Characterization of the triterpene saponins of the roots and rhizomes of blue cohosh (Caulophyllum thalictroides).

Seven triterpene saponins were isolated from n-butanol fractions of blue cohosh (Caulophyllum thalictroides) roots and rhizomes. Their structures were established by spectral ((1)H NMR, (13)C NMR, 2D-NMR, and APCI-MS) techniques and chemical reactions as hederagenin 3-O-alpha-L-arabinopyranoside (1); caulophyllogenin 3-O-alpha-L-arabinopyranoside (2); hederagenin 3-O-beta-D-glucopyranosyl-(1-->2)-alpha-L-arabinopyranoside (3); 3-O-alpha-L-arabinopyranosyl-hederagenin 28-O-alpha-L-rhamnopyranosyl-(1-->4)-beta-D-glucopyranosyl(1-->6)-beta-D-glucopyranoside (4); 3-O-alpha-L-arabinopyranosyl- caulophyllogenin 28-O-alpha-L-rhamnopyranosyl-(1-->4)-beta-D-glucopyranosyl(1-->6)-beta-D-glucopyranoside (5); 3-O-beta-D-glucopyranosyl-(1-->2)-alpha-L-arabinopyranosyl- echinocystic acid 28-O-alpha-L-rhamnopyranosyl-(1-->4)-beta-D-glucopyranosyl(1-->6)-beta-D-glucopyranoside (6); 3-O-beta-D-glucopyranosyl-(1-->2)-alpha-L-arabinopyranosyl-hederagenin 28-O-alpha-L-rhamnopyranosyl-(1-->4)-beta-D-glucopyranosyl(1-->6)-beta-D-glucopyranoside (7). All seven compounds were identified in this species for the first time.

Furanosesquiterpenoids of Commiphora myrrha.

An investigation on the gum exudates of Commiphora myrrha has led to the isolation of six sesquiterpenoids. On the basis of spectroscopic data interpretation, they were determined as two new furanosesquiterpenoids, rel-1S,2S-epoxy-4R-furanogermacr-10(15)-en-6-one (1) and rel-2R-methyl-5S-acetoxy-4R-furanogermacr-1(10)Z-en-6-one (2), and four known furanosesquiterpenoids, rel-3R-methoxy-4S-furanogermacra-1E,10(15)-dien-6-one (3), rel-2R-methoxy-4R-furanogermacr-1(10)E-en-6-one (4), furanogermacra-1(10)Z,4Z-dien-6-one, and curzerenone [6,7-dihydro-5beta-isopropenyl-3,6beta-dimethyl-6-vinylbenzofuran-4(5H)-one]. This is the first report of the relative stereochemistry for the known compounds 3 and 4. Compound 1 exhibited weak cytotoxic activity against a MCF-7 breast tumor cell line in a clonogenic assay, while the other five compounds were inactive in this assay.

Flavonol glycosides and novel iridoid glycoside from the leaves of Morinda citrifolia.

One new iridoid glycoside and five known flavonol glycosides have been isolated from the leaves of Morinda citrifolia. The new iridoid exists as an epimeric mixture in solution. Complete assignments of the proton and carbon chemical shifts for the individual epimers were accomplished on the basis of high-resolution 1D and 2D NMR data. Their antioxidative activities were measured. All of these compounds showed DPPH free radical scavenging activity at the concentration of 30 microM.

Iridoid glycosides from the leaves of Morinda citrifolia.

A new iridoid glucoside (1), named citrifolinoside A, was isolated from the leaves of Morinda citrifolia along with the known iridoids asperuloside and asperulosidic acid. The structure of 1 was established by interpretation and full assignments of NMR spectroscopic data.

Ecdysteroids of quinoa seeds (Chenopodium quinoa Willd.).

Quinoa (Chenopodium quinoa) is a hardy and nutritious Latin American pseudo-cereal. Studies on the seeds led to the isolation of five ecdysteroids using column chromatography. Their structures were determined as ecdysterone, makisterone A, 24-epi-makisterone A, 24(28)-dehydromakisterone A, and 20,26-dihydroxyecdysone by spectroscopic methods. This study demonstrates that quinoa seeds are a source of ecdysteroids, which were reported to be molting hormones in insects.

A new unusual iridoid with inhibition of activator protein-1 (AP-1) from the leaves of Morinda citrifolia L.

[structure in text] From the leaves of Morinda citrifolia, a new unusual iridoid, named citrifolinoside (1), showing significant inhibition of UVB-induced Activator Protein-1 (AP-1) activity in cell cultures, has been isolated. Its structure was elucidated on the basis of detailed high-field 1D and 2D spectral analysis.

Bioactive constituents from gum guggul (Commiphora wightii).

Bioactivity-directed fractionation and purification afforded cytotoxic components of Commiphora wightii. The exudates of C. wightii were extracted with EtOAc and the extract was subjected to repeated column chromatography. A fraction showing cytotoxic activity was characterized as a mixture of two ferulates with an unusual skeleton by spectral and chemical methods, including by NMR, GC-MS and chemical derivatization. This fraction also showed moderate scavenging effect against 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals.

Determination of allicin, S-allylcysteine and volatile metabolites of garlic in breath, plasma or simulated gastric fluids.

Various components of garlic and aged garlic extract, including allicin, S-allylcysteine (SAC) and volatile metabolites of allicin were determined in breath, plasma and simulated gastric fluids by HPLC, gas chromatography (GC) or HPLC- and GC-mass spectrometry (MS). Data indicate that allicin decomposes in stomach acid to release allyl sulfides, disulfides and other volatiles that are postulated to be metabolized by glutathione and/or S-adenosylmethionine to form allyl methyl sulfide. SAC can be absorbed by the body and can be determined in plasma by HPLC or HPLC-MS using atmospheric pressure chemical ionization (APCI)-MS.

Novel glycosides from noni (Morinda citrifolia).

Three new glycosides were isolated from the fruits of noni (Morinda citrifolia). Their structures were determined to be 6-O-(beta-D-glucopyranosyl)-1-O-octanoyl-beta-D-glucopyranose (1), 6-O-(beta-D-glucopyranosyl)-1-O-hexanoyl-beta-D-glucopyranose (2), and 3-methylbut-3-enyl 6-O-beta-D-glucopyranosyl-beta-D-glucopyranoside (3) using MS and NMR methods.

Modulation of bcl-2 and cytotoxicity by licochalcone-A, a novel estrogenic flavonoid.

Herbal therapies are commonly used by patients with cancer, despite little understanding about their clinical and biological activity. We recently demonstrated that the herbal combination PC-SPES, which contains licorice root, had potent estrogenic activity in vitro, in animals, and in patients with prostate cancer. Licochalcone-A (LA) is one flavonoid extracted from licorice root with antiparasitic and anti-tumor activity, but the effect on the human estrogen receptor and mechanism of anti-tumor activity is unknown. Recent studies demonstrated that the mechanism of cytotoxic effect by some estrogens may involve modulation of the anti-apoptotic protein bcl-2. In the present study, we determined if LA had estrogenic activity, anti-tumor activity, and modulated the apoptotic protein bcl-2 in human cell lines derived from acute leukemia, breast cancer, and prostate cancer. A yeast growth-based assay under the control of the human estrogen receptor (hER) demonstrated that LA was a phytoestrogen. A cell viability assay demonstrated that LA had anti-tumor activity in all cell lines tested and enhanced the effect of paclitaxel and vinblastine chemotherapy. LA induced apoptosis in MCF-7 and HL-60 cell lines, as demonstrated by cleavage of PARP, the substrate of ICE-like proteases. Immunoblot analysis demonstrated that LA decreased the anti-apoptotic protein bcl-2 and altered the bcl-2/bax ratio in favor of apoptosis. In contrast, the parent compound chalcone or estradiol did not decrease bc1-2 expression. Therefore, these data demonstrate that LA is a phytoestrogen with anti-tumor activity and is capable of modulating bcl-2 protein expression. The modulation of bcl-2 may be dependent on specific structural differences between LA and the parent compound chalcone and independent of LA estrogenicity.

Isolation and identification of stilbenes in two varieties of Polygonum cuspidatum.

The roots of two varieties of Polygonum cuspidatum (Hu Zhang and Mexican Bamboo) were analyzed for resveratrol and analogues. The roots of each variety were dried and ground into a powder. The powdered roots were then extracted with methanol and ethyl acetate. The ethyl acetate fraction of the Mexican Bamboo was then subjected to fractionation and purification using silica gel column chromatography and semipreparative HPLC. In addition to resveratrol (3,5,4'-trihydroxystilbene), three stilbene glucosides were identified by (1)H NMR, (13)C NMR, and MS. The stilbene glucosides were shown to be a piceatannol glucoside (3,5,3', 4'-tetrahydroxystilbene 4'-O-beta-D-glucopyranoside), resveratroloside (3,5,4'-trihydroxystilbene 4'-O-beta-D-glucopyranoside), and piceid (3,5,4'-trihydroxystilbene 3-O-beta-D-glucopyranoside). The levels of the piceatannol glucoside and piceid were twice as high in the Mexican Bamboo as compared to the Hu Zhang.

The determination of metabolites of garlic preparations in breath and human plasma.

Gas Chromatography-Mass Spectrometry (GC-MS) was the major technique used to determine various metabolites after consumption of dehydrated granular garlic and an enteric-coated garlic preparation, in breath, plasma, and simulated gastric fluids. A special short-path thermal desorption device was used as an introduction technique for the gas chromatograph for the determination of volatiles. These garlic preparations release allicin, which decomposes in stomach acid or with time in the intestine to release allyl sulfides, disulfides and other volatiles, some of which are postulated to be metabolized by glutathione and/or S-adenosylmethionine to form allyl methyl sulfide, the main sulfur containing volatile metabolite. S-Allylcysteine, a non-volatile bioactive component of aged garlic preparations, was determined in human plasma and urine by HPLC-MS using the negative ion atmospheric pressure chemical ionization mode (APcI)- MS. The technique of selected ion monitoring was used for quantitation. A synthetic internal standard of deuterated S-allylcysteine was added to the plasma or urine to ensure recovery and to obtain reliable quantitative data.

Thermal degradation of sulforaphane in aqueous solution.

Sulforaphane, a cancer chemopreventive agent identified from broccoli, was degraded in an aqueous solution at 50 and 100 degrees C. The reaction mixtures were extracted with methylene chloride and analyzed by gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS). Dimethyl disulfide, S-methyl methylthiosulfinate, S-methyl methylthiosulfonate, methyl (methylthio)methyl disulfide, 1,2,4-trithiolane, 4-isothiocyanato-1-(methylthio)-1-butene, and 3-butenyl isothiocyanate were identified as volatile decomposition products. After methylene chloride extraction, the aqueous layer was dried and silica gel column chromatography was used to separate and purify the nonvolatile decomposition products. The major thermal degradation compound was determined by (1)H NMR, (13)C NMR, and FAB-MS as N, N'-di(4-methylsulfinyl)butyl thiourea. A possible mechanism for the formation of these products is proposed.

Novel trisaccharide fatty acid ester identified from the fruits of Morinda citrifolia (Noni).

Two known glycosides and a novel trisaccharide fatty acid ester were isolated from the n-butanol-soluble fraction of the fruits of Morinda citrifolia (noni). Structure determination was carried out by spectral techniques such as MS, IR, NMR, and 2D-NMR. The novel trisaccharide fatty acid ester was elucidated as 2, 6-di-O-(beta-D-glucopyranosyl)-1-O-octanoyl-beta-D-glucopyranose. The known compounds were identified as rutin and asperulosidic acid.

2,2-Diphenyl-1-picrylhydrazyl radical-scavenging active components from Polygonum multiflorum thunb.

An activity-directed fractionation and purification process was used to identify the antioxidative components of Polygonum multiflorum Thunb. (PM). Dried root of PM was extracted with 95% ethanol and then separated into water, ethyl acetate, and hexane fractions. Among these only the ethyl acetate phase showed strong antioxidant activity by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) test when compared with water and hexane phases. The ethyl acetate fraction was then subjected to separation and purification using silica gel column chromatography and Sephadex LH-20 chromatography. Three compounds showing strong antioxidant activity were identified by spectral methods ((1)H NMR, (13)C NMR, and MS) and by comparison with authentic samples to be gallic acid, catechin, and 2,3,5, 4'-tetrahydroxystilbene 2-O-beta-D-glucopyranoside.

Effect of dietary 2(3)-tert-butyl-4-hydroxyanisole on the metabolism and action of estradiol and estrone in female CD-1 mice.

Administration of 0.75% 2(3)-tert-butyl-4-hydroxyanisole (BHA) in AIN-76A diet to female CD-1 mice for 3 weeks increased liver microsomal glucuronidation of estradiol, estrone, 4-aminophenol, and 4-nitrophenol by 103, 187, 162, and 92%, respectively (at pH 7.4). The overall rate of NADPH-dependent metabolism of estradiol and estrone by liver microsomes of BHA-treated animals as determined by substrate disappearance was increased by 20-40% over that by liver microsomes from control animals. The rate of 2-hydroxylation of estradiol and estrone (the major metabolic pathway) was increased by 24-38%, the rate of formation of 6alpha-hydroxyestradiol plus 6beta-hydroxyestradiol was increased by 90-115%, and the rate of 6beta-hydroxyestrone formation (a minor metabolite formed in liver microsomes from control mice) was increased by approximately 370% over controls. In contrast, BHA administration had little or no effect on the liver microsomal formation of 4- and 16alpha-hydroxylated estradiol and estrone metabolites. Measurable levels of estradiol and estrone were observed in the serum and uterus of ovariectomized CD-1 mice at 30 min after a single i.p. injection of 100 or 300 ng of estradiol or estrone, and these levels were decreased by 30-60% in animals fed a 0.75% BHA diet for 18 days prior to the injection of estrogen. Feeding a 0.75% BHA-supplemented diet to ovariectomized CD-1 mice for 18 days inhibited the uterotropic effect of estradiol or estrone (45 or 75 ng/mouse, i.p. once daily for 3 days) as compared to the response of animals fed the control diet. BHA administration also inhibited estradiol- or estrone-stimulated [3H]thymidine incorporation into uterine DNA. In conclusion, feeding a 0.75% BHA-supplemented diet to female CD-1 mice for 2-3 weeks increased the activities of liver microsomal enzymes that catalyze uridine 5'-diphosphoglucuronic acid-dependent glucuronidation and NADPH-dependent oxidation of estradiol and estrone, enhanced the in vivo metabolism of these estrogens, and inhibited their uterotropic action.

Triterpenoid saponins from Aster lingulatus.

Two new triterpenoid saponins named asterlingulatosides A and B were isolated from the whole plants of Aster lingulatus. Their structures were determined by spectroscopic data and chemical transformations to be 3-O-beta-D-glucopyranosyl-3 beta,16 alpha-dihydroxyolean-12-en-28-oic acid-28-O-alpha-L-O-arabinopyranoside and 3-O-beta-D-glucopyranosyl-3 beta,16 alpha-dihydroxyolean-12-en-28-oic acid-28-O-alpha-L-rhamnopyranosyl-(1-->2)-alpha-L-arabinopyranoside++ +. They showed inhibitory activity on DNA synthesis in human leukaemia HL-60 cells.

Effects of phenobarbital, dexamethasone, and 3-methylcholanthrene administration on the metabolism of 17 beta-estradiol by liver microsomes from female rats.

Female rats were treated with phenobarbital, dexamethasone, 3-methylcholanthrene, clofibrate, or isoniazid to induce different hepatic cytochromes P-450. The profile of hydroxylated metabolites of estradiol (E2) formed by liver microsomes was then determined using a new HPLC method for the separation of hydroxylated estrogen metabolites. Inhibition of liver microsomal E2 metabolism by monoclonal antibodies raised against specific cytochrome P-450 isozymes was also evaluated. Treatment of immature or adult female rats with phenobarbital caused a 3-fold increase in the 2-hydroxylation of E2 and a more than 5-fold increase in liver microsomal hydroxylation of E2 at the 4-, 6 alpha, 6 beta-, and 14 alpha-positions. Monoclonal antibody directed toward CYP2B1/2B2 completely inhibited the 6 alpha- and 6 beta-hydroxylation of E2 and partially inhibited the 2-hydroxylation of E2 by liver microsomes from phenobarbital-treated adult female rats. Antibodies directed toward CYP3A1/3A2 completely inhibited the 4- and 14 alpha-hydroxylation of E2 by these liver microsomes. Treatment of immature or adult female rats with dexamethasone resulted in a 2- to 3-fold increase in the microsomal 2-hydroxylation of E2 and a several-fold increase in the hydroxylation of E2 at the 4-, 6 beta-, 7 alpha-, and 14 alpha-positions. A substantial increase in the formation of two unidentified nonpolar metabolite peaks (UK1 and UK2) was also observed. A monoclonal antibody directed against CYP3A1/3A2 markedly inhibited the 2-, 4-, and 14 alpha-hydroxylation of E2 by liver microsomes from adult female rats treated with dexamethasone. Antibody directed against CYP2B1/2B2 inhibited only the 6 beta-hydroxylation of E2 by these microsomes. Treatment of immature or adult female rats with 3-methylcholanthrene resulted in a several-fold increase in the metabolism of E2 to 7 alpha-hydroxyestradiol (7 alpha-OH E2) and 15 alpha-OH E2, but there was a substantial decrease in the formation of 16 alpha-OH E2. Treatment with 3-methylcholanthrene caused a small increase in 2-hydroxylation (< or = 50%) in liver microsomes from immature or adult female rats, whereas a substantial increase in 6 alpha-hydroxylation was seen in liver microsomes from adult female rats. A monoclonal antibody directed toward CYP1A1 partially inhibited the 6 alpha-hydroxylation of E2 and the formation of the 7 alpha-OH E2/15 alpha-OH E2 peak by microsomes from adult female rats treated with 3-methylcholanthrene, but the 2-hydroxylation of E2 was not inhibited. Treatment of adult female rats with clofibrate increased the 2- and 4-hydroxylation of E2 by about 2-fold and by more than 6-fold, respectively. Isoniazid treatment had little or no effect on the metabolism of E2. The data demonstrate that prototype inducers of cytochrome P-450 can substantially alter the profile of hepatic E2 metabolism in female rats. Our results suggest that inducers of environmental relevance may also have an impact on E2 metabolism and homeostasis in humans.

High-performance liquid chromatography separation of hydroxylated estradiol metabolites: formation of estradiol metabolites by liver microsomes from male and female rats.

A high-performance liquid chromatography method has been described for the separation of estradiol (E2), estrone (E1) and 27 hydroxylated and keto derivatives of these estrogens. Chromatography of a mixture of 29 estrogen standards resulted in 20 different peaks. Solvent extraction followed by the chromatographic separation and quantification of radioactive metabolites was used for studies on the metabolism of [4-14C]E2 by liver microsomes from adult male and female rats. Liver microsomes from male rats metabolized [4-14C]E2 more rapidly and to a larger number of metabolites than liver microsomes from female rats. Under conditions in which less than 10% of the substrate was metabolized, major metabolites from liver microsomes of male rats cochromatographed with E1, 2-OH E2, 15 alpha-OH E2 and 16 alpha-OH E2, and major metabolites from liver microsomes of female rats cochromatographed with E1, 2-OH E2 and 16 alpha-OH E2. The identity of the metabolites was confirmed by mass spectrometry. Using liver microsomes from male rats and conditions in which more extensive metabolism of the substrate occurred, more than 15 additional metabolites of [4-14C]E2 were observed. Liver microsomes from male rats were many-fold more active than liver microsomes from female rats at catalyzing the 2-, 15 alpha- and 16 alpha-hydroxylation of E2. Our studies on the metabolism of [4-14C]E2 by rat liver microsomes indicate that the profile of E2 metabolites is dependent on the time of incubation, microsomal protein concentration and substrate concentration.

Antioxidative effect of polyphenol extract prepared from various Chinese teas.

METHODS. Twelve different types of Chinese teas, including green, semifermented, and black tea, were studied for their antioxidant activities and active components. Compositions of (-)-epicatechin, (-)-epigallocatechin, (-)-epicatechin gallate, (-)-epigallocatechin gallate, and gallic acid were identified by fast atom bombardment-mass spectrometry and high-performance liquid chromatography-mass spectrometry and quantified by high-performance liquid chromatography. Antioxidant activities in lard were measured by the Rancimat method. RESULTS. The results showed that both yields of polyphenol extract and antioxidant activities varied with different tea processing methods. It was found that (-)-epigallocatechin gallate, (-)-epigallocatechin, and (-)-epicatechin gallate inhibited soybean lipoxygenase at the IC50 values ranging from 10 to 20 microM.