Capillin, a major constituent of Artemisia capillaris Thunb. flower essential oil, induces apoptosis through the mitochondrial pathway in human leukemia HL-60 cells.

BACKGROUND: Natural products are one of the most important sources of drugs used in pharmaceutical therapeutics. Screening of several natural products in the search for novel anticancer agents against human leukemia HL-60 cells led us to identify potent apoptosis-inducing activity in the essential oil fraction from Artemisia capillaris Thunb. flower. METHODS: The cytotoxic effects of extracts were assessed on human leukemia HL-60 cells by XTT assay. Induction of apoptosis was assessed by analysis of DNA fragmentation and nuclear morphological change. The plant name was checked with the plant list website (http://www.theplantlist.org). RESULTS: A purified compound from the essential oil fraction from Artemisia capillaris Thunb. flower that potently inhibited cell growth in human leukemia HL-60 cells was identified as capillin. The cytotoxic effect of capillin in cells was associated with apoptosis. When HL-60 cells were treated with 10(-6) M capillin for 6 h, characteristic features of apoptosis such as DNA fragmentation and nuclear fragmentation were observed. Moreover, activation of c-Jun N-terminal kinase (JNK) was detected after treatment with capillin preceding the appearance of characteristic properties of apoptosis. Release of cytochrome c from mitochondria was also observed in HL-60 cells that had been treated with capillin. CONCLUSION: Capillin induces apoptosis in HL-60 cells via the mitochondrial apoptotic pathway, which might be controlled through JNK signaling. Our results indicate that capillin may be a potentially useful anticancer drug that could enhance therapeutic efficacy.

Altitudinal variation of flavonoid content in the leaves of Fallopia japonica and the needles of Larix kaempferi on Mt. Fuji.

Ultraviolet-B radiation is harmful to plants, and its intensity increases at altitude. So plants growing at high altitude possess UV protection systems. Flavonoid is known as a major UV protectant because it absorbs UV radiation and scavenges UV-induced free radicals in plant tissues. Japanese knotweed (Fallopia japonica) and Japanese larch (Larix kaempferi) grow at a wide range of altitudes on Mt. Fuji, the highest mountain in Japan, while the two plants harbor a homogeneous genetic structure. In the present study, a total of 14 flavonol 3-O-glycosides were isolated from both species. Furthermore, quantitative HPLC analyses revealed that flavonoid levels in the leaves of F. japonica and the needles of L. kaempferi increased with increasing altitude of their growing sites. The altitudinal trend of UV-absorbing antioxidants of herbal and woody plants was simultaneously revealed for the first time. These results suggest that both species have chemically acclimatized to high altitude regions, in which severe environmental conditions such as higher UV radiation exist.

New kaempferol 3,7-diglycosides from Asplenium ruta-muraria and Asplenium altajense.

A flavonoid was isolated from the fronds of Asplenium ruta-muraria and A. altajense (Aspleniaceae) collected in the Altai Mountains and adjacent area. The compound was identified as kaempferol 3-O-ß-[(6'''-E-caffeoylglucopyranosyl)-(1-->3)-glucopyranoside]-7-O-ß-glucopyranoside (1) by UV, 1H and 13C NMR spectroscopy, LC-MS, and acid and alkaline hydrolyses. Another flavonoid (2) was isolated from A. altajense, as a minor compound, together with 1 and identified as deacylated compound 1, i.e. kaempferol 3-O-laminaribioside-7-O-glucoside. They were found in nature for the first time.

New flavonol glycosides from the leaves and flowers of Primula sieboidii.

Three flavonol glycosides were isolated from the leaves of Primula sieboldii. They were identified as quercetin 3-O-ß-[xylopyranosyl-(1-->2)-ß- glucopyranosyl-(1-->6)-ß-glucopyranoside] (1), kaempferol 3-O-ß-[glucopyranosyl-(1-->2)-ß-glucopyranosyl-(1-->6)-ß-glucopyranoside] (2) and kaempferol 3- O-ß-[xylopyranosyl-(1-->2)-ß-glucopyranosyl-(1-->6)-ß-glucopyranoside] (3). Their chemical structures were determined by UV, 1H and 13C NMR spectroscopy, LC-MS and acid hydrolysis. Compounds 1 and 3 are found in nature for the first time. They were also detected in the flowers, together with two anthocyanins, malvidin 3,5-di-O-glucoside and a minor petunidin dihexoside.

Hinesol, a compound isolated from the essential oils of Atractylodes lancea rhizome, inhibits cell growth and induces apoptosis in human leukemia HL-60 cells.

Hinesol is a unique sesquiterpenoid isolated from the Chinese traditional medicine, Atractylodes lancea rhizome. In a previous study, we screened various natural products in human leukemia HL-60 cells and identified an essential oil fraction from A. lancea rhizome that exhibited apoptosis-inducing activity in these cells; hinesol was subsequently shown to be the compound responsible for this apoptosis-inducing activity. In this study, we describe the cytotoxic effects and molecular mechanisms of hinesol in HL-60 cells. The antitumor effect of hinesol was associated with apoptosis. When HL-60 cells were treated with hinesol, characteristic features of apoptosis, such as nuclear fragmentation and DNA fragmentation, were observed. These growth-inhibitory and apoptosis-inducing activities of hinesol in leukemia cells were much stronger than those of ß-eudesmol, another compound isolated from the essential oil fraction. Furthermore, hinesol induced activation of c-Jun N-terminal kinase (JNK), but not p38, prior to the onset of apoptosis. These results suggested that hinesol induced apoptosis through the JNK signaling pathway in HL-60 cells. Therefore, hinesol may represent a novel medicinal drug having indications in the treatment of various cancers, including leukemia.

Further characterization of foliar flavonoids in Crossostephium chinense and their geographic variation.

Foliar flavonoids of Crossostephium chinense in Japan and Taiwan were isolated and further characterized. Eighteen flavonoid aglycones, luteolin, apigenin, hispidulin, chrysoeriol, 5,7,4'-trihydroxy-6,3',5'-trimethoxyflavone, jaceosidin, cilsimaritin, quercetin 3-methyl ether, axillarin, chrysosplenol-D, cirsiliol, apometzgerin, 5,7,3'-trihydroxy-6,4',5'-trimethoxyflavone, luteolin 3',4'-dimethyl ether, cirsilineol, eupatilin, nepetin and 5,7,3',4'-tetrahydroxy-6,5'-dimethoxyflavone, were identified by UV, 1H and 13C NMR spectroscopic, LC-MS and HPLC comparisons w ith authentic samples. The compounds existed on the leaf surface. Four flavonoid glycosides, quercetin 3,7-di-O-glucoside, quercetin 3-O-rutinoside, luteolin 7-O-glucoside and apigenin 7-O-rutinoside, were also isolated as the intracellular flavonoids. It was shown by HPLC survey that variation of the species' flavonoids occurs among the collection sites.

Anthocyanins from the flowers of Nagai line of Japanese garden Iris (Iris ensata).

Six anthocyanins were isolated from the flowers of the Nagai line of Iris ensata Thunb. They were identified as petunidin and malvidin 3-O-beta-[(4"'-Z-p-coumaroyl-alpha-rhamnopyranosyl)-(1-->6)-beta-glucopyranoside]-5-O-beta-glucopyranosides (1 and 3) and their E-forms (2 and 4), and petunidin and malvidin 3-O-rutinoside-5-O-glucosides (5 and 6). Though the E-form of petunidin 3-O-[(4"'-p-coumaroylrhamnosyl)-(1-->6)-glucoside]-5-O-glucoside has been reported, its Z-form was found for the first time. The presence of Z- and E-forms of malvidin 3-O-[(4'''-p-coumaroylrhamnosyl)-(1-->6)-glucoside]-5-O-glucoside is also reported for the first time. Fifty-one cultivars of Nagai line and their wild form (I. ensata var. spontanea) were divided into four anthocyanin patterns, i.e. 1) the presence of 1-4, 2) the presence of 2 and 4, 3) the presence of 5 and 6, and 4) no anthocyanin.

Linkage mapping, molecular cloning and functional analysis of soybean gene Fg2 encoding flavonol 3-O-glucoside (1 → 6) rhamnosyltransferase.

There are substantial genotypic differences in the levels of flavonol glycosides (FGs) in soybean leaves. The first objective of this study was to identify and locate genes responsible for FG biosynthesis in the soybean genome. The second objective was to clone and verify the function of these candidate genes. Recombinant inbred lines (RILs) were developed by crossing the Kitakomachi and Koganejiro cultivars. The FGs were separated by high performance liquid chromatography (HPLC) and identified. The FGs of Koganejiro had rhamnose at the 6″-position of the glucose or galactose bound to the 3-position of kaempferol, whereas FGs of Kitakomachi were devoid of rhamnose. Among the 94 RILs, 53 RILs had HPLC peaks classified as Koganejiro type, and 41 RILs had peaks classified as Kitakomachi type. The segregation fitted a 1:1 ratio, suggesting that a single gene controls FG composition. SSR analysis, linkage mapping and genome database survey revealed a candidate gene in the molecular linkage group O (chromosome 10). The coding region of the gene from Koganejiro, designated as GmF3G6″Rt-a, is 1,392 bp long and encodes 464 amino acids, whereas the gene of Kitakomachi, GmF3G6″Rt-b, has a two-base deletion resulting in a truncated polypeptide consisting of 314 amino acids. The recombinant GmF3G6″Rt-a protein converted kaempferol 3-O-glucoside to kaempferol 3-O-rutinoside and utilized 3-O-glucosylated/galactosylated flavonols and UDP-rhamnose as substrates. GmF3G6″Rt-b protein had no activity. These results indicate that GmF3G6″Rt encodes a flavonol 3-O-glucoside (1 â†’ 6) rhamnosyltransferase and it probably corresponds to the Fg2 gene. GmF3G6″Rt was designated as UGT79A6 by the UGT Nomenclature Committee.

Genkwanin 4'-O-glucosyl-(1 --> 2)-rhamnoside from new chemotype of Asplenium normale in Japan.

New flavone glycoside, genkwanin 4'-O-ß-glucopyranosyl-(1 --> 2)-O-α-rhamnopyranoside was isolated from the fronds of new chemotype of Asplenium normale D. Don, together with two known C-glycosylflavones, vicenin-2 and lucenin-2. The chemical structure of the isolated glycoside was established by UV, LC-MS, characterization of acid hydrolysates, and 1H and 13C NMR spectroscopy.

New flavonol glycosides from the leaves of Triantha japonica and Tofieldia nuda.

Two new flavonol glycosides were isolated from the leaves of Triantha japonica, together with eight known flavonols, kaempferol 3-O-sophoroside, kaempferol 3-O-sambubioside, kaempferol 3-O-glucosyl-(1 --> 2)-[glucosyl-(1 --> 6)-glucoside], quercetin 3-O-sophoroside, quercetin 3-O-sambubioside, isorhamnetin 3-O-glucoside, isorhamnetin 3-O-sophoroside and isorhamnetin 3-O-sambubioside. The new compounds were identified as kaempferol 3-O-beta-xylopyranosyl-(1 --> 2)-[beta-glucopyranosyl-(1 --> 6)-beta-glucopyranoside] (1) and isorhamnetin 3-O-beta-xylopyranosyl-(1 --> 2)-[beta-glucopyranosyl-(1 --> 6)-beta-glucopyranoside] (3) by UV, LC-MS, acid hydrolysis, and 1H and 13C NMR spectroscopy. Another two new flavonol glycosides were isolated from theleaves of Tofieldia nuda, and identified as kaempferol 3-O-beta-glucopyranosyl-(1 --> 2)-[beta-glucopyranosyl-(1 --> 6)-beta-galactopyranoside] (4) and quercetin 3-O-beta-glucopyranosyl-(1 --> 2)-[beta-glucopyranosyl-(1 --> 6)-beta-galactopyranoside] (5). Though the genera Triantha and Tofieldia were treated as Tofieldia sensu lato, they were recently divided into two genera. It was shown by this survey that their flavonoid composition were also different to each other.

Kaempferol 3,7,4'-glycosides from the flowers of Clematis cultivars.

A new kaempferol glycoside, kaempferol 3-O-alpha-rhamnopyranosyl-(1 --> 6)-beta-glucopyranoside-7,4'-di-O-beta-glucopyranoside (1) was isolated from the flowers of Clematis cultivars "Jackmanii Superba" and "Fujimusume", together with the known compound kaempferol 3,7,4'-tri-O-beta-glucopyranoside (2). The chemical structures of the isolated kaemferol glycosides were established by UV, 1H and 13C NMR spectroscopy, LC-MS, and characterization of acid hydrolysates.

Identification of novel C-glycosylflavones and their contribution to flower colour of the Dutch iris cultivars.

Seventeen C-glycosylflavones including four novel ones, were isolated from the flowers of Dutch iris (Iris hollandica Hort. ex Todd.) cultivar 'Blue Diamond'. Four new C-glycosylflavones were identified as isovitexin 2″-O-(4″'-acetylrhamnoside) (F1), swertisin 2″-O-(4″'-acetylrhamnoside) (F2), isovitexin 2″-O-(4″'-acetylrhamnoside)-4'-O-glucoside (F3) and swertisin 2″-O-(4″'-acetylrhamnoside)-4'-O-glucoside (F4) by UV spectra, LC-MS and (1)H and (13)C NMR. Furthermore, to understand the contribution of flavones to flower colour, the relationships with flower colours of three Dutch iris cultivars and flavonoid components were examined. The degree of blueness in the bluish cultivars 'Blue Diamond' and 'Blue Magic' were higher than that of the violet cultivar, 'Yesterday', and it was suggested that the flower colour expression from violet to blue colour of Dutch iris cultivars depend on the high ratio of total flavone contents/total delphinidin contents (F/A ratio). In addition, in vitro examination was carried out by the isolated anthocyanin and flavone. The mixture solutions were prepared in respective F/A ratio of three Dutch iris cultivars and could essentially reconstruct their visible absorption spectra of flowers. In conclusion, it was confirmed that isolated flavones contribute to blueness due to intermolecular co-pigmentation with anthocyanins.

New flavonol triglycosides from the leaves of soybean cultivars.

Soybean (Glycine max) is a major crop in the world. Three new flavonol 3-O-glycosides, kaempferol 3-O-alpha-L-rhamnopyranosyl-(1 --> 4)-[alpha-L-rhamnopyranosyl-(1 --> 6)-beta-D-galactopyranoside] (1), kaempferol 3-O-alpha-L-rhamnopyranosyl-(1 --> 4)-[beta-D-glucopyranosyl-(1 --> 6)-beta-D-galactopyranoside] (4) and quercetin 3-O-beta-D-glucopyranosyl-(1--> 2)-[alpha-L-rhamnopyranosyl-(1 --> 6)-beta-galactopyranoside] (5) were isolated from the leaves of soybean cultivars, together with three known compounds, kaempferol 3-O-beta-D-glucopyranosyl-(1 --> 2)-[alpha-L-rhamnopyranosyl-(1--> 6)-beta-D-galactopyranoside] (2), kaempferol 3-O-beta-D-glucopyranosyl-(1 --> 2)-[alpha-L-rhamnopyranosyl-(1 --> 6)-beta-D-glucopyranoside] (3) and quercetin 3-O-beta-D-glucopyranosyl-(1 --> 2)-[alpha-L-rhamnopyranosyl-(1 --> 6)-beta-D-glucopyranoside] (6), and also common flavonoids. The isolated compounds possess similar structures and high water solubility, and so it was hard to isolate them (in particular 5 and 6) with a normal preparative HPLC system. Their final purification was achieved by a preparative HPLC system equipped with a recycle device.

Acylated delphinidin glycosides from violet and violet-blue flowers of Clematis cultivars and their coloration.

Three new acylated delphinidin glycosides, delphinidin 3-O-beta-[(2"-trans-caffeoylglucopyranosyl)-(1 --> 2)-(6"-succinylgalactopyranoside)]-7-O-beta-glucopyranoside (1), delphinidin 3-O-beta-[(2"-trans-caffeoylglucopyranosyl)-(1 --> 2)-(6"-trans-caffeoyl-tartaroyl-malonylgalactopyranoside)]-7-O-beta-glucopyranoside (2), and delphinidin 3-O-beta-[(2"-trans-caffeoylglucopyranosyl)-(1 --> 2)-(6"-trans-caffeoyl-tartaroyl-malonylgalactopyranoside)]-3'-O-beta-glucuronopyranoside (3), were isolated from the violet and violet-blue sepals of Clematis cultivars 'Jackmanii Superba' and 'Fujimusume'. The chemical structures of the isolated anthocyanins were determined by LC-MS, characterization of hydrolyzates, and UV, 1H and 13C NMR spectroscopy. The visible absorption spectra of these anthocyanins were compared with those of fresh sepals and crude extracts in pH 5.1 buffer solution. In addition, the co-pigment effect with some kaempferol glycosides and caffeoylglucose was examined.

Kaempferol tri- and tetraglycosides from the flowers of Clematis cultivar.

A new kaempferol glycoside, kaempferol 3-O-alpha-rhamnopyranosyl-(1 --> 2)-[alpha-rhamnopyranosyl-(1 --> 6)-beta-glucopyranoside]-7-O-beta-glucopyranoside (2) was isolated from the flowers of Clematis cultivar "Jackmanii Superba", together with a known kaempferol 3-O-alpha-rhamnopyranosyl-(1 --> 6)-beta-glucopyranoside-7-O-beta-glucopyranoside (1). The chemical structures of the isolated glycosides were established by UV, LC-MS, characterization of acid hydrolysates, and 1H and 13C NMR spectroscopy.

Key role of chemical hardness to compare 2,2-diphenyl-1-picrylhydrazyl radical scavenging power of flavone and flavonol O-glycoside and C-glycoside derivatives.

The antioxidant activities of flavonoids and their glycosides were measured with the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH radical, DPPH(·)) scavenging method. The results show that free hydroxyl flavonoids are not necessarily more active than O-glycoside. Quercetin and kaempferol showed higher activity than apigenin. The C- and O-glycosides of flavonoids generally showed higher radical scavenging activity than aglycones; however, kaempferol C3-O-glycoside (astragalin) showed higher activity than kaempferol. In the radical scavenging activity of flavonoids, it was expected that OH substitutions at C3 and C5 and catechol substitution at C2 of B ring and intramolecular hydrogen bonding between OH at C5 and ketone at C3 would increase the activity; however, the reasons have yet to be clarified. We here show that the radical scavenging activities of flavonoids are controlled by their absolute hardness (η) and absolute electronegativity (χ) as a electronic state. Kaempferol and quercetin provide high radical scavenging activity since (i) OH substitutions at C3 and C5 strikingly decrease η of flavones, (ii) OH substitutions at C3 and C7 decrease χ and η of flavones, and (iii) phenol or o-catechol substitution at C2 of B ring decrease χ of flavones. The coordinate r(χ, η) as the electron state must be small to increase the radical scavenging activity of flavonoids. The results show that chemically soft kaempferol and quercetin have higher DPPH radical scavenging activity than chemically hard genistein and daidzein.

Apigenin di- and trirhamnoside from Asplenium normale in Malaysia.

Two new flavone rhamnosides, apigenin 7-O-alpha-L-rhamnopyranosyl-(1-->4)-O-alpha-L-rhamnopyranoside and apigenin 7-O-alpha-L-rhamnopyranosyl-(1-->4)-O-alpha-L-rhamnopyranoside-4'-O-alpha-L-rhamnopyranoside were isolated from the fronds of Asplenium normale D. Don, together with two known C-glycosylflavones, vicenin-2 and lucenin-2. The chemical structures of the isolated glycosides were established by UV, LC-MS, characterization of acid hydrolysates, and 1H and 13C NMR spectroscopy.

Analysis of flavonoids in flower petals of soybean near-isogenic lines for flower and pubescence color genes.

W1, W3, W4, and Wm genes control flower color, whereas T and Td genes control pubescence color in soybean. W1, W3, Wm, and T are presumed to encode flavonoid 3'5'-hydroxylase (EC 1.14.13.88), dihydroflavonol 4-reductase (EC 1.1.1.219), flavonol synthase (EC 1.14.11.23), and flavonoid 3'-hydroxylase (EC 1.14.13.21), respectively. The objective of this study was to determine the structure of the primary anthocyanin, flavonol, and dihydroflavonol in flower petals. Primary component of anthocyanin in purple flower cultivars Clark (W1W1 w3w3 W4W4 WmWm TT TdTd) and Harosoy (W1W1 w3w3 W4W4 WmWm tt TdTd) was malvidin 3,5-di-O-glucoside with delphinidin 3,5-di-O-glucoside as a minor compound. Primary flavonol and dihydroflavonol were kaempferol 3-O-gentiobioside and aromadendrin 3-O-glucoside, respectively. Quantitative analysis of near-isogenic lines (NILs) for flower or pubescence color genes, Clark-w1 (white flower), Clark-w4 (near-white flower), Clark-W3w4 (dilute purple flower), Clark-t (gray pubescence), Clark-td (near-gray pubescence), Harosoy-wm (magenta flower), and Harosoy-T (tawny pubescence) was carried out. No anthocyanins were detected in Clark-w1 and Clark-w4, whereas a trace amount was detected in Clark-W3w4. Amount of flavonols and dihydroflavonol in NILs with w1 or w4 were largely similar to the NILs with purple flower suggesting that W1 and W4 affect only anthocyanin biosynthesis. Amount of flavonol glycosides was substantially reduced and dihydroflavonol was increased in Harosoy-wm suggesting that Wm is responsible for the production of flavonol from dihydroflavonol. The recessive wm allele reduces flavonol amount and inhibits co-pigmentation between anthocyanins and flavonols resulting in less bluer (magenta) flower color. Pubescence color genes, T or Td, had no apparent effect on flavonoid biosynthesis in flower petals.

An analysis of flavonoid compounds in leaves of Japonolirion (Petrosaviaceae).

Japonolirion, comprising Japonolirion osense Nakai, which occurs on serpentinite at two widely separated localities in Japan, has been considered as an isolated taxon, but more recently has been proved by molecular evidence to be a sister group to an achlorophyllous, mycoheterotrophic genus, Petrosavia. In an effort to research possible characters linking these groups, we analyzed the flavonoid compounds obtained from leaves of Japonolirion using UV spectra, mass spectrometry and 1H and 13C nuclear magnetic resonance, and acid hydrolysis of the original glycosides as well as direct thin layer chromatography and high performance liquid chromatography comparisons with authentic specimens. As a result, we identified seven flavonoids, of which two were major components identified as 6-C-glucosylquercetin 3-O-glucoside and isoorientin. The remaining five were minor components identified as 6-C-glucosylkaempferol 3-O-glucoside, quercetin 3-O-glucoside, quercetin 3-O-arabinoside, vicenin-2 and orientin. Both 6-C-glucosylquercetin 3-O-glucoside and 6-C-glucosylkaempferol 3-O-glucoside were recorded for the first time in nature. Because of their restricted occurrence in angiosperms, both C-glycosylflavonols and 3-O-glycosides of C-glycosylflavonols may be significant chemical markers for assessing relationships of J. osense.

Monoterpenoids and their glycosides from the leaf of thyme.

From the polar portion of the methanol extract of thyme (leaf of Thymus vulgaris; Labiatae), which has been used as an important stomachic, carminative, a component of prepared cough tea, and a spice, seven monoterpenoid glycosides were isolated together with two known monoterpenoids and three known monoterpenoid glucosides. Structures of the seven monoterpenoid glycosides were determined by spectral analysis.