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Journal of Theoretical and Experimental Biology (ISSN: 0972-9720), 7 (3): 127-134, 2011 2011 Elias Academic Publishers www.ejteb.


Galijeru as the Ayurvedic drug Vrshabhu: Assessing Admixture Problem and Proper Identification
Sateesh Suthari1, Srinivas Kota2, V. Anil Kumar2, P. Nataraj Kumar2, A. Sadanandam2 and Vatsavaya S. Raju1*
Plant Systematics Laboratory, Department of Botany, Kakatiya University, Warangal, Andhra Pradesh -506 009, India. 2 Department of Biotechnology, Kakatiya University, Warangal, Andhra Pradesh - 506 009, India.

Received: 30 December, 2010; revised received: 24 February, 2011.

There exists confusion on the true identity of tella and erra galijeru which constitute the ayurvedic crude drug Vrshabhu (as antioxidant, vermifuge, diuretic, uteralgia, oedema in the liver and spleen, for cough, rheumatism, etc.), green (leafy) vegetable or when the roots of these are used as substitute for punarnava (Boerhavia species). Galijeru is the Telugu vernacular used for the species of Eclipta (Asteraceae), Trianthema and Zaleya (Aizoaceae). Gunta (pit or shallow area) galijeru or galagara is Eclipta prostrata. The species and the infraspecific categories of Trianthema and Zaleya are discriminated by using the prefix tella (white, sveta/sweta, saphed, vellai, etc.), and [y]erra (lal, red) to Galijeru. Sweta punarnava also often has a mix-up with Alternanthera pungens (Amaranthaceae). These problems manifested largely due to (i) identification of the root drug plants with above ground parts, (ii) influx of allied, similar looking exotics, their naturalization and sympatric distribution with the actual-drug yielding plant species, and (iii) the want of knowledge of their distribution and nomenclature. The need to avoid the mix-up or adulteration for the efficacy of the drug is apparent. An attempt was made to characterize the ingredients (actual and adulterants) morphologically and with the aid of PCR. The genetic relatedness between the crude drug and its adulterants was evaluated by ISSR markers. Keywords: Ayurvedic drug, vrshabhu, admixture, morphological, molecular (ISSR marker) characterization.

The quality control of Ayurvedic products is a major problem at all levels. Either unintentional admixture arising from incorrect identification of source plants or deliberate adulteration leads to reduced efficacy of any Ayurvedic drug. The Ayurvedic Pharmacopoeia of India (Anonymous, 2004) enlists vrshabhu as the root drug (no. 64). It consists of the shade-dried roots of Trianthema portulacastrum L. (Aizoaceae), the vernacular name of which is desert horsepurslane (English), Galijeru (Telugu), etc. The problem arises when one attempts to collec the plant specimens based on vernaculars or when one has to supply the root material of this species employing the above ground parts. It is because, there exist two ecotypes in Trianthema portulacastrum, growing sympatrically (co-occur); the lay man in India calls them tella [white in English, saphed in Hindi, sweta in Marathi and Oriya, sveta in Sanskrit and Bengali, vellai in Tamil] and erra
*Corresponding author; Email address: satyavatsa@yahoo.co.in


Sateesh Suthari et al / Galijeru as the Ayurvedic drug Vrshabhu

[red in English, lal in Bengali, etc.] galijeru. Regardless whether there available separate names to distinguish these two forms in a locality or not, they are found throughout India. Therefore, it is the question of which form (ecotype) that constitutes the vrshabhu. There are other equally troublesome issues before the plant collector of Galijeru. There are plant species which grow sympatrically (in the same habitat) with Trianthema portulacastrum L. (Trianthema monogyna L.) and bear striking semblance (external appearance) in habit are Zaleya decandra (L.) Burm.f. (Trianthema decandra L.) and Zaleya pentandra (L.) C. Jeffrey (Trianthema pentandra L.) also of the same family, Aizoaceae, aside Alternanthera pungens L. of Amaranthaceae and Boerhavia diffusa L. of Nyctaginaceae. The last-mentioned is punarnava, with which Trianthema portulacastrum shares this common name and at times substituted. An attempt is made here to present the problems with the unintentional admixture of this plant drug and to provide a key to sort them out on external features. Of late, molecular tools are used to identify the admixtures with the raw drug trade of hepatoprotective Phyllanthus in India (Srirama et al., 2010). Therefore, ISSR (Inter Simple Sequence Repeat) markers are tried for a proper identification of the possible admixture. This effort is further supplemented by the distinctive secondary metabolites (micromolecules) present (reported) in these taxa. All these markers may help better to diagnose the adulterants of the plant drug, vrshabhu.

Materials and Methods

Plant Material The fresh plant materials from the field (different districts of the state of Andhra Pradesh) and those deposited at Madras Herbarium (MH) were studied for morphological and molecular characterization of vrshabhu. The identification of the plant species was based on standard floras and e-floras; the herbarium specimens of the taxa compared are all deposited at KUH (Kakatiya University Herbarium, Warangal, Andhra Pradesh).

Molecular Methods
DNA Isolation: The genomic DNA was isolated from freshly harvested young leaves of Alternanthera pungens, Boerhavia diffusa, Trianthema portulacastrum (flava, rubra) and Zaleya decandra by Mini Prep Method. DNA was diluted to uniform concentration of 10 ng/l. The ISSR-PCR was conducted in a reaction volume of 15 l containing 30 ng template DNA, 0.2 mol/L primer, 200 mol/L each dNTP, 10 mmol/L Tris-Cl (pH 8.3), 50 mmol/L KCl, 2.0 mmol/L MgCl2, and 1 U of Taq polymerase. The PCR amplification conditions were set as initial denaturation at 94C for 5 min, 40 cycles of denaturation at 94C for 1 min, annealing at 50C for 1 min, extension at 72C for 2 min, and a final extension at 72C for 7 min. The PCR was performed in 96-well plate thermal cycler (Biorad). The amplified products were mixed with loading dye (0.4 g/ml sucrose and 2.5 mg/ml bromophenol blue), resolved on 18 mg/ml agarose gel in 0.5 Tris borate EDTA (TBE) buffer under room temperature at constant voltage of 100 V, and detected by ethidium bromide staining (0.5 mg/ml). The gels were documented under ultraviolet light using gel documentation unit from Biorad. Each amplified product was scored as 1 or 0 depending on its presence or absence. The frequency of microsatellite polymorphism was calculated based on the presence or absence of common bands. The polymorphism information content (PIC) value was calculated as PIC = (1-Pi2)/ n, where n is the number of band positions analyzed and Pi is the frequency of i th pattern. The ability of the primers to distinguish between the two variants was assessed by calculating their resolving power (Rp) as Rp= b, where b is band informativeness, b = 1[2 (0.5pi)] and pi is the proportion of variants containing band i . The genetic associations among plants were evaluated using Jaccards similarity coefficient for pair-wise comparisons based on the proportions of shared bands produced by the select primers. Similarity matrices were generated using SIMQUAL sub-program of NTSYSPC software. Similarity coefficients were used for cluster analysis of species using SAHN
Journal of Theoretical and Experimental Biology (ISSN: 0972-9720), 7 (3): 127-134, 2011


Sateesh Suthari et al / Galijeru as the Ayurvedic drug Vrshabhu

sub-program of NTSYS-PC software while dendrogram was obtained using UPGMA, subprogram of NTSYS-PC software version 2.0 from Exeter software, NY, USA (Rohlf, 1993).

Results and Discussion

The present study was aimed at the morphological distinction as well as the molecular characterization of vrshabhu plant vis-a-vis its possible adulterants/admixtures at raw drug collection stage or later. 1. Morphological Characterization There are five taxa which are associated (co-occurring, sharing the same vernacular name, and resembling) with the drug plant vrshabhu. The following is the simple key to sort them employing the above ground parts. It is accompanied by the current nomenclature, brief description, nativity, distribution, phytochemical constituents reported, etc. for each taxon: 1 Leaves at nodes equal; flowers in heterogamous heads ... Eclipta prostrata + Leaves at nodes inequal; flowers not in heterogamous heads ................... 2 2(1) Ovary 1-locular; style 1, bifid ......3 + Ovary more than 1-locular; styles 1 or 2, entire ........4 3(2) Perianth and bracts scarious; perianth not coralline... Alternanthera pungens + Perianth and bracts not scarious; perianth coralline.............Boerhavia diffusa 4(2) Styles 2; stamens up to 10.............5 + Style 1; stamens more than 10.........Trianthema portulacastrum 5(4) Stamens 5...............Zaleya pentandra + Stamens 10.......... Zaleya decandra 1. Alternanthera pungens Kunth in H.B. & K., Nov. Gen. Sp. 2: 206. 1817. Achyranthes repens L., Sp. Pl.: 205. 1753. Alternanthera repens (L.) Link, Enum. Pl. Hort. Berol. 1: 154. 1821, non Gmelin, 1791- Fig. 1a. Spreading, mat-forming herb with woody base and vertical root stock; branches terete, villous-hairy. Leaves opposite-decussate, ovate to obovate; base truncate to cuneate, entire, mucronate. Inflorescence axillary, sessile. Flowers hermaphroditic; tepals dissimilar; stamens 5, alternating with 5 pseudo-staminodes; ovary compressed, style very short. Fruit lenticular; seed discoid, brown. A common naturalized weed of waste lands, along road sides, foot paths and open fields in most districts of Andhra Pradesh and throughout the peninsula. It is native of tropical America. Note: The young tops are a leafy vegetable in Tamil Nadu. It is called mulla ponnaganti in Telugu. 2. Boerhavia diffusa L., Sp. Pl.: 3. 1753. Boerhavia repens L., Sp. Pl.: 3. 1753. Hook.f., Fl. Brit. India 4: 709. 1885 (var. diffusa). Fig.1b. Diffuse herb; roots stocks fusiform, woody. Leaves opposite, inequal in pair, ovate to subcordate; base rounded, entire, obtuse to acute. Inflorescence chiefly axillary. Flowers small, in clustered cymes. Anthocarp club-shaped, five-ribbed, glandular-hairy; seeds black, smooth. A common weed of waste places, fields and road sides throughout the country.
Journal of Theoretical and Experimental Biology (ISSN: 0972-9720), 7 (3): 127-134, 2011


Sateesh Suthari et al / Galijeru as the Ayurvedic drug Vrshabhu

Figure 1: Vrshabhu and its adulterants. a - Alternanthera pungens; b Boerhavia diffusa; c Eclipta prostrata; d - Zaleya pentandra; e - Trianthema portulacastrum (green form); f T. portulacastrum (red form); g Z. decandra (green form); h Z. decandra (red form).

Note: It is known as punarnava (spreading hogweed). The entire plant, drug punarnava is reported to contain punarnavine 1 & 2 (quinolizidine alkaloids), punarnavoside, etc. The roots bear ecdysone, sitosterol, boeravinones A-F (rotenones), borhavine, lignan liriodendrin, etc. (cf. Daniel, 2006). 3. Eclipta prostrata (L.) L., Mant. Pl. 2: 286. 1771. Verbesina prostrata L., Sp. Pl.: 902. 1753. Verbesina alba L., Sp. Pl.: 902. 1753. Eclipta alba (L.) Hassk., Pl. Jav. Rar.: 528. 1848; Hook.f., Fl. Brit. India 3: 304. 1881. Eclipta erecta L., Mant. 2: 286. 1771.- Fig.1c. Annual prostrate herb with reddish stem and stiffly appressed-pilose tomentum. Leaves opposite, elliptic, strongly trinerved at base. Peduncles up to 4.5 cm. Heads white with
Journal of Theoretical and Experimental Biology (ISSN: 0972-9720), 7 (3): 127-134, 2011


Sateesh Suthari et al / Galijeru as the Ayurvedic drug Vrshabhu

numerous ray flowers. Achenes sparsely puberulous at top; pappus scales about 0.3 mm long. Common in wet places and undisturbed soils. Note: It is an essential ingredient of hair tonic Bringaraj. The herb possesses wedelolactone and demethylwedelolactone (the coumestan derivatives; hepatoprotective), ecliptine (alkaloid), ecliptal dithiophenes, ecalabosaponins I-IV, luteolin (flavone), etc. while the roots bear thiophene acetylenes (cf. Daniel, 2006). 4. Trianthema portulacastrum L., Sp. Pl.: 223. 1753; Gamble, Fl. Madras 1: 550. 1919. Trianthema monogyna L., Mant. 1: 69. 1767; Hook.f., Fl. Brit. India 2: 660. 1879. T. procumbens Mill., Gard. Dict. (ed.8) no.1.1768. T. obcordata Roxb., Fl. Ind. 2: 445. 1832. Fig. 1e, f.

A prostrate herb, glabrous. Stem often tinged purplish. Leaves opposite, inequal, broadly obovate, smaller ones oblong. Flowers axillary, sessile, solitary, pink or white. Stamens 10-20, inequal. Style 1, linear. Pyxidium of about 4 mm, with 3-5 black seeds of 2 mm. Native to tropical America; found almost throughout India as a weed of waste lands and delights in rubbish. It was described as Trianthema obcordata by Roxburgh (1832) with the Telugu vernaculars yurra-galjeror and bodo-pail-kura while J.S. Gamble collected it from Andhra Pradesh (Bezawada = Vijayawada) in August 1883. It is poisonous to live stock. While the leaves and tender tops are eaten as vegetable, the roots are a substitute for punarnava. Note: (i) Biodiversity: Two ecotypes occur in Trianthema portulacastrum namely, Flava: perianth white; stem, branches and leaves light green, called tella galijeru (Fig.1e) and Rubra: perianth light pink; stem and leaf-margin red, known as erra galijeru (Fig.1f) (Reddy, 1982; present study). (ii) Phytochemicals: C-methylflavone (flavonoid) and leptorumol (known from the fern genus Leptorumohra) were extracted with dichloromethane (Kokpol et al. 1997). Trianthenol, an antifungal tetraterpenoid, was isolated from the chloroform extract of the plant (Nawaz et al., 2001). The roots bear triantemine (C32H46O6N2) and ecdysterone - a phytoecdysterone of excellent application in insect control of crop plants as chemosterilant (Banerji et al., 1971; Daniel and Sabnis, 1986; Daniel, 2006), and (iii) Survival Strategy: Rubra ecotype is found to be more hardy (drought tolerant) and better adapted to aridity due to lesser water loss/higher water retention capacity, more protein content and proline accumulation over Flava. So, the rubra ecotype was found to be more aggressive and successful form because of the ecological adaptations and reproductive strategy, e.g. relatively higher seed germination percentage (Rao and Reddy, 1982; Mohammed and Sen, 1990). 5. Zaleya decandra (L.) Burm.f., Fl. Ind. 110. t. 31. f. 3. 1768. Trianthema decandra L., Mant. 1: 70. 1767; Cooke, Fl. Bombay 1: 555. 1903; Gamble, Fl. Madras 2: 551. 1919; Saldanha & Nicolson, Fl. Hassan Distr.: 94. 1976. Fig.1g, h. Prostrate herb. Leaves exstipulate, elliptic-lanceolate to elliptic-oblong, narrowed at base, acute at apex; petiole up to 1.2 cm long; bracteoles 2, small. Flowers in dense axillary, nearly sessile cymes. Perianth lobes pink or white, ovate, obtuse; stamens 10. Seeds 4, black with concentric rings.

It is an exotic weed, common on sandy or open places.

Note: Parallel to Flava and Rubra in Trianthema portulacastrum, this taxon also has two ecotypes: green (Fig.1g) and red (Fig.1h). Roxburgh (1832) provided the vernaculars tella galjeroo (Telugu) and gada-buni (Hindi) for it.

Journal of Theoretical and Experimental Biology (ISSN: 0972-9720), 7 (3): 127-134, 2011


Sateesh Suthari et al / Galijeru as the Ayurvedic drug Vrshabhu

6. Zaleya pentandra (L.) C. Jeffrey in Kew Bull. 14: 238. 1960. Trianthema pentandra L., Syst. Nat. ed. 12, 2: 297. 1767. Trianthema govindia Buch.-Ham. ex G. Don in Gen. Syst. 3: 72. 1834. Zaleya govindia (G. Don) N.C. Nair in Bull. Bot. Surv. India 8: 86. 1966. Fig.1d. A diffuse prostrate perennial. Stem glabrous. Leaves oblong-obovate, papillose; petiole 8-11 mm long, with a sheathing base. Flowers axillary, solitary, sometimes 2-4, 5 (rare), sessile, bract solitary, ovate-lanceolate, bracteoles 2-3 inequal. Calyx papillose, oblong-ovate. Stamens 5, alternating with sepals. Ovary ob-pyramidal, papillose; styles 2, slightly curved. Seeds 2, compressed, rugulose, black.

Note: It is found in Africa, Iran, Pakistan and India. The present report forms a new record for the state of Andhra Pradesh.
2. Molecular Characterization (a) Micromolecular: Although there was no attempt to characterize the drug vrshabhu and its possible adulterants in this regard, the available phytochemical literature (summarized above for each taxon) helps to identify the main elements. For example, while anthocyanins characterize Eclipta prostrata, betalains substitute them in the rest. A combination of the presence of micromolecules such as C-methylflavone, leptorumol, trianthenol and triantemine in Trianthema portulacastrum makes it distinctive from Zaleya species and Alternanthera pungens whereas Boerhavia erecta has none of these and instead bears punarnavine 1 and 2, punarnavoside, boeravinones A-F (rotenones), borhavine, lignan liriodendrin, etc. to mention some of these.
Table 1: Polymorphism with the five primers used against Vrshabhu and its four adulterants. ISSR Marker No. Primer 1 Primer 2 Primer 3 Primer 4 Primer 5 Total Alleles (48) 11 11 11 08 07 Polymorphic Alleles 10 11 11 08 07 Percentage Polymorphism 90.9 100 100 100 100


PIC 0.99 0.99 0.99 0.98 0.97

ISSR: Inter Simple Sequence Repeat; PIC: Polymorphism Information Content. (b) Macromolecuar: An attempt was made, for the first time, to characterize vrshabhu and the possible adulterants in India using ISSR markers in primers 1-5 (Table 1) against five taxa (Alternanthera pungens [Ap], Boerhavia diffusa [Bd], Trainthema portulacastrum flava [Tpg] and rubra [Tpr], and Zaleya decandra [Zd]). The DNA profiling was done for five primers in two slabs three species in one (Fig. 2a) and two in another (Fig. 2b). Based on numerical assessment of similarity (within genetic polymorphism evinced by the 48 alleles) and UPGMA, a phenogram was constructed (Fig. 2c). It is formed off two groups (Tpr-Tpg-Ap and Zd-Bd), with just 10% phenetic similarity between them. Of these, the first cluster (P1) is formed between the two ecotypes of Trianthema portulacastrum (vrshabhu) of Aizoaceae, evincing 63% similarity. The other phenon (P2) constituted of Alternanthera pungens of Amaranthaceae (part of betalain-bearing [potent class of antioxidants] Centrospermae/Chenopodiales), showing mere 17.8% similarity with P1. The third cluster (P3) is of two plant species of different families, though again of betalain-bearing stock (Zaleya decandra of Aizoaceae and Boerhavia diffusa of Nyctaginaceae), with mere 15.2% similarity.
Journal of Theoretical and Experimental Biology (ISSN: 0972-9720), 7 (3): 127-134, 2011


Sateesh Suthari et al / Galijeru as the Ayurvedic drug Vrshabhu

The present also establishes that the two species of Zaleya Burm.f. are different and generically distinct from Trianthema L.

Figure 2: Molecular characterization of vrshabhu and its adulterants. a and b DNA profiling primers 1-5 (as indicated in Table 1) and plants 1-5 (1 Trianthema portulacastrum [red], 2- T. portulacastrum [green], 3 Alternanthera pungens, 4 Zaleya decandra, 5 Boerhavia diffusa). M Marker. c Dendrogram for five taxa on Jaccards coefficeint and UPGMA.


Journal of Theoretical and Experimental Biology (ISSN: 0972-9720), 7 (3): 127-134, 2011


Sateesh Suthari et al / Galijeru as the Ayurvedic drug Vrshabhu

The plant drug vrshabhu is obtained from the dried root of Trianthema portulacastrum. The root material is collected based on the identity of above ground parts. Trianthema portulacastrum has similar foliage and appearance to that of punarnava and, therefore, T. portulacastrum and T. decandra (= Zaleya decandra) are usually misidentified or substituted for punarnava Boerhavia diffusa (Kokate et al., 2008). We observed Alternanthera pungens (mulla ponnaganti), Eclipta prostrata (gunta galagara/galijeru) and Zaleya pentandra (galijeru) are admixtured for Trianthema portulacastrum in view of their striking morphological semblance and sympatry. These are all adventive species from Africa and America, affecting the efficacy of the drug vrshabhu. A simple dichotomous key is provided to single out the true vrshabhu during the collection from the mix-up of herbs to resolve the problem of misidentification, mixing (possible admixture) and adulteration at field collection level or later in the trade. ISSR markers are found to provide distinct passport data for the ingredients of the mixtures or adulterants. The study also establishes the true identity of Zaleya pentandra which is overlooked or passed on in India as Z. decandra or Z. govindia (due to confusion in the existing taxonomic literature).

S. Suthari is obliged to Dr V.K. Dadhwal, Project Director, NCP (IGBP), Dr Sarnam Singh (Dy. Project Director, VCP), IIRS, Dehra Dun, for the financial assistance to visit Madras Herbarium, Coimbatore. The authors thank Dr G.V.S. Murthy, Joint Director, BSI, Southern Circle, Coimbatore, for permission to work at MH. The authors also thank the Heads of Department of Botany and Biotechnology, Kakatiya University, Warangal, India for facilities.

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Journal of Theoretical and Experimental Biology (ISSN: 0972-9720), 7 (3): 127-134, 2011