Macro-Microscopic evaluation , Physicochemical analysis and HPTLC Finger printing of Curculigo orchioides Gaertn . Rhizome

Introduction: Curculigo orchioides Gaertn. Family Hypoxydaceae (Amaryllidaceae) is a perennial herb with finger like rhizome. Its rhizomes have been significantly used in treatment of various ailments including cancer. Methods: Macro-microscopic analyses, physico-chemical studies and HPTLC finger printing of rhizomes of C. orchioides were performed according to pharmacopoeial procedures. Results: Microscopic analysis has shown rows of rectangular and conical cells in cork, thin walled cortex, parenchymatous cells, starch grains, crystal of calcium oxalate, mucilage cell and spiral xylem vessel in the rhizomes of C. orchioides. Physicochemical studies revealed loss on drying (10.469%), total ash (8.463%), acid insoluble ash (1.10%), water-soluble ash (2.163%), alcohol soluble extractive value (3.036%), and water-soluble extractive value (22.666%) of the raw drug. Ethanol extract of the plant were fingerprinted in petroleum ether and ethyl acetate (7.0:3.0) solvent system and scanned densitometrically after dipping in anisaldehyde-sulphuric acid reagent followed by heating at 105°C. Conclusion: These specific identities will be useful in identification and authentication of the raw drug in dried form.


INTRODUCTION
Curculigo orchioides Gaertn., syn.C. malabarica Wight., C. brevifolia Dryand. 1 and Hypoxis orchioides Kurz. 2 is well known medicinal plant known as Kalimusli in Hindi.It is a perennial herb with a rosette of sensible with linear lanceolate, membraneous leaves and bright yellow colour flowers, closed to ground. 2 The plant is native to India 3 and distributed from sub-tropical Himalayas, West Bengal, Assam, Konkan, West peninsula to Kanyakumari. 2 Its rhizomes are widely used as a demulcent, diuretic and aromatic tonic, aphrodisiac, in the treatment of leprosy and nervous disease. 4Rhizomes of C. orchioides have been reported for their medicinal properties like platelet regeneration, 5 antioxidant activity, 6 hepato-protective efficacy, 7 antipyretic activity 8 and immune stimulant properties. 9edic manuscripts described the therapeutic potential of C. orchioides and are well documented in the treatise of Ayurveda in the name of Talamuli.Accordingly, the synonyms and medicinal properties of C. orchioides are depicted in Sanskrit shlokas mentioned in Bhavprakash Nighantu (Shloka 183). 10hizomes of this plants has been described to possess medicinal properties as sweet, cooling, diuretic, aphrodisiac, anti-aging and considered as a Rasayana drug which can be used in male infertility, hemorrhoids, piles, which also increases Kapha and reduces Pitta daha (burning sensation), acts as stimulant and gives strength.C. orchioides has been reported with constituents 11 such as flavones, glycosides, steroids (sitosterol, stigmasterol and yuccagenin), 12 terpenoids, saponins, glucose, mannose, xylose, glucoronic acid, resin, tannin, fat, mucilaginous substances 13 and other secondary metabolites.Steam distilled fraction of C. orchioides has reported for its antibacterial activity against Gram-negative and Gram-positive pathogens. 14] With this background detailed quality control studies were undertaken for this traditional raw drug with the aim of developing standards of authenticity.

Collection and Identification
Dried rhizomes were collected from local Ayurveda pharmacy in Gwalior, Madhya Pradesh.The plant material was authenticated at Pharmacognosy department of SDM Center for Research in Ayurveda and Allied Sciences, Udupi, Kuthpady, Karnataka and (a specimen (725/16021205) is being maintained for future reference.The dried rhizomes were cleaned, coarsely powder and used for macroscopic and microscopical characterization, phytochemical analysis and HPTLC.

Macro-microscopic analysis
Macroscopic characters of fresh rhizome and powder were keenly observed under naked eyes to record the specific botanical characters.The external features of the test samples were documented using Canon IXUS digital camera.Rhizomes were preserved in formalin-acetic acid-alcohol preservative solution {5% formalin (5 ml), 5% acetic acid (5 ml) and 50% ethyl alcohol (90 ml)}, before 48 hours 19 of microscopic analysis.Very thin transverse sections of rhizome were obtained using sharp blade followed by safranine staining. 20Transverse sections were photographed using Zeiss AXIO trinocular microscope attached with Zeiss AxioCam camera under bright field light.Magnifications of figures are indicated in scale bars.For powder microscopy, 1g of coarse powder was sift through 80 pore size mesh.A pinch of powder was mixed with drops of choral hydrate on microscopic slides and mounted with a drop of glycerine-water.Slides were observed and characterized under Zeiss AXIO trinocular microscope.Magnifications are indicated by scale bars. 21hysico-chemical analysis: Physico-chemical characterization like loss on drying at 105ºC, total ash, acid insoluble ash, water soluble ash, ethanol and water soluble extractives values were determined as per Quality Standard of Indian Medicinal Plants.22

HPTLC Fingerprinting
One gram of powdered rhizomes were extracted with 10 ml ethanol and kept for cold percolation for 24 h and filtered.4, 8 and 12 µl of the plant extract were applied on a pre-coated silica gel F254 on aluminium plates to a band width of 7 mm using Linomat 5 TLC applicator.C. orchioides plate was developed in Petroleum ether: Ethyl acetate (7.0:3.0 v/v).The developed plate was visualized in UV 254 nm, 366 nm and then derivatised with anisaldehyde sulphuric acid reagent 23 and scanned under UV 254 nm, 366 nm and 620 nm post derivatisation.R f , colour of the spots and densitometric scan were recorded using CAMAG Scanner 4. [24][25]

DISCUSSION
C. orchioides rhizomes are mucilaginous in nature and well known Rasayana drug in Ayurveda System of Medicine.Morphological and anatomical standardization of herbal drugs needs the information from basic disciplines of plant sciences for identification of plant drug.Simultaneously for identification of chemical nature of plant in term of physicochemical analysis, qualitative and quantitative analysis for the detection of active constituent expertise are required.According to Kunle et al, 26 standardization of herbal drug is a series of protocols which assure the quality, efficiency and safety of plant drug.Macro-microscopy helps in the identification of plant characters anatomically and helps in identification of botanical background.Present study shows a clear zone of rectangular and tangentially elongated cork cells.Thin walled oval and spherical parenchymatous cells with small intercellular spaces were recorded.Parenchymatous cells were distributed throughout the section while some parenchymatous cells show the presence of bundles and needle like crystals of calcium oxalate.1][32] The values obtained in the study will serve as constants for quality standard measures for standardization of drug in the dried form.High performance thin layer chromatography (HPTLC) serves as quality assessment tool which helps in identification of variation in chemical composition plants.TLC identity is a part of every herbal monograph of international standards. 33HPTLC fingerprinting shows different R f values at different wavelength and reported values can be used as quality indicating fingerprint for Curculigo orchioides in the dried form.Findings reported in the present investigation are in support of API 34 and QSIMP 2012 2 .

CONCLUSION
Pharmacognostical characterization of the C. orchioides has been done as per pharmacopoeial methodology.Present study explores the botanical (in terms of macro-microscopic observations), physicochemical observation (in terms of total ash, AIA, WSA, ASE, WSE and loss on drying)    and HPTLC fingerprint profile can serve as excellent standard for the identification and authentication of drug in dried form.

Table 2 : R f values of all the samples
*L-Light, D-Dark, F-Fluorescence.