HPTLC Fingerprint Profile and Isolation of Marker Compound of Ruellia tuberosa

The present study was aimed to identification, isolation, and quantification of marker in R. tuberosa (Acanthaceae). HPTLC fingerprinting was carried out for various extract of root, stem, and leaf of R. tuberosa. From the HPTLC fingerprint the florescent band (under 366 nm) at Rf : 0.56 (mobile phase chloroform : toluene : ethyl acetate (6 : 3 : 1, v/v)) was found in leaf, root, and stem of R. tuberosa. So, the florescent band (under 366 nm) at Rf : 0.56 was isolated as marker compound RT-F2 from root of R. tuberosa. The marker compound RT-F2 was quantified by using HPTLC technique. The percentage (W/W) amount of RT-F2 was found to 40.0% and 44.6% in petroleum ether and ethyl acetate extract of R. tuberosa roots, respectively. Further study is suggested to characterization and biological nature of marker compound.


Introduction
Marker compound means chemical constituents within a medicinal 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 medicinals, because some medicinals have unknown active constituents and others have multiple active constituents.A chromatographic fingerprint of a herbal medicine is a chromatographic pattern of the extract of some common chemical components of pharmacologically active and/or chemical characteristics.By using chromatographic fingerprints, the authentication and identification of herbal medicines can be accurately conducted even if the amount and/or concentration of the chemically characteristic constituents is not exactly the same for different samples of drug.Hence it is very important to obtain reliable chromatographic fingerprints that represent pharmacologically active and chemically characteristic component of the herbal drug [1-5].
Ruellia tuberosa is an erect, suberect, or diffuse perennial herb up to 60-70 cm tall herb and belongs to family Acanthaceae, a native of Central America, introduced into Indian garden as ornament.It is used medicinally in West Indies, Central America, Guiana, and Peru.R. tuberosa is commonly known as "Cracker plant" [6][7][8].In Siddha system of medicine, leaves are given with liquid copal as remedy for gonorrhea and ear diseases [9], used in stomach cancer [10].Dried and ground roots in dose of two ounces cause abortion and also used in sore eyes [11].The herb also exhibits emetic activity and employed substitute of ipecac, also used in bladder stones and decoction of leaves used in treatment of Bronchitis [12].In Suriname's traditional medicine system, it is used as anthelmintic and also in management of joint pain and strained muscles.In folk medicine, it has been used as diuretic, antipyretic, antidiabetic, antidotal, thirst-quenching agent and analgesic and anti-hypertensive activity [13,14].Ruellia tuberosa is used as cooling in urinary problem, uterine fibroids [15,16].It has recently been incorporated as a component in a herbal drink in Taiwan [17].It has been experimentally proved to possess antioxidant [18], antimicrobial 2.2.Chemicals.All other reagents were analytical grade, purchased from Merck (Darmstadt, Germany).All UV-Vis measurements were recorded on a Shimadzu UV-1800.[27].Powdered air dried drug, weighing about 50 g, was extracted successively in soxhlet apparatus with the series of solvents of increasing polarity as follows: petroleum ether, toluene, chloroform, ethyl acetate, and methanol.Each time before extracting with the next solvent, the material was dried.All the extracts were filtered through Whatman filter paper and concentrated.Concentrated extracts were applied on the TLC plate as sample solution.[28,29] 2.4.1.TLC Conditions.TLC plate consists of 20 × 10 cm, precoated with silica gel 60 F254 TLC plates (E.Merck) (0.2 mm thickness) with aluminum sheet support.The spotting device was a CAMAG Linomat V Automatic Sample Spotter (Camag Muttenz, Switzerland); the syringe, 100 μL (from Hamilton); the developing chamber was a CAMAG glass twin trough chamber (20 × 10 cm); the densitometer consisted of a CAMAG TLC scanner 3 linked to WINCATS software.Mobile phase was chloroform : toluene : ethyl acetate (6 : 3 : 1, v/v).Saturation time for mobile phase was 2 hours.

Procedure.
Various extracts of roots, leaf, and stem of R. tuberosa were applied on TLC plate and the plate was developed in chloroform : toluene : ethyl acetate (6 : 3 : 1, v/v) solvent system to a distance of 8 cm.The plates were dried at room temperature in air.The plate was scanned at 254 nm (Figure 1) and 366 nm (Figure 2) before spraying and at 600 nm (Figure 3) after spraying with detection reagent (Anisaldehyde sulfuric acid reagent and plate was heated at 110 • C for 5 minutes).The R f values and color of the resolved bands were noted.(200 g) was extracted with petroleum ether and ethyl acetate (500 mL) separately in soxhlet apparatus for 2 days.Then the extracts were concentrated by distilling the solvent and concentrated extracts were subjected to repetitive preparative thin layer chromatography using Silica Gel G as stationary phase (20 × 20 cm glass plates) and chloroform : toluene : ethyl acetate (6 : 3 : 1 v/v/v) as mobile phase.Fluorescents bands under 366 nm at R f value 0.56 were identified RT-F2 compound.RT-F2 bands were scraped.RT-F2 was separated from silica Gel G by treating with methanol and Chloroform mixture (1 : 1), filtered through Whatman filter paper, and filtrates were combined, concentrated, and dried.Isolated compounds were subjected to TLC and HPTLC, UV spectroscopy (Figure 4), IR spectroscopy (Figure 5), and Mass spectroscopy (Figure 6).

Quantification of RT-F2 in Petroleum Ether and Ethyl Acetate Extract Using HPTLC Method
Standard Stock Solution.A solution of F2 compound (500 μg/mL) was prepared in chloroform.

Sample Preparation
Ethyl Acetate Extract.Stock solution of sample 2 mg/mL of extract was prepared in chloroform.Petroleum Ether Extract.Stock solution of sample 1 mg/mL of extract was prepared in chloroform.
Calibration Curve.From the standard stock solution 2.5-12.5 μL solutions were applied on precoated plate of Silica Gel G, to produce the range of 1.25-6.25 μg of RT-F2 per spot, respectively (Figure 7).Calibration curve is given in Figure 11.
Sample.A 10 μL of each extract was applied.
Stationary Phase.The stationary phase was Precoated plate, Silica Gel G 60 F254.

Development. Plate was developed in a twin trough chamber.
Detection.We spray with Anisaldehyde sulfuric acid reagent and heat at 110 • C for 5 minutes.
The plate was scanned at 366 nm under fluorescent mode before spraying and at 600 nm (Figure 7) after spraying.The Chromatograph of RT-F2 standard compound (Figure 8), petroleum ether extracts (Figure 9), and ethyl acetate extract (Figure 10) were reported.

Results and Discussion
3.1.HPTLC Fingerprint Profile.HPTLC fingerprint showed that purple colored band (after derivatisation) (Figure 3) at R f : 0.56 was found in leaf, root, and stem of R. tuberosa.The florescent band (under 366 nm) at R f : 0.56 was selected as marker compound and identified as RT-F2.

Isolation and Characterization of Marker RT-F2
Compound.Data from fingerprinting results provide information about presence of major terpenoid in petroleum ether extract and ethyl acetate extract of root, targeted for isolation.

Quantification of RT-F2 in Petroleum
Ether and Ethyl Acetate Extract Using HPTLC. Figure 7 shows the HPTLC chromatogram of standard RT-F2 compound, petroleum ether extract and ethyl acetate extract.
The percentage (W/W) amount of RT-F2 was found to 40.0% and 44.6% in petroleum ether and ethyl acetate extract of R. tuberosa roots, respectively (Tables 1 and 2).

Conclusion
Herbal medicines are composed of many constituents and are therefore very capable of variation.Hence it is very important to obtain reliable chromatographic fingerprints that represent pharmacologically active and chemically characteristic components of the herbal medicine.HPTLC fingerprinting profile is very important parameter of herbal drug standardization for the proper identification of medicinal plants.A TLC densitometric method for the quantification of isolated marker compound RT-F2 was established in petroleum ether and ethyl acetate extract of roots of R. tuberosa.The present HPTLC fingerprinting profile can be used as a diagnostic tool to identity and to determine the quality and purity of the R. tuberosa in future studies.

2. 1 .
Plant Material.Fresh plant of Ruellia tuberosa was collected from the campus of The M. S. University of Baroda in the month of August 2008.Plant was authenticated at Botany Department of The M. S. University.Voucher specimen (PHR/HDT/DC-RT-08) was stored in herbarium of our laboratory.Roots were separated and sun dried separately.Dried plant material was powdered.