Abstract
In this study “RAPD” molecular marker was employed for the identification of Sennaangustifolia, Sennaacutifolia, Sennatora and Sennasophera. 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.
Keywords: Adulteration, Senna, Herbal medicine, PCR
1. Introduction
Herbal medicine has been enjoying renaissance among customers throughout the world. The World Health Organization estimates that 80% of the world’s population utilizes traditional medicines for healing and curing diseases (http://www.worldwildlife.org/what/globalmarkets/wildlifetrade/faqs-medicinalplant.html). Medicinal plants cover a wide range of plant taxa and closely related species. There is an increasing international market for medicinal plants, which are used both for herbal medicine and for pharmaceutical products. Accurate and rapid authentication of plants and their respective adulterants is difficult to achieve at the scale of international trade in medicinal plants. The natural medicines are much safer than synthetic drugs, have gained popularity in recent years, leading to a tremendous growth of phyto-pharmaceutical usage. However, herbal medicines can be potentially toxic to human health and sometimes may cause unknown effects. The recent investigations have revealed that many plants used in traditional and folk medicine are potentially toxic and mutagenic (Matthews et al., 2003; Ferreira-Machado et al., 2004). Due to the complex nature and inherent variability of the chemical constituents of plant-based drugs, it is difficult to establish quality control parameters. Due to the popularity of herbal drugs globally, their adulteration/substantiation aspects are gaining importance at the commercial level. Pharmaceutical companies are procuring materials from traders, who are getting these materials from untrained persons from rural and/or forest areas. This has given rise to wide-spread adulteration/substitution, leading to poor quality of herbal formulations.
Misidentification of herbs can be non-intentional (processed plant parts are inherently difficult to distinguish) or intentional (profit-driven merchants sometimes substitute expensive herbs with less-expensive look-alike ones (Kiran et al., 2010). Adulteration can occur due to ignorance or intentional 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 the plant material to the final product, is a need of the hour. The general approaches to herb identification are dependent on morphological (Carlsward et al., 1997), anatomical (Stern et al., 1994), chemical (Kite et al., 2009; Li et al., 2010) and molecular (Li et al., 2005) techniques. However, traditional taxonomic studies require expertise of experienced professional taxonomists. In the case where diagnostic morphological traits of the given specimen are lacking, it becomes difficult even for specialists to recognize a species correctly. Genetic analysis has a 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 affected by age, physiological conditions and environmental factors. It also helps in the identification of useful genotypes which are likely to improve efficacy of standard drug formulations; even the plant extract used in the herbal-drug formulations can be authenticated by DNA-based methods (Novak et 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 throughout south India and other parts of the country. It is a branching shrub with a height up to 1.8 m. The seeds are creamish to brown in color, obovate–oblong, dicotyledonous and medium size (3–4 × 1.5–2.0 × 4–5 mm) with weight 2.65 g per 100 seeds approximately. It has complex mixture of several active constituents such as dianthrone glycosides, free anthraquinones (aloe-emodin, chrysophanol and rhein) and anthraquinone glycosides. S. angustifolia contributes significantly to commercial drugs and has been investigated for various therapeutic preparations in several parts of the world in various ways such as anti-mutagenic, anti-genotoxic and anti-fungal (Silva et al., 2008). The other species of Senna of same family such as Senna sophera, Senna acutifolia and Senna tora have medicinal value and are found in Indian sub continent, and widely used as folk medicine for the treatment of numerous diseases. The phytochemicals of various species of Senna are different and some are common to each other which depend on its genotype and environmental conditions. However, their identification based on these markers is difficult as they are influenced by environmental conditions. The search for and development of herbal medicines is rapidly increasing worldwide, therefore, practical and accurate authentication methods are needed (Sucher and Carles, 2008; Yao et al., 2009; Song et al., 2009) to maintain quality and efficacy of herbal formulations.
Random amplified polymorphic DNA (RAPD) is a simple and 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 number of medicinal species from their close relatives or adulterants, including Echinacea species (Nieri et al., 2003), turmeric (Sasikumar et al., 2004), Astragali radix (Na et al., 2004), Dendrobium officinale (Ding et al., 2005), Typhonium species (Acharya et 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 cultivars and varieties (Qi et al., 2008), Desmodium species (Irshad et al., 2009), Glycyrrhiza glabra and its adulterant (Khan et al., 2009), Piper nigrum (Khan et al., 2010b), Cuscuta reflexa and Cuscuta chinensis (Khan et al., 2010a), and Ruta graveolens and its adulterant (Khan et al., 2011). The Senna species viz., S. angustifolia, S. acutifolia, S. tora and S. sophera are immensely used in Ayurvedic prescription and their accurate identification is urgent need in herbal industry to maintain the efficacy and quality of herbal formulations. Our major objective therefore, was to develop DNA based marker “RAPD” for accurate 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) Hyderabad. The samples for authentication were purchased from the local markets of Khari Baoli, Delhi, India. The material was identified at National Institute of Science Communication and Information (NISCAIR), New Delhi.
2.2. Reagents and chemicals
The stock solution concentration were CTAB 3% (w/v), 1 M Tris–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 β-mercaptoethanol. The extraction 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 β-mercaptoethanol were added freshly prepared in the extraction buffer at the time of genomic DNA extraction. All PCR chemicals were supplied by Bengalore 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 CTAB method (Khan et al., 2007). The dried leaves were ground into 800 μl extraction buffer in pestle-mortar and taken into micro-tube. The suspension was gently mixed and incubated at 65 °C for 20 min with occasional mixing. The suspension was then cooled to room temperature and an equal volume of chloroform: isoamyl alcohol (24:1) was added. The mixture was centrifuged at 12,000 rpm for 5 min. The clear upper aqueous phase was then transferred to a new tube and added 2/3 volume of ice-cooled isopropanol and incubated at −20 °C for 30 min. The nucleic acid was collected by centrifugation at 10,000 rpm for 10 min. The resulting pellet was washed 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 temperature 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 purity were determined by measuring the absorbance of diluted DNA solution at 260 nm and 280 nm. The quality of the DNA was determined using agarose gel electrophoresis stained with ethidium bromide.
2.4. RAPD analysis
RAPD reaction was performed according to the method developed by McClelland et al. (1995). The total volume of reaction was performed in 25 μl. PCR reaction for RAPD analysis consisted of 15 mM MgCl2, 2.5 μl 10× buffer, 2 μl 2 mM dNTPs (mix), 0.5 U Taq polymerase in buffer, 25 ng/μl of each primer, 30 ng/μl plant DNA and sterile water up to 18 μl. For DNA amplification 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 followed by 45 cycles of 1 min at 94 °C, 30 s at 36 °C, and 1 min at 72 °C, then terminating with 5 min at 72 °C. The RAPD fragments were separated on 1.2% agarose gel by electrophoresis in 1× TAE buffer for 2 h 30 min at 60 V. The gel was stained with ethidium bromide (0.5 μg/ml) and photographed under ultraviolet light using a chemiluminescence Imaging System (UVP Bioimaging systems, CA, USA). To overcome the reproducibility problems, the experiments were repeated thrice.
2.5. Data analysis
The RAPD bands were scored as present (1) or absent (0), each of which was treated as an independent character regardless of its intensity. Only prominent and reproducible bands obtained for 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. A dendrogram was constructed using the unweighted pair group method with arithmetic average (UPGMA) with the SAHN module of NTSYS-pc to show a phenetic representation of genetic relationships as revealed by the similarity coefficient (Sneath and Sokal, 1973).
3. Results
Four species of Senna were chosen to test the reliability of quality control using RAPD technique. In the local market samples, the adulteration was found among these species. The dried leaves of these species are more or less similar in morphology to each other and easily adulterated (Fig. 1). RAPD technique was carried out in replicates (three) using genomic DNA with 32 decamer primers for reproducibility of the results. The selected primers amplified DNA fragments across the 4 species studied, with the number of amplified fragments varied from 3 (OPC-3) to 10 (OPC-4, OPC-17, OPC-19, OPC-19 and OPC-20), and the amplicon size varied from 500 to 2500 bp. Out of 42 loci detected, the polymorphic, monomorphic and unique loci were 24, 2 and 16, respectively. All Senna species were discriminated by the presence or absence of unique fragment in RAPD profile. The total number of unique fragments specific to each species with different primers is summarized in Figs. 2–4 and Table 1.
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.
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.
Table 1.
The unique bands and common for S. angustifolia, S. acutifolia, S. sophera and S. tora obtained with 6 decamer 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 | – | |
3.1. Cluster analysis
A dendrogram based on UPGMA analysis, the 4 Senna species clustered into two groups (group I and II), with Jaccard’s similarity coefficient ranging from 0.1034 to 0.7273 (Fig. 5, Table 2). First group comprised S. anustifolia and S. acutifolia which had 72.73% similarity whereas second group comprised S. sophera and S. tora which had 23.68% similarity to each other.
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 |
4. Discussion
According to WHO general guidelines for methodologies on research and evaluation of traditional medicines, first step is assuring quality, safety and efficacy of traditional medicines for correct identification. In the present article S. angustifolia was chosen for the identification through RAPD technique. In the local market samples, S. tora, S. sophera and S. acutifolia were found as adulterant when identified at NISCAIR, New Delhi. For accuracy of the results, the high quality and purity of genomic DNA free from secondary metabolites was isolated from these species by modified CTAB method (Khan et al., 2007). For RAPD reaction, it was necessary to standardize the following variables for successful amplification with 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 parameters constant. MgCl2 of 1.5 mM concentration was proved best in 25 μl reaction volume. For most amplification reactions 0.5–1.5 units of the enzyme were used. Initially amplification reactions 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 polymerase per 25 μl reaction volume, which gave better amplification. In all PCR trials the annealing temperature 36 °C has been used which was determined with gradient PCR. DNA denaturation is a critical step in DNA amplification reactions. For most DNA amplification reactions incubation time for DNA denaturation is 1 min at 94 °C.
In the present investigation, 6 RAPD primers produced 24 polymorphic bands that unambiguously discriminated Senna species including S. angustifolia, S. acutifolia, S. sophera and S. tora, respectively. This RAPD marker exhibited 57.14% polymorphism among these species with 6 decamer primers. Our results indicated the presence of wide genetic variability among different Senna species. Variations in the DNA sequences lead to the polymorphism and greater polymorphism is indicative of greater genetic diversity. All four species of Senna were differentiated from each other based on unique band obtained in PCR amplification. S. angustifolia had unique bands; 1, 1, 0, 0, 0 and 1 with primers OPC-3, 4, 17, 18, 19 and 20, respectively (Table 1). Similarly, other species viz., 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 were reproducible when PCR reaction was repeated thrice. Among the different primers utilized, OPC-3 and OPC-4 produced maximum number of polymorphic bands and may be used in future for the identification of these Senna species. The cluster analysis clearly showed the genetic divergence among these species. These four species are clustered into two groups: First group had S. angustifolia and S. acutifolia which had high similarity (72.73%) to each other as compared to the second group which had S. sophera and S. tora (Table 2 and Fig. 5). However, the S. angustifolia and S. acutifolia have morphological similarities in leaves to a great extent and in dried state it is very difficult to differentiate to each other, but in RAPD analysis both species showed more genetic divergence.
The advantages of this technique are its rapidity, simplicity and avoidance of any need such as genetic information about the plant prior to the commencement of the experiment. These characters are especially advantageous for the identification of any herbal drugs because of little DNA existing in the dried material. The significance of the present work is that a single primer can differentiate genuine as well as adulterant samples as reported in our study. More reproducible DNA markers such as sequence characterized amplified regions (SCAR) can be developed in further studies, which would provide an alternative 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 useful tool to supplement the distinctness, uniformity and stability analysis for plant samples to maintain their identity for the protection in the future. Our study clearly indicated that RAPD markers could be used effectively to authenticate genuine as well as their respective adulterant samples sold in the local herbal markets.
Acknowledgement
This work was supported by Central Council Research for Unani Medicine (CCRUM) New Delhi, Govt. of India.
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