Design, semisynthesis, and evaluation of O-acyl derivatives of (-)-epigallocatechin-3-gallate as antitumor agents

The partially purified catechin fraction isolated from green tea extract was treated with a variety of acylating agents (acyl anhydrides/chloride) to obtain (-)-epigallocatechin-3-gallate (EGCG) O-acyl derivatives in 20-25.4% yields. The (-)-EGCG O-acyl derivatives were characterized by physical data and spectral studies. These compounds were evaluated for their antitumor activity by use of a two-stage carcinogenesis model in 7,12-dimethylbenz[a]anthracene (DMBA)/12-O-tetradecanoylphorbol 13-acetate (TPA)--induced cancer in Swiss albino mice. The study showed that there was a significant decrease in the antitumor activity with the increase in size and branching of the chain length of acyl groups. The results indicated that these O-acyl derivatives of (-)-EGCG have the potential to be developed as cancer chemopreventive agents. Keywords: Green tea; catechins; (-)-EGCG O-acyl derivatives; antitumor activity.     

Isolation,structural elucidation,and inhibitory effects of terpenoid and lipid constituents from sunflower pollen on Epstein-Barr virus early antigen induced by tumor promoter,TPA

Eight fatty acid esters of triterpene alcohols (1-8), four free triterpene alcohols (9, 12, 17, and 18), four diterpene acids (19-22), two tocopherol-related compounds (23 and 24), four estolides (25-28), three syn-alkane-4,6-diols (29-31), one 1,3-dioxoalkanoic acid (32), and one aliphatic ketone (33), along with the mixture of free fatty acids, were isolated from the diethyl ether extract of the pollen grains of sunflower (Helianthus annuus). Among these compounds, 14 (2-8, 12, 23, 25-28, and 33) were new naturally occurring compounds, and their structures were determined on the basis of spectroscopic methods. Twenty-four terpenoids and lipids (1-4, 6-9, 12, and 19-33) and six free triterpene triols (10, 11, and 13-16), derived from their fatty acid esters (2, 3, and 5-8) by alkaline hydrolysis, were evaluated with respect to their inhibitory effects on the induction of Epstein-Barr virus early antigen (EBV-EA) induced by the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), in Raji cells, which is known to be a primary screening test for antitumor promoters. Among the 30 compounds tested, 21 compounds possessing a di- or a polycyclic ring system in the molecule (1-4, 6-16, and 19-24) showed potent inhibitory effects on EBV-EA induction (91-100% inhibition at 1 x 10(3) mol ratio/TPA).     

Inhibitory effects of cucurbitane glycosides and other triterpenoids from the fruit of Momordica grosvenori on epstein-barr virus early antigen induced by tumor promoter 12-O-tetradecanoylphorbol-13-acetate

Two new triterpene benzoates, 5-dehydrokarounidiol dibenzoate (1) and karounidiol dibenzoate (2), and two new triterpene glycosides, 5alpha,6alpha-epoxymogroside IE(1) (8) and 11-oxomogroside A(1) (9), along with 15 known triterpenoids (one triterpene benzoate, 3; three triterpene mono-ols, 4-6; one triterpene aglycon, 7; and 10 triterpene glycosides, 10-19), were isolated from the ethanol extract of the fruit of Momordica grosvenori. The structures of 1, 2, 8, and 9 were determined on the basis of spectroscopic and chemical methods. Among the known triterpene glycosides, mogroside I E(1) (12) was a new naturally occurring compound. Eighteen triterpenoids (2-19) and 11-oxomogrol (20), a hydrolysis product of 9, were evaluated with respect to their inhibitory effects on the induction of Epstein-Barr virus early antigen (EBV-EA) by 12-O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells, which is known to be a primary screening test for antitumor promoters. All of the compounds tested showed potent inhibitory effects on EBV-EA induction (70-100% inhibition at 1 x 10(3) mol ratio/TPA).     

Flavonoid constituents of Chorizanthe diffusa with potential cancer chemopreventive activity

An ethyl acetate-soluble extract of Chorizanthe diffusa was found to exhibit significant antioxidant activity, as judged by scavenging stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radicals and inhibition of 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced free radical formation with cultured HL-60 cells. Bioassay-directed fractionation of this extract using the DPPH antioxidant assay as a monitor led to the isolation of five structurally related flavonoids (1-5), including the novel compound 5,8,3',4',5'-pentahydroxy-3, 7-dimethoxyflavone (1). Isolates 1-5 demonstrated varying degrees of antioxidant or antimutagenic activity. Two of the compounds, 5,7,3', 4'-tetrahydroxy-3-methoxyflavone (2) and quercetin (4), were subsequently found to inhibit carcinogen-induced preneoplastic lesions in a mouse mammary organ culture model. Inhibitory activity of this type is known to correlate with cancer chemopreventive effects in full-term models of tumorigenesis.     

Sterol ferulates, sterols, and 5-alk(en)ylresorcinols from wheat, rye, and corn bran oils and their inhibitory effects on Epstein-Barr virus activation

Sterol ferulate, free sterol, and 5-alk(en)ylresorcinol constituents of wheat, rye, and corn bran oils were studied. Among the sterol ferulates, one novel compound, 24-methylenecholestanol ferulate (7), along with six known compounds, namely, 24-methylcholestanol ferulate (1), 24-methylcholesterol ferulate (2), 2-methyllathosterol ferulate (3), stigmastanol ferulate (4), sitosterol ferulate (5), and schottenol ferulate (6), were isolated and characterized. Five known free sterols, namely, 24-methylcholesterol (8), stigmastanol (9), sitosterol (10), schottenol (11), and stigmasterol (12), were isolated and identified. 5-Alk(en)ylresorcinols were found in wheat and rye bran oils but not in corn bran oil. Of these, one new compound, 5-n-(2'-oxo-14'-Z-heneicosenyl) resorcinol (19), and seven known compounds, namely, 5-n-heptadecyl- (13), 5-n-nonadecyl- (14), 5-n-heneicosyl- (15), 5-n-tricosyl- (16), 5-n-pentacosyl- (17), 5-n-(14'-Z-nonadecenyl)- (18), and 5-n-(2'-oxoheneicosyl)resorcinols (20), were isolated and characterized. These compounds were evaluated with respect to their inhibitory effects on the induction of Epstein-Barr virus early antigen (EBV-EA) by 12-O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells, which is known to be a primary screening test for antitumor promoters. Four compounds, 1, 2, 4, and 11, showed potent inhibitory effects on EBV-EA induction.     

Spectrophotometric determination of phenolic compounds by enzymatic and chemical methods--a comparison of structure--activity relationship

A spectrophotometric determination of phenolic compounds by a peroxidase-catalyzed enzymatic (PE) method and the Folin-Ciocalteu (FC) chemical method was compared for their structure-activity relationship. In the PE method, the reaction time of 19 phenolic compounds with different chemical structures was found to be within 15 min, with those having bulky substituents showing slower reactivity. The responses of the phenolic compounds toward the PE method in terms of molar absorbance were positively correlated with the nucleophilicity of their corresponding phenoxyl radicals. An increase in the nucleophilicity by substitution of methoxyl and hydroxyl (electron-donating) groups enhanced the responses; while a decrease in nucleophilicity by substitution of the allyl carboxylic (electron-withdrawing) groups lowered the responses of phenolic compounds toward the enzymatic assay. The responses of phenolic compounds to the enzymatic method were found to be independent of the degree of hydroxylation while those to the FC assay were affected by both the position and degree of hydroxylation. Interfering chemicals found in the FC assay, including vitamin C, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (a vitamin E analogue), and tert-butylhydroquinone (a commercial antioxidant) were insensitive to the enzymatic assay. The PE method appears to have a higher specificity toward phenolic compounds and subject to less interferences from other antioxidants than the FC method.     

Attenuation of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced gap junctional intercellular communication (GJIC) inhibition in MCF-10A cells by c9,t11-conjugated linoleic acid

The protective effect of c9,t11-conjugated linoleic acid (CLA) on the inhibition of gap junctional intercellular communication (GJIC) was examined in a human mammary epithelial cell line (MCF-10A) treated with 12-O-tetradecanoylphorbol-13-acetate (TPA), relative to t10,c12-CLA isomer. TPA inhibited GJIC in a dose-dependent and reversible manner and was associated with connexin 43 phosphorylation. Pretreatment of 20 μM c9,t11-CLA for 24 h prior to 60 nM TPA for 1 h prevented the inhibition of GJIC by reducing the phosphorylation of connexin 43 via suppressing extracellular signal-regulated kinases (ERK1/2) activation. Reactive oxygen species (ROS) accumulation by TPA was attenuated by c9,t11-CLA. The efficacy of c9,t11-CLA in protecting inhibition of GJIC, connexin 43 phosphorylation, and ROS production was superior to that of t10,c12-CLA. These results suggest that c9,t11-CLA, including t10,c12-CLA, prevents the carcinogenesis of MCF-10A cells by protecting down-regulation of GJIC during the cancer promotion stage, and lack of their toxicities could be an excellent indicator for the chemoprevention of breast cancer.     

Azaphilones, furanoisophthalides, and amino acids from the extracts of Monascus pilosus-fermented rice (red-mold rice) and their chemopreventive effects

Six azaphilones, monascin (1), ankaflavin (2), rubropunctatin (3), monascorburin (4), rubropunctamine (5), and monascorburamine (6), two furanoisophthalides, xanthomonasin A (7) and xanthomonasin B (8), and two amino acids, (+)-monascumic acid (9) and (-)-monascumic acid (10), isolated from the extracts of Monascus pilosus-fermented rice (red-mold rice) were evaluated for their inhibitory effects on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation in mice, on the induction of Epstein-Barr virus early antigen (EBV-EA) by TPA in Raji cells, and on the activation of (+/-)-(E)-methyl-2[(E)-hydroxy-imino]-5-nitro-6-methoxy-3-hexemide (NOR 1), a nitric oxide (NO) donor. Among the compounds tested, seven compounds (1-6 and 10) on TPA-induced inflammation, and six compounds (1, 3-5, 9, and 10) on EBV-EA activation, exhibited potent inhibitory effects. All of the compounds tested showed moderate inhibitory effects on NOR 1 activation.     

Transformation of bioactive compounds by Fusarium sacchari fungus isolated from the soil-cultivated ginseng

Ginsenoside bioactive compounds, namely, compound K (C-K), compound Mx (C-Mx), and ginsenoside Mc (G-Mc), were the metabolites of ginsenosides Rb 1, Rb 2, Rb 3, and Rc by intestinal microflora of humans or rats, microorganisms, and enzymes, and C-K showed beneficial effects in vitro and in vivo as an antitumoral agent. The objective of this work was to explore an efficient procedure for biotransformation of these bioactive compounds. Thus, a filamentous fungus, Fusarium sacchari, was first obtained from the soil-cultivated ginseng, which was verified to possess a potent capacity of transformation of C-K, C-Mx, and G-Mc. The optimal biotransformation conditions of F. sacchari with C-K, C-Mx, and G-Mc were obtained as follows: transforming temperature, 30 degrees C; transforming time, 6 days; rotary speed, 160 rpm; pH of the medium, 5.5. HPLC analysis indicated that these three bioactive compounds were key metabolites and their structures were confirmed by (1)H and (13)C NMR analysis. Moreover, the in vitro antitumor activities of C-K, C-Mx, and G-Mc and the in vivo antitumor activities of the transformed product mainly containing these compounds were also evaluated. Among C-K, C-Mx, and G-Mc, C-K exhibited the most potent antitumor activities. The in vivo study showed that the transformed products by F. sacchari had much more antitumor activity than those of commonly used ginsenoside Rg3 and Paclitaxel.     

Antitumor principle constituents of Myrica rubra Var. acuminata

Myrica rubra var. acuminata is a native shrub widely distributed and used as folk medicine in Taiwan for stomach disorders and diarrhea. Column chromatography combined with cytotoxic bioassay-guided fractionation was performed to isolate the antitumor principles from fresh leaves of M. rubravar. acuminata. The 20% MeOH eluate fraction of M. rubra var. acuminata inhibited the viability of HeLa and P-388 cells in an in vitro assay and an in vivo P-388 tumor-bearing CDF(1) mouse model. The percent increase in life span (%ILS) of 20% MeOH eluate fraction was greater than 125%. (-)-Epigallocatechin 3-O-gallate (1) and prodelphinidin A-2,3'-O-gallate (2) were isolated from D-20 as the antitumor principle components. Both compounds can inhibit the growth of HeLa cells, but 1 had lower cytotoxic effects in normal cervical fibroblasts than did 2. Moreover, pretreatment with a caspase-3 specific inhibitor prevented 1- and 2-induced poly(ADP-ribose) polymerase cleavage. In view of these results, we suggest that 1 and 2 can induce apoptosis in HeLa cells and that activation of caspase-3 may provide a mechanistic explanation for their cytotoxic effects. Therefore, we suggest that the 20% MeOH eluate fraction extract is good for health and that M. rubra var. acuminata is an economically valuable plant.     

Cytotoxic and antitumor activity of momilactone B from rice hulls

The cytotoxic and antitumor activity of methanolic extract of rice hulls (MERH) were evaluated by the MTT-dye reduction assay against human colon cancer cells and the colonic aberrant crypt foci (ACF) assay in 1,2-dimethylhydrazine (DMH)-injected F344 male rats, respectively. MERH was found to be highly cytotoxic, with IC50 values of 0.5 microg/mL in vitro. Forty weeks of MERH supplementation (50 mg/kg of body weight/day) reduced colonic pre-neoplastic ACF formation by 35% (p < 0.01). An active compound, momilactone B, was isolated from MERH by silica gel chromatography, Sephadex LH-20 chromatography, and HPLC. The cytotoxic activity of momilactone B was evaluated by the MTT-dye reduction, lactate dehydrogenase (LDH), and colony-forming ability assays in human colon cancer HT-29 and SW620 cells. The results indicated that momilactone B from rice hulls might be a new candidate for chemotherapeutic agent against human colon cancer.     

Flavonoid Glycosides from the Seeds of Litchi chinensis

Seven flavonoid glycosides, including one new (1) and five previously uncharacterized (3-7), were obtained from the seeds of lychee ( Litchi chinensis Sonn. cv. Heiye) by means of repetitive column chromatography and high-performance liquid chromatography (HPLC) preparation. They were identified as litchioside D (1), (-)-pinocembrin 7-O-neohesperidoside (2), (-)-pinocembrin 7-O-rutinoside (3), taxifolin 4'-O-β-d-glucopyranoside (4), kaempferol 7-O-neohesperidoside (5), tamarixetin 3-O-rutinoside (6), and phlorizin (7) on the basis of spectroscopic analysis and comparison of their data to the values reported in the literatures. Among them, compounds 1, 4, and 5 showed in vitro antitumor activity against A549, LAC, Hep-G2, and HeLa cell lines in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay.     

Isolation and identification of compounds responsible for antioxidant capacity of Euryale ferox seeds

Euryale ferox seed is consumed medicinally or for food in China. The present study revealed it to contain significant antioxidant activity, which may be associated with its medical applications as a proteinuria inhibitor of diabetic nephropathy. This study resulted in the identification of 3 new sesquineolignans, named euryalins A-C (1-3), and 16 known compounds, which were all first isolated from this plant apart from 5,7,4-trihydroxy-flavanone. The antioxidant potential of the partial isolates was evaluated using the DPPH radical scavenging assay and mesangial cellular assay. Compounds 2, rel-(2α,3β)-7-O-methylcedrusin (4), syringylglycerol-8-O-4-(sinapyl alcohol) ether (5), and (+)-syringaresinol (7) were found to be most active on DPPH assay, whereas compounds 2, 4, 7, (1R,2R,5R,6S)-2-(3,4-dimethoxyphenyl)-6-(3,4-dihydroxyphenyl)-3,7-dioxabicyclo[3.3.0]octane, and buddlenol E could significantly inhibit high glucose-stimulated reactive oxygen species production in mesangial cells. The results suggested that E. ferox seed could be considered as an excellent source of natural antioxidants and is useful in the prevention of diabetic nephropathy.     

Phenolic profile and hydrophilic antioxidant capacity as chemotaxonomic markers of tomato varieties

Tomatoes (Solanum lycopersicum L.), the second most important vegetable crop worldwide, are a key component in the so-called "Mediterranean diet", which is strongly associated with a reduced risk of chronic degenerative diseases. In this work, we evaluate the differences in the total and individual polyphenol content and hydrophilic antioxidant capacity of seven varieties of tomato cultivated in Vegas Bajas del Guadiana, Badajoz (Spain), which were collected from two consecutive harvests (2008-2009). Hydrophilic antioxidant capacity was evaluated using the TEAC assay, while the Folin-Ciocalteau assay with a previous cleanup was used to establish total polyphenol content. The method was optimized and validated. Individual polyphenols were quantified using liquid chromatography/electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) on a triple quadrupole. All compounds were found to be significantly different when analysis of variance was performed. Results from the principal component analysis show that phenolic compounds and hydrophilic antioxidant capacity were responsible for the differences among tomato samples according to variety.     

Anti-oxidant constituents of the roots and stolons of licorice (Glycyrrhiza glabra)

As part of a search for new cancer chemopreventive agents, a new chalcone derivative (1), a novel group of neolignan lipid esters (2), and seven known phenolic compounds (formononetin, glabridin, hemileiocarpin, hispaglabridin B, isoliquiritigenin, 4'-O-methylglabridin, and paratocarpin B) (3-9) were isolated from the roots and stolons of licorice (Glycyrrhiza glabra). The structures of compound 1 and the individual components of isolate 2 were elucidated using various spectroscopic and chemical methods. All isolates were tested in an authentic peroxynitrite anti-oxidant assay. Of these compounds, hispaglabridin B (6), isoliquiritigenin (7), and paratocarpin B (9) were found to be the most potent anti-oxidant agents. Furthermore, isoliquiritigenin (7) was demonstrated to prevent the incidence of 1,2-dimethylhydrazine-induced colon and lung tumors in mice when administered at a dose of 300 mg/kg.     

Isolation and Caenorhabditis elegans lifespan assay of flavonoids from onion

The main flavonoids were isolated from three selected onion cultivars. Three phenolic compounds were obtained by reverse-phase HPLC, and their structures were elucidated by multiple NMR measurements. There were two known compounds, quercetin and quercetin 3'-O-β-D-glucopyranoside (Q3'G), and one novel compound, quercetin 3-O-β-D-glucopyranoside-(4→1)-β-d-glucopyranoside (Q3M), which was identified in onion for the first time. These flavonoids were found to be more abundant in the onion peel than in the flesh or core. Their antioxidative activities were tested using the DPPH method, and their antiaging activities were evaluated using a Caenorhabditis elegans lifespan assay. No direct correlation was found between antioxidative activity and antiaging activity. Quercetin showed the highest antioxidative activity, whereas Q3M showed the strongest antiaging activity among these flavonoids, which might be related to its high hydrophilicity.     

Antioxidant Activities of Extracts from Teas Prepared from Medicinal Plants, Morus alba L., Camellia sinensis L., and Cudrania tricuspidata , and Their Volatile Components

The antioxidant activity of essences of teas prepared from mulberry ( Morus alba L.), Camellia sinensis L., and Cudrania tricuspidata (Carr.) Burea plant was examined using two antioxidant assays. Selected volatile chemicals identified in these plants were also tested for antioxidant activity. All extracts exhibited antioxidant activity with a clear dose response in the aldehyde/carboxylic acid and the malonaldehyde/gas chromatography (MA/GC) assays. Antioxidant activity of extracts at the level of 500 μg/mL ranged from 77.02 ± 0.51% (stems of Burea plant) to 52.57 ± 0.92% (fermented tea of Camellia and stems of Mulberry tea) in the aldehydes/carboxylic acid assay. Their antioxidant activity at the level of 160 μg/mL ranged from 76.17 ± 0.27% (roots of Burea plant) to 59.32 ± 0.27% (stems of Mulberry tea) in the MA/GC assay. Among the positively identified compounds (11 terpenes and terpenoids, 15 alkyl compounds, 26 nitrogen containing heterocyclic compounds, 9 oxygen containing heterocyclic compounds, 18 aromatic compounds, 7 lactones, 6 acids, and 4 miscellaneous compounds), eugenol, 2,5-dihydroxyl acetophenone, and isoeugenol exhibited antioxidant activity comparable to that of BHT in both assays. Vanillin and 2-acetylpyrrole showed potent antioxidant activity in the aldehydes/carboxylic acid assay but only moderate activity in the MA/GC assay. These results suggest that consumption of antioxidant-rich beverages prepared from these plants may be beneficial to human health.     

Purification and characterization of the antitumor protein from Chinese tartary buckwheat (Fagopyrum tataricum Gaertn.) water-soluble extracts

A novel antitumor protein, coded as TBWSP31, was isolated from tartary buckwheat water-soluble extracts and purified by DEAE-Sepharose Fast Flow anion exchange, Sephadex G-100 gel filtration, and Sephacryl S-200 gel filtration column chromatography. TBWSP31 was identified to a homogeneous fraction by native PAGE. The antitumor effect of TBWSP31 against human mammary cancer cell Bcap37 was measured by an MTT assay. TBWSP31 showed higher antitumor activity, and time- and concentration-dependent effects were observed. SDS-PAGE analysis showed that TBWSP31 is composed of a single polypeptide with an approximate molecular weight of 57 kDa. TBWSP31 was rich in Glx (Gln+Glu), Arg, and Asx (Asp+Asn) according to amino acid analysis. Secondary structural analysis by CD spectroscopy revealed that TBWSP31 has the following: alpha-helix (33.9%), beta-sheet (22.8%), beta-turn (11.3%), and random coil (32.0%).     

Synthesis and fungicidal activities of novel indene-substituted oxime ether strobilurins

Nineteen novel indene-substituted oxime ether strobilurins, which used an indene group to stabilize the ( E)-styryl group in SYP-Z071 (an unsaturated oxime strobilurin fungicide under development by the Shenyang Research Institute of Chemical Industry), were designed and synthesized. The biological assay results showed that all compounds possessed good or excellent fungicidal activities. It was found that most of the compounds showed higher fungicidal activities against Pyricularia oryzae, Phytophthora infestans, Erysiphe graminis, and Colletotrichum lagenarium than SYP-Z071 at the tested concentration. The biological assay results also indicated that most of the compounds exhibited higher in vivo fungicidal activities against cucumber Pseudoperonospora cubensis and C. lagenarium than the commercial fungicides trifloxystrobin and kresoxim-methyl at a concentration of 6.25 mg/L. Furthermore, it was found that alpha-(methoxyimino)- N-methylphenylacetamide oxime ethers 6m- s exhibited a broad spectrum and remarkably higher activities against all tested fungi. Especially, the 6-methylindene-substituted compound 6p was identified as the most promising candidate for further study.