Abstract: As part of the gene bank activity of CMPR many collections of Adhatoda zeylanica Medic. were made from varying agroecological regions in South India. These collections could be grouped into four morphotypes based on growth habits and other morphological characters. These four morphotypes were evaluated for their chemical and pharmacognostical characters and significant differences among the morphotypes were noticed. Among quality characters vasicine content, fingerprint profiles, stomatal index, leaf architecture and venation pattern showed significant variation indicating the predominance of additive gene effects. A reverse phase HPLC method for the quantitative determination of vasicine in the morphotypes was developed based on which variation in the vasicine content was observed. Thin layer chromatographic analysis showed variation in the chemical composition of these morphotypes. These studies indicated variability in the chemical composition and pharmacognostic characters among the morphotypes of A. zeylanica. Two of the morphotypes containing significantly higher vasicine indicated the presence of chemical diversity, providing adequate scope for selection of superior chemotypes having high therapeutic value and economic benefit.
INTRODUCTION
Adhatoda zeylanica Medic. (Common name-Vasaka), a perennial shrub of the family Acanthaceae, is a highly reputed Ayurvedic medicinal plant used in the treatment of cough, bronchitis, asthma, tuberculosis and recommended for other ailments of the respiratory system (Sivarajan and Balachandran, 1994). Leaves, flowers and roots of this plant are used in herbal drugs against cancer (Pandey, 2002). Different methods of isolation and estimation of active constituents from A. zeylanica have been suggested (Narayana et al., 1995; Johri and Zutchi, 2000; Srivastava et al., 2001) and its seasonal variations were also reported (Arambewela et al., 1988; Das and Chowdhury, 2005). Bagchi et al. (2003) reported that A. zeylanica showed higher content of vasicine compared to A. beddomei all throughout the year. Work done in various aspects of A. zeylanica such as morphology, anatomy, diseases and pests, chemical constituents, propagation, tissue culture, toxicology, intellectual property rights, phytochemical studies, pharmacological studies, medicinal uses, antimicrobial activity, traditional knowledge and quantitative standards have been reviewed by George et al. (2006). Raw material of Vasaka, consisting mainly of leaves, is very much in demand in the herbal pharmaceutical industry. Plant materials, mostly collected from natural sources, are mostly used in the preparation of the herbal drugs. However, such materials show a wide range of variation in quality. Attempts have been made to cultivate the plant in several parts of India, but so far large-scale cultivation to meet the requirements of the pharmaceutical industry has not been started. Variation in the quality of the plant material is observed, which may be due to the seasonal variations of the secondary metabolites and the difference in the agro-ecological conditions. Apart from such factors, other reasons for the difference in quality may include the intra-specific variation, as there exists different morphotypes of A. zeylanica.
In the present work comparative phytochemical and pharmacognostic characters of the different morphotypes of A. zeylanica collected from South India were studied taxonomically and authenticated and maintained in the germplasm bank of CMPR. The study on the germplasm collection of the morphotypes of A. zeylanica led to the grouping of the collections into four morphotypes viz. large type (AZ1), small type (AZ2), spreading type (AZ3) and intermediate type (AZ4). Considering the therapeutic and economic aspects of A. zeylanica and its diversity in India, it is essential to assess the extent of diversity at chemical and pharmacognostic levels. Such an analysis can provide scope for the selection of superior genotypes and chemical varieties (chemovars) for future use.
MATERIALS AND METHODS
Plant Material
Accessions of A. zeylanica were collected from different phytogeographical
zones of south India. Based on the macro and micromorphological characters,
four distinctly different morphotypes (AZ1, AZ2, AZ3 and AZ4) were identified
and subjected to detailed chemical and pharmacognostical studies. Leaves from
four accessions belonging to each of the morphotypes maintained in the germplasm
bank (Collection No. 01169, 02342, 02340, 02341 respectively), Arya Vaidya Sala,
Kottakkal, Kerala, India, were collected in September 2004. The voucher specimens
of the plant materials are archived in the Botany Department (Centre for Medicinal
Plants Research, Arya Vaidya Sala, Kottakkal, Kerala, India). The plant material
was dried at 50°C for 48 h and powdered. The powdered material was stored
in glass bottles protected from light and moisture at room temperature.
Extraction
Powdered leaves (20 g) of each of the morphotypes were mixed thoroughly
with 20 mL 10% ammonia solution and then refluxed with ethanol (3x200 mL) for
1 h over water bath at 60°C. The individual extracts were combined, filtered
and concentrated to dryness under reduced pressure below 60°C. The concentrate
was dissolved in ethanol and used for the analysis.
TLC Analysis
Thin Layer Chromatographic analyses of the extracts were performed on silica
gel 60 F254 TLC plates (10x10 cm; Merck, Darmstadt, Germany). Aliquots (10 μL)
of the extracts were applied on the plates as bands. Plates were developed in
TLC chamber previously saturated (30 min) with the mobile phase, chloroform:
methanol (9:1 v/v). The chromatogram was developed to 80 mm and then dried to
remove the solvents. The plates were visualized under UV 254 and 365 nm and
then sprayed with Dragendorff’s reagent.
HPLC Analysis Standard Preparation
Stock solution of vasicine (Natural Remedies, Bangalore, India) was prepared
in ethanol (Analytical Grade; Merck (India) Limited) at 1000 μg mL-1.
The HPLC analyses were carried out at 25°C on a Shimadzu (Kyoto, Japan) LC-10AT liquid chromatograph equipped with a Phenomenex Luna (Torrance, CA, USA) RP18 column (250x4.60 mm i.d.; 5 μm) with a Phenomenex guard column (4x2 mm i.d.; 5 μm). The samples were injected using a 20 μL loop (Rheodyne Rohnet Park, CA, USA). The mobile phase consisted of methanol: water (40:60) and the separation was performed by using isocratic elution at a flow rate of 1 mL min-1. The chromatograms were run for 35 min. Shimadzu SPD-M10A photodiode array detector was used at 298 nm for detection. The data was processed on a Shimadzu LC Workstation Class-VP System.
Method Validation Studies
Samples for validation analyses were obtained by the same procedure as described
previously. A vasicine solution in three different concentrations was added
to the AZ1 extract to evaluate the method’s performance. The method was
validated for precision, accuracy, limit of detection and of quantification,
linearity range and sensitivity. Intra and inter-day variability measurements
were used to determine the method’s precision and accuracy. The intra-day
precision of four individual samples was examined one day and inter-day precision
was determined on five independent days. The intra and inter-day accuracy was
determined by the same procedure. Linearity was determined using the vasicine
reference standard, at different levels of concentration, each level in triplicate.
In order to determine the accuracy of the method, known amount of vasicine was added in three concentrations (50, 150 and 250 μg mL-1) to AZ1 leaves prior to extraction. Recovery rates were calculated by the following equation:
R (%) = [Average of the amount of vasicine quantified in the sample]/ [Average of the amount of vasicine added to the sample] x 100
Quantitative Analysis of Vasicine
The total vasicine content was determined by external standard method. The
standard solutions were prepared in ethanol (50-250 μg mL-1).
The vasicine content was found out from the calibration curve of the standard
plotted between the concentration and area by calculating the area under curve
of the sample.
Anatomy and Micromorphology
The leaves of the morphotypes were processed as per the standard procedures
for histological studies (Johansen, 1940). Measurements were taken using ocular
and stage micrometers. Photomicrographs were taken using Canon digital camera
attached to Zeiss microscope.
RESULTS AND DISCUSSION
Different mobile phases for the separation of A. zeylanica extracts were tested, using silica gel TLC plates. The mobile phase that had the best resolution and separation was chloroform: methanol (9:1). The TLC fingerprint profiles of all the four morphotypes of A. zeylanica were compared. The extracts were separated into individual components using appropriate solvent systems after performing trials with a wide range of solvents. The solvent system for TLC separation was selected based on the efficiency of separation and degree of resolution of the system. The Rf (Retardation factor) was calculated for identifying the spots. The TLC fingerprint profile comprises of the bands resolved, Rf values and colour/fluorescence when scanned at UV 254 and 365 nm (Table 1). Derivatisation of the TLC plates with Dragendorff’s reagent was done and visualized. Among the four morphotypes studied, AZ1 and AZ2 showed more similarity in the TLC profiles. Like wise similarity was observed between AZ3 and AZ4. Overall, the TLC fingerprint profiles showed significant differences, which indicated variation in their chemical constituents.
The HPLC fingerprint profiles of all the four morphotypes of A. zeylanica were compared (Fig. 1). Among the four morphotypes, AZ1 and AZ2 showed similar profiles both qualitatively and quantitatively, so also similarity existed between AZ3 and AZ4. The amount of vasicine present in the plant extracts was calculated by external standard method and the morphotypes showed variation in vasicine content.
| Table 1: | TLC fingerprint profile of the four morphotypes of A. zeylanica |
| Table 2: | Vasicine content of the four morphotypes of A. zeylanica |
| n = 5, *Mean±SD | |
AZ4 (2.57% w/w) and AZ3 (2.35% w/w) contain higher amounts of vasicine compared to AZ1 (1.41% w/w) and AZ2 (1.22% w/w) (Table 2). The reported yield of alkaloids from different samples in India ranged from 0.541-1.105% w/w on dry basis (Anonymous, 2003). The method validation was done by performing precision, accuracy, limit of detection (LOD) and quantification (LOQ), linearity range and sensitivity studies (Table 3).
| Fig. 1: | HPLC Chromatograms of standard vasicine and the four morphotypes of A. zeylanica |
| Fig. 2: | Stomatal pattern of morphotypes of A. zeylanica |
All the four morphotypes of A. zeylanica studied, have diacytic stomata and variations were observed in the characters of epidermal and subsidiary cells. The epidermal cells of AZ1 and AZ2 are similar in size and shape, having straight cell walls. The epidermal cells are larger in AZ3 and AZ4, than those of AZ1 and AZ2 with slightly wavy and prominently wavy cell walls respectively (Fig. 2). Highest stomatal index was observed in AZ3, where as AZ1 and AZ2 showed almost equal stomatal index and AZ4 had the lowest. Variation in the number of veins in the midrib region of the morphotypes were noticed; five in AZ1 and AZ2 and three in AZ3 and AZ4. The study also indicated that AZ2 closely resembles AZ1 while the other two morphotypes are distinctly different (Table 4).
| Table 3: | Method validation parameters for the estimation of vasicine in A. zeylanica |
| Table 4: | Micro morphology and anatomy of A. zeylanica morphotypes |
| n = 5, *Mean±SD | |
There were significant differences among the morphotypes for different chemical and pharmacognostical characters. The chemical variation could be characterized from HPLC and TLC fingerprint profiles, vasicine content and presence of compounds. Among the pharmacognostic characters, variations were observed in stomatal index, leaf architecture and venation pattern. The variability in the morphotypes collected from various parts of South India, indicate that they are locally adapted genotypes. Such variations provide adequate scope for selection of chemically superior morphotypes (chemovars). The two morphotypes with high vasicine content are such superior chemotypes that can be further evaluated and recommended for commercial cultivation. Adequate pharmacological evaluation should be done before their use in the manufacture of herbal and ayurvedic formulations.
The small morphotype (AZ2) is often mistakenly treated as A. beddomei and the small variety is the one preferred in the Ayurvedic medicine preparation. However this is only a morphological variant and is not the A. beddomei described originally by Hooker (1885). This is not in anyway superior to the larger morphotype; in fact it contains lesser amounts of vasicine compared to A. zeylanica (Bagchi et al., 2003). The original A. beddomei is a highly endangered and extremely rare species and is not cultivated at all.
At CMPR further studies on biomass production, yield per unit area and other agrotechnological aspects are in progress. AZ3 seems to be a chemotype that may be useful for herbal drug preparations and thus needs to be promoted and popularized in future.
ACKNOWLEDGMENTS
We are thankful to the Department of Biotechnology, New Delhi and Sir Dorabji Tata Trust, Mumbai for providing the financial support and the management of AVS for providing the facilities and encouragement to undertake this study.