ical screening of various groups of phytoconstituents such as al-kaloids, flavonoids, phenolic acids, sterols, cardiac glycosides,tannins and so on by using standard procedure.30,24

2.8. HPTLC studies

Qualitative densitometric analyses (HPTLC) was performed forthe development of characteristic fingerprinting profile for petro-leum ether (60�e80�) extract and essential oil of leaf of Piper betlecultivars. 10 ml of the sample solution (10 mg/mL for petroleumether extract; 5% solution in petroleum ether for essential oil) wasapplied and plates were developed in solvent system [(hexane:ethyl acetate; 8.8:1.2) for petroleum ether extract, (toluene: ethylacetate; 7:3)] for essential oil. Developed plates were scanned atvarious wavelengths.

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3. Results and discussion

3.1. Macroscopic characters

The detailed macroscopical description was studied and shownin (Table 1) (Fig. 1).

3.2. Leaf constants

Quantitative microscopy is an important tool for identifyingcharacteristic of leaf, stomatal number, vein islet and vein termi-nation numbers are present in (Table 2) and (Fig. 1a, b and d).

Fig. 1. Different Piper betel L. cultivars, Adaxial view and leaf constant measurement photogra,b) Apostomatic and hypostomatic tetra-cyticstomatal complex, face view of the adaxial andcrystal, Balloon like epidermal cells; d) Vein termination, vein islet and f) Conjugation tube likEpidermal cells; RC) Rosette crystals; BE) Balloon like epidermal cells; VT) Vein terminatio

3.3. Anatomy

The comparative anatomy of eight cultivars of Piper betleshowed structural similarities (Fig. 2). All the cultivars of Piper betleleaves shows thick cuticle on the upper epidermis and thin on thelower epidermis. Piper betle leaves showed barrel shaped fourlayered upper and two layered lower epidermis (8m� 4m) (Fig. 2a, gand i). Sclerenchymatous cells for support present above theschizogenous secretory canal (Fig. 2a and b). The Schizogenoussecretory canal on the adaxial surface was present in all the culti-vars of the species (Fig. 2a). Distinct phloem tissue can be seen onthe ventral surface and well developed xylem tissue towardsthe dorsal surface of the midrib. Xylem consist of tracheids

aphs; A) Desi; B) Bangla; C) Kali; D) Haldia; E) Sanci; F)Meetha; G) Birkoli and H)Magahi;abaxial sides of the epidermis (40�), epidermal cells, subsidiary cells; c,e) Rosette typee structure, glandular trichomes. Abbreviations: ST) Stomata; SBC) Subsidiary cells; EC)n; VI) Vein islet; COS) Conjugation tube like structure and GT) Glandular trichome.

Fig. 2. Transverse section of leaf of different Piper betle cultivars; A) Magahi; B) Meetha; C) Sanci; D) Haldia; E) Kali; F) Bilkori; G) Bangla and H) Desi. Enlarged view of transversesection of leaf of Piper betle figure 2G): a, b) Showing the presence of SC e Sclerenchymatous cells; SZ e Schizogenous secretory canal; T e Trichomes; UE e Upper epidermal cells(Two to three layer); c) Showing the presence of tracheoid idioblasts like structure; MXY e Metaxylem; PXY e Protoxylem; PH e Phloem with SBS e Sclerenchymatous bundlesheath; CVB e Collateral vascular bundle and V e Vessels; d) UPP e Upper palisade parenchyma, LPP e Lower palisade parenchyma; OC e Oil cell; e) SPF e Sclerenchymatouspericycle fibre; f) OC e Oil reserves/oil cells, CO e Collenchymatous cells, PO e Prism of calcium oxalate; g) LE e Lower epidermal cells (side view), TT e Tector trichomes; h) PC/G e

Polygonal Parenchymatous cells or ground tissue with oil cell; and i) LE e Lower epidermal cells (one to two layer), ground tissue/Parenchymatous cells.

A.A. Khan et al. / Pharmacognosy Journal 5 (2013) 176e183 179

(3.96e10.45 m), xylem parenchyma, protoxylem, and metaxylemtowards lower periphery. One conjoint collateral vascular bundle isencircled with parenchymatous cells with sclerenchymatousbundle sheath (Fig. 2c). Tracheoid idioblasts like structure have

been noticed from the vessels elements of midrib and petiole(Fig. 2c). It is also characterized by presence of polygonal paren-chymatous cells with oil cell was observed (Fig. 2h). The leaves arehypostomatic, tetra-cyticstomatal complexes are commonwhich is

Fig. 3. Powder characters showing the presence of ST) Stomata (40�, 100�); OC/OR/SC) Oil cells/oil reserves/ secretary cells; ROC) Ruptured oil cells; LEC) Lower epidermal cells;UEC) Upper epidermal cells; VE) Spiral vessel element; SVF) Spiral vessel fibre; PO) Prism of calcium oxalate and TOC) Translucent oil cells. Multicellular glandular trichomes (40�)of Piper betle L. cultivars a) Meetha; b) Magahi c, d, e) Sanci, f,g) Desi; h,i) Bangla; j,k) Bilkori; l,m) Kali and n) Haldia. Uniseriate (r, v) and multicellular tector trichomes (o, p, q, s, t, u).

A.A. Khan et al. / Pharmacognosy Journal 5 (2013) 176e183180

the characteristic feature of the Piperaceae.31,32 Hypostomatictetra-cytic stomata in lower epidermis and apostomatic in upperepidermis of Piper betle leaves (Fig. 1a and b). Some times stomatalcomplexes are cyclocytic but twin stomata and just aposed type are

also seen (Fig. 1c 10�) and (Fig. 1e 100�). Hypostomatic, tetra-cyticstomatal complexes in lower epidermis and apostomatic inupper with heterogenous mesophyll, were previously reported.They have a multiple epidermis in both the adaxial and abaxial

Table 2Leaf constant of leaves of Piper betle cultivars {Mean � SD}.

Cultivars Stomatal no. Stomatal index Vein termination no. Vein islet no.

Magahi 19 � 1.6a 27 � 3.8 11 � 1.9 6.0 � 1.1Haldia 16 � 2.4 21 � 2.1 12 � 3.0 5.0 � 1.0Bangla 9.0 � 1.6 25 � 2.0 15 � 3.9 7.0 � 1.0Desi 12 � 1.8 20 � 0.9 10 � 1.4 6.0 � 0.5Sanci 17 � 1.8 29 � 1.4 13 � 1.9 5.0 � 0.9Birkoli 12 � 1.8 24 � 1.4 11 � 3.2 6.0 � 0.8Meetha 17 � 1.2 19 � 1.4 20 � 2.2 6.0 � 1.1Kali 10 � 1.2 28 � 1.4 14 � 2.1 7.0 � 1.1

a Each value is the mean � standard deviation of triplicate determinations.

Table 3Moisture & Ash values of leaves of Piper betle cultivars {Mean � SD}.

Cultivars Moisture content Total ash Water soluble ash Acid insoluble ash

Magahi 3.1 � 0.1a 3.0 � 0.1 1.3 � 0.1 0.2 � 0.05Haldia 4.4 � 0.2 2.0 � 0.1 1.7 � 0.15 0.8 � 0.1Bangla 6.0 � 0.3 2.8 � 0.1 1.0 � 0.1 0.3 � 0.1Desi 11.0 � 0.1 3.2 � 0.1 1.2 � 0.15 0.7 � 0.1Sanci 5.5 � 0.3 3.6 � 0.3 1.1 � 0.15 0.5 � 0.2Birkoli 7.0 � 0.1 2.3 � 0.1 1.4 � 0.15 0.1 � 0.05Meetha 8.0 � 0.2 2.8 � 0.3 1.9 � 0.1 0.5 � 0.15Kali 8.5 � 0.3 2.9 � 0.2 1.3 � 0.1 0.4 � 0.1

a Each value is the mean � standard deviation of triplicate determinations.

Table 4Extractive values of leaves of Piper betle cultivars with successive extraction.(Soxhlation) {% (w/w) � SD}.

Cultivars Pet-ether(60e80 �C)

Ethyl acetate Chloroform Methanol

Magahi 2.8 � 0.1a 3.5 � 0.15 4.0 � 0.15 2.1 � 0.1Haldia 3.0 � 0.26 4.4 � 0.17 2.1 � 0.15 9.4 � 0.20Bangla 4.6 � 0.15 2.2 � 0.15 13 � 0.1 12.4 � 0.25Desi 3.2 � 0.15 15.4 � 0.25 2.0 � 0.1 8.1 � 0.15Sanci 2.0 � 0.15 1.0 � 0.15 5.2 � 0.4 14.5 � 0.35Bilkori 2.8 � 0.3 7.0 � 0.15 5.6 � 0.4 7.5 � 0.30Meetha 4.2 � 0.15 6.1 � 0.15 3.7 � 0.15 2.5 � 0.11Kali 4.9 � 0.4 4.4 � 0.15 2.6 � 0.1 12.4 � 0.26

a Each value is the mean � standard deviation of triplicate determinations.

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surfaces with secretory glandular trichomes and tector trichomesrespectively15,18,19 Conjugation tube like structures is present be-tween the twin stomata in Sanci cultivar (Fig. 1f). The apical cell oftrichome is slightly pointed or clavately shaped are multicellularand uniseriate and are more restricted in the midrib and petioleregion (Fig. 2b, g and i; Fig. 3oev). Cultivar Magahi leaves has morestomatal number (Table 2) and trichome frequency (Fig. 2A)

Table 5Behaviour of powdered leaves on treatment with different chemical reagents.

Reagents Bangla Magahi Haldia Desi

1N NaOH (aq.) Brown Brown Brown Brown1N NaOH (alk.) Dark brown Dark brown Dark brown Dark brownH2SO4 (dil.) Yellow Pale yellow Brown Light brownHCl (dil.) Buff colour Light buff Light buff Buff colourHCl (Conc.) Dark brown Brown Dark brown Dark brownAcetic acid Olive green Light green Dark brown Olive green5% Fecl3 Brownish yellow Yellow Yellow Brownish yell5% Iodine Maroon red Maroon red Blood red Maroon redAmmonia Dark brown Dark brown Brownish green Dark brown1N HNO3 Yellow Dark yellow Brick red Yellow

Abbreviations: aq) Aqueous; alk) Alkaline; 1N) Normal; dil) Dilute; Conc.) Concentrated

frequency than the other cultivars. All the cultivars of the speciesstudied have a heterogenous mesophyll with a single palisade pa-renchyma layer (Fig. 2d) and three to four compactly arrangedspongy parenchyma is also observed. Themargin is similar in all thecultivars studied where the sub-epidermal cell layer is collenchy-matous at the midrib (Fig. 2f). Calcium oxalate crystals are mostlyprismatic types (Fig. 2f and 3L) and oil reserves (Fig. 2d, f and h;Fig. 3C, D, F, I and K) are observed in the cortical collenchymas andin parenchyma regions of the petiole.

3.4. Powder study

The most common features of powder of leaves show presenceof glandular trichomes which have uniseriate and multicellularapical cell and a short pedicel (Fig. 3aen) are found in the subepidermal cells on both sides. Non-glandular trichomes (tectortrichomes) are multicellular and uniseriate are more restricted atthe midrib and petiole regions (Fig. 3oev). Oil reserves {yellow(Fig. 3I, F), reddish yellow (Fig. 3K) and red in colour (Fig. 3C, D)found in the cortical collenchyma and in parenchyma regions of thepetiole. The powder study of leaves also shows the presence ofruptured oil cells (Fig. 3E), epidermal cells (Fig. 3F, H), xylem vesselelement (Fig. 3J), stomata (Fig. 3A and B), spiral xylem fibers(Fig. 3G) and prismatic calcium oxalate monocrystals with a pyra-midal base occur in parenchymatous and collenchymatous petiolecells. (Figs. 2f and 3L). Balloon like epidermal cells and rosette typecrystals are observed (Fig. 1C). These characteristics are observedand are reported for the first time in these cultivars.

3.5. Physicochemical parameters

Physicochemical parameters like percentage of moisture con-tent, total ash, acid insoluble ash, water soluble ash values weredetermined. Ash values of a drug give an idea of the earthy matteror the inorganic composition and other impurities present alongwith the drug. Piper betle cultivars showed lower content of watersoluble and acid insoluble ash followed by total ash. Materialscontain free water, the relative amount being dependent uponthe physical and chemical properties of the material. The dryingprocess stops the degradation processes caused by yeasts or en-zymes, prevents the development of microorganisms and oxida-tion and hydrolysis reactions. The moisture content in fresh plantsleaves varies from 60% to 80%. The drying process reducesthe moisture content to 5%e12%. In Desi cultivar moisture contentwas high 11.0 � 0.1 followed by other cultivars (Table 3). Theextractive values are primarily useful for the determination ofexhausted or adulterated drug. The water soluble extractive waslow. (Table 4).

Sanci Bilkori Meetha Kali

Brown Brown Brown BrownDark brown Dark brown Dark brown Dark brownDirty white Pale Buff colour BuffBlackish colour Buff colour Buff colour Off whiteStraw colour Yellow Brownish Green Dark greenLight green Light green Light green Green

ow Yellowish brown Yellow Yellow Yellowish greenDark reddish Maroon Blood red Maroon redBrown Brownish green Light brown YellowYellow Yellow Yellow Orange

.

Fig. 4. Qualitative HPTLC fingerprint profiles of Piper betle cultivars A) Petroleum ether extract (at 200 nm) B) Essential oils (250 nm).

A.A. Khan et al. / Pharmacognosy Journal 5 (2013) 176e183182

3.6. Preliminary phytochemical screening and HPTLC studies

Behaviour of powdered leaves of Piper betle cultivars on treat-ment with different chemical reagents are furnished in Table 5.Preliminary phytochemical screening of methanolic extractrevealed the presence of carbohydrates, protein, amino acids, tri-terpenoids, steroids, glycosides, flavonoids and absence of alka-loids, fixed oils, saponins. HPTLC studies performed on petroleumether extract and essential oil shows differences in track profile ofall the cultivars (Fig. 4). Further there is need to explore thechemical differences in the volatile oil content as well as in otherextracts. “Chemical Markers” for each cultivar would help in theirdifferentiation quickly.

In brief the leaves of Piper betle cultivars are hypostomatic, tetra-cytic (Fig. 1a, b) stomatal complex which seems to be characteristicof Piperaceae family. Oil cells occur in the parenchyma, collen-chyma and vascular tissues of the petiole and midrib. In the blade,they can appear in the mesophyll or sub epidermis. The oil secre-tory cells are common in Piperaceae.32 Prismatic calcium oxalatemonocrystals with a pyramidal base occur in parenchymatous andcollenchymatous petiole cells.33 Tracheoid idioblasts like structurewere previously reported.34 Conjugation tube like structure waspresent between twin stomata in Sanci cultivar (Fig. 1F). Schizo-genous oil cavity was observedwith secretory epithelial layer at themidrib and in petiole35 (Fig. 2a). According to Cristina, translucentsecretions are found in newly sectioned leaf of Piper species36

(Fig. 3D).As there is no pharmacognostical work on record of this tradi-

tionally much valued drug, the present work was taken up with aview to lay down standards, which could be useful to detect theauthenticity of this medicinally useful plant. Macro and micromorphological standards discussed here can be considered asidentifying parameters to authenticate the drug.

Conflicts of interest

All authors have none to declare.

Acknowledgement

This work was supported by grant from the CSIR, (Council ofScientific and Industrial Research), Government of India, NewDelhi. (09/554 (0030)/2011 EMR-1). The authors are very muchthankful to Dr. S. Jha (Professor) and Dr. K. Jayaram (AssociateProfessor), Dept. of Pharmaceutical Sciences, Birla Institute ofTechnology, Mesra, Ranchi, Jharkhand, India, for valuable supportduring research work.

References

1. Mazura MDP, Nuziah H, Rasadah MA, Kiong LS. Evaluation of Piper betle onplatelet activating factor (PAF) receptor binding activities. Malayas J Sci.2007;26:79e83.

2. Sarkar A, Sen R, Saha P, Ganguly S, Mandal G, Chatterjee M. An ethanolic extractof leaves of Piper betle (Paan) Linn. mediates its antileishmanial activity viaapoptosis. Parasitol Res. 2008;102:1249e1255.

3. Lakshmi A, Kumaratunga KGA, Kalyani D. Studies on Piper betle of Srilanka.J Natl Sci Found Sri. 2005;33:133e139.

4. Evans PH, Browers WS, Funk EJ. Identification of fungicidal and nematocidalcomponents in the leaves of Piper betle (Piperaceae). J Agric Food Chem.1984;32:1254e1256.

5. Balasubrahmanyam VR, Rawat AKS. Notes on economic plants e betel vine.Econ Bot. 1990;44:540e543.

6. Rawat AKS, Tripathi RD, Khan AJ, Balasubrahmanyam VR. Essential oilcomposition as markers for identification of Piper betle L. cultivars. Biochem SystEcol. 1989;17:35e38.

7. Rathee JS, Patro BS, Mula S, Gamre S, Chattopadhyay S. Antioxidant activity ofPiper betel leaf extract and its constituents. J Agric Food Chem. 2006;54:9046e9054.

8. Ali I, Khan FG, Suri KA, et al. In vitro antifungal activity of hydroxychavicolisolated from Piper betle L. Ann Clin Microbiol Antimicrob. 2010;9:1e9.

9. Jahir AK, Naveen K. Evaluation of antibacterial properties of extracts of Piperbetel leaf. J Pharma Biomed Sci. 2011;11:1e3.

10. Pin KY, Chuah AL, Rashih AA, et al. Antioxidant and anti-inflammatory activ-ities of extracts of betel leaves (Piper betle) from solvents with different po-larities. J Trop For Sci. 2010;22:448e455.

11. Sayanti B, Mahesh S, Susri R, et al. Radio protective property of the ethanolicextract of Piper betel leaf. J Radiat Res. 2005;46:167e171.

12. Kanjwani DG, Marathe TP, Chiplunkar SV, Sathaye SS. Evaluation of immuno-modulatory activity of methanolic extract of Piper betel. Scand J Immunol.2008;67:589e593.

13. Bajpai V, Sharma D, Kumar B, Madhusudanan KP. Profiling of Piper betle Linn.cultivars by direct analysis in real time mass spectrometric technique. BiomedChromatogr. 2010;24:1283e1286.

14. Guha P. Betel leaf: the neglected green gold of India. J Hum Ecol. 2006;19:87e93.

15. Periyanayagam K, Jagadeesan M, Karimani S, Vetriselvam T. Pharmacognosticaland phyto-physicochemical profile of the leaves of Piper betle Var. Pachaikodi(Piperaceae) valuable assessment of its quality. Asia Pac J Trop Biomed. 2012:S506eS510.

16. Pradhan D, Suri KA, Pradhan DK, Biswasroy P. Golden heart of the nature: Piperbetle L. J Pharmacog Phytochem. 2013;1:147e167.

17. Vijayasankar R, Ahmed MG, Ikhlas AK. An investigation of the vegetativeanatomy of Piper sarmentosum, and a comparison with the anatomy of Piperbetle (Piperaceae). Am J Plant Sci. 2012;3:1135e1144.

18. Sheeta Lakshmi B, Naidu KC. Comparative morphoanatomy of Piper betel L.cultivars in India. Scholar research library. Ann Biol Res. 2010;1:128e134.

19. Vasuki K, Senthamarai R, Shri Vijaya Kirubha T, Balasubramanian P,Selvadurai S. Pharmacognostical studies on leaf of Piper betle. Der PharmaciaLettre. 2011;3:232e235.

20. Wallis TE. Text Book of Pharmacognosy. New Delhi: CBS; 1989:356e549.21. Dutta AC, Dutta TC. Botany for Degree Student. 6th ed. India: Oxford University

Press; 1997:724.22. Evans WC, ed. Trease and Evans Pharmacognosy. 15th ed. London: Saunders;

2003:585.23. Johansen DA. Plant Micro Technique. 1st ed. New York: McGraw Hill; 1940:

183e203.24. Kokate CK, ed. Practical Pharmacognosy. 1st ed. Pune: Vallabh Prakashan; 1986:

112e120.25. Kokate CK, ed. Practical Pharmacognosy. 3rd ed. Pune: Vallabh Prakashan; 2003:

105e109.

A.A. Khan et al. / Pharmacognosy Journal 5 (2013) 176e183 183

26. Evans WC. Trease and Evans Pharmacognosy. 15th ed. London: Baillere Tindall;1983:538e547.

27. Iyenger MA, Nayar SJK. Pharmacognosy Lab Manual. Berlin: Springer Verlag;1994:16e18.

28. Indian Pharmacopoeia, Government of India, Ministry of Health and HumanWelfare. 4th ed. New Delhi: Controller of Publications; 1996:A53eA54.

29. WHO/PHARM/92.559/rev.1. Quality Control Methods for Medicinal Plant Mate-rials. Geneva: Organisation Mondiale de la sante; 1992:22e34.

30. Harborne JB. Methods of extraction and isolation, Phytochemical Methods. 3rded. London: Chapman & Hall; 1998:60e66.

31. Judd WS, Campbell CS, Kellogg EA, Stevens PF, Donoghue JM. Plant Systematics:A Phylogenetic Approach. 3rd ed. Sunderland: Sinauer Associates Inc; 1999:258.

32. Metcalfe CR, Chalk L. Anatomy of the Dicotyledons e Leaves, Stem and Wood inRelation to Taxonomy with Notes on Economic Uses. Oxford: Clarendon Press;1957:1500.

33. Souza LA, Moscheta IS, Oliveira JHG. Comparative morphology and anatomy ofthe leaf and stem of Peperomia dahlstedtii C. DC., Ottonia martiana Miq. AndPiper diospyrifolium Kunth (Piperaceae). Gayana Bot. 2004;61:6e17.

34. Fahn A. Plant Anatomy. 3rd ed. Oxford: Pergamon Press; 1982:105e129.35. Charles BB. An Introduction to Plant Structure and Development. 2nd ed. Cam-

bridge: Cambridge University Press; 2010:258e271.36. Marinho CR, Adilson AZ, Marilia CV. Secretory cells in Piper umbellatum

(Piperaceae) leaves: a new example for the development of idioblasts. Flora.2011;206:1052e1062.