Elucidation of β-sitosterol from Benincasa hispida Seeds , Carissa congesta Roots and Polyalthia longifolia Leaves by High Performance Thin Layer Chromatography

Background: Fruits of Benincasa hispida (BH) is regarded as Valliphala due to its vast plethora of medicinal properties, Carissa congesta (CC) is an imperative local plant particularly in rural communities and Polyalthia longifolia (PL) is an ornamentally significant traditionally relevance plant in India system. β-sitosterol, an active constituent identified from enormous plants has been reported to possess excellent amount of pharmacotherapeutic potential by number of researchers. Objective: In the recent studies, the research team focuses on determining the percentage of the β-sitosterol present in the BH seeds, CC roots petroleum ether extracts as well as PL leaves ethanolic extract by chromatographic technique in harmony with High Performance Thin Layer Chromatography. Materials and Methods: Respective parts of BH, CC and PL plants were shade-dried and extracted by appropriate extraction methods followed by identification of β-sitosterol from the extracts by High Performance Thin Layer Chromatography after preliminary phytochemical screening extracts for the constituents. Results: The amount of β-sitosterol present in the BH seeds, CC roots and PL leaves extracts was found to be 23.00, 5.94 and 1.81 % w/w respectively. Research studies elucidated a peak that coincided with standard peak of β-sitosterol suggesting the presence of constituent in the extracts. Conclusion: Thus, extracts contains important constituent of β-sitosterol in BH, CC and PL.


INTRODUCTION
Plant kingdom is regarded as an enormous reservoir of biologically active molecules but least fraction of plants with medicinal importance and activity are explored until date.Phytochemistry is one of the burgeoning fields of research, which has providing immense tool development in correlating data of the plant extracts.The progress has aided immensely by the development of rapid and accurate methods of screening plants for particular constituents present in the extracts. 1Isolated and characterized phytoconstituents present in the plants have paved the way in contributing to healthcare and income to the consumer as well as government in terms of well-being and cheap price respectively. 2nincasa hispida (Thunb.)Cogn.has been reported as Kushmanda Avaleha, Vasakhanda, Khanda and Rasayana in classical medicine in India. 3,4Rasayana properties of this plant species is attributed in offering immune protection and is advised during the later years of life at forty-five in both the sexes. 510] Carissa congesta is well known in local people in India among the tribal communities due to its multiple properties as well as whole plant is powdered, mixed with cow's milk and given in diabetes. 11,12The plant has been known to produce pentacyclic triterpenoids and glycosides yielding oderside H, digitoxigenin and sugars like D-glucose and D-digitalose. 13The roots have yielded a various volatile principles like 2-acetylphenol, lignans as carinol, and a mixture of sesquiterpenes as carissone and carindone.8][19] Numbers of chemical constituents has been identified from the leaves such as azafluorene alkaloid and three new Aporphine N-oxide alkaloids. 20ctivity guided fractionation has elucidated presence of quercetin, quercetin-3-o-β-glucopyranoside, kaemperfol-3-o-α-rhamnopyranosyl-β-glucopyranoside,kaempferol-3-o-α-rhamnopyranosyl-(1-6)-β-gluco-pyranoside, rutin, β-sitosterol and allantoin from butanol fraction.Ethanolic extract has been found to contain bulbocapnin and steroids like β-sitosterol, stigmasterol and campesterol constituents. 21wever, literature reports several methods for scrutinaztion of β-sitosterol from various plants species, no studies on HPTLC guided fraction for β-sitosterol from these plant extracts has been noticed.Selection of BH, CC and PL species for present research studies was based on its easily availability in local market and enormous pharmacological potential.Minute doses of β-sitosterol have been reported to increase the in vitro proliferative activity of T-lymphocytes as well as acting as chemopreventive agent in colon and breast cancer. 22,23aking pictograph of above viewpoints, our research article focuses on β-sitosterol percentage estimation in the BH seeds, CC roots petroleum ether, and further on PL leaves ethanolic extracts.

Part A: Collection, authentication and extraction
[26]

Part B: Reagents and Biomarker
Standard biomarker used for identification purpose in chromatographic studies was procured from Sigma-Aldrich Private Ltd., Bangalore, India, and solvents from Merck Ltd., Mumbai, India.

Graphical Abstract
Uses: Benincasa hispida, Carissa congesta (CC) and Polyalthia longifolia are significantly important plant in India system.These plants are well known among trial communities.The plants were shade-dried and extracted by appropriate extraction methods followed by identification of β-sitosterol from the extracts by High Performance Thin Layer Chromatography.

METHODS
Our research studies enlist the same flow pattern as we have reported in our previous studies for lupeol identification in these extracts.[26] (a) Thin Layer Chromatography (TLC) Mobile Phase: Hexane: Ethyl Acetate (8.3:1.7-BH and PL) and (7: 3-CC) Dilution: The standard and the sample were dissolved in ethanol and was filtered using Whatman Filter paper no.41 before spotting on TLC plate.
Saturation: Chamber was saturated for 30 mins.

(b) High Performance Thin Layer Chromatography (HPTLC)
The HPTLC was performed at Radiant Research Services Private Limited, Bangalore, India.The HPTLC plates (20x10 cm) coated with silica gel 60 F254 were used and scanning of the developed plates was performed at 336 nm (before derivatization) and 550 nm (after derivatization).
The standard and the sample were prepared by dissolving 5.16 mg and 47.5 mg in 10 ml of ethanol each.Spots of 3 µg/l, 6 µg/l, 9 µg/l and 12 µg/l were applied on plates per extract.

Percentage of β-sitosterol = sample area x standard dilution x purity
x 100/ standard area x sample dilution x 100

(a) Extraction and Preliminary phytochemical analysis
[26] (b) Thin Layer Chromatography (TLC) In TLC, Chromatographic analysis of extracts revealed that Rf value for standard β-sitosterol and the extracts (BH, CC and PL) were found to be 0.72 to 0.77 which confirms the presence of the constituent in these plants (Figures 1 and 2).

(c) High Performance Thin Layer Chromatography (HPTLC)
HPTLC reports interpreted that BH, CC and PL extracts showed well-resolved spots at tracks 5 and 6 in

DISCUSSION
A plant extract contains vast plethora of phytoconstituents correlated to their enormous pharmacological activity.
Chromatography means "colour writing" which is a useful analytical method of organic and inorganic substances, fractionation of complex mixtures, separation of closely related compounds and in the isolation of unstable substances.Qualitative measurements on plant extracts are determinations of amounts of components present in it.[29][30][31][32] The technique uses TLC plate coated with fine microparticles of silica is termed as HPTLC.The analytical technique helped us in separating and identification of the active β-sitosterol component from these extracts.HPTLC is sophisticated powerful visualization technique preferred in the detection of the multiple phytoconstituents from herbal extracts due to its reliability, rapidity, surety, reproducibility and linearity. 27Large number of samples could be analyzed in a single run simultaneously.4][35] Thus, the results depicted in TLC and HPTLC experimentation played a vital role in our research helping us to identify the β-sitosterol with their percentage in the plant extracts.

CONCLUSION
β-sitosterol has been noticed to play an important role in various plant extracts signifying its pharmacotherapeutic potential.Our research studies directs the field of the pharmacognosist budding professionals towards the identified constituent in the these extracts of BH, CC and PL which were confirmed by simple methods like TLC and HPTLC.The current research studies paves the pathway for research thrust scientists to consider the other probable identified constituents in the extracts and correlate there utilities on pharmacological models of different therapeutic categories.

CONFLICT OF INTEREST
We declare no conflict of Interest.