The present study was aimed to identification, isolation, and quantification of marker in
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 [
Fresh plant of
All other reagents were analytical grade, purchased from Merck (Darmstadt, Germany). All UV-Vis measurements were recorded on a Shimadzu UV-1800.
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.
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
Various extracts of roots, leaf, and stem of
HPTLC fingerprint of various extracts of
HPTLC fingerprint of various extracts
HPTLC fingerprint of various extracts of
The dried powder of root (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
UV spectra of RT-F2 compound (
IR spectra of RT-F2 compound.
Mass spectra of RT-F2 compound.
A solution of F2 compound (500
Stock solution of sample 2 mg/mL of extract was prepared in chloroform. Stock solution of sample 1 mg/mL of extract was prepared in chloroform.
From the standard stock solution 2.5–12.5
Densitogram of RT-F2 compound at 600 nm, where T1, T2, T3, T6, and T7 are 1.25, 2.5, 3.75, 5.0, and 6.25 concentration of standard RT-F2 (
A 10
The mobile phase was chloroform : toluene : ethyl acetate (6 : 3 : 1).
The stationary phase was Precoated plate, Silica Gel G 60 F254.
The applicator phase was CAMAG LINOMAT 5.
Plate was developed in a twin trough chamber.
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
Chromatogram of RT-F2 standard compound at 600 nm.
Chromatogram of petroleum ether extract at 600 nm.
Chromatogram of ethyl acetate extract at 600 nm.
Calibration curve of RT-F2 compound.
HPTLC fingerprint showed that purple colored band (after derivatisation) (Figure
Data from fingerprinting results provide information about presence of major terpenoid in petroleum ether extract and ethyl acetate extract of root, targeted for isolation.
Isolated compound F2 has sticky type of nature. It gives violet purple color with Anisaldehyde sulfuric acid reagent and Liebermann-Burchard reagent.
Anisaldehyde sulfuric acid reagent (heat at 105°C for 5 minutes. 3409, 1622. (m/z) 279, 167, 149, 113, 83, 55.
Figure
The percentage (W/W) amount of RT-F2 was found to 40.0% and 44.6% in petroleum ether and ethyl acetate extract of
Calibration curve data for RT- F2 compound of HPTLC method.
Track | Concentration of RT-F2 | Height of peak | Calculated RT-F2 | Area of peak | Calculated RT-F2 | |
---|---|---|---|---|---|---|
1 | 0.57 | 1.250 | 51.72 | 641.96 | ||
2 | 0.56 | 2.500 | 48.37 | 1005.73 | ||
3 | 0.56 | 3.750 | 71.26 | 1446.38 | ||
4 | 0.56 | Unknown* | 73.75 | 3.758 | 1558.45 | 4.006 |
5 | 0.57 | Unknown** | 158.38 | >6.875 | 3246.91 | >6.875 |
6 | 0.57 | 5.000 | 86.79 | 1945.75 | ||
7 | 0.58 | 6.250 | 110.10 | 2313.74 |
*Petroleum ether extract, **Ethyl acetate extract
Regression equation (Height)
Regression equation (Area)
Quantification of RT-F2 compound.
Stationary phase | Precoated Silica Gel 60 GF254 |
Mobile phase | Chloroform : toluene : ethyl acetate (6 : 3 : 1) |
Calibration range of F2 | 2.5–12.5 |
Detection | Anisaldehyde sulphuric acid reagent heated at 110°C for 5 min and detected at 600 nm. |
Regression equation | |
0.99862 (area wise) |
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