A COMPARATIVE PHARMACOGNOSTIC EVALUATION OF ANATOMICAL AND CHEMICAL CHARACTERS OF NARDOSTACHYS JATAMANSI (D.DON) DC. (JATAMANSA) AND VALERIANA MOONI ARN. EX C.B.CLARKE (LANKA THUWARALA) Abstract Nardostachys jatamansi (Caprifoliaceae), known as Jatamansa, is an important plant in Ayurveda and traditional medicine in Sri Lanka. The dried rhizome of the plant is an important ingredient in many drugs. This plant does not grow in Sri Lanka. The practice

Nardostachys jatamansi (Caprifoliaceae), known as Jatamansa, is an important plant in Ayurveda and traditional medicine in Sri Lanka. The dried rhizome of the plant is an important ingredient in many drugs. This plant does not grow in Sri Lanka. The practice has been to import it from India However, currently its export has been banned as it has become an endangered species in India . An island wide market survey carried out by authors revealed, that the material sold as Jatamansa is substituted by materials other than its recommended substitute, Valeriana wallichii to an extent ranging from 66% to 100%. Substitution with materials other than recommended substitute might adversely affect the efficacy and safety of the drugs manufactured. As V. wallichii also does not grow naturally in Sri Lanka, the present study was carried out to compare pharmacognostic properties of V. mooni the only species of Valeriana found in Sri Lanka with N. jatamansi to evaluate the possibility of replacing N. jatamansi with V. mooni and to establish comparative quality standards of the two plant species which will enable to identify raw material at the point of purchasing. Preliminary phytochemical screening indicated the presence of alkaloids, flavonoids and hydrolysable tannins in both species. The TLC analysis of the ethanol extract of the rhizomes, and GCMS analysis of their essential oils indicated that both species possess similar chemical compounds. The pharmacologically important principal sesquiterpene Jatamansone, was found even in higher amount in V. mooni (25.6%) than in N. jatamansi (8.9%). However clinical trials are needed to decide on the possibility of substituting Indian N. jatamansi with local V. mooni. The findings of the present study have added a new member to the potential list of medicinal plants and identified as an important endangered plant with the need for conservation, hence develop suitable propagation systems and cultivation.


Introduction
Herbal medicine is getting popular among people around the world. The use of accurate raw material is essential to produce high quality drugs with expected therapeutic values. With respect to quality control, correct identification of raw material in fresh, dry or powder state is of prime importance (Springfield et al., 2005). Misidentification and subsequent substitution with incorrect material might lead to severe health problems or even to death (Chaudhury & Rafei, 2002). Therefore, implementation of rigorous standardization procedure for raw material identification using botanical and chemical features will ensure high standards of the product with desired therapeutic values.

Nardostachys jatamansi of family
Caprifoliaceae is an important medicinal herb in Ayurveda which is heavily used in Sri Lanka (Pandey, 1991), (Jadhav et al., 2009). This species is not recorded in Sri Lanka and importing of the material from India has been also banned since 1997as it has become endangered in India. In Ayurveda practice, many exotics are being used mistakenly or as substitute when the original plant recommended does not grow in the country or when the amount of locally available raw materials is insufficient to meet the increasing daily demand. A survey carried out by authors among drug raw material dealers in five representative provinces and some Ayurveda and some traditional physicians in the country revealed that majority of them use subterranean part of a wild plant called Balanophora fungosa (Balanophoraceae) instead of N. jatamansi.
This plant is not recommended as a substitute of N.jatamansi in any Ayurveda pharmacopeia. Therefore finding a possible substitutes for such rare and expensive raw material is vital therapeutically as well as economically.
Valeriana mooni (Caprifoliaceae) is an erect perennial critically endangered herb, growing in high altitude areas of Sri Lanka and Tamil Nadu, India. Being globally threatened, V. mooni is an untouched medicinal plant. If V. mooni could be used as a substitute for N. jatamansi, it would minimize the unwanted bad effects which might be present in manufactured drugs due to the use of wrong raw material such as Balanophora fungosa (Balanophoraceae). However no reports are available regarding pharmacognostic properties of V. mooni. Even though V. mooni appears to be a safe substitute and possibly effective, it is not available in the market. Lack of investigations and documentation on pharmacognostic properties of V. mooni might be the reason for its unavailability in the crude drug market of the country. Therefore this species has hardly earned attention of conservationists and other researchers.

Material
Raw, dried rhizomes of V. mooni collected from Pattipola herbal garden and market samples (dried rhizomes) of N. jatamansi were used in the present study. Collected samples were identified using their macro morphological and organoleptic characteristics, following standard methods (WHO, 1998). Then the identification was confirmed by comparing samples collected with the authenticated specimens deposited at drug museum of the Bandaranayake Ayurvedic Research Institute. Air dried plant materials were used for anatomical and chemical studies.

Anatomical studies
Free hand sections of dried rhizomes of N. jatamansi and V. mooni soaked in water for ten minutes were taken using a sharp razor blade. Subsequently, sections were stained by double stained permanent slide method (Prasad & Prasad 1986). Anatomical features of the stained sections were studied using compound light microscope (model Kyowa, Biolux-12) and photomicrographs were taken (Sony cyber-shot digital camera).

Preparation of counter stained permanent slides of freehand sections
Selected sections were successively soaked in 30%, and 50% alcohol (for 5 minutes each) and subsequently stained with 1% safranin in 50% alcohol. Excess stain was washed off using 50% alcohol and subsequently, sections were dehydrated by treating successively with 70%, 90% alcohol (for 1 minute each), followed by 3 changes of absolute alcohol, keeping 1 minute interval at each step. Counterstaining was done using light green SF in clove oil for 2 minutes. Excessive stain was removed using absolute alcohol immediately. Sections were washed with clearing solution (clove oil) for 2 minutes. Clearing solution was removed by dipping sections for few minutes in xylene. After clearing in xylene for two changes, they were mounted on slides with a drop of Canada balsam (Prasad & Prasad 1986).

Powder Microscopy
Dried rhizomes were powdered using a grinder (Kinematica -CH-6014, Switzerland) and passed through steel sieve mesh No 355 (Retsch ®, AS 200, Germany). Powder microscopic studies were carried out following standard method (Trease & Evans, 1983;Khandelwal, 2005). Small amount of powder of both plants were separately transferred to a drop of Chloral hydrate on cleaned glass slide. After mixing and placing a cover glass, the slide was heated carefully over a small flame of micro burner until the air bubbles were completely removed. Ten microscopic fields (10x10 each) per sample were examined under compound light microscope and the relevant parameters were recorded. Photomicrographs of xylem components in the sample were taken using Sony cyber-shot digital camera.

Preliminary phytochemical screening
Qualitative chemical tests for identifying various phyto-constituents present were carried out following standard techniques (Trease & Evans 1983;WHO 1998;Khandelwal, 2005). Partially ground plant material (10 g) was kept in contact with 100 mL of 70% ethanol in a stoppard conical flask at room temperature for a period of 36 hours with frequent agitation (Tiwari et al., 2011).
The extracts of plant materials prepared as above were subjected to a preliminary phytochemical analysis to detect the different chemical compounds present (Fong et al., 1986).
Commercially available pre-coated (Merck) analytical high performance silica plates (Silica gel 60A, 20x20 cm, thickness of 0.25 mm) were used after prewashing them by eluting methanol once. Subsequently, plates were air dried and activated at 110 °C for 30 minutes prior to use. The samples were applied to plates, and the plates were developed separately using peroleum ether (PE): ethyl acetate (EAc) 1:1; PE:EAc 4:1; EAc:Acetone 4:1 and dichloromethane (DCM) as solvent systems. The chromatograms were visualized under UV (366 nm) and by spraying with anisaldehyde sulphuric reagent.

Detection of valepotriates
The powdered rhizomes were extracted in a soxhlet apparatus using Dichloromethane. The sample was spotted on TLC plates and developed using toluene:ethyl acetate (3:1) as the mobile phase (Wagner et al, 1984). Chromatograms were visualized by spraying with HCl -glacial acetic acid(GAA) reagent followed by heating at 110 o C.

Extraction of oil using hydro distillation
Ground rhizomes were subjected to hydro-distillation using a Clevenger apparatus (3340024-Borosil ® ) for 6 hours. The heating was stopped and the volume of oil collected in the graduated tube was read after 10 min. The yield of the oil was calculated as milliliters per kilogram of drug (v/w). The total volume of oil and water containing any dissolved oil in the clevenger arm was extracted with hexane in a separating funnel. The hexane layer was dried over anhydrous sodium sulphate and evaporated in a rotary evaporator under vacuum at 40 ºC and the last traces of hexane were removed by stream of nitrogen. The oil was stored at 4°C in dark coloured air-tight containers and used within one week for Gas chromatography/ Mass spectrometry (GC/MS) analysis.
The oil (0.1 mL) was dissolved in 2 mL of hexane and 2µl sample was injected to the GC/MS. The detected compounds were identified by comparing with National Institute of Standard and Technology (NIST), USA mass spectra data base and from retention times and mass spectra of standard compounds. The analysis of essential oils was carried out using GC/MS of Agilent Technologies, Model 7890A operating in the EI (Electron Impact) mode with an ionization energy of 70ev equipped with a split injector. Helium used as a carrier gas at the flow rate of 0.8 mL/min, while HP -5 MS (30 m x 0.25 mm, 0.25 μm) capillary volume was used. The initial temperature was programmed at 40 °C at the rate of 5°C/min and 250 °C at the rate of 2 °C/min The injector temperature was 275 °C. In both species, ray parenchyma are clear in some areas and phloem appears as patches of small cells. In V. mooni circular shape comparatively large pith remains alive with compact parenchymatouse cells ( Figure 6) while it becomes stellete shape, necrotic and is margined by medullary cork layer in N. jatamansi ( Figure 5).

Macroscopic and organoleptic characteristics of two raw materials
V. mooni is comparable with N. jatamansi in many powder characters. Both powders are yellowish brown, fibrous, aromatic and bitter in taste. Starch granules, sclerides and fibers are abundant in both species (Figures 7 and  8). Special features are summarized in Table 1.   The results for both plants were similar with positive results for alkaloids, flavonoids and hydrolysable tannins.
When considering the TLC profiles which were used to determine the valepotriates in two plant species, both species exhibited four fluorescent bands at Rf = 0.8, 0.7, 0.5 and 0.3 under UV 366 nm and five common bands having Rf =0.82, 0.8, 0.65, 0.62, 0.37 (pinkish brown) with HCL/GAA spray reagent ( Figure 10).
As evident from the TLC finger prints, it could be suggested that the chemical constituents present in V. mooni and N. jatamansi are comparable.

Comparative analysis of the essential oils of two plant material
The oil extracted using hydrodistilation of the rhizomes exhibited a variation in their oraganolepticity (Table 3).

GCMS analysis
The oils, hydro distilled from the two plant materials were used for the GC/MS analysis.
Only a small number of peaks could be identified by comparing their mass spectrum with those in NIST library data bases (NIST 2011) suggesting that GC condition need to be optimized for better resolution. Table 4 gives the formulae, retention time (RT) and the relative concentrations (in percentage) of the compounds identified in GCMS analysis with a matching factor >90%.

Discussion
Results of the present study show that N. jatamansi and V. mooni which belong to the same family exhibit many similar features in their pharmacognostic characters. Therefore it could be suggested that there is a possibility of using V. mooni as a substitute for N. jatamansi.
In the TLC profiles, extracts of N. jatamansi and V. mooni indicated a close resemblance exhibiting similar bands in both species.
In GCMS analysis of N. jatamansi and V. mooni demonstrated a similarity in the chemical constitution and their essential oils. Both species had substantial quantities of Jatamansone, (V. mooni -25.65%, N. jatamansi-8.93%) which has number of pharmacological actions, was recorded as the principal sesquiterpene and as the marker compound in jatamansa oil (Jha et al., 2012). The presence of even a higher percentage (25.65% when compared to 8.93% in N. jatamansi) of this key compound in oil extracts of V. mooni provide evidence for its suitability to use as a substitute for N. jatamansi. In addition to Jatamansone, patchouli alcohol which is used in some drugs and also in perfumery industry was also detected in N. jatamansi (Long, 2000).
Moreover, macroscopic and microscopic characters (organoleptic, anatomical and powder microscopic characters) together with banding pattern in TLC of N. jatamansi and V. mooni presented in this study could be used in raw material identification at the time of purchase and this will minimize the risk of unintentional use of incorrect plant species in drug industry.

Conclusion
Phytochemical screening, TLC and GCMS analyses indicate that the chemistry of two species is similar but not identical. Therefore, it could be recommended that V. mooni be considered as a substitute for N. jatamansi with further clinical studies.
Moreover, V. mooni could be considered as a new addition to the list of medicinal plants in Sri Lanka. Findings of this study will support the possible medicinal usage of this plant which in turn will ensure that it will be propagated, cultivated and conserved. The data produced in the present investigation will also be helpful in the preparation of monograph of the herb for the inclusion in various pharmacopeias.