Flavonol glycoside production in callus cultures of Epimedium diphyllum.

Callus cultures of Epimedium diphyllum produced a large amount of epimedoside A in addition to a small amount of diphylloside B, ikarisoside C, epimedoside E, diglycosides of des-O-methylanhydroicaritin (8-gamma, gamma-dimethylallylkaempfero). Icariin, epimedins A-C, which are glycosides of anhydroicaritin, were also produced in the callus cultures. Contents of the flavonol glycosides in callus tissue were higher than those of mother plants, but the composition of each flavonol glycoside mixture in the callus cultures was different from that of the original plants. The time-course experiments showed that an inverse relationship existed between cell growth and flavonol glycoside production. Effects of hormonal factors on cell growth and flavonol glycoside production indicated that 2,4-dichlorophenoxyacetic acid was needed for the production of flavonol glycosides.     

New anti-malarial flavonol glycoside from Hydrangeae Dulcis Folium.

Bioassay-guided fractionation of the MeOH extract of Hydrangeae Dulcis Folium resulted in isolation of a new flavonol glycoside and two known congeners as anti-malarial principles. These flavonol glycosides showed characteristic proliferation inhibition of Plasmodium falciparum at significantly low concentration without showing any cytotoxicity. In addition, several naturally occurring flavonol glycosides were also shown to exert similar anti-malarial behavior.     

Flavonol triglycosides from Blackstonia peroliata.

Three new flavonol triglycosides quercetin, kaempferol and isorhamnetin 3-rhamnosyl(1----2)galactoside-7-glucosides have been isolated from leaves and stems of Blackstonia perfoliata. This species together with three other genera of the tribe Gentianeae, subtribe Chlorae: Centaurium, Coutoubea and Eustoma, is unusual in producing flavonol glycosides instead of C-glycosyl flavones, the more characteristic flavonoid constituents of the Gentianaceae.     

Novel flavonol 3-sulfotransferase. Purification, kinetic properties, and partial amino acid sequence.

A flavonol sulfotransferase (EC 2.8.2.-), which catalyzes the transfer of the sulfate group from 3'-phosphoadenosine 5'-phosphosulfate to the 3-hydroxyl group of flavonol aglycones, has been purified to apparent homogeneity from Flaveria chloraefolia. The specific activity of flavonol 3-sulfotransferase was enriched 2000-fold, as compared with the homogenate, with a recovery of 9%. The molecular mass of the native and denatured enzyme was found to be 34.5 kDa, suggesting that the active from of the enzyme is a monomer. The enzyme exhibited expressed specificity for position 3 of flavonol aglycones, showed two activity optima at pH 6.0 and 8.5, did not require divalent cations, and was not inhibited by either EDTA or sulfhydryl group reagents. The results of substrate interaction kinetics and product inhibition are consistent with an Ordered Bi Bi mechanism where 3'-phosphoadenosine 5'-phosphosulfate is the first substrate to bind to the enzyme and 3'-phosphoadenosine 5'-phosphate is the final product to be released. The amino acid sequence of two peptides representing 17 and 33 amino acids showed no significant sequence similarity with the amino acid sequences reported for animal sulfotransferases. Antibodies raised against F. chloraefolia 3-sulfotransferase were found to cross-react with the 3'- and 4'-sulfotransferase activities of the same plant, suggesting that the three enzymes are structurally related.     

诸葛菜茎叶中黄酮类化合物的研究

诸葛菜茎叶乙醇提取物用Ｍｇ＋ＨＣｌ,Ｚｎ＋ＨＣｌ,１％ＦｅＣｌ３－乙醇液,１％ＮａＯＨ进行显色反应,呈现黄酮类化合物性质特征颜色。又以槲皮素,山柰酚,异鼠李素为对照品,采用ＨＰＬＣ法分析测定了其茎叶中黄酮醇的含量。结果表明干品中总黄酮含量（以甙元计）为０．５６８％。     

普通鸡冠花序中黄酮类化合物的研究

红色普通鸡冠（Ｃｅｌｏｓｉａ ａｒｇｅｎｔｅａ Ｌ．,ｒｅｄ ｆｌｏｗｅｒ）花序乙醇提取物用Ｍｇ＋ＨＣｌ,Ｚｎ＋ＨＣｌ,１％ＦｅＣｌ３－乙醇液,２％ＡｌＣｌ３－乙醇液,１％ＮａＯＨ进行显色反应,呈现黄酮类化合物性质特征颜色。又以槲皮素、山奈酚、异鼠李素为对照品,采用ＨＰＬＣ法测定分析了不同花期花序中黄酮醇的含量。结果表明,晚期花序干品中总黄酮含量（以甙元计）为０．７６１％。     

普通鸡冠花序中黄酮类化合物的研究

红色普通鸡冠(Celosia argentea L., red flower)花序乙醇提取物用Mg+HCl,Zn+HCl,1%FeCl3-乙醇液,2%AlCl3-乙醇液,1%NaOH进行显色反应,呈现黄酮类化合物性质特征颜色。又以槲皮素、山奈酚、异鼠李素为对照品,采用HPLC法测定分析了不同花期花序中黄酮醇的含量。结果表明,晚期花序干品中总黄酮含量(以甙元计)为0.761%。     

Urakunoside, a new tetraglycosyl kaempferol from petals of the Wabisuke camellia cv. Tarokaja

A new tetraglycosyl flavonol, 3-O-[2-O-xylosyl-6-O-(3-O-glucosyl-rhamnosyl) glucosyl] kaempferol was isolated from pale purplish-pink petals of Wabisuke camellia cv. Tarokaja with three known flavonols. It was named urakunoside after the species name of Tarokaja, Camellia uraku. Urakunoside was a major flavonol component in the Tarokaja petals, but was not detected in petals of Tarokaja's presumed ancestor species.     

Two flavonol glycosides from seeds of Camellia sinensis.

Two novel flavonol triglycosides, camelliaside A and B, have been isolated from seeds of Camellia sinensis. The structures were determined to be kaempferol 3-O-[2-O-beta-D- galactopyranosyl-6-O-alpha-L-rhamnopyranosyl]-beta-D-glucopyranoside and kaempferol 3-O-[2-O-beta- D-xylopyranosyl-6-O-alpha-L-rhamnopyranosyl]-beta-D-glucopyranoside on the basis of spectroscopic, chemical and enzymatic studies. These types of interglycosidic linkages, Gal(1----2)[Rha(1----6)]Glc and Xyl(1----2)[Rha(1----6)]Glc, have not been reported previously in flavone and flavonol glycosides.     

Chemosystematic studies in the chrysobalanaceae. I. Flavonoids in Parinari

A survey of flavonoids in 31 Asian, African and Neotropical species of Parinari showed a predominance of flavonol glycosides based on myricetin, quercertin, and kaemp-ferol. The African taxa split into two groups based on the presence or absence of myricetin glycosides. The Neotropical taxa, a complex of closely related species, are chemically very similar to each other and lack myre?etin, as does one group of African species. The Asian taxa are similar to the Neotropical ones in their flavonoid patterns and lack of myricetin glycosides. The presence of myricetin considered a primitive flavonoid character, suggest that te African species pro-ducing this flavonol represent a primitive nucleus eastward and westward ex-pansion to two myricetin-lacking phytogeographic lines. This hypothesis is in agreement with current proposals for geographic evolution in the Chrysobalanaceae.     

Localization of partially methylated flavonol glucosides in Chrysosplenium americanum. II. Immunofluorescence

Chrysosplenium americanum Schwein. ex Hooker (Saxifragaceae) accumulates a variety of partially methylated flavonol glucosides. Specific antibodies to tri- and tetramethylated flavonol-2′-O-glucosides, located using fluorescein isothiocyanate (FITC) goat antirabbit antibody, were used for the localization of the flavonol glucosides in leaf epidermis, cross sections and protoplasts. The results indicated that flavonoid accumulation occurred mainly in the walls of epidermal cells and, to a much lesser extent, in mesophyll cell walls. The weak fluorescence observed in the vacuoles of protoplasts suggested a minor role of this compartment in the accumulation process. The significance of flavonoid deposition within epidermal cell walls is discussed in relation to the lipophilic nature of these compounds and their possible role in the physiology of the plant.     

Functional expression and mutational analysis of flavonol synthase from Citrus unshiu.

Flavonols are produced by the desaturation of flavanols catalyzed by flavonol synthase. The enzyme belongs to the class of intermolecular dioxygenases which depend on molecular oxygen and FeII/2-oxoglutarate for activity, and have been in focus of structural studies recently. Flavonol synthase cDNAs were cloned from six plant species, but none of the enzymes had been studied in detail. Therefore, a cDNA from Citrus unshiu (Satsuma mandarin) designated as flavonol synthase was expressed in Escherichia coli, and the purified recombinant enzyme was subjected to kinetic and mutational chacterizations. The integrity of the recombinant synthase was revealed by a molecular ion from MALDI-TOF mass spectrometry at m/z 37888 +/- 40 (as compared to 37899 Da calculated for the translated polypeptide), and by partial N-terminal sequencing. Maximal flavonol synthase activity was observed in the range of pH 5-6 with dihydroquercetin as substrate and a temperature optimum at about 37 degrees C. Km values of 272, 11 and 36 micro m were determined for dihydroquercetin, FeII and 2-oxoglutarate, respectively, with a sixfold higher affinity to dihydrokaempferol (Km 45 micro m). Flavonol synthase polypeptides share an overall sequence similarity of 85% (47% identity), whereas only 30-60% similarity were apparent with other dioxygenases. Like the other dioxygenases of this class, Citrus flavonol synthase cDNA encodes eight strictly conserved amino-acid residues which include two histidines (His221, His277) and one acidic amino acid (Asp223) residue for FeII-coordination, an arginine (Arg287) proposed to bind 2-oxoglutarate, and four amino acids (Gly68, His75, Gly261, Pro207) with no obvious functionality. Replacements of Gly68 and Gly261 by alanine reduced the catalytic activity by 95%, while the exchange of these Gly residues for proline completely abolished the enzyme activity. Alternatively, the substitution of Pro207 by glycine hardly affected the activity. The data suggest that Gly68 and Gly261, at least, are required for proper folding of the flavonol synthase polypeptide.     

The effect of nitrogen and phosphorus deficiency on flavonol accumulation in plant tissues

The flavonol content of Arabidopsis thaliana and tomato seedlings was assessed in conditions of reduced nitrogen or phosphorus availability. In both systems, a significant inverse relationship was observed between nutrient availability and flavonol accumulation, with nitrogen limitation promoting the greatest increase in flavonols. A trial was established to determine the effects of decreased nitrogen and phosphorus availability on the flavonol content of leaf and fruit tissues of tomato plants (Lycopersicon esculentum cv. Chaser) in a commercial situation. Nutrients were supplied by a hydroponic system with nutrient regimes designed to provide the highest and lowest nitrogen and phosphorus levels with which it is possible to support plant growth and fruit set. Fruiting was abundant and tomato fruits were harvested at mature green, breaker and red stages of ripening; leaves were also harvested from the tops of the plants. All tissues were analysed for flavonol content using reversed‐phase high‐performance liquid chromatography. Flavonol accumulation in the leaves of mature tomato plants was found to increase significantly in response to nitrogen stress, whereas phosphorus deficiency did not elicit this response. Reduced nitrogen availability had no consistent effect on the flavonol content of tomato fruits. Phosphorus deficiency elicited an increase in flavonol content in early stages of ripening. Effects of nutrient stress on the flavonol content of tomato fruits were lost as ripening progressed. The findings suggest that nutrient status may be employed to manipulate the flavonol content of vegetative tissues but cannot be used to elevate the flavonol content of tomato fruit.     

Flavonoids from seeds of Camellia semiserrata Chi. and their estrogenic activity

Two new flavonol glycosides and three known flavonoids were isolated from seeds of Camellia semiserrata Chi. The structures of these new flavonol glycosides were established as kaempferol 3-O-[(2',3',4'-triacetyl)-alpha-L-rhamnopyranosyl(1-->3)(2',4'-diacetyl)-alpha-L-rhamnopyranosyl (1-->6)-beta-D-glucopyranoside] and kaempferol 3-O-[(3',4'-diacetyl)-alpha-L-rhamnopyranosyl(1-->3)(2',4'-diacetyl)-alpha-L-rhamnopyranosyl(1-->6)-beta-D-glucopyranoside] by spectroscopic methods. The estrogenic activity of these compounds was investigated by a recombinant yeast screening assay.     

Effects of total flavonoids and flavonol glycosides from Epimedium koreanum Nakai on the proliferation and differentiation of primary osteoblasts.

In a bioassay-guided drug screening for anti-osteoporosis activity, eight flavonol glycosides were isolated from Epimedium koreanum Nakai, which is traditionally widely used in China for the treatment of impotence and osteoporosis. The effects of total flavonoids and flavonol glycosides on the proliferation and differentiation of rat calvarial osteoblast-like cells were evaluated by the MTT method and measuring the activity of alkaline phosphatase (ALP activity). Total flavonoids (1.2 x10(-2) to 6.0 x10(-7) mg/ml) and flavonol glycosides (2.0 x10(-5) to 1.0 x10(-9) mol/l) exhibited a strong inhibition on the proliferation of primary osteoblasts at most concentrations. However, the total flavonoids and icariin significantly promoted the differentiation of primary osteoblasts. The results suggested that flavonoids from E. koreanum Nakai may improve the development of osteoblasts by promoting the ALP activity; and icariin might be one of the active constituents facilitating the differentiation of osteoblasts.     

Organ-specific transcription of putative flavonol synthase genes of grapevine and effects of plant hormones and shading on flavonol biosynthesis in grape berry skins

In order to investigate the control mechanism of flavonol biosynthesis of grapevine, we obtained five genomic sequences (FLS1 to FLS5) of putative flavonol synthase genes from Vitis vinifera cv. Cabernet Sauvignon. The mRNA of five FLSs accumulated in flower buds and flowers, while the mRNA of FLS2, FLS4, and FLS5 accumulated in small berry skins and then decreased toward veraison. At the ripening stage, the mRNA of only FLS4 and FLS5 accumulated again. This change in mRNA accumulation did not contradict the flavonol accumulation in the berry skins. Shading of the berries completely inhibited the increase in flavonol content and mRNA accumulation of FLS4, but did not affect the mRNA accumulation of FLS5. The effects of light and plant hormones on flavonol accumulation were different from those on anthocyanin accumulation. Thus flavonol biosynthesis appears to be under a different control system from that of anthocyanin biosynthesis.     

Comprehensive analysis of flavonols in Ginkgo biloba products by ultra-high-performance liquid chromatography coupled with ultra-violet detection and time-of-flight mass spectrometry

The aim of this study was to survey the flavonol compositions of Ginkgo biloba products, especially those on the Japanese market. Sixteen food products, six medicinal products, and raw Ginkgo biloba leaves were examined by ultra-high-performance liquid chromatography coupled with ultra-violet detection and time-of-flight mass spectrometry. Eleven flavonol glycosides, three biflavones, and a flavonol aglycone were qualified by analysis of accurate mass spectra. The quantitative data obtained were then applied to multivariate data analysis, and the flavonol compositions of the food and medicinal products were classified into four groups. Most of the food products were classified into the same group as the medicinal products, which contained high percentages of flavonol glycosides. On the other hand, some food products contained high percentages of biflavones or an aglycone.     

Complementary action of jasmonic acid on salicylic acid in mediating fungal elicitor-induced flavonol glycoside accumulation of Ginkgo biloba cells

The antagonistic action between jasmonic acid (JA) and salicylic acid (SA) in plant defence responses has been well documented. However, their relationship in secondary metabolite production is largely unknown. Here, we report that PB90, a protein elicitor from Phytophthora boehmeriae , triggers JA generation, SA accumulation and flavonol glycoside production of Ginkgo biloba cells. JA inhibitors suppress not only PB90-triggered JA generation, but also the elicitor-induced flavonol glycoside production. However, the elicitor can still enhance flavonol glycoside production even though the JA generation is totally inhibited. Over-expression of SA hydrolase gene NahG not only abolishes SA accumulation, but also suppresses the elicitor-induced flavonol glycoside production when JA signalling is inhibited. Interestingly, expression of NahG does not inhibit the elicitor-induced flavonol glycoside accumulation in the absence of JA inhibitors. Moreover, JA levels are significantly enhanced when SA accumulation is impaired in the transgenic cells. Together, the data suggest that both JA and SA are involved in PB90-induced flavonol glycoside production. Furthermore, we demonstrate that JA signalling might be enhanced to substitute for SA to mediate the elicitor-induced flavonol glycoside accumulation when SA signalling is impaired, which reveals an unusual complementary relationship between JA and SA in mediating plant secondary metabolite production.