A COMPARATIVE PHARMACOGNOSTICAL EVALUATION OF RAW AND TRADITIONALLY SHODHITA VACHA (ACORUS CALAMUS LINN.) RHIZOMES

Abstract

Acorus calamus Linn. (Vacha) is a highly valued medicinal plant not only in traditional system, but also in western medicine. Though it is a non poisonous drug, Shodhana (purificatory procedure) has been indicated in Ayurveda prior to its use. Even in folklore practice, the rhizomes are processed in media like milk (Ksheera) and whey of curd (Mastu) prior to medicinal use. In the present study, pharmacognostical and preliminary phytochemical analysis of A. calamus was carried out in comparison with Ksheera and Mastu processed A. calamus rhizomes in order to know if there are any gross differences occurring after Shodhana. Few changes in oil globules were observed in the transverse sections of Shodhita (processed) samples in comparison to non-processed. Further thin layer chromatography revealed that Shodhana procedure did not affect the β-asarone qualitatively. Various parameters like macro, micro and physiochemical standards of this study will be helpful in authenticating Shodhita Vacha and will also serve as reference material for further scientific investigations. [PUBLICATION ABSTRACT]

Headnote

ABSTRACT

Acorus calamus Linn. (Vacha) is a highly valued medicinal plant not only in traditional system, but also in western medicine. Though it is a non poisonous drug, Shodhana (purificatory procedure) has been indicated in Ayurveda prior to its use. Even in folklore practice, the rhizomes are processed in media like milk (Ksheera) and whey of curd (Mastu) prior to medicinal use. In the present study, pharmacognostical and preliminary phytochemical analysis of A. calamus was carried out in comparison with Ksheera and Mastu processed A. calamus rhizomes in order to know if there are any gross differences occurring after Shodhana. Few changes in oil globules were observed in the transverse sections of Shodhita (processed) samples in comparison to non-processed. Further thin layer chromatography revealed that Shodhana procedure did not affect the β-asarone qualitatively. Various parameters like macro, micro and physiochemical standards of this study will be helpful in authenticating Shodhita Vacha and will also serve as reference material for further scientific investigations.

KEY WORDS: Vacha, Acorus calamus, Shodhana, β-asarone, Mastu, Ksheera

INTRODUCTION

Vacha (Acorus calamus Linn.) is a semiaquatic, perennial and aromatic herb which is found ascending up to an altitude of 2,200 m in the Himalayas. It is commonly known as sweet flag and is a valued medicinal plant in Ayurveda (Gupta, 2004) and as well as other traditional systems (Sharma, 2000; Savitha Bhat et al., 2011). In the classical literature the morphological characteristics of Vacha are described by the synonyms like Ugragandha, Golomi, Shadgrantha, Shataparvika etc., owing to its aromatic and rhizomatous nature (Shastry, 2001). The rhizome is the main useful part and is the genuine source of 'Vacha' in commerce [Figure-1] (Khare, 2007). Major chemical constituents of the rhizome are Asarone, Calamene, Calamenenol, Calameone, α - pinene, Camphene, Eugenol etc., among which β-asarone is the most researched one (Ernest Guenther, 1976). Acorus has shown significant effects on CNS and other systems which proves its utility in diseases like Apasmara (epilepsy), Unmada (schizophrenia), Vibandha (constipation), Adhmana (Tympanitis), Shoola (Colic), Karnasrava (otitis media) etc (Yende et al., 2008; Sharma PV, 2004).

In Ayurveda, Shodhana has been advocated not only for poisonous drugs but also for non poisonous drugs like Haridra (Curcuma longa Linn.), Hingu (Ferula narthex Boiss.), Chitraka (Plumbago zeylanica Linn.) and Lashuna (Allium sativum Linn.) using different media like Gomutra (Cow's urine), Godugdha (Cow's milk) etc., as per the nature of the drugs (Ramnarayan Vaidya, 1982; Ramachandra Reddy, 2005). It seems that Shodhana was carried out with an intension of not only purifying the drugs but also to alter their pharmacological effects (Kamble et al., 2008). This was also evidenced in one of our pharmacological study in which Shodhita Vacha samples showed better anti-convulsant activity than non-purified one (Savitha Bhat et al., 2012).

Similarly, even though Vacha is not considered as a poisonous drug, classical texts

like Chakradatta advises Shodhana of Vacha using Gomutra, Mundi kwatha (Decoction of Sphaeranthus indicus Linn.), Panchapallava kwatha (Decoction of a group of five tender leaves) and Gandhodaka (Decoction of group of aromatic herbs) (Ramanath Dwivedi, 2005). Ayurvedic pharmacopeia of India also recommends the use of Vacha after Shodhana (Anonymous, 2007).Other than the classical method; there are other folklore methods which are simple and economically viable. One of the methods practised in certain parts of Kerala is soaking rhizomes of Vacha in Dadhi Mastu (whey) overnight. Another method seen practised in certain coastal regions of Karnataka is soaking of Vacha in Goksheera (cow's milk) overnight.

Many research works have been carried out on pharmacognosy of Acorus calamus (Datta, 1950; Dipali Dey et al., 2005; Narayana Aiyar, 1957) but no work has been reported on the pharmacognostical aspects of Shodhita Vacha (processed Vacha). Hence this study intends to explore the pharmacognostical differences present in raw (unpurified) Vacha and Ksheera and Mastu Shodhita Vacha in order to lay certain standards which can serve as a future reference material.

MATERIALS AND METHODS:

Collection of plant material:

Fresh rhizomes of Acorus calamus were collected from the forest areas of Yelagiri Hills, Tamil Nadu, India in matured condition, in the month of November as per Ayurvedic criteria for collecting rhizomes (Indradeo Tripathi, 2003). After proper identification and authentication by Dr. Harisha C. R., Head, Pharmacognosy Laboratory, IPGT & RA, Jamnagar, the voucher specimen was deposited in the institute's Pharmacognosy department vide voucher specimen No. PhM. 6002. The leaves attached to the rhizomes were cut and separated. It was then rubbed by a gunny cloth to remove the roots and old leaf scars. Later the rhizomes were washed thoroughly in water to remove the soil adhered to it and dried in partial shade for 10 days.

Method of Shodhana:

Mature and long rhizomes of Acorus were selected, cut into pieces of one inch length and equally partitioned into three groups of 200 g each. The first group consisted of raw Vacha and was marked as sample RV. The second group was soaked in 2 litres of pasteurised Goksheera overnight (9 hours), washed with warm water (47°C) the next day and dried in sunlight for 6 days and marked as sample KV. The third group was soaked in 2 litres of Dadhi Mastu overnight (9hours), later washed in warm water (50°C) and dried in sunlight for 6 days. It was marked as sample MV.

Macroscopic and microscopic evaluation:

Macroscopical evaluation of raw Vacha rhizomes in both fresh as well as dry state in comparison with Shodhita samples was carried out as per standard procedure (Evans WC, 2002). Thin free hand transverse sections of dry rhizomes of raw and Shodhita samples were taken to evaluate both microscopical characteristics and histochemical reactions (Khandelwal KR, 2000). Further the samples were coarsely powdered with the help of a pulveriser, passed through sieve no 60 and were used for powder microscopy (Kokate, 2003). Both stained and unstained specimens were used to identify and confirm the microscopic structures (Anonymous, 2008a). Photomicrographs were taken using Carl Zeiss binocular microscope.

Phytochemical evaluation:

Physicochemical analysis, namely loss on drying at 105°C, ash value, acid insoluble ash, water soluble extractive value, alcohol soluble extractive value, pH value as well as qualitative test for various functional groups like alkaloids, glycosides etc were also carried out for all the three samples. Heavy metal analysis and pesticide residue analysis was done only for raw Vacha to check the contamination (Anonymous, 2008b). Histochemical tests were carried out by treating the transverse sections of all the samples using specific reagent to detect the colour changes and localization of chemicals. Fluorescence analysis was carried out with the powder of the rhizome sieved through 60 mesh and treated with various reagents. The supernatants were examined under day light and ultraviolet light (234 nm and 366 nm) (Krishnamurthy, 1988; Anonymous, 1998; Maluventhan, 2010).

Thin layer chromatography:

Methanol extracts of RV, KV and MV were subjected to thin layer chromatography and compared with β-asarone (1 mg dissolved in 2 ml of methanol) standard marker compound. Silica gel G plate of thickness 0.3 mm activated at 105°C for 30 minutes was used as stationary phase and Toluene:Ethyl acetate (9:1) as mobile phase (Anonymous, 2007). 10 Nl of test solution and 5 Nl of standard solution were applied on the TLC plate and the plates were developed in the solvent phase till the solvent front run was 9.6 cm. They were visualized under UV light at 254 nm and 366 nm, also after derivatization with Vanillin - Sulphuric acid reagent followed by heating for ten minutes at 105°C.

RESULTS AND DISCUSSION:

Macroscopic characters:

Raw Vacha (RV):

The fresh rhizome was woody, horizontal, creeping partially underground, varying in length from 25 cm-30 cm, vertically slightly compressed from 1.8-2.5 cm in diameter. It was rarely straight, much branched with thick long adventitious roots arising from the lower side. The dried rhizome was brownish in colour, tortuous, branched, sub cylindrical, 1.2- 1.8 cm in thickness, having distinct nodes and internodes. The nodes were broad with dry, fibrous, persistent, triangular, transverse leaf scars often attached to the upper side. The internodes were ridged, 7-10 mm in diameter. The under surface of the rhizomes are provided with irregularly arranged, slightly elevated round root scars and short fragment of roots [Figure 2]. Fracture short, granular and porous, emitted strong aromatic odour and had a pungent taste. The fracture surface exhibited cream coloured interior with a central and peripheral region marked by a faint endodermal line.

Ksheera Shodhita Vacha (KV):

KV was pale brownish in colour, with an average diameter of 1.8-2.5 cm, had a pleasing aromatic odour with a reduction in its pungent taste. Fracture short, granular, porous and the fractured surface was whitish cream in colour. No gross changes were observed in other macroscopical characteristics.

Mastu Shodhita Vacha (MV):

MV was also pale brown in colour, pungent and slightly sour in taste, with an average diameter of 2-2.5cm and had mixed odour of both raw Vacha and sour whey. Fracture short, granular and porous and the exposed surface exhibited a dull white interior. Other macroscopical features were similar to raw Vacha.

Microscopic features:

Important microscopic characteristics observed in the transverse sections of RV, KV, and MV rhizomes [Figures 3, 4 & 5] have been given in table - 1. The few changes observed in the structures, taste and oil globules may be due to the method of soaking in different media where the rhizomes tend to acquire the properties of the media used.

Powder microscopy:

The powder of sample RV was brown in colour with strong aromatic odour and pungent taste. Patches of parenchyma cells, ruptured spheroid oil cells, scattered pale yellow oil globules and oleoresin content were seen in addition to abundant simple, spherical to ovoid starch granules. Also lignified, scalariform and pitted vessels, fibres of fibrovascular bundles, occasional fragments of the epidermis and cork tissue and occasional hairs of the leaf scars in case of unpeeled rhizome were also observed.

The powder of sample KV was pale brownish in colour, having mild aromatic odour with both pungent and astringent taste. Abundant starch grains, patches of parenchyma cells and pale yellow oil globules similar to sample RV were observed. In addition, small translucent white coloured oil globules were also seen which might be acquired by the sample from the milk used as medium. Other characteristics were same as sample RV. The powder of sample MV was also pale brownish in colour, having mixed aroma of raw Vacha and whey along with pungent and sour taste. Occasional dull brown coloured cork tissue patches, oleoresin content and oil globules were seen scattered but were comparatively less in number when compared to raw Vacha. Other characteristics observed were similar to raw Vacha.

Phytochemical evaluation:

Extractive values of raw and Shodhita Vacha samples have been tabulated in table - 2. Higher percentage of loss on drying as seen in sample MV showed that Vacha after Mastu Shodhana contained components having more moisture holding capacity. The ash value of sample MV was comparatively more indicating inorganic residue remaining after incineration. There were also indications of presence of acid insoluble particles like silica in sample MV. Water soluble extractive values showed an increase in both Shodhita samples indicating the presence of polar constituents like sugars, acids, glycosides acquired by the media used in Shodhana (Yogesh Patel et al., 2010). Functional groups like carbohydrates, flavonoids, steroids, glycosides, alkaloids and tannins were present in all the three samples. Saponin content which was absent in raw Vacha was observed in Mastu and Ksheera Shodhita Vacha [Table -3]. Occasionally saponin content was detected in cow milk based on the type of its feed. Since the source of the milk was same for both groups, saponins might have been imbibed by the drug from the milk during the soaking process. Histo- chemical tests also showed the presence of starch, tannin, glycosides etc [Table - 4]. Heavy metals like mercury, lead, arsenic, cadmium and pesticides like lindane, aldrin, hexa-chlorobenzene and endosulfan were not detected indicating the safety of the drug. Fluorescence analysis of the sample powder showed the presence of florescence compounds and specific colour variations with various reagents which are tabulated in tables-5, 6 & 7. In comparison to RV there was a slight variation in colour pattern of KV and MV, which may be due to imbibing of media components during processing. Since no reported data regarding florescence analysis of processed Acorus is available, florescence analysis of present study will serve as reference value for future studies.

Shodhana procedure did not affect the β- asarone qualitatively as revealed by thin layer chromatography in which all the three samples showed similar spots with Rf value 0.44 which was the only spot obtained from TLC of marker β-asarone. Under short UV, both MV and KV showed extra spots with different Rf values [Figure - 6], this may be due to some active principle acquired by the sample during Shodhana. Similarly under long UV, sample MV showed one spot and KV showed two spots lesser than that of RV [Figure- 7], the reason for this might be movement of some active principles from the drug into media during Shodhana [Table 8]. The derivatization with vanillin-sulphuric acid exhibited same number of spots in all the three samples indicating that the steroidal compounds were not altered by Shodhana procedure [Figure- 8].

CONCLUSION

In the present investigation, there were no marked changes in the anatomy between raw Vacha and Ksheera or Mastu Shodhita Vacha rhizomes except for a slight reduction in the number of oil globules in Mastu Shodhita Vacha. There were only few variations in organoleptic characters which were implied to the process of Shodhana. Various parameters like macro, micro and physiochemical standards observed in this article will be helpful in authenticating Shodhita Vacha and will also serve as reference material in future research.

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