Microscopical Evaluation, Phytochemical Analysis and HPTLC Fingerprinting of Tuber of Actinoscirpus grossus (L.f.) Goetgh. & D.A.Simpson

Actinoscirpus grossus (L.f.) Goetgh. & D.A.Simpson (Cyperaceae), is a Perennial with long stolons and rhizomes ending in small tubers. It is popularly known as Kasheruk in Sanskrit. The plant is traditionally used as anti-diarrheal, anti-emetic, and tonic to the liver. In order to do the detail standardization of plant macro-microscopical observation, phytochemical analysis and HPTLC Finger printing of tuber was performed according to pharmacopoeia procedure. Microscopic analysis has showed thick-walled polygonal epidermal cells of young root stalk in surface view, elongated phloem parenchyma filled with starch grains, spiral to annular vessel fragments and simple starch grains scattered all over the powder. Phytochemical analysis showed presence of carbohydrate, coumarins, flavanoids, steroid, tannin, and terpenoid. Ethanol extract of plant were fingerprinted in toluene: ethyl acetate (7:3). The developed plates were visualized in UV 254, 366, and then derivatised with vanillin sulphuric acid and scanned under UV 254 and 366 nm. These specific identities will be useful in identification and authentication of the raw drug.

of herbal products is affected badly by practice of substitution and adulteration, therefore systematic identification of drugs and their substitutes is an essential step while producing standardized herbal products.Standardization of herbal drug is not an easy task as numerous factors influence the bio efficacy and reproducible therapeutic effect in order to obtain quality oriented herbal products. 8In the present study standardization of tuber extract A. grossus was performed.Preliminary phytochemical study, powder microscopy and HPTLC of tuber extract of plant are documented.

Plant materials
The authenticated sample of plant tuber powder of A. grossus was purchased from Vaidya Hukam Chand Arogyadham Gharaunda, Haryana India.The voucher specimen (No. 495/14101822) was deposited at the Pharmacognosy Laboratory of S.D.M Centre for Research in Ayurveda and Allied Sciences, Udupi for future reference.For powder microscopy, the sample was sift through mesh 60; the powder was stored in glass vial for the phytochemical examination and HPTLC study.simple and pitted fibres, thick and thin walled parenchyma with or without yellowish brown contents, elongated phloem parenchyma filled with starch grains, spiral to annular vessel fragments and simple starch grains scattered all over the powder [Figure 1].HPTLC photo documentation of alcoholic extract of A. grossus showed 7, 13 and 14 spots with characteristics R f value and colors under short UV, long UV and white light after derivatisation respectively [Table 1].Spots with R f value 0.27,0.36and 0.81 were detected in all three-detection method though in different colors.HPTLC densitometric scan at 254nm showed maximum number of detectable spots counting to 11 with different percentage area.HPTLC with photo documentation and densitometric scan can be used as finger print of chemical component present in ethanol extract of A. grossus [Figure 2,3].Powdered tuber extract of plant A. grossus showed presence of carbohydrate, coumarins, flavonoids, steroid, tannin, and terpenoid [Table 2].

DISCUSSION
The plant A. grossus is a principal weed of four South East Asian countries.It is well known herb which is used in Indian System of medicine.Medicinal plant materials are being adulterated due to many reasons.The practice of substitution and adulteration will badly affect the therapeutic activity of herbal products.Microscopical analysis of plants is invaluable for assuring the identity of the material and as initial screening test for impurities. 15Authentication and quality assessment of herbal materials deals with the pharmacognosy that is based on macroscopic and microscopic features.A big quantum of research works in the area of authentication of the correct plant source has been undertaken to provide means

Powder microscopy
A pinch of powder was warmed with drops of chloral hydrate on a microscopic slide and mounted in glycerin.Slides observed under microscope and diagnostic characters were observed and photographed using Zeiss AXIO trinocular microscope attached with Zeiss AxioCam camera under bright field light.Magnifications of the figures are indicated by the scale-bars. 9

Preliminary phytochemical analysis
Preliminary phytochemical investigation was done with the coarse powder to detect the presence of carbohydrate, coumarins, flavonoids, steroid, tannin, and terpenoid in ethanolic extract. 10TLC 1g of powder was extracted with 10 ml of ethanol, 4, 8 and 12 µl of the above extract was applied on a pre-coated silica gel F254 on aluminum plates to a band width of 7 mm using Linomat 5 TLC applicator.The plate was developed in Toluene: Ethyl acetate (7:3).[13][14]

RESULTS
7][18] Sophisticated modern research tools for evaluation of the plant drugs are available but microscopic method is still one of the simplest and cheapest methods to establish the correct identity of the source of material. 19According to the world health organization (WHO) the macroscopic and microscopic description of a plant is the first step to establish the identity before any tests are undertaken.Preliminary phytochemical analysis showed presence of carbohydrate, coumarins, flavonoids, steroid, tannin, and terpenoid which may help in the hepatoprotective activity of plant.(HPTLC) is one of the sophisticated instrumental techniques for qualitative and quantitative analysis of the herbs and herbal drugs. 21,22Marker compound means chemical constituents within a medicinal plant that can be used to verify its potency or identity.For sometimes, the marker compounds may be described as active ingredients or chemicals that confirm the correct botanical identity of the starting material.It is very difficult to identify correct marker compounds for all traditional medicines, because some medicines have unknown active constituents and others have multiple active constituents.][25] One of the drawbacks of herbal medicine is lack of standardization and quality control profiles of correct identification of species.The mistaken substitution and misclassification of species is real danger in preparation and administration of herbal medicine.The features available in the powder form of the given specimens are useful to distinguish the samples even in a mixture form. 26,27TLC is one of the sophisticated instrumental techniques for qualitative and quantitative analysis of the herbs and herbal drugs.HPTLC serves as quality assessment tool which helps in identification of variation in chemical composition in plants.HPTLC fingerprinting shows different R f values can be used as quality indicating finger printing for A. grossus in the dried form.High-performance thin-layer chromatography (HPTLC) is a form of thin-layer chromatography (TLC) that provides superior separation using optimized coating material, novel procedures for mobile-phase feeding, layer conditioning, and improved sample application.It promotes for higher separation efficiencies, shorter analysis time, lower amounts of mobile phase, and efficient data acquisition and processing. 20Among the modern analytical tools HPTLC is a powerful analytical method equally suitable for qualitative analytical tasks HPTLC produces visible chromatogram complex information about the entire sample and multiple samples are seen simultaneously, so that reference and test samples can be compared for identification.The analysis and quality control of herbal medicines are moving a step ahead towards an integrative and comprehensive direction, to tackle the complex nature of herbal medicines.High-performance thin layer chromatography