Occurrence of curcuminoids in Curcuma longa : A quality standardization by HPTLC

A simple high performance thin layer chromatographic (HPTLC) method has been developed for the simultaneous determination of the pharmacologically important active curcuminoids viz. curcumin, demethoxycurcumin and bis-demethoxycurcumin in Curcuma longa L. The assay combines the separation and quantification of the analytes on silica gel 60 GF254 HPTLC plates with visualization under UV and scanning at 425 nm. Using this technique, the alkaloidal content of different parts of the title plant has been determined.


Introduction
The identities of curcumin, demethoxycurcumin and bis -demethoxycurcumin were confirmed by comparison of their spectral data with those previously reported (Govindarajan et al., 1980).

Extraction of plant material for analysis
Air dried (35-50°C) rhizomes of seven germplasm of C. longa and market turmeric powder samples (1 g each) were ultrasonically extracted separately in 20 mL HPLC grade methanol for 15 min (3 times) and filtered through Whatman No. 42 filter paper after each extraction. Extracts were concentrated under vacuum and finally made up to 20 mL with HPLC grade methanol and ready for HPTLC analysis.

Chromatographic conditions
Chromatography was performed on glass-backed silica gel 60 GF254 HPTLC layers (20 x 20 cm, 300 µm layer thickness) prepared using a Camag (Multenz, Switzerland) TLC plate auto-coater. Methanolic solutions of samples and standard compounds curcumin, demethoxycurcumin and bis-demethoxycurcumin of known concentrations were applied to the layers as 7 mm wide bands positioned 15 mm from the bottom and 20 mm from the side of the plate, using a Camag Linomat 5 automated TLC applicator with the nitrogen flow providing a delivery speed of 150 nL/s from the syringe. These parameters were kept constant throughout the analysis of samples.

Detection and quantification of the alkaloids
After sample application plates were developed in a Camag twin trough glass tank pre-saturated with the mobile phase chloroform:methanol (48:2, v/v) for one hour. It was then poured in twin trough glass solvent development chamber well in advance to allow complete saturation which was further enhanced by keeping one filter paper along one wall of the twin trough chamber. The plate was then kept in a chamber and solvent front was allowed to develop at the height of 7 cm on the plate. The TLC runs were made under laboratory conditions of 25 ± 5°C and 50% relative humidity. After drying, the spots were visualized under Camag UV cabinet (254 and 366 nm). Quantitative analysis of the compounds was done by scanning the plates using Camag TLC scanner model 3 equipped with Wincats software (Camag) applying the following conditions: slit width 6 x 0.45 mm, wavelength (λmax) 425 nm, absorption-reflection scan mode. The identifycation of curcumin, demethoxycurcumin and bisdemethoxycurcumin in rhizomes were confirmed by superimposing the UV spectra of samples and standards within the same Rf window. In order to prepare calibration curves, stock solutions of curcumin, demethoxycurcumin and bis-demethoxycurcumin (1 mg/5 mL each) were prepared and various volumes of these solutions were analyzed by HPTLC exactly as described above. Then calibration curves of peak area vs. concentration were prepared.

Results and Discussion
Different compositions of the mobile phase for HPTLC analysis were tested in order to obtain high resolution and reproducible peaks. The desired aim was achieved using chloroform:methanol (48:2, v/v) as the mobile phase. The wavelength of 425 nm was found to be optimal for the highest sensitivity (Figure 1). The calibration curves for the alkaloids curcumin, demethoxycurcumin and bis-demethoxycurcumin were linear in the range 100-1,000 ng ( Table I). The accuracy of the determination of the recovery rate was determined by triplicate analyses of the rhizomes spiked with three different concentrations of stock solution of curcumin, demethoxycurcumin and bis-demethoxycurcumin. The recovery rates were 97.3, 92.9 and 95.4% for curcumin, demethoxycurcumin and bis-demethoxycurcumin, respectively.
For the quantitative determination of curcumin, demethoxycurcumin and bis-demethoxycurcumin, the analyses of turmeric rhizome specimens of C. longa were repeated three times. The average content of curcumin, demethoxycurcumin and bis-demethoxycurcumin in the rhizomes are given in Table II. It is clear that three alkaloids were present in two cultivars viz. Kalimpong and PTS-43 having their maximum concentrations in the Kalimpong cultivar.

Conclusion
The HPTLC method for the simultaneous analysis of curcumin, demethoxycurcumin and bis-demethoxycurcumin from C. longa reported here is very simple, sensitive, economic and suitable for rapid screening of large number of plant samples. Moreover, this analysis can be performed without any special sample pretreatment and 15 samples can be analyzed on a single TLC laver (20 x 20 cm).