Sulfuric acid hydrolysis of steroidal glycosides of Amber fenugreek was studied by capillary gas chromatographic analysis of diosgenin [(25R)-spirost-5-en-3-ol] and isomeric spirostadiene artifacts from 100 mg samples of seed material. Following extraction with 80% ethanol, highest recoveries of diosgenin occurred when hydrolyses were conducted in sulfuric acid, prepared at 1 molar (M) concentration in water containing 60-80% 2-propanol. Compared to a previous method with aqueous hydrochloric acid, the selected conditions of hydrolysis at 100 degrees C for 2 h with sulfuric acid in 70% 2-propanol reduced diene formation but did not completely eliminate these artifacts. Extraction of steroidal saponins with various alcohol/water mixtures prior to sulfuric acid hydrolysis gave similar recoveries of diosgenin. Application of the quantitative method to experimental samples of Amber, Quatro, and ZT-5 fenugreek, using 10 mg subsamples of crushed seed that had been defatted with petroleum ether and dried at 60 degrees C, gave diosgenin levels of 0.55, 0.42, and 0.75%, respectively. Levels of smilagenin and sarsasapogenin were very low in hydrolyzed seed extracts from ZT-5, a Canadian breeder line of fenugreek.

Mixtures of steroidal sapogenins isolated from the seed and combined aerial parts (foliage) of fenugreek (cultivar Amber) have been examined by capillary column gas chromatography with mass spectral and flame ionization detectors. Diosgenin [(25R)-spirost-5-en-3β-ol] was the major component in seed and foliage extracts hydrolyzed with hydrochloric acid. Yamogenin also appeared to be present. Tigogenin, neotigogenin, smilagenin, and sarsasapogenin were identified in the extracts. Dihydroxy steroidal sapogenins, tentatively identified as yuccagenin, gitogenin, and neogitogenin, were detected as minor components in hydrolyzed extracts from seed. A gas chromatographic method based on the use of an internal standard and a column of HP-5MS was developed to determine diosgenin in Amber fenugreek. Expressed on a dry weight basis, mean levels of diosgenin in seed were 0.54%. In field-grown foliage sampled during a season at 9, 15, and 19 weeks postseeding, diosgenin levels were 0.16, 0.07, and 0.07%, respectively. Keywords: Steroidal sapogenins; spirostanols; fenugreek; diosgenin; quantitation; seed; foliage

A collection of 10 accessions of fenugreek (Trigonella foenum-graecum L.), an annual legume, was grown during two summers at three plot locations in western Canada to assess whether genetic (accession) and environmental factors (site and year of production) influenced levels of diosgenin, a steroidal sapogenin. The 60 harvested seed samples, each analyzed by single determinations on three subsamples of defatted and dried seed material, were hydrolyzed by a microscale procedure in water containing 2-propanol (70%) and sulfuric acid (1 M). The extracts were analyzed by capillary gas chromatography with 6-methyldiosgenin as internal standard. Diosgenin levels from mature seeds ranged from 0.28 to 0.92% (28-92 microg/10 mg). Analysis of variance on combined diosgenin levels from the three sites and two years revealed that accession, accession x year, and site x year effects were significant for diosgenin content, whereas site, year, and site x accession effects were not. Four accessions, CN 19062, CN 19067, CN 19070, and CN 19071, were identified with high levels of diosgenin on the basis of the 2-year data set. In these accessions, mean levels of diosgenin plus yamogenin from seven site years were estimated at 0.70, 0.98, 0.84, and 0.87%, respectively.

Chromatographic fractionation of crude extracts (C8 extracts) from the protein-enriched flour of commercial field peas (Pisum sativum L.) has been shown here to yield peptide mixtures related to the pea albumin 1b (PA1b) family of cysteine-rich plant peptides. The mixtures were obtained initially by flash chromatography with silica gel. Following elution of soyasaponins and lysolecithins, the end fractions obtained with the use of two flash chromatographic solvent systems displayed activity in a flour disk antifeedant bioassay with the rice weevil [Sitophilus oryzae (L.)]. Chemical properties of these mixtures were compared by thin-layer chromatography, high-performance liquid chromatography (HPLC), IR, MS, and amino acid analyses. The major peptides of C8 extracts, with average masses of 3752, 3757, and 3805 Da, were isolated by anion exchange chromatography. Samples enriched in the peptide of mass 3752 were isolated by cation exchange chromatography. Reduction plus alkylation experiments in combination with electrospray ionization mass spectrometry showed that C8 extracts contained about 10 peptides and, like PA1b, each peptide possessed six cysteine residues (three disulfide bonds). Disulfide bond reduction with 2-mercaptoethanol destroyed the antifeedant activity. The native peptides of C8 extracts were found to be resolved into nine peaks with XTerra HPLC columns operating at alkaline pH. These columns were employed to assess the distribution of pea peptides in the isolated fractions, with photodiode array and electrospray detection.

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