Cucurbitacins detection

Thin-layer chromatography (TLC) is a good method for detecting the presence of cucurbitacins in a plant extract. The best results to date have been achieved using high-performance TLC (HPTLC) plates [41] with silica gel and chloroform-methanol (95 10) (Table 1) or toluene-ethyl acetate (25 75) as mobile phases [3,11]. Reversed-phase (RP) HPTLC plates with methanol-water (7 3) have also been used to separate cucurbitacins [3]. The compounds are easily detected with vanillin-sulphuric acid or vanillin-phosphoric acid reagents and ultraviolet light (UV) at 365 nm [11]. 

Analytical high-performance liquid chromatography (HPLC) produces a good level of separation between cucurbitacins with similar chemical structures [41], The most effective set-up involves the use of an RP system as the stationary phase with a Cis column, acetonitrile-water mixtures as the mobile phase, and UV detection at 230 nm [11,42]. Thus, using a Ci8 column and acetonitrile-water (20 80) to (50 50) for 45 min (linear gradient at 2.0 ml/min), Bauer and Wagner [11] analyzed seven... 

Jacobs et al. [1] used two-dimensional NMR spectroscopy to assign the H and 13C chemical shifts of cucurbitacin B, establishing these values as a benchmark for analyzing the spectra of other cucurbitacins isolated from the same source. In fact, the characteristic groups present in all cucurbitacins are easily detectable by means of NMR and typically include the presence of carbonyls at C-ll and C-22, which appear between 212-213 ppm, as well as hydroxyls at C-16(a) and C-20( 3), which appear at approximately 71-73 ppm and 77-79 ppm, respectively. Other characteristic patterns of substitution such as the presence of acetoxyl groups at C-2 and C-25 have also been observed. In this case, the acetylation of the hydroxyl at C-2 modifies both the C-l and C-3 values. Indeed, when a carbonyl is present, this latter change involves a major shift, namely from 213 to 205 ppm. In the case of a second double... 

The TLC in Fig. 3a with solvent (a) shows in VIS in the -range between =0.65 and I =0.85, 5-6 violet/light blue zones. In the same range are detectable also the cucurbitacins T3 and T4 with distinct pink colour (very low concentration). The colour differences of the other zones may be due to overlapping of other triterpenoids. 

In the TLC of Fig. 3c with solvent (b) detected at UV 366 nm appear the cucurbitacins of the extracts and the reference with distinct characteristic carmin red fluorescent colour as reported in the literature... 

The authentication of the Radix Trichosanthes kirilowii or Tr. rosthornii extracts can be confirmed by the TLC and HPLC-detection of the characteristic cucurbitacins. 

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