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Standardization of the Plant Epaltes Pygmaea DC. (Asteraceae)

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Standardization of the Plant Epaltes Pygmaea DC. (Asteraceae) K. Amala et al. / Journal of Pharmacy Research 2016,10(10),674-682 Research Article Available online through ISSN: 0974-6943 http://jprsolutions.info Standardization of the plant Epaltes pygmaea DC. (Asteraceae) K. Amala*1 , R. Ilavarasan2, S. Amerjothy3 1,2Captain Srinivasa Murti Regional Ayurveda Drug Development Institute (CCRAS), Anna Hospital Campus, Arumbakkam, Chennai –600 106, India. 2Department of Plant Biology & Plant Biotechnology, Presidency College, Chennai. India. Received on:17-09-2016; Revised on: 22-10-2016; Accepted on: 03-11-2016 ABSTRACT Background: To establish the pharmacognostic parameters using pharmacopoeial standards for correct identity of whole plant of Epaltes pygmaea DC. (Asteraceae). Methods: In pharmacognostic studies different types of evaluations were carried out that focus on taxonomy of the species, macro- and microscopic characters, physico-chemical parameters, fluorescence analysis, preliminary phytochemical screening and HPTLC finger print compared with marker compound stigmosterol. Results and discussion: The taxonomically it is a small annual herb, 8 to 20 cm high, minutely winged branched stem with aromatic roots, leaves are alternate, linear, lanceolate to oblong, flower pink, solitary, terminal, heterogamous. Microscopically the plant showed the presence of bi or tri-radiate wings of the stem, leaves microscopy indicated the presence of anisocytic type of stomata, thin walled with small spindle shaped cells of leaf epidermis, powder microscopy showed vessel elements with short tail, vertical chain of elongated parenchyma cells with prominent simple pits, large, spherical and spiny pollen grains. Plant extracts showed the presence of flavonoids, steroids, phenols, tannin and sugars. The standard marker stigmosterol detected with Rf 0.54 in alcohol extract has been confirmed by TLC/HPTLC finger print. Conclusions: The study provides the scientific data for the proper identification and establishment of standards for the use of Epaltes pygmaea. These information will also be helpful to differentiate Epaltes pygmaea from closely related other species. KEY WORDS: Epaltes pygmaea; Asteraceae; Pharmacognosy; HPTLC finger printing. 1. INTRODUCTION: The genus Epaltes Cass. of the family Asteraceae found in the glucoside.8 Recent studies have reported that the alcohol and tropics, about nine species are recorded in the worldwide, only about aqueous extract of E.pygmaea possesses hepatoprotective activity two species reported in India.1-3 The genus Epaltes is traditionally against paracetamol-induced liver damage in rats and also have potent used in Sri Lanka to cure various ailments like urethral discharges, diuretic activity.9 jaundice, acute dyspepsia, diaphoretic, diuretic and stimulating expectorant.4 One among the uncovered medicinal weed of this genus A survey of the literature revealed that detailed pharmacognostical is Epaltes pygmaea DC. is a small annual herb, 8 to 20 cm high, studies are lacking in E. pygmaea, the present paper attempts to minutely winged branched stem with aromatic roots, leaves are undertake the pharmacognostic and phytochemical screening on alternate, linear, lanceolate to oblong, flower pink, solitary, terminal, successive extracts and fluorescence behavior of powder as well as heterogamous. It is found in Sri Lanka, India, Java and China, mostly presence of marker compounds stigmosterol has been confirmed by found in Sri Lanka and a limited extend in South India, especially TLC/HPTLC finger print profile for proper identification and quality towards the coast, gregarious in low lying ground by river banks and standardization of this plant. The pharmacognostic characteristics paddy field after harvesting in clayey soil.5-7 The chemical constituents of this plant are reported here for the first time. of this plant were lupeol acetate, stigmasterol, stigmasterol acetate, apigenin, luteolin, apigenin-7-0-glucoside and luteolin-7-0- 2. MATERIALS AND METHOD: *Corresponding author. 2.1. Plant material Dr. K. Amala The healthy fresh whole plant of E. pygmaea was collected during Captain Srinivasa Murti Regional Ayurveda Drug flowering in the month of February, 2010 from Thirunelveli District of Development Institute (CCRAS), Tamil Nadu and authenticated by Dr. V. Chelladurai, retired Research Anna Hospital Campus, Arumbakkam, Officer, Survey of Medicinal Plant Unit-Siddha, Palayamkottai, Tamil Chennai –600 106, India. Nadu, India. Voucher specimens (00630) of the plant was deposited Journal of Pharmacy Research Vol.10 Issue 10 October 2016 674-682 K. Amala et al. / Journal of Pharmacy Research 2016,10(10),674-682 at the pharmacognosy department of Captain Srinivasa Murti Drug 2.6. TLC/HPTLC Research Institute for Ayurveda, Chennai for further documentation Sample preparation: 1 g of powdered sample was accurately and reference. Care was taken to select healthy plants and normal weighed and exhaustively extracted by methanol (100 ml) using organs. The required samples of different plant organs were excised Soxhlet apparatus and filtered and made up to 10 ml in standard flask and fixed in FAA (Formalin-5ml + Acetic acid-5ml + 70% Ethyl alcohol- separately. The standard solution of stigmosterol was prepared by 90ml) immediately after collection The materials were left in FAA for dissolving 10 mg of stigmosterol in methanol and made up to 10 ml in more than 48 hours and then dehydrated through a graded series of a standard flask. tertiary-butyl alcohol as per standard protocol.10 The paraffin wax embedded specimens (10 to 12 µm) were sectioned with the help of 5 μl and 10 μl of each of the test solutions of E. pygmaea and 10 μl of rotary microtome. The sections were processed and stained with standard solution containing 10 μg of stigmosterol were applied on toluidine blue as per reported method of O’Brein et al.11 TLC Figure of size 20 x 10 cm as 6 mm bands using CAMAG TLC autosampler 4 applicator. The Figure was developed in the solvent For studying the stomatal morphology, venation pattern and trichome of toluene: ethyl acetate (9:1, v/v) in a CAMAG twin trough chamber distribution, paradermal sectioning was done.10 Powder microscopic up to a distance of 8.0 cm at a temperature of 27±2°C. The Figures studies were carried out by examining the powder of the plant samples was air dried, scanned at a wavelength of 520 nm using CAMAG as well as by macerating them in Jeffery’s maceration fluid.10, 12 TLC scanner (030618) with CATS V 4.06 software. The peak area of stigmosterol was recorded. The Figure was then dipped in vanillin- 2.2. Photomicrographs sulphuric acid reagent (1 g vanillin dissolved in 95: 5 ml of ethanol: Photomicrography of the examined cells and tissues were taken at sulphuric acid mixture) and heated in an oven at 105°C till the different magnifications using a Nikon lab photo -2 microscopic unit appearance of colour of the spots.19-20 Rf, colour of the spots and under polarized light microscope. For quantitative microscopy, the densitomatric scan were recorded. cleared tissues were thoroughly washed and stained with safranin and toluidine blue as per the procedure given by Wallis.13 3. RESULTS AND DISCUSSIONS: 2.3. Chemicals and solvents 3.1. Macroscopic All chemicals and solvents used in these studies were of analytical A small annual glabrous herb, 8 to 20 cm high branched from the base, minutely winged branched stem with aromatic roots. Leaves grade obtained from E.Merck, Mumbai. Stigmosterol standard was alternate, linear, lanceolate to oblong, sessile, glabrous, entire or isolated from Captain Srinivasa Murti Drug Research Institute for distantly toothed, base cuneate, decurrent, margin sparsely dentate, Ayurveda, Arumbakkam, Chennai – 600 106. apex obtuse. Heads pink to purple, solitary, terminal, 5 mm across, heterogamous, consisting of both bisexual and female florets, 2.4. Physico-chemical parameters Involucre bract smaller than the flowers, Ray florets multiseriate, Powder of the dried plant material was used for physico-chemical female fertile. Corolla filiform, 2 to 3 toothed, much smaller than style, analysis for determining the total ash, acid insoluble ash, water soluble ovary linearly oblong, 0.4 mm. Disc florets bisexual, tubular, sterile. ash, alcohol and water solubility and loss on drying. Successive Achenes minute, ellipsoid, ribbed (Figure 1&2). extractive values using Soxhlet extraction method were determined as per WHO guidelines.14 The fluorescence analysis of the powdered drug as well as plant extracts was carried out according to method given by Chase and Pratt.15 2.5. Phytochemical analysis Phytochemical tests were performed as per the standard methods.16-18 The air dried and coarsely powdered plant material (4 g) was extracted successively by using soxhlet apparatus with different solvents of increasing polarity i.e. n- hexane, chloroform, ethyl acetate and alcohol. The marc was dried each time before Figure 1. Epaltes pygmaea DC. - Figure 2. Epaltes pygmaea extraction with next solvent. Colour of the extracts was observed External profile of the plant in DC. - Growth habit and floral and percentage yield was calculated (Table 1). natural habitat exomorphic features . Journal of Pharmacy Research Vol.10 Issue 10 October 2016 674-682 K. Amala et al. / Journal of Pharmacy Research 2016,10(10),674-682 3.2. Microscopic epidermal cells are narrow and possess thick, wavy anticlinal walls (Figure 4.a,b). 3.2.1. Leaf Transverse Section of leaf passing through the midrib is neither distinct nor prominent; it has a narrow vertical adaxial groove and the abaxial part is slightly raised. The midrib has a small, conical vascular bundle which is collateral. It consists of three vertical rows of wide, angular thick walled xylem elements and a wide mass of abaxial phloem. There is a small group of sclerenchyma element situated at the protoxylem end. The lamina has smooth and undulate surfaces. Both the adaxial and abaxial epidermal layers are thin with small spindle shaped cells. The mesophyll tissue is not differentiated into distinct palisade and spongy parenchyma zones. There are about six layers of spherical parenchyma. The structure of the leaf margins is similar to that of the middle part of the lamina (Figure 3.a-c).
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