An improved HPLC method for quantitative determination of six triterpenes in Centella asiatica extracts and commercial products.

An improved HPLC qualitative and quantitative method of six triterpenes (asiaticoside, madecassoside, asiatic acid, madecassic acid, terminolic acid, and asiaticoside-B) in Centella asiatica (raw plant material and preparations) is described in this paper. After 50 minutes the six active triterpenes were separated and detected in the methanolic extract at a limit of 0.01 microg/ml. The method uses a Phenomenex Aqua 5mu C18 (200 A) column as the stationary phase, a gradient mobile phase of water (0.1% TFA), acetonitrile (0.1% TFA), and methyl tert-butyl ether (0.1% TFA), and UV detection at 206 nm. The correlation coefficients for the calibration curves and the recovery rates ranged from 0.995 to 0.999 and from 98.39% to 100.02%, respectively. The qualitative and quantitative results are discussed.

Analysis of sesquiterpenes in Valeriana officinalis by capillary electrophoresis.

A capillary electrophoresis (CE) method permitting the determination of the main sesquiterpenes in Valeriana officinalis has been developed. A separation of valerenic acid and its hydroxy and acetoxy derivatives, three compounds characteristic for the species, was achieved using a 40 mM phosphate-borate buffer at pH 8.5, which contained 10% isopropanol as organic modifier. Applied temperature and voltage were 35 degrees C and 17.5 kV, respectively. This setup allowed a baseline separation of the three compounds within 8 min, with a detection limit of 5.8 micrograms/ml or less. Out of six market products analyzed, only one contained a detectable amount of the marker compounds, with 0.54% of hydroxyvalerenic acid and 0.13% valerenic acid, respectively. The quantitative results were comparable to those obtained by HPLC.

Antigiardial activity of isoflavones from Dalbergia frutescens bark.

Several isoflavones [formononetin (1), castanin (5), odoratin (6), glycitein (7), pseudobaptogenin (8), fujikinetin (9), and cuneatin (10)] were isolated from Dalbergia frutescens, and their antiprotozoal activities were determined against Giardia intestinalis. Among these compounds, formononetin (1) was the most potent antigiardial agent, with an IC(50) value of 30 ng/mL (approximately 0.1 microM), as compared to the value for metronidazole, the current drug of choice, of 100 ng/mL (approximately 0.6 microM). Three isoflavones closely related to formononetin [daidzein (2), biochanin A (3) and genistein (4)] were also evaluated, but they were at least 100 times less active than 1. Formononetin (1) may thus be an interesting lead for development of new antigiardial agents or as a probe for a new mechanistic target.

Phytotoxic lignans of Leucophyllum frutescens.

Bioassay-guided fractionation of the hexane:ethyl acetate (1:1) extract of the leaves of Leucophyllum frutescens (Berl.) I.M.Johnst (Scrophulariaceae) led to the isolation of its phytotoxic constituents diayangambin (1), epiyangambin (2), diasesartemin (3) and epiashantin (4). Phytotoxicity was demonstrated as inhibition of seed germination of Agrostis stolonifera cv. penncross (Poaceae) and inhibition of development of Lactuca sativa L. (Asteraceae) seedlings in a microassay using 24-well plates. Compound 1 was the most phytotoxic to L. sativa, showing strong inhibitory activity at 110 microM. Compound 1 was more active than 2 and 3 in inhibiting the growth of A. stolonifera with I(50) values of 160, 670 and 930 microM, respectively. At a concentration of 500 microM, these compounds inhibited all phases of onion root cell division. This is the first demonstration of antimitotic activity of these furofuran lignans, and the first report of their isolation from this species.

Microbial production of a crisnatol metabolite.

Microbial metabolism studies of crisnatol (1), a new DNA intercalator, has resulted in the isolation and characterization of a major metabolite identified as the C-1 hydroxylated crisnatol (2). The structure of the metabolite was established by comparison of its spectral data to that of crisnatol. Complete 13C-NMR assignments for crisnatol and its C-1 hydroxylated metabolite were also made.