Original article

Pharmacognostic standardization of leaves of Melaleuca leucadendron

Gagan Shah a,*, Amit Chawla b, Uttam Singh Baghel b, Sandeep Rahar b,Prabh Simran Singh b, Ravi Kumar Dhawan c

aDepartment of Pharmacognosy, Khalsa College of Pharmacy, Amritsar 143001, IndiabDepartment of Pharmaceutical Chemistry, Khalsa College of Pharmacy, Amritsar 143001, IndiacDepartment of Pharmacology, Khalsa College of Pharmacy, Amritsar 143001, India

a r t i c l e i n f o

Article history:Received 11 May 2013Accepted 17 July 2013Available online 21 August 2013

Keywords:Melaleuca leucadendronStandardization parametersPhytochemical screening

a b s t r a c t

Introduction: Essential oil of Melaleuca leucadendron used as antimycotic and antimicrobial. The oil alsoused as antiinflammation and antifungal.Methods: The present study will assist in standardization for quality, purity and sample identification.Various standardization parameters like morphological characters, microscopic evaluation, physico-chemical evaluations) preliminary phytochemical screening and TLC chromatographic profile of theextract were carried out and the qualitative parameters were reported.Result: In the pharmacognostical studies, in an attempt to standardize the leaves ofM. leucadendron havebeen shown which will be definitely useful to the future scientist for the identification of the plants.Conclusion: These studies provide referential information for correct identification and standardizationof this plant material.

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

1. Introduction

TheWorldHealth Organization estimates that 80% of theworld’spopulation relies onherbalmedicine.Meanwhile, the use of herbs inthe United States is expanding rapidly, to the point where herbalproducts are readily found in most pharmacies and supermarkets.From 1990 to 1997, as the use of complementary and alternativemedicine rose from 34 to 42%, herbal use quadrupled from 3 to 12%.It is worth remembering that these rapid changes have come notthrough themedical profession, but by popular demand. The publichas discovered that naturalmedicines oftenprovide a safe, effective,and economical alternative to pharmaceuticals, and research vali-dates this finding. The majority of those who use herbal and high-dose vitamin products fail to tell their physicians. Either they as-sume that these products are harmless andnotworthmentioningorthey fear being ridiculed by doctors sceptical about their use. Thesesame doctors, however, must begin to familiarize themselves withthe subject. Aside from the advantages of the natural products,herbedrug interactions are a growing concern: almost one in fiveprescription drug users also using supplements.1 India has a richheritage of traditional medicines and the traditional health care

systems have been flourishing for many centuries. It mainly consistof threemajor systemsnamely Ayurveda, Siddha andUnani systemsof Medicine.2 In almost all the traditional systems of medicine, thequality control aspect has been considered from its inspection itselfby the Rishis and later by the Vaidya and Hakims. However, inmodern concept it requires necessary changes in their approach.Quality control and quality assurance is an integral part of tradi-tional medicines, which ensures that it delivers the required quan-tity of quality medicament.3 Essential oil ofMelaleuca leucadendronpossessed antimicrobial and antifungal activities.4 chloroform andmethanol extracts of the fruits ofM. leucadendron strongly inhibitedhistamine release fromratmast cells inducedbycompound48/80orconcanavalin A. Ursolic acid, a triterpene, was the most activecompound contained in the chloroform extract and two stilbenes,piceatannol and oxyresveratrol, were isolated as active compoundsfrom the methanol extract.5 New lupane-type nortriterpene and 13known compounds from the leaves of M. leucadendron. Based onchemical and spectral methods, the structure of the new compoundwas elucidated as 28-norlup-20(29)-ene-3beta,17beta-diol, whilethe known compounds were identified as (2E,6E)-farnesol, phytol,squalene, alloaromadendrene, ledene, palustrol, viridiflorol, ledol,betulinaldehyde, betulinic acid, 3beta-acetyl-lup-20(29)-en-28-oicacid, 3-oxolup-20(29)-en-28-oic acid, and platanic acid.6 Four newtriterpenes, eupha-7,24-diene-3beta,22beta-diol (1), 20-taraxastene-3alpha,28-diol (2), 3alpha,27-dihydroxy-28, 20beta-tarax-astanolide (3), and 3alpha-hydroxy-13(18)-oleanene-27, 28-dioic

* Corresponding author.E-mail addresses: gaganshah83@gmail.com, gaganshah83@yahoo.com

(G. Shah).

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Pharmacognosy Journal 5 (2013) 143e148

acid (4) have been isolated from the heartwood ofM. leucadendron.The structures and stereochemistry of 1e4havebeendetermined byspectroscopic analysis, with compounds 3 and 4 being investigatedin the forms of their diacetate (3a) and dimethyl (4a) derivative,respectively.7 The essential oil ofM. leucadendron. (tea tree) againstthe Gram-negative bacterium Escherichia coli AG100, the Gram-positive bacterium Staphylococcus aureus NCTC 8325, and theyeast Candida albicans has been using a range of methods. Theessential oil of M. leucadendron. (tea tree) exhibits broad-spectrumantimicrobial activity.8 The tree oil reduces histamine-inducedskin inflammation.9 In vitro susceptibility of oral bacteria to M.leucadendron oil and found that a range of oral bacterial are sus-ceptible to tea tree oil. Investigation was also made on the in vitroantifungal activity of the components of M. leucadendron oil andfound active.10 However no scientific standards or pharmacognosticparameters are yet available to determine the quality of this crudedrug. Thus the present study was designed to evaluate the phar-macognostic parameters of M. leucadendron leaves.

2. Materials and methods

Fresh leaves of. M. leucadendron were collected from MPDA,Doddabetta and Sims Park at Coonoor in Nilgiris respectively in themonth of February. The collected material were identified,confirmed and authenticated by botanist Dr. D. Suresh Baburaj,Botanical survey of India, Central Council for Research in Home-opathy, Govt. Arts College campus, Ootacamund.

2.1. Phytochemical screening

The various extracts of M. leucadendron were subjected toqualitative chemical examination.11,12

2.2. Thin layer chromatographic profile

TLC glass plates (5�15 cm), 0.25 mm thick were prepared usingsilica gel G. The plates were activated at 110 �C for 30 min. The TLCprofiles of the extracts were studied using different solvent sys-tems. TLC plates were developed in TLC chamber. Thin layer chro-matograms were visualized under 254/366 nm UV light and iniodine chamber. Spraying reagent 5% methanolic-sulphuric acid isused.

2.3. Organoleptic evaluation

Organoleptic evaluation of leaves was done by observing fruitsand seeds with naked eyes.

2.4. Microscopic and histological techniques of leaves

2.4.1. Study of transverse sectionsThe leaves of M. leucadendronwere boiled with water until soft.

Free hand sections of both fruits and seeds were cut transferred onslides cleared by warming with chloral hydrate and mounted inglycerin. The lignified and cellulosic tissues were distinguishedusing differential staining techniques.13

2.4.2. PhotomicrographyMicroscopic evaluation of tissues was supplemented with mi-

crographs. Photographs of differentmagnificationswere takenwithNikon Labpot 2 microscopic unit. For normal observations brightfield was used. For the study of crystals, starch grain and lignifiedcells, polarized light was employed. Since these structures havebirefringent property, under polarized light they appear bright

against dark background. Magnifications of the figures are indi-cated by the scale-bars.14

2.4.3. Powder microscopyA few drops of chloral hydrate solutionwas added to a sample of

powered plant material on a slide, covered with a glass slip andheated gently over a microbunsen. Vigorous boiling was avoided.The slide was examined under the microscope. When the clearingprocess is completed a drop of glycerol solution was added whichwill prevent crystallization of the mounting agent on cooling.

2.5. Physicochemical parameters

Physicochemical analysis i.e., alcohol (90% ethanol) and watersoluble extractive values, total ash, acid-insoluble ash, and loss ondrying of the powdered drug were determined.15,16

2.6. Phytochemical screening

The various extracts of M. leucadendron were subjected toqualitative chemical examination.11,17

2.7. Quantitative microscopy

Numbers of leaf measurements were used to study microscopicfeatures not easily characterized by general microscopy. Theseincluded e stomatal number vein islet number e veinlet termina-tion number e stomatal index, palisade ratio. The average numberof palisade cells beneath each upper epidermal cell is termed thepalisade ratio.18

2.8. Evaluation of volatile oil

Volatile oil was evaluated for optical rotation and refractive in-dex as per the standard procedure.19

3. Results

3.1. Organoleptic features of leaves (Fig. 1)

3.2. Microscopic features of the leaves

3.2.1. Anatomy of leafIn TS view, the leaf is flat with more or less uniform lamina and

fairly prominent midrib (Fig. 2). The lateral veins are thick andprominent, lent do not project beyond the surface of the lamina(Fig. 3). The marginal part is thick and semicircular (Fig. 4). Thefollowing parts of the leaf are seen.

Condition Fresh adult leaves

Colour Dark greenOdour CharacteristicShape LanceolateDimensions Length e 10e15 cm, Width e 2e4 cmLeaf base ExstipulateMargin EntireApex AcuminateBase SymmetricalSurface GlabrousTexture CoriaceousVenation Pinnate lateral veins anastomose near

the margin to a continuous linePetiole Short and twisted

G. Shah et al. / Pharmacognosy Journal 5 (2013) 143e148144

3.2.1.1. Epidermal layers. The epidermal layers of adaxial andabaxial sides are very thin and less prominent. The cells arehemispherical with prominent cuticle. The layer is less than 5 mmthick. The upper epidermis is apostomatic the lower epidermis isstomatiferous.

3.2.1.2. Mesophyll tissues. The palisade tissue occurs both in theadaxial and abaxial regions, so that the lamina is isobilateral. Bothadaxial and abaxial palisade zones are two layered measuring50 mm in height. The cells are short rectangular and chlor-enchymatous. In between the adaxial and abaxial palisade zones ina wide, non chlorenchymatous parenchymatous zone. The cells areangular and compact, the cell walls thin; some of the cells havetannin contents (Fig. 4).

3.2.1.3. Vascular bundles. The vascular system consists of a largemidrib bundle and many smaller laminar bundles. The midribbundle is elliptical in outline measuring 300 mm vertically and200 mm horizontally. It consists of a wide band of parallel lines ofxylem elements and an arc of phloem elements (Fig. 2). The entirevascular bundle is surrounded by sclerenchyma bundle sheaths;the sheath is thick in the upper and lower regions and along theirlateral parts. When viewed under the polarized light microscope,

the sclerenchyma sheath appears bright under dark background,indicating the liquefied nature of the cells (Fig. 5).

The lateral veins decrease in size gradually towards the margins.They are similar to the midrib bundle. They have collateral xylemand phloem surrounded by sclerenchymatous bundle sheath(Figs. 3 and 4).

3.2.1.4. Secretary canals (Fig. 6). Wide, circular secretary canals areabundant in the mesophyll. The canals originate by separation of agroup of cells forming a central cavity; the cells bordering the cavity

Fig. 1. Organoleptic features of leaves.

Fig. 2. T.S. of leaf through midrib with lamina. [Ads e adaxial side, MR e midrib, Sc e

sclerenchyma, SC e secretory cavity].

Fig. 3. T.S. of leaf through lateral vein with lamina. [Abp e abaxial palisade cells, Adp e

adaxial palisade cells, LV e lateral vein, MP e middle layer of parenchyma cells, Pa e

parenchyma cell, Ph e phloem, Sc e sclerenchyma, SC e secretory cavity, X e xylem].

Fig. 4. T.S. of leaf margin. [Abp e abaxial palisade cells, Adp e adaxial palisade cells,LM e leaf margin, MB e marginal bundle, Pa e parenchyma cell, SC e secretory cavity,VB e vascular bundle].

Fig. 5. T.S of leaf showing crystals abatting the sclerenchyma sheath of vascularstandard. [Cr e crystal, Cu e cuticle, SC e Secretory cavity, X e xylem].

G. Shah et al. / Pharmacognosy Journal 5 (2013) 143e148 145

develop in to a layer of spindle shaped cells called epithelial cellswhich is secretary in function. The cavities are 90 mm in diameter.

3.2.1.5. Cell inclusions. Calcium oxalate crystals are abundant in theleaf. Primatic types of crystals are densely packed along the lateralveins, especially abutting the sclerenchyma sheath of the vascularstrands (Fig. 7 and Fig. 8). Druses or crystal masses are located inthe mesophyll tissues.

3.3. Powder microscopy

Broken pieces of laminawere seen in the powder. They show thevein-islets whichwere four or five angled, large and distinct (Fig. 9),

the broadening veins were thick and consist of fibres. Fibres wereabundant in the powder; they were long, thick walled and lignified.The fibres were either straight or bent at one end or both ends(Fig. 10). They range in length from 500 to 800 mm.

Fig. 6. Paradermal section showing adaxial epidermis with secretory cavity. [AdE e

adaxial e SC e secretory cavity, Ve e vein].

Fig. 7. T.S. of showing sclerenchyma bundle sheath, crystals and starch grains. [BSc e

bundle sheath cells, Cr e crystal, SG e starch grains].

Fig. 8. Prismatic crystal in the abaxial palisade mesophyll and starch grains in themiddle parenchyma cells. [Cr e crystal, Ph e phloem, SG e starch grains].

Fig. 9. A fragment of lamina showing distinct vein-islets. [VI e vein-islets].

Fig. 10. Fibres of the veins (under polarized light microscope). [Fi e fibres].

Fig. 11. Powder of the leaf showing epidermal morphology. [Epidermal cells, St e

stomata].

Fig. 12. Oil bodies. [SC e secretory cells].

Table 1Physical parameters of leaves of Melaleuca leucadendron.

Physical parameters % (With reference to air dried drug)

Total Ash 5.8824Acid Insoluble Ash 1.432Water Soluble Ash 1.903Sulphated Ash 2.742Alcohol soluble extractive 20.160Water soluble extractive 12.320

G. Shah et al. / Pharmacognosy Journal 5 (2013) 143e148146

Epidermal cells and stomatawere seen in small broken pieces ofepidermis (Fig. 11). The epidermal cells were small and polyhedralin shape. The anticlinal walls of the cells were thick and straight.Secretory cavities were sporadically seen in the epidermis. Stomatawere abundant on both epidermal layers. The stomata were

anomocytic type (Fig. 10). Some of the stomata were largemeasuring 30� 40 mm; others were smaller measuring 20� 30 mm.Most of the layered stomata have closed, dark stomatal aperture.The powder contains large spherical cells containing dark oil bodieswere frequently seen in the powder (Fig. 12). The oil containingcells were 100 mm in diameter.

3.4. Physical parameters

The physical parameters of powdered leaves of M. leucadendronwere evaluated as shown in (Table 1).

3.5. Thin layer chromatography of volatile oils

The volatile oil obtained from the leaves ofM. leucadendronweresubjected to TLC in toluene: ethyl acetate (93:7) as mobile phase.The colour of the spots and Rf values are given in Table 2 and TLCChromatogram is given in (Fig. 13).

3.6. Preliminary phytochemical screening

The preliminary phytochemical investigation of the petroleumether, chloroform extracts, methanol and aqueous extracts of M.leucadendron leaves shows the presence of carbohydrates, steroids,flavonoids, phenolic compounds and tannins (Table 3).

3.7. Quantitative microscopy (Table 4)

The quantitative microscopy of the M. leucadendron leaves asshown in (Table 4).

3.8. Evaluation of volatile oil

Volatile oil was evaluated for optical rotation and refractive in-dex as per the standard procedure as shown in (Table 5).

4. Discussion

To ensure reproducible quality of herbal products, proper con-trol of starting material is important. The first step towardsensuring quality of starting material is authentication. Thus, inrecent years there has been a rapid increase in the standardizationof selected medicinal plants of potential therapeutic significance.Despite the modern techniques, identification of plant drugs bypharmacognostic studies is more reliable. As a part of standardi-zation, the macroscopical examination of leaves ofM. leucadendron

Table 2TLC chromatographic profile.

Absorbent Mobile phase Spraying reagent No. ofspots

Rf values of Melaleucaleucadendron

Colour ofthe spots

Due to presence of

Silica gel Toluene:ethylacetate (93:7)

Vanillin Sulphuricacid

1 0.21 Blue e

2 036 Green e

3 0.48 Blue Cineole4 0.62 Pink e

5 0.79 Orange e

6 0.98 Yellow e

Fig. 13. TLC chromatogram.

Table 3Phytochemical screening of the extracts of the Melaleuca leucadendron leaves.

Phytochemicals Petroleumether extract

Chloroformextract

Methanolextract

Aqueousextract

Alkaloids d d d d

Carbohydrate þ þ þ þProteins & amino

acidsd d d d

Phytosterols d þ þ d

Phenoliccompoundsand tannins

d d þ þ

Saponins d d þ þTriterpenoids d þ þ d

Flavonoids d d þ d

Table 4Leaf constants of Melaleuca leucadendron.

Stomatal Number Upper Epidermis ¼ 180e201e225Lower Epidermis ¼ 194e224e266

Vein islet number Range e 15.4e21.7, Average e 18.67Veinlet termination number Range e 18.8e23.5, Average e 20.3Stomatal index Upper Epidermis e 3.9e4.4e5.9

Lower Epidermis e 9.4e10.5e11.7Palisade ratio Upper Epidermis e 5.5e7.4e6.9

Lower Epidermis e 4.2e5.5e6.3

Table 5Evaluation of volatile oil of the leaves of Melaleucaleucadendron.

Optical rotation þ0.7�

Refractive index 1.4553

G. Shah et al. / Pharmacognosy Journal 5 (2013) 143e148 147

was studied. Macroscopical evaluation is a technique of qualitativeevaluation based on the study of morphological and sensory pro-files of drugs. Themacroscopical characters of the fruits of plant canserve as diagnostic parameters. The microscopic evaluation ofleaves ofM. leucadendron and extractive values, ash values and losson drying of the powdered drug and phytochemical screening ofthe extract have been carried out which would be of considerableuse in the identification of this drug. Percentages of the extractivevalues, ash value and loss on drying were calculated with referenceto the air-dried drug. The percent extractives in different solventsindicate the quantity and nature of constituents in the extracts. Theextractive values are also helpful in estimation of specific constit-uents soluble in particular solvent. Thin layer chromatography(TLC) was examined in short UV (254 nm) and long UV (366 nm)which is particularly valuable for the preliminary separation anddetermination of plant constituents. Numbers of leaf measure-ments were used to study microscopic features not easily charac-terized by general microscopy. This finding is useful to supplementthe existing information with regard to identification and stan-dardization ofM. leucadendron leaves even in the powdered form ofthe plant drug to distinguish it from drug and adulterant. Thesestudies also suggest that the observed pharmacognostic andphysiochemical parameters are of great value in the quality controland formulation development.

5. Conclusion

The present study may be useful to supplement the informationwith regard to its standardization and identification and in carryingout further research and its use in Ayurveda system of medicine.

Conflicts of interest

All authors have none to declare.

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