Isolation of Nematicidal Polyacetylenes from Carthamus tinctorius L.

Two nematicidal polyacetylenes, 3-cis,11-trans- and 3-trans,11-trans-trideca-1,3,11-triene-5,7,9-triyne, were isolated from flowers of Carthamus tinctorius L., by column chromatography and high speed liquid chromatography under the dark condition.

The nematicidal activities of 3-cis,11-trans- and 3-trans,11-trans-isomer to Aphelenchoides besseyi were 80% at 10 ppm and 95% at 1 ppm, respectively.     

不同产地红花的挥发油成分

不同产地红花的挥发油成分郭美丽张汉明张芝玉苏中武（第二军医大学药学院,上海２００４３３）Ｔｈｅｃｏｎｓｔｉｔｕｅｎｔｓｏｆｅｓｅｎｔｉａｌｏｉｌｆｒｏｍｓａｆｆｌｏｗｅｒ（ＣａｒｔｈａｍｕｓｔｉｎｃｔｏｒｉｕｓＬ．）ｉｎｄｉｆｆｅｒｅｎｔｌｏｃａｌｉ...     

Adaptation of safflower genotypes Carthamus tinctorius L.

Summary Adaptation reactions of 33 genotypes of safflower Carthamus tinctorius L. were studied under 7 different climatic conditions. The genotpyes were divided into two sets. Set I consisted of 15 genotpyes selected from the local populations. Set II had 15 introduced and local varieties. Three control genotpyes, Ute, Ferio, and Local Arak, were common to both sets.Genotpye-environment interaction was not significant for Set I but it was highly significant for Set II. Three environmental indices were obtained and used in the adaptation analyses of the genotpyes in Set II. One of the environmental indices, designated EI, was dependent on the genotypes of Set II. The other two indices, designated EI-1 and EI-2, were independent of the genotpyes of Set II. The methods of Eberhart and Russell (1966) were used in analyses of adaptation by Index EI and the methods of Freeman and Perkins (1971) for Indices EI-1 and EI-2.The mean square associated with genotype-environment interaction was partitioned into two components, heterogeneity of regression and its residual, under EI-1 and EI-2. Both components were highly significant for both cases. However, the mean square of heterogeneity of regression was equal to its residual under EI-1 and even smaller than its residual under EI-2. These observations indicate that a major part of genotpye-environment interaction can not be accounted for by differences in the regressions of the individual genotypes. As well as this overall test, individual regression analyses for single genotypes were also considered. None of the genotypes had significant regression mean square under EI-1. Only two introduced genotypes had significant regression mean squares when EI-2 was used. The overall test of equality of the slopes of the regression for the genotypes of Set II was rejected at the 1% level under EI. This test indicated that genotypes of Set II were significantly different in their association with the EI. The significant differences among the genotypes of Set II were also shown by an F test of the pooled deviation mean square divided by the pooled error mean square. Individual regression analyses for single genotpyes of Set II were considered under EI. Mixed adaptation reactions were observed for different genotypes. Among 18 genotypes of Set II, regression mean squares were significant for only 10 genotpyes. Therefore, it appeared that the dependent environmental indices are more useful than the independent environmental indices when statistical theory of regression is used in the analysis of adaptation. Observations in the present study were not in agreement with the hypothesis that the relation between the performance of different genotypes in the various environments and some measure of these environments is linear or nearly so.Among the 12 introduced genotypes, only one, Ute, was identified as stable and high-yielding. Among the 15 selected from the locally adapted populations, eleven did not differ significantly from Ute in mean yield but four exceeded Ute significantly in mean productivity. The present study thus indicates that the Iranian safflower breeding project has been successful in identifying genotpyes which give high and stable yields under diverse environmental conditions. It does not indicate that introduced and exotic germplasms are unimportant in the breeding projects; it is quite possible that still more desirable genotypes can be developed by incorporating introduced genetic variability into the local germplasm.Associate Professors and Plant Breeder     

Dysregulation of KSHV replication by extracts from Carthamus tinctorius L.

Carthamus tinctorius L. (CT) is traditionally used to reduce ailments from diseases of the musculoskeletal system and connective tissue and diseases of blood circulation and the cardiovascular system. Flower extracts from CT are known to have antibacterial activity, anti-inflammatory activity, and to inhibit tumor promotion in mouse skin carcinogenesis. In order to discover new antiviral agents from CT extracts, we tested whether CT extracts contain antiviral activity against gammaherpesvirus infection. This study demonstrated that treatment with CT extracts disrupted KSHV latency in the viral-infected host cells, iSLK-BAC16. n-Hexane and EtOH fractions of CT extracts critically affected at least two stages of the KHSV life-cycle by abnormally inducing KSHV lytic reactivation and by severely preventing KSHV virion release from the viral host cells. In addition to the effects on KSHV itself, CT extract treatments induced cellular modifications by dysregulating cell-cycle and producing strong cytotoxicity. This study demonstrated for the first time that CT extracts have antiviral activities that could be applied to development of new anti-gammaherpesviral agents.     

Polyacetylenes from Immature Seeds of Safflower (Carthamus tinctorius L.)

Six polyacetylenes have been isolated from immature seeds of safflower (Carthamus tinctorius L.) by thin-layer chromatography. They were identified as 1,11-tridecadiene-3,5,7,9-tetrayne, 1,3,11-tridecatriene-5,7,9-triyne, 1,3,5,11-tridecatetraene-7,9-diyne, 1-tridecene-3,5,7,9,11-pentayne, 1,3-tridecadiene-5,7,9,11-tetrayne and 1,3,5-tridecatriene-7,9,11-triyne from the results of their spectroscopic and chemical analyses. Three of these polyacetylenes had not been isolated from Carthamus tinctorius L.

Changes in the polyacetylene content during maturation were followed by the measurement of ultraviolet absorbance. While 1,3,11-tridecatriene-5,7,9-triyne and 1,3,5,11-tridecatetraene-7,9-diyne had already occurred abundantly at the day of flowering, the amounts of the other polyacetylenes reached the maximum values at the fourth to sixth days after flowering. Although the total amounts of the six polyacetylenes were about 0.8 mmole/g lipid at the fourth day after flowering, no polyacetylene was detected in the mature seeds.     

Biointeraction between precarthamin and cell components in florets ofCarthamus tinctorius L.

The relative rate of precarthamin extractability and floret protein inactivation by organic solvents were compared. Upon trituration of the floral tissues ofCarthamus tinctorius L., usually less extractable precarthamin is released at a markedly high level, while the releasability rate is changed conspicuously by macerating the florets in the test solvents of increased concentrations; the amount of releasable precarthamin also varies according to the solvents used. 30% (v/v) acetone promotes the pigment solubility about twice, whereas, its capacity decreases abruptly in reverse proportion to the increment of acetone content. Methanol accelerates the precarthamin release, but the rate is very low. Ethanol acts in far lesser extent. The data indicate that these varied aspects are more or less attributed to the inactivation of possible factors dissociating precarthamin from cellular components in freezed safflower florets.     

Proliferation and differentiation from endosperms of Carthamus tinctorius

The endosperms of Carthamus tinctorius cv. HUS-305, excised at globular to heart-shaped stages of zygotic embryo development, were cultured on Murashige and Skoog’s medium (MS) supplemented with different concentrations of 6-benzylaminopurine (BAP), kinetin, thidiazuron (TDZ), 2,4-dichlorophenoxyacetic acid (2,4-D) or α-naphthalene-acetic acid (NAA). The highest incidence of callusing was on 2,4-D supplemented media. However, embryos differentiated only from the calli developed on media supplemented with BAP, kinetin or TDZ with the last eliciting maximum embryogenic response. The addition of a reduced nitrogen source, casein hydrolysate to MS medium supplemented with BAP and/or NAA, did not stimulate the response. However, adenine sulphate (100 mg dm⁻³) promoted the induction of somatic embryos. Upon transfer to MS basal medium or the same supplemented with 0.61 μM gibberellic acid (GA₃), plumular poles of few embryos elongated resulting in the development of shoots.     

EMS-induced cytomictic variability in safflower (Carthamus tinctorius L.)

Seeds of safflower (Carthamus tinctorius L.) were subjected to three treatment durations (3h, 5h and 7h) of 0.5 % Ethyl Methane Sulphonate (EMS). Microsporogenesis was carried out in the control as well as in the treated materials. EMS treated plants showed interesting feature of partial inter-meiocyte chromatin migration through channel formation, beak formation or direct cell fusion. Another interesting feature noticed during the study was the fusion among tetrads due to wall dissolution. The phenomenon of cytomixis was recorded at nearly all the stages of microsporogenesis connecting from a few to several meiocytes. Other abnormalities such as laggards, precocious movement, bridge and non-disjunction of chromosomes were also recorded but in very low frequencies. The phenomenon of cytomixis increased along with the increase in treatment duration of EMS. Cells with these types of cytomictic disturbances may probably result in uneven formation of gametes or zygote, heterogenous sized pollen grains or even loss of fertility in future.     

The enzymatic mobilization of bound precarthamine from the flower florets ofCarthamus tinctorius L.

Floret pieces prepared from the methanol-treated and air-dried flowers of safflower (Carthamus tinctorius L.) were subjected to an enzymatic hydrolysis in a buffered solution and the rate of precarthamine solubilization was assessed spectrophotometrically. The amount of precarthamine was risen by treating the floral pieces with a β-glucosidase (EC 3.2.1.21), supporting a previous assumption that precarthamine is in the floral tissues associated with cellular components. The enzyme activity was reduced by various inhibitors. On the basis of the experimental data, it is proposed that the precarthamine may be bound through 0-β-glucosyl linkage(s).     

Purification and characterization of precarthamin decarboxylase from the yellow petals of Carthamus tinctorius L

Carthamin, a red quinochalcone pigment in safflower (Carthamus tinctorius L.), is enzymatically converted from a yellow precursor, precarthamin. The enzyme, which catalyzes the oxidative decarboxylation of precarthamin to carthamin, was purified to apparent homogeneity from yellow petals of safflower and named precarthamin decarboxylase. The molecular mass of the denatured enzyme was estimated as 33 kDa by SDS-PAGE. The molecular mass of the native enzyme was determined by gel filtration chromatography to be 24 kDa; thus, the native enzyme is a monomer. The optimum pH of the enzyme was 5.0. The enzyme activity was inhibited by Mn2+, Fe2+, and Cu2+ and sharply decreased at temperatures higher than 50 degrees C for 10 min. The activation energy and the Arrhenius frequency factor of the enzyme reaction were 19.7 kcal mol(-1) and 9.94 x 10(11) s(-1), respectively. The saturation curve of precarthamin showed that the enzyme follows Michaelis-Menten kinetics. The Km and Vmax of the enzyme were calculated as 164 microM and 29.2 nmol/ min, respectively. The turnover number (kcat) of the enzyme was calculated as 1.42 x 10(2) s(-1). The enzyme activity was severely inhibited by reducing agents such as glutathione and DTT at pH 5.0, suggesting that a disulfide bond may play an important role in enzyme function.     

红花降糖活性成分谱效关系研究

红花为菊科植物红花(Carthamus tinctorius L.)的干燥花,具有活血通经,散瘀止痛等功能。为研究其降糖活性成分及谱效关系,本文确定了红花降糖活性部位;建立了HPLC指纹图谱,通过对照品指认其中7个主要成分后,用灰色关联度法和正交偏最小二乘法分析,揭示了这7个共有峰协同发挥蛋白酪氨酸磷酸酯酶1B抑制作用,其中羟基红花黄色素A(HSYA,6号峰)和6-羟基山柰酚-3,6-二-O-葡萄糖基-7-O-葡萄糖苷(3号峰)是贡献最大的成分。含量测定显示10批红花中羟基红花黄色素A及山柰素的含量均在1.67%~1.94%和0.09%~0.12%之间,该研究为红花在糖尿病药物开发领域的应用提供了依据。     

Studies of the delta 12 desaturase of Carthamus tinctorius L

The delta 12 desaturase of developing safflower seeds responsible for the conversion of an oleoyl moiety to the linoleoyl moiety of phospholipids was further characterized. The protein concentration of the microsomal preparation, the oleoyl-CoA concentration (the primary substrate), short incubation periods, and the addition of lysophospholipids must be controlled to obtain optimal desaturation. No evidence could be obtained to implicate cytochrome b5 as the intermediate electron carrier. Attempts to solubilize the desaturase with a variety of detergents and chaotropic reagents were not successful. Brief exposure of the microsomal preparation to trypsin resulted in rapid loss of activity. The overall evidence would suggest that the delta 12 desaturase requires a reductant (NADPH), a NADPH:electron carrier reductase, an electron carrier, a specific desaturase, and an acyltransferase with oleoyl-CoA as the substrate to acylate lysophospholipid to the active oleoyl phospholipids (presumably phosphatidylcholine or phosphatidylethanolamine). The complexity of this system suggests that purification of the components and a reassembling of the purified components will be difficult.     

Improvements in rooting regenerated safflower (Carthamus tinctorius L.) shoots

A continuing obstacle for regenerating safflower (Carthamus tinctorius L.) plants from cultured explants or callus has been a reliable method for rooting shoots. For shoots directly regenerated from primary explants, 76% of shoots rooted after a 7-d exposure to 10 mg/1 indole-3-butyric acid. Auxin source, concentration or exposure time did not greatly affect root formation or morphology, but strongly affected callus production. Shoots infected with Agrobacterium rhizogenes produced massive numbers of fibrous roots, but shoots did not elongate or survive transfer to soil. Shoot hyperhydricity symptoms were reduced by including 1 g/1 activated charcoal in rooting media. The optimal protocol for inducing root formation consisted of a 7-d exposure to 10 mg/l indole-3-butyric acid in root induction media, followed by incubation in media containing 15 g/l sucrose and 1 g/1 activated charcoal for 21 d.Abbreviations IBA indole-3-butyric acid - MS Murashige and Skoog (1962) medium - NAA anaphthalene acetic acid - POP 2,3,5-trichloro--phenoxypropionic acid     

Anther, pollen and tapetum development in safflower, Carthamus tinctorius L

In safflower, the anther wall at maturity consists of a single epidermis, an endothecium, a middle layer and the tapetum. The tapetum consists mainly of a single layer of cells. However, this single-layer appearance is punctuated by loci having ‘two-celled’ groupings due to additional periclinal divisions in some tapetal cells. Meiotic division in microsporocytes gives rise to tetrads of microspores. The primexine is formed around the protoplasts of microspores while they are still enveloped within the callose wall. Just prior to microgametogenesis, the microspores enlarge through the process of vacuolation, and the exine wall pattern becomes established. Microgametogenesis results in the formation of 3-celled pollen grains. The two elongated sperm cells appear to be connected. The exine wall is highly sculptured with a distinct tectum, columellae, a foot layer, an endexine and a thin intine. Similar to other members of the Asteraceae family, the tapetum is of the invasive type. The most novel finding of this study is that in addition to the presence of invasive tapetal cells, a small population of ‘non-invasive’ tapetal cells is also present. The tapetal cells next to the anther locules in direct contact with the microspores become invasive and start to grow into the space between developing microspores. These tapetal cells synthesize tryphine and eventually degenerate at the time of gametogenesis releasing their content into the anther locules. A smaller population of non-invasive tapetal cells is formed as a result of periclinal divisions at the time of tapetum differentiation. These cells are not exposed to the anther locules until the degeneration of the invasive tapetal cells. The non-invasive tapetal cells have a different cell fate as they synthesize pollenkitt. This material is responsible for allowing some pollen grains to adhere to each other and to the anther wall after anther dehiscence. This observation explains the out-crossing ability of Carthamus species and varieties in nature.     

应用RAMP分子标记研究红花资源遗传多样性

采用RAMP（Random amplified microsatellite polymorphism）对原产于42个国家的84份红花材料进行遗传多样性分析。结果表明,被测材料间RAMP标记多态性较高。16个引物组合所产生的122条DNA扩增片段中,有118条具有多态性,PPB为96.7%。PIC的变化范围为0.580～0.978,平均值0.874。每个引物可扩增出4～11个DNA片段,平均获得7.6个DNA片断,其中7.4个具有多态性,遗传相似系数（GS）的变化范围0.338～0.907,平均值为0.665。基于GS的聚类结果可以将所有84份材料完全分开,并划分为6类,聚类结果与材料的地理分布有一定关系,来源于亚洲和美洲的材料多样性相对比较丰富,所有来自中国的材料被聚为一大类。据此认为,红花种质资源在分子水平上确实存在较大遗传差异,RAMP分子标记是评价红花资源遗传多样性的有效方法。     

Inheritance of flower color and spininess in safflower (Carthamus tinctorius L.)

Safflower (Carthamus tinctorius L.) flowers are used for coloring and flavoring food and also as fresh-cut and dried flowers. The most important characteristics which contribute to the ornamental value of safflower are flower color and spinelessness. The objective of this study was to determine the inheritance mode and the number of genes controlling spininess and flower color in some Iranian genotypes of safflower. The results indicated that the existence of spines on the leaves and bracts of safflower is controlled by a single dominant gene in which the spiny phenotype was completely dominant to spineless. In some crosses, flower color was controlled by two epistatic loci each with two alleles, resulting in a ratio of 13:3 in the segregating F2 population for plants with orange and yellow flowers. Also, other mechanisms of genetic control, such as duplicate dominance and duplicate recessive types of epistasis, were observed for flower color in other crosses that led to ratios of 7:9 and 15:1 for plants with orange and yellow flowers, respectively. The results suggest that for ornamental use or in the food dying industry, genotypes with orange or yellow flowers and without spines on the leaves and bracts can be produced.     

Serotobenine,a Novel Phenolic Amide from Safflower Seeds (Carthamus tinctorius L.)

The structure of a new indole derivative, serotobenine (1), from sfflower meal (Carthamus tinctorius> L.) is proposed based on ¹H- and ¹³C-NMR spectral data and X-ray crystallography. The known compounds N-feruloyltryptamine (2) and N-(p-coumaroyl)tryptamine (3) were also isolated and identified.