Determination of diarylheptanoids from Alpinia officinarum (Lesser Galangal) by HPLC with photodiode array and electrochemical detection.

Normal-phase column chromatography followed by semi-preparative reversed-phase HPLC has been used to isolate, from the rhizomes of Alpinia officinarum, five diarylheptanoids identified as 5-hydroxy-7-(4"-hydroxy-3"-methoxyphenyl)-1-phenyl-3-heptanone, 5-methoxy-7-(4"-hydroxy-3"-methoxyphenyl)-1-phenyl-3-heptanone, 7-(4"-hydroxyphenyl)-1-phenylhept-4-en-3-one, 7-(4"-hydroxy-3"-methoxyphenyl)-1-phenyl-hept-4-en-3-one, 1,7-diphenylhept-4-en-3-one. The levels of these five diarylheptanoids in root material were determined quantitatively by HPLC with UV detection and the assay methods so developed were simple, rapid and accurate. Four of the diarylheptanoids could also be detected by HPLC with electrochemical detection (ECD) in the oxidative mode, and ECD was found to have a higher sensitivity than photodiode array detection.

A sesquiterpenelactone from Inula britannica induces anti-tumor effects dependent on Bcl-2 phosphorylation.

BACKGROUND: The over-expression of the anti-apoptotic protein Bcl-2 in cancer is associated with resistance to chemotherapeutic drugs. The phosphorylation of Bcl-2 is one mechanism by which anti-microtubule agents, such as paclitaxel or docetaxel, may inactivate Bcl-2. Although initially active in clinical studies, current anti-microtubule agents are only temporarily effective and the discovery of new agents is warranted. MATERIALS AND METHODS: We isolated and identified two known sesquiterpenelactones, O, O-diacetylbritannilactone (OODABL) and O-acetylbritaanilactone (OABL) from the flowers of the medicinal plant Inula britannica and studied their mechanism of anti-tumor effects. To determine the biological significance of Bcl-2 phosphorylation, we used a baby rat kidney (BRK-p53) cell line that was transformed with EIA and a temperature-sensitive mutant p53. The BRK-p53 cell line was transfected with either a vector with wild type Bcl-2 or a vector in which Bcl-2 had mutations in the paclitaxel phosphorylation sites (pcDNA3.1 V5/His Bcl-2 S70, 87A). RESULTS: OODABL and OABL induced phosphorylation of Bcl-2 in breast, ovary and prostate cancer cell lines and induced G2/M cell cycle arrest. Using the BRK cells with mutant Bcl-2 (BRK-Bcl-2-mt) and control (BRK-Bcl-2-wt), we found that OODABL induced phosphorylation of Bcl-2 at sites similar to paclitaxel. Phosphorylation of Bcl-2 was important for OODABL-induced cytotoxicity, since the abrogation of phosphorylation in BRK-Bcl-2-mt cells decreased OODABL-induced cytotoxicity. CONCLUSION: We concluded that OODABL is cytotoxic in multiple tumor cell lines, and the cytotoxicity is dependent, at least in part, on the phosphorylation of Bcl-2.

Inhibiting progression of coronary calcification using Aged Garlic Extract in patients receiving statin therapy: a preliminary study.

BACKGROUND: Aged Garlic Extract (AGE) reduces multiple cardiovascular risk factors, including blood pressure, cholesterol, platelet aggregation and adhesion, while stimulating nitric oxide generation in endothelial cells. However, no study has evaluated the ability of AGE to inhibit vascular calcification, a marker of plaque formation in human coronary arteries. OBJECTIVE: To assess the efficacy of Aged Garlic Extract (AGE) on changing the rate of atherosclerosis progression as compared to placebo. DESIGN: A placebo-controlled, double-blind, randomized pilot study to determine whether the atherosclerotic plaque burden detected by electron beam tomography (EBT) will change at a different rate under the influence of AGE as compared to placebo. Twenty-three patients were enrolled, and 19 patients completed the study protocol. AGE 4 ml or the equivalent amount of placebo was given to subjects. Duration of the study was 1 year. S-allylcysteine (SAC), one of the active compound of AGE, was measured in the blood as a compliance marker. RESULTS: The mean change of the calcium score (volumetric method) for the AGE group (n = 9) was 7.5 +/- 9.4% over 1 year. The placebo group (n = 10) demonstrated an average increase in calcium scores of 22.2 +/- 18.5%, significantly greater than the treated cohort (P = 0.046). There were no significant differences in individual cholesterol parameters or C reactive protein between the groups. In patients randomized to AGE, there was a nonsignificant trend for improving cholesterol/high-density lipoprotein ratio (P = 0.07) and homocysteine level (P = 0.08). CONCLUSIONS: This small pilot study indicates the potential ability of AGE to inhibit the rate of progression of coronary calcification, as compared to placebo over 1 year. Should these findings be extended and confirmed in larger studies, garlic may prove useful for patients who are at high risk of future cardiovascular events.

Prospective, multicenter, randomized phase II trial of the herbal supplement, PC-SPES, and diethylstilbestrol in patients with androgen-independent prostate cancer.

PURPOSE: To evaluate the herbal combination, PC-SPES, and diethylstilbestrol (DES) in patients with androgen independent prostate cancer (AIPC). PATIENTS AND METHODS: A randomized phase II study was conducted with cross-over design. Patients were randomly assigned to receive either three PC-SPES capsules orally three times a day or DES 3 mg orally once a day. Prophylactic warfarin was administered. At clinical or prostate-specific antigen progression, patients received the other therapy. The study closed prematurely after PC-SPES was withdrawn from the market. Chemical analyses were performed on multiple lots of PC-SPES. RESULTS: Ninety patients were enrolled, of whom 85 were assessable for response. Prostate-specific antigen declines > or = 50% were noted in 40% (95% CI, 25% to 56%) with PC-SPES, and 24% (95% CI, 12% to 39%) with DES. Median response duration was not reached with PC-SPES, and was 3.8 months with DES. Median time to progression for randomly assigned patients was 5.5 months for PC-SPES and 2.9 months for DES. Common toxicities included mild fatigue, gynecomastia, and mastodynia. Five thromboembolic events occurred (one PC-SPES, four DES). Responses in the cross-over phase were inconclusive. Four lots of PC-SPES had measurable quantities of DES, ranging from 0.01% to 3.1% of the dose used in the DES arm. Ethinyl estradiol was also detected in PC-SPES lots. CONCLUSION: PC-SPES and DES demonstrate activity in AIPC and are well tolerated. However, the synthetic estrogens, DES and ethinyl estradiol, were detected in various lots of PC-SPES, including those used in this trial. Clinical trials that utilize herbal therapies must account for issues of purity and consistency.

Analysis of theaflavins and thearubigins from black tea extract by MALDI-TOF mass spectrometry.

Black tea contains two major groups of pigments, theaflavins (TFs) and thearubigins (TRs). TFs contain a bis-flavan substituted 1,2-dihydroxy-3,4-benzotropolone moiety. Unlike the TFs, TRs have not yet been characterized. The chemical structure of the TRs remains a mystery. The present paper reports our effort to study the structure of TFs and TRs using delayed pulsed ion extraction of ions generated via the matrix-assisted laser desorption ionization (MALDI) technique, on line with a Linear time-of-flight (TOF) mass spectrometer. Spectra of standard TFs show not only pseudomolecular ions but also ions resulting from fragmentation. The analysis of MALDI-TOF spectra of black tea fractions shows the structure of some TRs, which are similar to those of TFs because the same loss of mass is observed.

Characterization of flavonols in cranberry (Vaccinium macrocarpon) powder.

Flavonoids were extracted from cranberry powder with acetone and ethyl acetate and subsequently fractionated with Sephadex LH-20 column chromatography. The fraction eluted with a 60% methanol solution was composed primarily of phenolic constituents with maximum absorbance at 340 nm. A high-performance liquid chromatography procedure was developed, which resolved 22 distinct peaks with UV/vis and mass spectra corresponding to flavonol glycoside conjugates. Six new constituents not previously reported in cranberry or in cranberry products were determined through NMR spectroscopy to be myricetin-3-beta-xylopyranoside, quercetin-3-beta-glucoside, quercetin-3-alpha-arabinopyranoside, 3'-methoxyquercetin-3-alpha-xylopyranoside, quercetin-3-O-(6' '-p-coumaroyl)-beta-galactoside, and quercetin-3-O-(6' '-benzoyl)-beta-galactoside. Quercetin-3-O-(6' '-p-coumaroyl)-beta-galactoside and quercetin-3-O-(6' '-benzoyl)-beta-galactoside represent a new class of cranberry flavonol compounds with three conjugated components consisting of a flavonol, sugar, and carboxylic acid (benzoic or hydroxycinnamic acids). This is also the first report identifying quercetin-3-arabinoside in both furanose and pyranose forms in cranberry. Elucidation of specific flavonol glycosides in cranberry is significant since the specificity of the sugar moiety may play a role in the bioavailability of the flavonol glycosides in vivo.

Enzymatic synthesis of tea theaflavin derivatives and their anti-inflammatory and cytotoxic activities.

Derivatives based on a benzotropolone skeleton (9-26) have been prepared by the enzymatic coupling (horseradish peroxidase/H2O2) of selected pairs of compounds (1-8), one with a vic-trihydroxyphenyl moiety, and the other with an ortho-dihydroxyphenyl structure. Some of these compounds have been found to inhibit TPA-induced mice ear edema, nitric oxide (NO) synthesis, and arachidonic acid release by LPS-stimulated RAW 264.7 cells. Their cytotoxic activities against KYSE 150 and 510 human esophageal squamous cell carcinoma and HT 29 human colon cancer cells were also evaluated.

Identification of six phenylpropanoids from garlic skin as major antioxidants.

The extract of garlic skins (peels) showed strong antioxidant activity, and some responsible constituents were isolated and identified. Garlic (Allium sativum L.) has been used as an herbal medicine, but there is no report on the health benefits of the skin or peel. In this study, the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity of garlic skin extract was evaluated. Using chromatographic techniques, the active constituents were isolated and subsequently identified. Analyses by high-performance liquid chromatography coupled with a photodiode array detector (HPLC-PDA) suggested that these compounds were phenylpropanoids, which had a characteristic absorbance at 300-320 nm. Liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance analyses allowed the chemical structures of the isolated constituents to be postulated. The proposed compounds were subsequently synthesized and compared with the constituents in the extract using HPLC-PDA and LC-MS. N-trans-Coumaroyloctopamine, N-trans-feruloyloctopamine, guaiacylglycerol-beta-ferulic acid ether, and guaiacylglycerol-beta-caffeic acid ether were identified as were trans-coumaric acid and trans-ferulic acid. Also, the antioxidant activities of these compounds were determined.

Chemical studies of the antioxidant mechanism of tea catechins: radical reaction products of epicatechin with peroxyl radicals.

Tea catechins, an important class of polyphenols, have been shown to have antioxidant activity and are thought to act as antioxidants in biological systems. However, the mechanisms of their antioxidant reactions remain unclear. The objective of this study was to characterize the reaction products of epicatechin with peroxyl radicals generated by thermolysis of the azo initiator azo-bisisobutyrylnitrile (AIBN). Structural elucidation of these products can provide insights into specific mechanisms of antioxidant reactions. Eight reaction products were isolated and identified using high-field 1D and 2D NMR spectral analysis. The observation of these compounds confirmed that the B-ring is the initial site for formation of reaction products in the peroxyl radical oxidant system.

New unusual iridoids from the leaves of noni (Morinda citrifolia L.) show inhibitory effect on ultraviolet B-induced transcriptional activator protein-1 (AP-1) activity.

A novel iridoid dimer in whose structure the two iridoid units are connected by a rare ether group, together with two new unusual iridoids showing significant inhibition of UVB-induced Activator Protein-1 (AP-1) activity in cell cultures, have been isolated from the leaves of noni (Morinda citrifolia L.). Their structures were determined on the basis of detailed high-field 1D and 2D spectral analysis. Their inhibitory effect on UVB-induced transcriptional Activator Protein-1 (AP-1) activity are also discussed.

Antifungal constituents from the seeds of Allium fistulosum L.

A new unsaturated fatty acid monoglyceride (1), glycerol mono-(E)-8,11,12-trihydroxy-9-octadecenoate, was isolated from the seeds of Allium fistulosum L. along with five known compounds: tianshic acid (2), 4-(2-formyl-5-hydroxymethylpyrrol-1-yl) butyric acid (3), p-hydroxybenzoic acid (4), vanillic acid (5), and daucosterol (6). The structures of 1-3 were established by interpretation and full assignments of NMR spectroscopic data. Both 1 and 2 were found to inhibit the growth of Phytophtohora capsici on V8 media.

Sphingolipid and other constituents from almond nuts (Prunus amygdalus Batsch).

One sphingolipid, 1-O-beta-D-glucopyranosyl-(2S,3R,4E,8Z)-2-[(2R)-2-hydroxyhexadecanoylamino]-4,8-octadecadiene-1,3-diol, and four other constituents, beta-sitosterol, daucosterol, uridine, and adenosine, have been isolated from the nuts of almond (Prunus amygdalus). Complete assignments of the proton and carbon chemical shifts for the sphingolipid were accomplished on the basis of high-resolution 1D and 2D NMR data. All of these compounds are being reported from almond nuts (P. amygdalus)for the first time.

Chemical studies on antioxidant mechanism of tea catechins: analysis of radical reaction products of catechin and epicatechin with 2,2-diphenyl-1-picrylhydrazyl.

Tea catechins, an important class of polyphenols, have been shown to have wide spectrum of antitumor activity believed to be due mainly to their antioxidative effect. In this study, the radical scavenging behavior of catechins on 2,2-diphenyl-1-picrylhydrazyl (DPPH) was studied. Two reaction products of (+)-catechin, and two reaction products of (-)-epicatechin were purified and identified. Their structures were determined on the basis of detailed high-field 1-D and 2-D NMR spectral analysis. Structure elucidation of these products can provide insights into specific mechanisms of antioxidant reactions. A possible mechanism of the formation of reaction products is suggested.

Antioxidative phenolic compounds isolated from almond skins (Prunus amygdalus Batsch).

Nine phenolic compounds were isolated from the ethyl acetate and n-butanol fractions of almond (Prunus amygdalus) skins. On the basis of NMR data, MS data, and comparison with the literature, these compounds were identified as 3'-O-methylquercetin 3-O-beta-D-glucopyranoside (1); 3'-O-methylquercetin 3-O-beta-D-galactopyranoside (2); 3'-O-methylquercetin 3-O-alpha-L-rhamnopyranosyl-(1-->6)-beta-D-glucopyranoside (3); kaempferol 3-O-alpha-L-rhamnopyranosyl-(1-->6)-beta-D-glucopyranoside (4); naringenin 7-O-beta-D-glucopyranoside (5); catechin (6); protocatechuic acid (7); vanillic acid (8); and p-hydroxybenzoic acid (9). All of these compounds have been isolated from almond skins for the first time. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activities for compounds 1-9 were determined. Compounds 6 and 7 show very strong DPPH radical scavenging activity. Compounds 1-3, 5, 8, and 9 show strong activity, whereas compound 4 has very weak activity.

Novel polyphenol molecule isolated from licorice root (Glycrrhiza glabra) induces apoptosis, G2/M cell cycle arrest, and Bcl-2 phosphorylation in tumor cell lines.

Herbal therapies are commonly used by patients with cancer, despite little understanding about biologically active chemical derivatives. We recently demonstrated that the herbal combination PC-SPES, which contains licorice root, had anti-prostate cancer activity attributable to estrogen(s) that produced a chemical castration. A recent study also demonstrated that licorice root alone decreased circulating testosterone in men. Other studies demonstrated antitumor activity of PC-SPES in vitro associated with decreased expression of the anti-apoptotic protein Bcl-2 and in patients independent of chemical castration, suggesting that other mechanisms of antitumor activity exist separate from chemical castration. In the present study, we assessed licorice root extract for effects on Bcl-2 to identify novel cytotoxic derivatives. Licorice root extract induced Bcl-2 phosphorylation as demonstrated by immunoblot and G2/M cell cycle arrest, similarly to clinically used antimicrotubule agents such as paclitaxel. Bioassay-directed fractionations resulted in a biologically active fraction for Bcl-2 phosphorylation. HPLC separation followed by mass spectrometry and NMR identified 6 compounds. Only one molecule was responsible for Bcl-2 phosphorylation; it was identified as 1-(2,4-dihydroxyphenyl)-3-hydroxy-3-(4'-hydroxyphenyl) 1-propanone (beta-hydroxy-DHP). The effect on Bcl-2 was structure specific, because alpha-hydroxy-DHP, 1-(2,4-dihydroxyphenyl)-2-hydroxy-3-(4'-hydroxyphenyl) 1-propanone, in contrast to beta-hydroxy-DHP, was not capable of Bcl-2 phosphorylation. Pure beta-hydroxy-DHP induced Bcl-2 phosphorylation in breast and prostate tumor cells, G2/M cell cycle arrest, apoptosis demonstrated by Annexin V and TUNEL assay, decreased cell viability demonstrated by a tetrazolium (MTT) assay, and altered microtubule structure. Therefore, these data demonstrate that licorice root contains beta-hydroxy-DHP, which induced Bcl-2 phosphorylation, apoptosis, and G2/M cell cycle arrest, in breast and prostate tumor cells, similarly to the action of more complex (MW >800) antimicrotubule agents used clinically.

Triterpene saponins from debittered quinoa (Chenopodium quinoa) seeds.

Twelve triterpene saponins have been isolated from the debittered seeds of quinoa (Chenopodium quinoa), and their structures were characterized on the basis of hydrolysis and spectral data, especially NMR evidence. Among them, three compounds, including 3-O-beta-D-glucuropyranosyl oleanolic acid (1), 3-O-beta-D-glucopyranosyl-(1-->3)-alpha-L-arabinopyranosyl hederagenin (2), and the new compound 3-O-beta-D-glucopyranosyl-(1-->3)-alpha-L-arabinopyranosyl-30-O-methyl spergulagenate 28-O-beta-D-glucopyranosyl ester (3), are identified for the first time from quinoa seeds. The other isolated saponins have been previously reported in quinoa.

New prenylated benzoic acid and other constituents from almond hulls (Prunus amygdalus Batsch).

One new prenylated benzoic acid derivative, 3-prenyl-4-O-beta-D-glucopyranosyloxy-4-hydroxylbenzoic acid, and three known constituents, catechin, protocatechuic acid, and ursolic acid, have been isolated from the hulls of almond (Prunus amygdalus). Complete assignments of the proton and carbon chemical shifts for the new prenylated benzoic acid derivative were accomplished on the basis of high-resolution 1D and 2D nuclear magnetic resonance data. All of these compounds except ursolic acid are being reported from almond hulls (P. amygdalus) for the first time.