HISTOCHEMICAL AND PHYTOCHEMICAL MARKERS FOR THE AUTHENTICATION OF AYURVEDIC RAW DRUG HALLAKAM (KAEMPFERIA ROTUNDA) AND ITS MARKETED ADULTERANT

HISTOCHEMICAL AND PHYTOCHEMICAL MARKERS FOR THE AUTHENTICATION OF AYURVEDIC RAW DRUG HALLAKAM (KAEMPFERIA ROTUNDA) AND ITS MARKETED ADULTERANT

K. Sereena, U. Prakash Kumar and A. B. Rema Shree*

Drug Standardization Division, Centre for Medicinal Plants Research (CMPR), Kottakkal Arya Vaidya Sala, Kottakkal – 676503, Malappuram, Kerala, India

ABSTRACT

Comparative pharmacognostic and phytochemical studies are the reliable source to identify the genuine raw drug from its adulterants. This paper deals with the characterization of the repute ayurvedic drug hallakam from its substitute/adulterants. Ayurvedic experts equated rhizomes of Kaempferia rotunda L. of Zingiberaceae as hallakam and in certain market samples Lagenandra toxicaria Dalz. is also sold as  hallakam. The distinguishing pharmacognostic and phytochemical characters evolved from the study help to detect the genuine and market adulterant of hallakam

GCMS

INTRODUCTION: Hallakam is mentioned in Amarakosam ¹ and given several synonyms like Sauganthika (aromatic), hallakam (which attracts bee), Raktasandhyakam (reddish), Kalhara (water plant) etc. The drug is stomachic, anti-inflammatory to wounds and bruices, improve complexion, cure burning sensation, mental disorders and insomnia ². The rhizome is used for the preparation of many Ayurvedic formulations. From the reported synonyms, hallakam is equated with Kaempferia rotunda by Nadkarni ², Chopra et al ³.

Moosad, (Amarakosam¹) equated hallakam with chengazhinirkizhangu (K. rotunda) of Kerala physicians. However Rheede ⁴ equated this with Malankua (Zingiber zerumbet). Nicolson ⁵ also equated it with Zingiber zerumbet. According to Sivarajan and Balachandran⁶, hallakam is equated with K. rotunda and they reported that in practice L. toxicaria of Araceae is also used in Kerala. The controversies do exist in the case of hallakam by physicians.

Hence the present author made a thorough classical literature survey to identify the genuine plant. According to Dalhan a famous commentator of Sushrutasamhita ⁷, the synonym kalhara is one of the varieties of utpala (water lilly). Kalhara has been mentioned in another famous lexicon Bhavaprakashnighantu ⁸ written around 16 AD being included in the pushpavargha. The Ayurvedic expert Thakur Balwant Singh in his Glossary of vegetable drugs ⁹ has suggested that Sougandhika is considered to be a variety of Utpala.

So, Hallakam evidently denotes water lilly, even though there has been different opinion on this. Some of the Ayurvedic experts of Kerala have considered ‘Chengazhi’ (K. rotunda) and another water plant L. toxicaria as Hallakam. This paper deals with the characterization of the repute ayurvedic drug Hallakam from its substitute/adulterants. The distinguishing characters evolved from the study help to detect the genuine and substitute of Hallakam.

MATERIALS AND METHODS:

Anatomical studies: Plant materials for the present study were collected from Herb Garden, Arya Vaidya Sala, Kottakkal. The materials for anatomical study were fixed in Formalin: Acetic acid: Alcohol mixture (FAA). Histological and histochemical staining was carried out according to Johansen ¹⁰.

Photomicrographs were taken using Canon G3 camera attached to Zeiss microscope. Polarization microscopic studies were highly useful to locate and distinguish the types of crystals and minerals present in the useful parts. The characters were observed under Motic BA 400 polarization microscope. Fluorescent microscopic studies of the useful parts were done with the help of UV light. Observations were done under Leica DM 1000 LED fluorescent microscope and photographs were taken with the help of a digital camera. For examining the cell structure in powder form, material were powdered and sieved and mounted under glycerol and saffranin to study the nature and identification of particles.

Determination of Quantitative Data: Physicochemical parameters such as water soluble extractive, alcohol soluble extractive, percentage of total ash and acid insoluble ash were estimated according to the standard procedures ¹¹.

Phytochemical Studies: The dried root tuber and rhizomes were powdered and 5 g each K. rotunda and L. toxicaria were kept in 100 ml each of petroleum ether for overnight. The extract was then filtered and the solvent was evaporated under reduced pressure in a rotary evaporator. The residue was then dissolved in 10 ml of petroleum ether and subjected to TLC and GCMS profiling. Essential oils were distilled using Clevenger apparatus to perform GCMS.

GCMS analysis: GCMS is one of the widely used methods for analysis of volatile compounds present in herbal drugs Gas chromatography-mass spectrometry was carried out on an Agilent GC-MS 6850 under electron impact ionization (70 eV). The interface temperature was 230°C, and the MS scan range was 50-800 atomic mass units (AMU). The separation of constituents was done on HP5 - MS capillary column (30 m x 0.25 mm internal diameter). The carrier gas used was helium at a flow rate of 1 ml/min. The oven temperature was 60°C to 250°C with a constant increase of 5°C. The injection was performed in split mode (1:100) at 250°C.

RESULTS AND DISCUSSION:

Morphological characters: Morphological characters of these two species are entirely different. In K. rotunda rhizome is very fleshy and having 3 - 3.5 cm in length and 1.5 - 1.75 cm in diameter. Each tuber has the shape of club with a bulged lower portion and a stalk like cylindrical upper portion. But in the market, it is obtaining in dried transversely cut pieces (Fig. 1 A-C).

        (A) PLANT                                     (B) FRESH RHIZOME

(C) DRIED PIECES OF RHIZOME

FIG. 1 A-C: KAEMPFERIA ROTUNDA

In L. toxicaria rhizomes are long simple or branched, very thick, 2 - 2.5 cm in diameter, spongy and on drying dark brown in color; nodes and internodes are very prominent and internodes are very short; alternately arranged leaf scars are seen at the nodes (Fig. 1 D-F).

        (A) PLANT                               (B) FRESH RHIZOME

(C) DRIED PIECES OF RHIZOME

FIG. 1 D-F: LAGENANDRA TOXICARA

From the thorough classical literature survey to identify the genuine hallakam using the synonyms, it is aggreable in the case of K. rotunda ie., one of the characteristic feature of hallakam is souganthika. It is applicable only to K. rotunda and no aromatic smell is observed in L. toxicaria.

Anatomical characters: Preliminary pharmacognostic studies of K. rotunda, was carried out by Nambiar et al ¹². There are no reports regarding the comparative pharmacognostic characterization of K. rotunda and L. toxicaria. From the present study both the rhizomes showed significant anatomical characters. Both the rhizomes are almost circular in outline (Fig. 2 A & F). Periderm is well developed with broad cork in K. rotunda when compared to that of L. toxicaria (Fig. 2 B & G). Though K. rotunda belongs to monocot a correct demarcation between the outer and inner zone by endodermis like layer is present and it is termed as endodermoidal layer.

Rema Shree et al., ¹³ reported endodermidal layer in Zingiber species whereas these layer is not observed in L. toxicaria. Nature of vascular bundles shows much difference in these two species. It is typical monocot type in K. rotunda and amphivasal type in L. toxicaria (Fig. 2 C & H).  Its number and distribution also shows differences. Difference is observed in the case of nature of crystals i.e., large number of raphides of calcium oxalate are observed in L. toxicaria but in K. rotunda, it is sandy crystal type. Though starch grains and oleoresin cells are present in both the species, difference is seen in their size, shape and number. Oleoresins and starch grains are more in K. rotunda. Starch grains oval in shape in K. rotunda whereas it is elongated and finger like in L. toxicaria (Fig. 2 D, E, I & J). Comparative anatomical features of these two plants listed in Table 1.

FIG. 2 A-E: MICROSCOPY OF K. ROTUNDA RHIZOME. A: CS OF GROUND PLANE X 40. B: CORK REGION ENLARGED X 400. C: VASCULAR BUNDLE ENLARGED X 400. D & E: HSTOCHEMICAL LOCALIZATION OF STARCH & OLEORESIN CELLS X 200& 400. F-J: MICROSCOPY OF L. TOXICARIA. F: CS OF GROUND PLANE X 40. G: CORK REGION ENLARGED X 400. H: VASCULAR BUNDLE ENLARGED X 400. I & J: HSTOCHEMICAL STAINING OF STARCH & OLEORESIN CELLS X 200& 400. ck: Cork, vb: Vascular bundle, ph: Phloem, sg: Starch grain, oc: Oleoresin cells, xy: Xylem

TABLE 1: COMPARATIVE ANATOMICAL CHARACTERS

Polarization microscopy: Polarization microscopic studies revealed the presence, position and shape of sandy crystals and lignified cells. In K. rotunda presence of sandy crystals observed in cork, outer zone and inner zone and lignified layer under endodermal layer; show polarization (Fig. 3 A), whereas in L. toxicaria raphide crystals of calcium oxalate in the ground tissue and tracheids with lignified walls also showed polarization (Fig. 3 D).

Fluorescent microscopy: Fluorescent microscopic studies showed auto fluorescent with yellow color. In K. rotunda sandy crystals in the outer cortical region, endodermoidal layer were showed yellow fluorescence (Fig. 3 B & C), whereas in L. toxicaria outer cork layer and the xylem tracheids showed yellow fluorescence (Fig. 3 E & F).

Powder microscopy: In K. rotunda powder microscopy shows trachieds with spiral and reticulate thickening and large and oval shaped starch grains, groups of longitudinally cut xylem parenchyma and oleoresin containing cells and sandy crystals (Fig. 4 A-F). Whereas in L. toxicaria powder shows tracheids with spiral and reticulate thickenings, bundle of raphide crystals and scattered needles of calcium oxalate crystals, fragments of parenchyma with finger like starch grains and masses of reddish brown oleoresin depositions (Fig. 4. G-L).

FIG. 3 A: POLARIZATION MICROSCOPY OF K. ROTUNDA RHIZOME X 40

FIG. 3 B-C: FLOURESCENT MICROSCOPY OF K. ROTUNDA SHOWING ENDODERMOIDAL LAYER AND SANDY CRYTAL X 400. ct: Cortex, end: Endodermoidal layer, sc: Sandy crystal

FIG. 3 E: POLARIZATION MICROSCOPY OF L. TOXICARIA RHIZOME x 40

FIG. 3 E-F: FLOURESCENT MICROSCOPY OF L. TOXICARIA SHOWING CORK AND VASCULASR BUNDLE x400. ck: cork,  vb, vascular bundle

FIG. 4 A-F: POWDER MICROSCOPY OF K. ROTUNDA RHIZOME. A-C: GROUPS OF TRACHIEDS WITH SPIRAL THICKENING X 40. D: FRAGMENTS OF LONGITUDINALLY CUT FIBERS WITH TRACHIEDS X 400. E: STARCH GRAINS X 600. F: OLEORESIN CONTAINING CELLS X 400.

FIG. 4 G-L: POWDER MICROSCOPY OF L. TOXICARIA RHIZOME. G-I: GROUPS OF TRACHIEDS WITH SPIRAL THICKENING X 40. J: FRAGMENTS OF LONGITUDINALLY CUT FIBERS WITH TRACHIEDS X 400. K: STARCH GRAINS AND NEEDLES OF RAPHIDE CRYTSALS X 600. L: BUNDLES OF RAPHIDE CRYSTALS OF CALCIUM OXALATE X 600.

Chemical studies: Phytochemical studies of K. rotunda has been attributed to contain flavonoids, crotepoxide, chalcones, quercetin, flavonols, β- sitosterol, stigmasterol, syringic acid, protocatechuic acid and some hydrocarbons ¹⁴.  Woerdenbag et al., ¹⁵ reported the presence of volatile constituents in K. rotunda like benzyl benzoate (69.7%), n-pentadecane (22.9%) and camphene (9.1%). Methanol extract of the rhizome oil of L. toxicaria was subjected to GCMS analysis and the chemical constituents identified as Methyl ester of 2-hydroxy benzoic acid, Diethyl phathalate, Oleic acid, Palmitic acid ethyl ester and Dioctyl phthalate. Diethyl phathalate was found to be the major constituent (89.461%) ¹⁶.

In the present study comparing the physicochemical parameters like Moisture content, Water soluble extractive, Alcohol soluble extractive, Ash value and Acid insoluble ash, except the acid insoluble ash all other characters are higher in L. toxicaria than in K. rotunda (Table 2 & Fig. 5).

TABLE 2: PHYSICOCHEMICAL PARAMETRES (%)

FIG. 5: PERCENTAGE OF PHYSICOCHEMICAL CHARACTERS OF K. ROTUNDA AND L. TOXICARIA

From the GCMS analysis carried out by the author using petroleum ether extracts by cold maceration of dried and powdered samples of K. rotunda and L. toxicaria showed entirely different compounds in their essential oil. The major compounds identified in K. rotunda are n-dodecane, hexadecane, stearaldehyde, dodecanoic acid, kauren-ol whereas in L. toxicaria, we were able to identify a single compound 3-eicosyne from their GCMS analysis (Table 3 & Fig. 6 & 7). Essential oils were distilled using Clevenger apparatus to perform GCMS, and found that K. rotunda having fairly good amount of oil. But in the case of L. toxicaria the quantity was very less to perform GCMS analysis. Hence, GCMS performed only in volatile oil of K. rotunda. In the present study, we could able to quantify and identify 13 compounds from the essential oil of fresh sample of K. rotunda using GCMS. Among the compounds bornyl acetate and benzyl benzoate was found to be major constituents in the oil (Table 4 & Fig. 8).

TABLE 3: COMPOUNDS IDENTIFIED USING MASS SPECTROSCOPIC STUDIES

FIG. 6: FULL SCAN GCMS SPECTRUM OF K. ROTUNDA

FIG. 6: FULL SCAN GCMS SPECTRUM OF L. TOXICARIA

TABLE 4: COMPOUNDS IDENTIFIED IN K. ROTUNDA USING MASS SPECTROSCOPIC STUDIES

FIG. 8: GCMS PROFILE OF ESSENTIAL OIL OF K. ROTUNDA

From the available literature and present study shows that L. toxicaria don’t have any similar therapeutic activity mentioned for hallakam in classical texts and moreover it is reported to be a poisonous plant ¹⁷, though it is having similarities with the synonym i.e., Water lilly (kalhara) and hallakam (powerful odour which attracts bee) etc, it is not the genuine source plant of hallakam.

CONCLUSION: From the present study the anatomical and histochemical characters observed between these two plants shows differences in their nature of cells and cell inclusions. Phytochemical studies between these two plants shows much difference in their chemical constituents. So, it is a very helpful tool to distinguish the genuine dried raw drug from the adulterants and it is concluded that L. toxicaria is a clear case of adulterant.

ACKNOWLEDGEMENT: The authors are grateful to Kerala State Council for Science Technology & Environment (KSCSTE), Govt. of Kerala and Arya Vaidya Sala, Kottakkal for providing support to carry out this work. We are thankful to Dr. Indira Balachandran, Project Director, CMPR, Arya Vaidya Sala, Kottakkal for the necessary guidance.

REFERENCES:

1. Amarakosam with Parameswari Vyakhyanam. Vachaspati T.C Paramesvaran Moosad. Kerala Sahitya Acadamy, Thrissur, 2008.
2. Nadkarni KM. Indian Materia Medica. Popular Prakashan Pvt. Ltd., Bombay, Maharashtra, India. Vol. 1,
3. Chopra RN, Nayar SL and Chopra IC: Glossary of Indian Medicinal Plants. Council of Scientific and Industrial Research (CSIR), New Delhi, India, 1956.
4. Rheede HA and Van: Hortus Indicus Malabaricus. Amsterdam, 1692: I- XII. 11.
5. Nicolson, Dan H, Suresh CR and Manilal KS: An interpretation of Van Rheede’s Hortus Malabaricus, Konigstein, 1988.
6. Sivarajan VV and Balachandran I: Ayurvedic drugs and their Plant Sources. IBH Publishing Co. Pvt. Ltd, New Delhi, 1994.
7. Sushrutasamhita with Dalhana’s commentary and English translations by P. V. Sharma. Chaukhambha visvabharati, Varanasi & Delhi, 2005.
8. Bhavaprakasanighantu of sri Bhavamisra. Commentary by K. C Chunekar and edited by Dr. G S Pandey. Chaukambha Bharat Academy, Varanasi, 1982.
9. Singh TB and Chunekar KC: Glossary of vegetable drugs in Brihatrayi. Chaukambha Sanskrit series office, Varanasi, 1972.
10. Johansen DA. Plant Micro technique. McGraw – Hill, New York, USA, 1940.
11. The Ayurvedic Pharmacopoeia of India. Govt. of India, Part-1 Vol. III, 2001.
12. Nambiar VPK, Jayanthi A and Sabu TK: Pharmacognostical studies on Bhoomichampaka (Kaempferia rotunda) Aryavaidyan 2000; 14: 13-27.
13. Rema Shree AB, Sherlija KK, Unnikrishnan K and Ravindran PN: Histological studies on ginger (Zingiber officinale Rosc). Phytomorphology 1997; 47: 67-75.
14. Pai BR, Rao NN and Wariyar NS: Occurrence of crotepoxide in Kaempferia rotunda. J. Chem 1970; 8: 468.
15. Woerdenbag HJ, Windono T, Bos R, Riswan S and Quax WJ: Composition of the essential oils of Kaempferia rotunda and Kaempferia anguistifolia Roscoe rhizomes from Indonesia. J. Flav. Frag 2004; 19: 145-148.
16. Selvakumari PAS and De Britto AJ: GCMS Analysis of the Rhizome oil of Lagenandra toxicaria Malaysian J. Sci. 2008; 27: 89-99.
17. The Wealth of India- Raw Materials. CSIR, New Delhi, India, 1991: Vol. V, H- K; 315.
    

    ---