Saudi Journal of Biological Sciences (2011) 18, 287–292

King Saud University

Saudi Journal of Biological Sciences

www.ksu.edu.sawww.sciencedirect.com

Authentication of the medicinal plant Senna angustifoliaby RAPD profiling

Salim Khana,*, Khanda Jabeen Mirza

b, Fahad Al-Qurainy

a, Malik Zainul Abdin

b

a Department of Botany and Microbiology, College of Science, King Saud University, Riyadh 11451, Kingdom of Saudi Arabiab Centre for Transgenic Plant Development, Department of Biotechnology, Faculty of Science, Jamia Hamdard,Hamdard Nagar, New Delhi 110062, India

Received 28 December 2010; revised 27 February 2011; accepted 21 March 2011Available online 25 March 2011

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KEYWORDS

Adulteration;

Senna;

Herbal medicine;

PCR

Corresponding author. Tel.

-mail address: salimkhan17@

19-562X ª 2011 King Saud

sevier B.V. All rights reserve

er review under responsibilit

i:10.1016/j.sjbs.2011.03.001

Production and h

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yahoo.c

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Abstract In this study ‘‘RAPD’’ molecular marker was employed for the identification of Senna

angustifolia, Senna acutifolia, Senna tora and Senna sophera. Total 32 decamer primers were

screened in amplification with genomic DNA extracted from all species, of which 6 primers yielded

species-specific reproducible bands. Out of 42 loci detected, the polymorphic, monomorphic and

unique loci were 24, 2 and 16, respectively. Based on dendrogram and similarity matrix, 4 species

were differentiated from each other and showed more divergence. Thus, this technique may prove

and to contribute the identification of these species of Senna having similar morphology sold in the

local markets.ª 2011 King Saud University. Production and hosting by Elsevier B.V. All rights reserved.

1. Introduction

Herbal medicine has been enjoying renaissance among custom-ers throughout the world. The World Health Organization

estimates that 80% of the world’s population utilizestraditional medicines for healing and curing diseases (http://www.worldwildlife.org/what/globalmarkets/wildlifetrade/

14675876; fax: +966 4678301.

o.in (S. Khan).

y. Production and hosting by

Saud University.

lsevier

faqs-medicinalplant.html). Medicinal plants cover a wide

range of plant taxa and closely related species. There is anincreasing international market for medicinal plants, whichare used both for herbal medicine and for pharmaceuticalproducts. Accurate and rapid authentication of plants and

their respective adulterants is difficult to achieve at the scaleof international trade in medicinal plants. The naturalmedicines are much safer than synthetic drugs, have gained

popularity in recent years, leading to a tremendous growthof phyto-pharmaceutical usage. However, herbal medicinescan be potentially toxic to human health and sometimes may

cause unknown effects. The recent investigations have revealedthat many plants used in traditional and folk medicine arepotentially toxic and mutagenic (Matthews et al., 2003; Ferre-ira-Machado et al., 2004). Due to the complex nature and

inherent variability of the chemical constituents of plant-baseddrugs, it is difficult to establish quality control parameters.Due to the popularity of herbal drugs globally, their adultera-

tion/substantiation aspects are gaining importance at the

288 S. Khan et al.

commercial level. Pharmaceutical companies are procuring

materials from traders, who are getting these materials fromuntrained persons from rural and/or forest areas. This has gi-ven rise to wide-spread adulteration/substitution, leading topoor quality of herbal formulations.

Misidentification of herbs can be non-intentional (pro-cessed plant parts are inherently difficult to distinguish) orintentional (profit-driven merchants sometimes substitute

expensive herbs with less-expensive look-alike ones (Kiranet al., 2010). Adulteration can occur due to ignorance or inten-tional substitution with cheaper plant material and may cause

damage to human body (Jordan et al., 2010). Therefore,authentication at various stages, from the harvesting of theplant material to the final product, is a need of the hour.

The general approaches to herb identification are dependenton morphological (Carlsward et al., 1997), anatomical (Sternet al., 1994), chemical (Kite et al., 2009; Li et al., 2010) andmolecular (Li et al., 2005) techniques. However, traditional

taxonomic studies require expertise of experienced professionaltaxonomists. In the case where diagnostic morphological traitsof the given specimen are lacking, it becomes difficult even for

specialists to recognize a species correctly. Genetic analysis hasa promising role in resolving disputes of taxonomic identities,relations and authentication of the species in question, as the

genetic composition is unique for each species and is not af-fected by age, physiological conditions and environmental fac-tors. It also helps in the identification of useful genotypeswhich are likely to improve efficacy of standard drug formula-

tions; even the plant extract used in the herbal-drug formula-tions can be authenticated by DNA-based methods (Novaket al., 2007). Therefore, DNA-based methods have gained wide

acceptance in quality control to authenticate crude materials.Formerly included in Cassia L., Senna (Senna angustifolia)

(family: Caesalpiniaceae) is widely used in Unani medicine and

also has been adopted by the pharmacopoeias of the world(Trease and Evans, 1983). It is found in abundance throughoutsouth India and other parts of the country. It is a branching

shrub with a height up to 1.8 m. The seeds are creamish tobrown in color, obovate–oblong, dicotyledonous and mediumsize (3–4 · 1.5–2.0 · 4–5 mm) with weight 2.65 g per 100 seedsapproximately. It has complex mixture of several active con-

stituents such as dianthrone glycosides, free anthraquinones(aloe-emodin, chrysophanol and rhein) and anthraquinoneglycosides. S. angustifolia contributes significantly to commer-

cial drugs and has been investigated for various therapeuticpreparations in several parts of the world in various ways suchas anti-mutagenic, anti-genotoxic and anti-fungal (Silva et al.,

2008). The other species of Senna of same family such as Sennasophera, Senna acutifolia and Senna tora have medicinal valueand are found in Indian sub continent, and widely used as folk

medicine for the treatment of numerous diseases. The phyto-chemicals of various species of Senna are different and someare common to each other which depend on its genotype andenvironmental conditions. However, their identification based

on these markers is difficult as they are influenced by environ-mental conditions. The search for and development of herbalmedicines is rapidly increasing worldwide, therefore, practical

and accurate authentication methods are needed (Sucher andCarles, 2008; Yao et al., 2009; Song et al., 2009) to maintainquality and efficacy of herbal formulations.

Random amplified polymorphic DNA (RAPD) is a simpleand cost ineffective PCR based method as compared to other

DNA based markers. Due to such property of this technique, it

has been widely used for the differentiation of a large numberof medicinal species from their close relatives or adulterants,including Echinacea species (Nieri et al., 2003), turmeric (Sas-ikumar et al., 2004), Astragali radix (Na et al., 2004), Dendro-

bium officinale (Ding et al., 2005), Typhonium species (Acharyaet al., 2005), Dendrobium species and its products (Zhang et al.,2005), Tinospora cordifolia (Rout, 2006), Mimosae tenuiflorae

cortex (Rivera-Arce et al., 2007), Rahmannia glutinosa culti-vars and varieties (Qi et al., 2008), Desmodium species (Irshadet al., 2009), Glycyrrhiza glabra and its adulterant (Khan et al.,

2009), Piper nigrum (Khan et al., 2010b), Cuscuta reflexa andCuscuta chinensis (Khan et al., 2010a), and Ruta graveolensand its adulterant (Khan et al., 2011). The Senna species viz.,

S. angustifolia, S. acutifolia, S. tora and S. sophera are immen-sely used in Ayurvedic prescription and their accurate identifi-cation is urgent need in herbal industry to maintain the efficacyand quality of herbal formulations. Our major objective there-

fore, was to develop DNA based marker ‘‘RAPD’’ for accu-rate identification of Senna species in the local markets.

2. Materials and methods

2.1. Plant materials

The genuine sample of S. angustifolia was provided by Central

Council for Research in Unani Medicine (CCRUM) Hydera-bad. The samples for authentication were purchased fromthe local markets of Khari Baoli, Delhi, India. The material

was identified at National Institute of Science Communicationand Information (NISCAIR), New Delhi.

2.2. Reagents and chemicals

The stock solution concentration were CTAB 3% (w/v), 1 MTris–HCl (pH 8), 0.5 M EDTA (pH 8), 5 M NaCl, absolute

ethanol, chloroform-IAA (24:1 [v/v]), polyvinylpyrrolidone(PVP) (40,000 mol. wt.) (Sigma) and b-mercaptoethanol. Theextraction buffer consisted of CTAB 3% (w/v), 100 mM

Tris–HCl (pH 8), 25 mM EDTA (pH 8), and 2 M NaCl,respectively. PVP and b-mercaptoethanol were added freshlyprepared in the extraction buffer at the time of genomicDNA extraction. All PCR chemicals were supplied by Benga-

lore Genei Pvt. Ltd. (Bangalore, India) except for primers,which were supplied by Genetix Biotech Asia Ltd. (Bangalore,India).

2.3. DNA extraction

DNA was isolated from dried leaves using a modified CTABmethod (Khan et al., 2007). The dried leaves were ground into800 ll extraction buffer in pestle-mortar and taken into micro-

tube. The suspension was gently mixed and incubated at 65 �Cfor 20 min with occasional mixing. The suspension was thencooled to room temperature and an equal volume of chloro-form: isoamyl alcohol (24:1) was added. The mixture was

centrifuged at 12,000 rpm for 5 min. The clear upper aqueousphase was then transferred to a new tube and added 2/3volume of ice-cooled isopropanol and incubated at �20 �Cfor 30 min. The nucleic acid was collected by centrifugationat 10,000 rpm for 10 min. The resulting pellet was washed

Figure 1 Morphological slides of four Senna species: (a) S. angustifolia, (b) S. acutifolia, (c) S. tora, (d) S. sophera.

Figure 2 RAPD analysis carried out with primers: OPC-3 (lanes:

1–8), OPC-4 (lanes: 9–16). S. angustifolia (lanes: 1, 2, 9, 10), S.

acutifolia (lanes: 3, 4, 11, 12), S. sophera (lanes: 5, 6, 13, 14), S. tora

(lanes: 7, 8, 15, 16). 1 kb and 100 bp are DNA ladder.

Authentication of the medicinal plant Senna angustifolia by RAPD profiling 289

twice with 80% ethanol. The pellet was air-dried under a sterile

laminar hood and the nucleic acid was dissolved in TE buffer(10 mM Tris buffer pH 8 and 1 mM EDTA) at room temper-ature and stored at 4 �C until used. The RNA from crude

DNA was eliminated by treating the sample with RNase A(10 mg/ml) for 30 min at 37 �C. DNA concentration and pur-ity were determined by measuring the absorbance of diluted

DNA solution at 260 nm and 280 nm. The quality of theDNA was determined using agarose gel electrophoresis stainedwith ethidium bromide.

2.4. RAPD analysis

RAPD reaction was performed according to the method devel-

oped by McClelland et al. (1995). The total volume of reactionwas performed in 25 ll. PCR reaction for RAPD analysis con-sisted of 15 mM MgCl2, 2.5 ll 10· buffer, 2 ll 2 mM dNTPs

(mix), 0.5 U Taq polymerase in buffer, 25 ng/ll of each primer,30 ng/ll plant DNA and sterile water up to 18 ll. For DNAamplification a Techne thermal cycler was programmed for 1

cycle of 3 min at 94 �C, 30 s at 36 �C, and 1 min at 72 �C fol-lowed by 45 cycles of 1 min at 94 �C, 30 s at 36 �C, and 1 minat 72 �C, then terminating with 5 min at 72 �C. The RAPDfragments were separated on 1.2% agarose gel by electropho-

resis in 1· TAE buffer for 2 h 30 min at 60 V. The gel wasstained with ethidium bromide (0.5 lg/ml) and photographedunder ultraviolet light using a chemiluminescence Imaging Sys-

tem (UVP Bioimaging systems, CA, USA). To overcome thereproducibility problems, the experiments were repeatedthrice.

2.5. Data analysis

The RAPD bands were scored as present (1) or absent (0), eachof which was treated as an independent character regardless ofits intensity. Only prominent and reproducible bands obtainedfor each RAPD primer were considered. By comparing the

banding patterns of species for a primer, species-specific bands

were identified. Faint or unclear bands were not considered. Adendrogram was constructed using the unweighted pair groupmethod with arithmetic average (UPGMA) with the SAHN

module of NTSYS-pc to show a phenetic representation of ge-netic relationships as revealed by the similarity coefficient(Sneath and Sokal, 1973).

3. Results

Four species of Senna were chosen to test the reliability ofquality control using RAPD technique. In the local marketsamples, the adulteration was found among these species.

The dried leaves of these species are more or less similar in

Table 1 The unique bands and common for S. angustifolia, S.

acutifolia, S. sophera and S. tora obtained with 6 decamer

290 S. Khan et al.

morphology to each other and easily adulterated (Fig. 1).

RAPD technique was carried out in replicates (three) usinggenomic DNA with 32 decamer primers for reproducibilityof the results. The selected primers amplified DNA fragmentsacross the 4 species studied, with the number of amplified frag-

ments varied from 3 (OPC-3) to 10 (OPC-4, OPC-17, OPC-19,OPC-19 and OPC-20), and the amplicon size varied from 500to 2500 bp. Out of 42 loci detected, the polymorphic, mono-

morphic and unique loci were 24, 2 and 16, respectively. AllSenna species were discriminated by the presence or absenceof unique fragment in RAPD profile. The total number of un-

ique fragments specific to each species with different primers issummarized in Figs. 2–4 and Table 1.

Figure 3 RAPD analysis carried out with primers: OPC-17

(lanes: 1–8), OPC-18 (lanes: 9–16). S. angustifolia (lanes: 1, 2, 9,

10), S. acutifolia (lanes: 3, 4, 11, 12), S. sophera (lanes: 5, 6, 13, 14),

S. tora (lanes: 7, 8, 15, 16). 1 kb and 100 bp are DNA ladder.

Figure 4 RAPD analysis carried out with primers: OPC-19

(lanes: 1–8), OPC-20 (lanes: 9–16). S. angustifolia (lanes: 1, 2, 9,

10), S. acutifolia (lane; 3, 4, 11, 12), S. sophera (lanes: 5, 6, 13, 14),

S. tora (lanes: 7, 8, 15, 16). 1 kb and 100 bp are DNA ladder.

3.1. Cluster analysis

A dendrogram based on UPGMA analysis, the 4 Senna speciesclustered into two groups (group I and II), with Jaccard’s sim-

ilarity coefficient ranging from 0.1034 to 0.7273 (Fig. 5, Table2). First group comprised S. anustifolia and S. acutifolia whichhad 72.73% similarity whereas second group comprised S. sop-

hera and S. tora which had 23.68% similarity to each other.

primers in PCR amplification.

Primers Species (specific unique bands (bp)

S. angustifolia S. acutifolia S. sophera S. tora

OPC-3 (Fig. 2) 2600 – – –

– – 2000 –

– – 1400 –

– – 1200 1200

1100 – 1100 –

– – 1000 1000

900 900 – –

800 800 800 800

– – – 700

600 600 – –

– – 500 –

OPC-4 (Fig. 2) – – – 2400

– – – 2300

– – 2000 2000

– – 1500 1500

– – 1400 1400

1200 1200 – –

1000 1000 1000 1000

– – 800 800

700 700 – –

650 – – –

600 600 – –

OPC-17 (Fig. 3) – – 2400 –

– – – 2300

1800 1800 – –

– – 1400 –

OPC-18 (Fig. 3) – – 2800 –

2500 – – 2500

– – 2400 2400

1800 – 1800 –

1700 – 1700 1700

– 1600 – 1600

– – – 1500

1000 1000 1000 –

900 900 900 900

800 800 – –

OPC-19 (Fig. 4) 2500 2500 2500 2500

– – – 1800

1000 1000 – –

– – 900 –

800 800 – –

OPC-20 (Fig. 4) – – 2500 –

– – 2300 –

– – – 2000

1400 – – –

– – 1100 –

800 800 800 –

700 700 – –

– – 650 –

Figure 5 UPGMA dendrogram showing clustering of 4 Senna species based on RAPD data.

Table 2 Genetic similarity matrix based on RAPD data

among 4 Senna species estimated according to Jaccard’s

method.

S. angustifolia S. acutifolia S. sophera S. tora

S. angustifolia 1.0000

S. acutifolia 0.7273 1.0000

S. sophera 0.2368 0.1714 1.0000

S. tora 0.1875 0.1034 0.3226 1.0000

Authentication of the medicinal plant Senna angustifolia by RAPD profiling 291

4. Discussion

According to WHO general guidelines for methodologies onresearch and evaluation of traditional medicines, first step isassuring quality, safety and efficacy of traditional medicines

for correct identification. In the present article S. angustifoliawas chosen for the identification through RAPD technique.In the local market samples, S. tora, S. sophera andS. acutifolia were found as adulterant when identified at NISC-

AIR, New Delhi. For accuracy of the results, the high qualityand purity of genomic DNA free from secondary metaboliteswas isolated from these species by modified CTAB method

(Khan et al., 2007). For RAPD reaction, it was necessary tostandardize the following variables for successful amplificationwith PCR: RAPD amplification is not reproducible below a

certain concentration of genomic DNA and produces ‘smears’or results in poor resolution, if DNA concentration is high,series of dilutions were made to check good amplification.PCR trials were undertaken with different concentrations of

MgCl2 (0.5 mM, 1 mM and 1.5 mM) keeping all other param-eters constant. MgCl2 of 1.5 mM concentration was provedbest in 25 ll reaction volume. For most amplification reactions

0.5–1.5 units of the enzyme were used. Initially amplificationreactions were carried out with 0.9 units of Taq polymerase,however, this was not found to work to generate better results.

Therefore the quantity was reduced to 0.5 units of Taq poly-merase per 25 ll reaction volume, which gave better amplifica-tion. In all PCR trials the annealing temperature 36 �C has

been used which was determined with gradient PCR. DNAdenaturation is a critical step in DNA amplification reactions.For most DNA amplification reactions incubation time forDNA denaturation is 1 min at 94 �C.

In the present investigation, 6 RAPD primers produced 24polymorphic bands that unambiguously discriminated Senna

species including S. angustifolia, S. acutifolia, S. sophera andS. tora, respectively. This RAPD marker exhibited 57.14%polymorphism among these species with 6 decamer primers.

Our results indicated the presence of wide genetic variabilityamong different Senna species. Variations in the DNAsequences lead to the polymorphism and greater polymor-phism is indicative of greater genetic diversity. All four species

of Senna were differentiated from each other based on uniqueband obtained in PCR amplification. S. angustifolia had un-ique bands; 1, 1, 0, 0, 0 and 1 with primers OPC-3, 4, 17, 18,

19 and 20, respectively (Table 1). Similarly, other speciesviz., S. acutifolia, S. sophera and S. tora had unique bands;(0), (3,0,2,2,1,4) and (1,2,1,2,1,1), respectively with primers

(OPC-3, 4, 17, 18, 19 and 20) (Table 1). These bands werereproducible when PCR reaction was repeated thrice. Amongthe different primers utilized, OPC-3 and OPC-4 produced

maximum number of polymorphic bands and may be used infuture for the identification of these Senna species. The clusteranalysis clearly showed the genetic divergence among thesespecies. These four species are clustered into two groups: First

group had S. angustifolia and S. acutifolia which had high sim-ilarity (72.73%) to each other as compared to the second groupwhich had S. sophera and S. tora (Table 2 and Fig. 5). How-

ever, the S. angustifolia and S. acutifolia have morphologicalsimilarities in leaves to a great extent and in dried state it isvery difficult to differentiate to each other, but in RAPD anal-

ysis both species showed more genetic divergence.The advantages of this technique are its rapidity, simplicity

and avoidance of any need such as genetic information aboutthe plant prior to the commencement of the experiment. These

characters are especially advantageous for the identification ofany herbal drugs because of little DNA existing in the driedmaterial. The significance of the present work is that a single

primer can differentiate genuine as well as adulterant samplesas reported in our study. More reproducible DNA markerssuch as sequence characterized amplified regions (SCAR) can

be developed in further studies, which would provide an alter-native tool to monitor the quality of these herbal drugs. Thus,our study revealed that RAPD markers can be used for the

identification of commercial Senna species and could be a use-ful tool to supplement the distinctness, uniformity and stabilityanalysis for plant samples to maintain their identity for theprotection in the future. Our study clearly indicated that

292 S. Khan et al.

RAPD markers could be used effectively to authenticate gen-

uine as well as their respective adulterant samples sold in thelocal herbal markets.

Acknowledgement

This work was supported by Central Council Research for

Unani Medicine (CCRUM) New Delhi, Govt. of India.

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