18-nor-Podocarpanes and podocarpanes from the Bark of Taiwania cryptomerioides

Seven nor- and podocarpane-type diterpenes were isolated from the bark of Taiwania cryptomerioides Hayata, including three 18-nor-podocarpanes: 18-nor-1beta,4alpha,14-trihydroxy-13-methoxy-8,11,13-podocarpatriene (1), 18-nor-1beta,4alpha,13,14-tetrahydroxy-8,11,13-podocarpatrien-7-one (2), 18-nor-1beta,4alpha,14-trihydroxy-13-methoxy-8,11,13-podocarpatrien-7-one (3), 1beta,14,19-trihydroxy-13-methoxy-8,11,13-podocarpatrien-7-one (4), 1beta,13,14,18-tetrahydroxy-8,11,13-podocarpatrien-7-one (5), 18-acetoxy-1beta,13,14-trihydroxy-8,11,13-podocarpatrien-7-one (6), and 1beta,14,18-trihydroxy-13-methoxy-8,11,13-podocarpatrien-7-one (7). Their structures were determined by application of 1D and 2D NMR spectroscopy and other techniques. Podocarpane-type diterpenes do not occur extensively in nature, and the presumed oxidative enzyme in this plant will be of interest to identify.     

Chemical Constituents from the Whole Plant of Gaultheria itoana Hayata

Two new diterpenoids, 14,18‐dihydroxyabieta‐8,11,13‐trien‐7‐one ( 1 ) and 13‐acetyl‐14,18‐dihydroxy‐podocarpa‐8,11,13‐triene ( 2 ), together with eight known compounds, i.e., gaultheric acid ( 3 ), vanillic acid ( 4 ), 4‐hydroxybenzoic acid ( 5 ), cinnamic acid ( 6 ), stearic acid ( 7 ), palmitic acid ( 8 ), β‐sitosterol ( 9 ), and stigmasterol ( 10 ), were isolated from the MeOH extract of the whole plant of Gaultheria itoana Hayata (Ericaceae). The structures of the new constituents were elucidated by spectroscopic methods (UV, IR, and 1D‐ and 2D‐NMR) and by mass spectrometry (HR‐ESI‐MS). Among them, 1 and 2 were demonstrated to exhibit significant cytotoxic activity against the LNCaP cell line.     

Diterpene constituents of leaves from Juniperus brevifolia

The dichloromethane extract from leaves of Juniperus brevifolia, through chromatographic fractionations yield six compounds: 3beta-hydroxy-abieta-8,11,13-trien-7-one, 18-hydroxy-sandaracopimara-8(14),15-dien-7-one, sandaracopimara-8(14),15-dien-18-yl formate; and the first examples of sandaracopimaranes and abieta-8,11,13-triene diterpenoids with a large aliphatic chain on C-18, abieta-8,11,13-trien-18-yl hexadecanoate, 7-oxoabieta-8,11,13-trien-18-yl hexadecanoate, sandaracopimara-8(14),15-dien-18-yl hexadecanoate. Moreover fifteen known compounds were also isolated, some of them for the first time identified on Juniperus genus. The compound abieta-8,11,13-trien-18-yl formate is reported for the first time as a natural product. All the structures were established by spectroscopic methods. 2D NMR techniques have allowed the revision of certain previously reported (13)C NMR assignments. Studies on the isolated new compounds showed those possessing a diterpenol ester of a long-chain fatty acid present lipophilicity very distinct from other diterpenoid compounds.     

贡蕉胚性细胞悬浮系的建立和植株再生

鲜食蕉品种的高度不育性和多倍性制约了用传统育种方法培育生产实践中所需的新品种 ,建立稳定的胚性细胞悬浮系是香蕉生物技术育种的前提。以目前国内尚未建立该体系的鲜食蕉品种贡蕉 (AA)未成熟雄花序的第 1～ 15位花梳为外植体 ,对胚性细胞悬浮系的建立和植株再生体系进行了优化。结果表明 ,5～ 6个月的培养后可获得分生小球体和浅黄色、松散易碎的胚性愈伤组织。 9μmol/L 2,4 D对外植体愈伤组织的诱导效果最好 ,诱导率为 40.96 % ,胚性愈伤组织诱导率可达7.45 % ,其中5.79%的胚性愈伤组织来源于第 6～12号位置的花梳。胚性愈伤组织悬浮培养后 ,通过 3个月的筛选和继代培养 ,可得到均质的胚性细胞悬浮系。该培养体系合适继代周期为 15d ,继代时合适的起始接种量为每 30mL培养基加 2mLPCVECS。培养 6个月的胚性细胞在体细胞胚诱导培养基中培养15d后可见到白色半透明体细胞胚的发生 ,体细胞胚诱导率为 2 80× 10³个 mLPCV。成熟体细胞胚的萌发率为 17 2 8% ,其中发育成正常的再生植株的百分率为 14 16 %。     

Anti-tumor promoting diterpenes from the stem bark of Thuja standishii (Cupressaceae).

Three new labdane-type diterpenoids, labda-8(17),13-dien-15,12R-olid-19-oic acid (1), 12S-hydroxylabda-8(17),13(16),14-trien-19-oic acid (2) and 13-ethoxylabda-8(17),11,14-trien-19-oic acid (3), along with known diterpenoids, trans-communic acid (4), totarol (5), 12-methoxyabieta-8,11,13-trien-11-ol (6), and 7 alpha,8 alpha-epoxy-6 alpha-hydroxyabieta-9(11),13-dien-12-one (7) were isolated from the stem bark of Thuja standishii. The structures of 1--3 were established by spectroscopic methods and chemical conversion. These compounds together with standishinal (8), 12-hydroxy-6,7-seco-abieta-8,11,13-trien-6,7-dial (9) and 6 alpha-hydroxysugiol (10) were tested for their inhibitory effects on Epstein--Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), as a test for potential cancer chemopreventive agents. Compound 10 showed strong inhibitory effect on EBV-EA induction (100% inhibition at 1000 mol ratio/TPA), and compounds 2 and 6 showed moderate inhibitory effects on EBV-EA induction. In addition, 15-oxolabda-8(17),11Z,13E-trien-19-oic acid (11) was found to exhibit the anti-tumor promoting activity in two-stage mouse skin carcinogenesis test using 7,12-dimethylbenz[a]anthracene and TPA.     

Initiation and culture of potato tuber callus tissue with picloram

Callus cultures of 7 potato cultivars were initiated from tuber tissue and maintained on Gelrite-solidified media with 1–20 M picloram as the only PGR. Ten M picloram was the optimal concentration for callus induction. By 4–6 weeks after explanting, there was sufficient callus produced for subculture to maintenance media which contained 1–20 M picloram as the only PGR. When grown in the dark at 25°C, subcultured callus typically increased 10-fold in wet weight in 4–5 weeks. The callus produced was friable and a light grey to cream color. Callus cultures were used to establish cell suspension cultures. Callus and cell suspension cultures have been maintained for over 2 years on the picloram containing media.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige-Skoog - NAA naphthaleneacetic acid - PGR plant growth regulator Research paper #9053 of the Idaho Agricultural Experiment Station.     

Molecular Cloning and Construction of agp Gene Deletion-mutant in Cyanobacterium Synechocystis sp. PCC 6803

Nine compounds were isolated from Elsholtzia blanda (Benth.) Benth. Their　structures were identified with spectral and chemical methods as follows: 5,6-dihydro-6-styry-2-pyrone (1), friedelin (2), 4-hydroxy-3-methoxystyrene (3), 5,2′-dimethoxy-6,7-methylene dioxyflavanone (4), 5-hydroxy-7-methoxy-6-O-［α- L -rhamnopyranosyl(1→2)-β- D -fucopyranosyl］ flavone glycoside (5), 5,5′-dihydroxy-7-acetoxyl-6,8,3″,3″-tetramethylpyran　(3′,4′)　flavone (6), 5,5′-dihydroxy-7-(α-methyl) butyroxyl-6,8,3″,3″-tetramethylpyran (3′,4′) flavone (7), 5,5′-dihydroxy-6,7-methylenedioxy-8,3″,3″-trimethylpyran　(3′,4′)　flavone (8), glucosyringic acid (9). Among them, 6, 7 and 8 are new compounds, named as sifanghaoine Ⅰ,Ⅱ and Ⅲ, respectively.     

Alkaloid production in Vernonia cinerea: Callus, cell suspension and root cultures

Fast-growing callus, cell suspension and root cultures of Vernonia cinerea, a medicinal plant, were analyzed for the presence of alkaloids. Callus and root cultures were established from young leaf explants in Murashige and Skoog (MS) basal media supplemented with combinations of auxins and cytokinins, whereas cell suspension cultures were established from callus cultures. Maximum biomass of callus, cell suspension and root cultures were obtained in the medium supplemented with 1 mg/L alpha-naphthaleneacetic acid (NAA) and 5 mg/L benzylaminopurine (BA), 1.0 mg/L NAA and 0.1 mg/L BA and 1.5 mg/L NAA, respectively. The 5-week-old callus cultures resulted in maximum biomass and alkaloid contents (750 microg/g). Cell suspension growth and alkaloid contents were maximal in 20-day-old cultures and alkaloid contents were 1.15 mg/g. A 0.2-g sample of root tissue regenerated in semi-solid medium upon transfer to liquid MS medium containing 1.5 mg/L NAA regenerated a maximum increase in biomass of 6.3-fold over a period of 5 weeks. The highest root growth and alkaloid contents of 2 mg/g dry weight were obtained in 5-week-old cultures. Maximum alkaloid contents were obtained in root cultures in vitro compared to all others including the alkaloid content of in vivo obtained with aerial parts and roots (800 microg/g and 1.2 mg/g dry weight, respectively) of V. cinerea.     

The 13C-NMR Features of Natural Taxane Diterpenoid Compounds

The paper reviewed the 13C-NMR features of natural taxane diterpenoids according to their carbon-skeleton types. In the 13C-NMR it is easy to distinguish the 6/8/6 and 5/7/6 membered rings by observation of the 13C-NMR data of C-1 and C-15. The remarkable differences of the resonance of C -13 and C-12 were found. In addition, based on various chemical environments man y obscured chemical shifts of carbons can be distinguished, such as the oxygenat ed tertiary carbons of C-2, C-5, C-7, C-9 and C-13, and sometimes between C-9 an d C-10, as well as between aliphatic quaternary carbons of C-8 and C-15, and sec ondary carbons of C-6 and C-14. All the above-mentioned characteristics are helpful for structural elucidation and assignments of the carbon signals of taxoids.     

民族药四数九里香的化学成分及其细胞毒活性研究

为了阐明民族药四数九里香Murraya tetramera Huang的药效物质基础,课题组使用色谱技术从其醇提取物中分离得14个化合物;运用~1H NMR、¹³C NMR波谱方法和文献数据对比鉴定为正24烷酸(1)、9,13,17,21-tetramethyl-5-docosenoic acid(2)、3-O-β-D-吡喃葡萄糖基-山奈酚甙(3)、补骨脂素(4)、紫苏醛(5)、槲皮素(6)、methyl salicylate glucoside(7)、(9S,10R,11E,13R,15Z)-9,10,13-trihydroxyoctadeca-11,15-dienoic Acid(8)、2-isopropyl-5-methylphenol(9)、β-胡萝卜甘(10)、7-羟基香豆素(11)、七叶内酯(12)、β-谷甾醇醋酸酯(13)、山奈酚(14)。除了化合物4、6外,其他化合物为首次从该植物中分离得到。同时,研究各化合物对5株肿瘤细胞的体外生长抑制作用。与阳性对照组比较,结果表明:化合物3、4、5、6、7、8、10、11、12、13、14对白血病HL60显现良好的细胞毒活性;化合物1～14对肺腺癌A549的的细胞毒活性低于阳性对照组;化合物6、13、14对肝癌SMMC7721显现良好的细胞毒活性;化合物1、2、3、6、7、9、11、12对乳腺癌MCF7显现良好的细胞毒活性;化合物1～14对结肠癌SW480的细胞毒活性低于阳性对照组。     

Chemical Constituents from Roots of Jatropha curcas

Thirteen compounds were isolated from the roots of datropha curcas L. Combining the determination of physico-chemical constants and spectral analyses (IR, 1H-NMR, 13C-NMR, EIMS, FABMS), the structures of the compounds were identified as 5α-stigmastane-3, 6-dione (1), nobiletin (2), β-sitosterol (3), taraxerol (4), 2S-tetracosanoic acid glyceride-1(5),5-hydroxy-6,7-dimethoxycoumarin (6), jatropholone A (7), jatropholone B (8), 6-methoxy-7-hydroxycoumarin (9), caniojane (10), 3-hydroxy-4-methoxybenzaldehyde (11), 3-methoxy-4-hydroxybenzoic acid (12) and daucosterol (13). Among them, compound 5 is a new compound which has never been reported in China and abroad, compound 1, 2, 9, 10, 11, 12 were first time isolated from the plant, 7 and 8 are a pair of stereoisomers which can be inverted in dilute basic solution. 10 is a diterpenoid containing peroxide bridge.     

Chemical Analysis and Plant Sources of Maytansinoids

By high performance liquid chromatography (HPLC) and mass spectrometry (MS), maytansine, maytanprine and maytanbutine, three anticancer maytansinoids were successfully isolated and identified from ten species of plants belonging to Celastraceae. These plants are: Maytenus graciliramula, M. austroyunnanensis, M. orbiculata, M. hookeri Loes var. longiradiata, M. inflata, M. berberoides, M. esquirolii, M. variabilis, M. royleana and M. senegalensis. This result shows that the plant resources containing maytansinoids in China are rather aboundant.     

Specific and abundant secretion of a novel hydroxyproline-rich glycoprotein from salt-adapted winged bean cells

Winged bean callus was adapted to increasing concentrations of NaCl by sequential transfer to medium with 0, 0.5, 1.0, 1.5, and 2.0% (w/v) NaCl. When the culture media, after cell suspension cultures of callus adapted to 0.5 (SA-0.5), 1.0 (SA-1.0), 1.5 (SA-1.5), or 2.0% (w/v) NaCl (SA-2.0), were analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis, six specific or enhanced polypeptide bands (SAP1, -2, -3, -4, -5, and -6) were observed. SAP1, with a molecular weight of 84,000, was abundantly secreted in suspension cultures of SA-1.0 and SA-1.5, and was observed as the most striking polypeptide band. The SAP1 yield was about 4 mg/g cells fresh weight. SAP1 was abundantly secreted after the suspension culture of SA-1.0 in the presence of AlCl₃, but little was secreted in the presence of KCl, LiCl, CaCl₂, MgCl₂, mannitol, sucrose, or abscisic acid. SAP1 was purified from the culture medium after suspension culture of SA-1.0 in the presence of 1.0% (w/v) NaCl. Two steps, ammonium sulfate fractionation and CM-cellulose chromatography, were sufficient for purification to homogeneity. Finally, about 5 mg of SAP1 could be isolated from 7 g of fresh callus cells. Of the amino-terminal 32 amino acid residues of SAP1, 10 and 5 were found to be hydroxyproline and proline, respectively. SAP1 on an acrylamide gel was stained by the periodic acid-Schiff method. It is interesting that SAP1 has pentahydroxyproline blocks (Hyp₅) instead of tetrahydroxyproline blocks (Hyp₄) common to many hydroxyproline-rich glycoproteins in dicotyledons. Thus, this novel hydroxyproline-rich glycoprotein was shown to be abundantly secreted from NaCl-adapted winged bean cells.     

Efficient callus formation and plant regeneration of goosegrass [<Emphasis Type="Italic">Eleusine indica </Emphasis>(L.) Gaertn.]

Efficient methods in totipotent callus formation, cell suspension culture establishment and whole-plant regeneration have been developed for the goosegrass [ Eleusine indica (L.) Gaertn.] and its dinitroaniline-resistant biotypes. The optimum medium for inducing morphogenic calli consisted of N6 basal salts and B5 vitamins supplemented with 1-2 mg l(-1) 2,4-dichlorophenoxyacetic acid (2,4-D), 2 mg l(-1) glycine, 100 mg l(-1) asparagine, 100 mg l(-1) casein hydrolysate, 30 g l(-1) sucrose and 0.6% agar, pH 5.7. The presence of organogenic and embryogenic structures in these calli was histologically documented. Cell suspension cultures derived from young calli were established in a liquid medium with the same composition. Morphogenic structures of direct shoots and somatic embryos were grown into rooted plantlets on medium containing MS basal salts, B5 vitamins, 1 mg l(-1) kinetin (Kn) and 0.1 mg l(-1) indole-3-acetic acid (IAA), 3% sucrose, 0.6% agar, pH 5.7. Calli derived from the R-biotype of E. indica possessed a high resistance to trifluralin (dinitroaniline herbicide) and cross-resistance to a structurally non-related herbicide, amiprophosmethyl (phosphorothioamidate herbicide), as did the original resistant plants. Embryogenic cell suspension culture was a better source of E. indica protoplasts than callus or mesophyll tissue. The enzyme solution containing 1.5% cellulase Onozuka R-10, 0.5% driselase, 1% pectolyase Y-23, 0.5% hemicellulase and N(6) mineral salts with an additional 0.2 M KCl and 0.1 M CaCl(2) (pH 5.4-5.5) was used for protoplast isolation. The purified protoplasts were cultivated in KM8p liquid medium supplemented with 2 mg l(-1) 2,4-D and 0.2 mg l(-1) Kn.     

Synthesis of 7-substituted-5-androstene derivatives promoted by SmI2

The 7-substituted-5-androstene derivatives 2a-10a and 2b-10b were prepared by reaction of 3beta,17beta-di(tert-butyldimethylsilyloxy)-5-androsten-7-one 1 with different organic halides. The resulting 7alpha- and 7beta-isomers were carefully separated by column chromatography. The structural assignments of the 7alpha- and 7beta-isomers were determined by 13C-NMR.     

Biosynthesis of ribosome-inactivating proteins from callus and cell suspension cultures of Mirabilis expansa (Ruiz &Pavon)

 Callus and cell suspension cultures from the little known Andean crop Mirabilis expansa were developed and maintained on Murashige and Skoog medium supplemented with 2,4-dichlorophenoxyacetic acid (1 mg/l) and kinetin (0.1 mg/l). Callus and cell suspension cultures were screened with antibodies raised against ME1 (27.5 kDa) and ME2 (27 kDa), two ribosome-inactivating proteins (RIPs) previously found in roots of M. expansa. A 29-kDa protein found in callus and cell suspensions reacted strongly with ME1 antibodies. The 29-kDa protein, named MEC, was purified to homogeneity by ammonium sulfate precipitation and cation exchange perfusion chromatography. Amino acid N-terminal sequencing revealed close homology between MEC and ME1. The MEC amino acid sequence examined was highly conserved among RIPs from widely different sources. This new RIP was immunolocalized to the cell walls of callus and cell suspension cultures. Received: 20 August 1999 / Revision received: 10 December 1999 / Accepted: 21 December 1999     

Concerning the chemical synthesis of 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one, a novel regulator of cholesterol metabolism

A four-step synthesis of 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one (I) from 7-dehydrocholesterol is described. This synthesis, which is efficient and suitable for kilogram scale work, was carried out in a 33% overall average yield (39% overall best yield). A major byproduct of the hydrolysis of 3 beta-benzoyloxy-14 alpha,15 alpha-epoxy-5 alpha-cholest-7-ene to I was found to be the ring C aromatic sterol 12-methyl-18-nor-5 alpha-cholesta-8,11,13-trien-3 beta-ol. Several other intermediates and byproducts of these reactions were also identified. All new sterols were characterized by 1H- and 13C-NMR.     

Plant regeneration from protoplasts isolated from embryogenic suspension cultures of creeping bentgrass (Agrostis palustris Huds.)

A rapidly growing embryogenic suspension culture cell line of creeping bentgrass cv Penncross (Agrostis palustris Huds.) was established from callus derived from the culture of mature seeds. High concentrations of 2,4-D were required for the induction of callus (3 mg/1) as well as for the maintenance of the cell Une (2 mg/1) on modified B5 medium of Gamborg. Protoplasts isolated from the suspension cultures were successfully cultured in Murashige and Skoog's basal medium supplemented with only 0.1 mg/1 2,4-D. Although protoplast plating efficiency was rather low (0.36%), 30% of the protocalli formed normal green plants that were successfully established in soil.Abbreviations BA 6, benzylaminopurine - 2,4-D 2,4 dichlorophenoxyacetic acid - MES 2, (N-morpholino)-ethane sulfonic acid - MS Murashige and Skoog medium (1962) - B5 Gamborg medium (1968)     

NMR assignments of the major cannabinoids and cannabiflavonoids isolated from flowers of Cannabis sativa

The complete 1H- and 13C-NMR assignments of the major Cannabis constituents, delta9-tetrahydrocannabinol, tetrahydrocannabinolic acid, delta8-tetrahydrocannabinol, cannabigerol, cannabinol, cannabidiol, cannabidiolic acid, cannflavin A and cannflavin B have been determined on the basis of one- and two-dimensional NMR spectra including 1H- and 13C-NMR, 1H-1H-COSY, HMQC and HMBC. The substitution of carboxylic acid on the cannabinoid nucleus (as in tetrahydrocannabinolic acid and cannabidiolic acid) has a large effect on the chemical shift of H-1" of the C5 side chain and 2'-OH. It was also observed that carboxylic acid substitution reduces intermolecular hydrogen bonding resulting in a sharpening of the H-5' signal in cannabinolic acid in deuterated chloroform. The additional aromaticity of cannabinol causes the two angular methyl groups (H-8 and H-9) to show identical 1H-NMR shifts, which indicates that the two aromatic rings are in one plane in contrast to the other cannabinoids. For the cannabiflavonoids, the unambiguous assignments of C-3' and C-4' of cannflavin A and B were determined by HMBC spectra.     

思茅松松香中的一个新二萜

从思茅松（Pinus kesiya var.langbianensis）的松香中分离得到一个新奇的松香烷二萜化合物——思茅松素,经现代波谱分析将其化学结构确定为13（14）-烯-8,12-环氧-1-松香酸（1）,同时分离得到5个已知化合物,分别为abiet-8,11,13-trien-15-hydroxy-18-oic acid（2）;pimarol（3a）;iso-pimarol（3b）;abiet-trien-18．oic acid（4）;15．hydroxy abietic acid（5）。