崇左金花茶花朵和叶片类黄酮UPLC-Q-TOF-MS分析

以崇左金花茶（Camellia chuangtsoensis）为材料,利用超高效液相色谱-四极杆-飞行时间质谱（UPLC-Q-TOF-MS）联用技术定性定量分析其花朵（花瓣、雄蕊）和叶片（老叶、新叶）中类黄酮成分与含量。结果表明,崇左金花茶中共检测到14种类黄酮成分,木犀草素、木犀草素-7-O-芸香糖苷、槲皮素-3,7-O-二葡萄糖苷、芸香柚皮苷、圣草素和染料木苷为山茶属金花茶组植物中首次发现,其中槲皮素-3,7-O-二葡萄糖苷、芸香柚皮苷、圣草素和染料木苷主要存在于花朵中,木犀草素和木犀草素-7-O-芸香糖苷在花朵中含量高于叶片,雄蕊中高于花瓣;槲皮素-3-O-葡萄糖苷、槲皮素-7-O-葡萄糖苷、槲皮素-3-O-芸香糖苷和山柰酚-3-O-葡萄糖苷为金花茶组植物叶片中首次发现,其叶片中含量远低于花朵,老叶中远低于新叶,雄蕊中远低于花瓣;儿茶素和表儿茶素在花朵中含量高于叶片,雄蕊中高于花瓣;槲皮素和山萘酚在花朵和叶片中含量均较低。崇左金花茶花瓣和雄蕊中含量较高的类黄酮为儿茶素类、木犀草素类和槲皮素类,主要是表儿茶素、木犀草素和槲皮素-3-O-葡萄糖苷;叶片中为儿茶素类和木犀草素类,主要是表儿茶素、木犀草素和木犀草素-7-O-芸香糖苷。崇左金花茶花瓣和雄蕊中儿茶素类、木犀草素类及类黄酮总量均高于叶片,且雄蕊高于花瓣;花瓣和雄蕊中槲皮素类远高于叶片,且花瓣中远高于雄蕊。     

三种山茶属金花茶组植物花朵类黄酮成分研究

该研究以山茶属金花茶组的金花茶、凹脉金花茶和崇左金花茶为材料,利用超高效液相色谱-四极杆-飞行时间质谱联用技术定性定量分析其花朵中类黄酮成分与含量。结果表明:三种植物中检测到15种类黄酮,其中天竺葵素-3-O-葡萄糖苷、木犀草素、木犀草素-7-O-芸香糖苷、槲皮素-3,7-O-二葡萄糖苷、芸香柚皮苷、圣草素和染料木苷为金花茶组首次发现;槲皮素-3-O-葡萄糖苷、槲皮素-7-O-葡萄糖苷、槲皮素-3-O-芸香糖苷和山萘酚-3-O-葡萄糖苷为凹脉金花茶和崇左金花茶中首次发现。儿茶素、表儿茶素、槲皮素-3-O-葡萄糖苷、槲皮素-7-O-葡萄糖苷、槲皮素-3-O-芸香糖苷和山萘酚-3-O-葡萄糖苷为三个物种主体成分;天竺葵素-3-O-葡萄糖苷为金花茶特有,槲皮素-3,7-O-二葡萄糖苷为崇左金花茶特有;木犀草素-7-O-芸香糖苷主要存在于金花茶和崇左金花茶;木犀草素主要存在于凹脉金花茶和崇左金花茶。类黄酮类型主要为儿茶素类、槲皮素类、木犀草素类和山萘酚类;崇左金花茶中槲皮素类、木犀草素类及类黄酮总量远高于金花茶和凹脉金花茶,凹脉金花茶和崇左金花茶儿茶素类高于金花茶,金花茶和崇左金花茶山萘酚类高于凹脉金花茶。     

叶下珠中黄酮类化学成分及其生物活性

利用色谱分离方法与现代波谱分析技术,对叶下珠乙酸乙酯提取物的化学成分进行了系统的研究。从中分离鉴定了14个黄酮类化合物,分别为槲皮素-3-(4"-O-乙酰基)-O-α-L-鼠李糖-7-O-α-L-鼠李糖苷(1)、槲皮素-7-O-α-L-鼠李糖苷(2)、槲皮素-3-O-α-L-鼠李糖苷(3)、槲皮素-3-O-β-D-葡萄糖苷(4)、芸香甙(5)、槲皮素(6)、山柰酚(7)、山柰酚-3-O-α-L-鼠李糖苷(8)、木犀草素(9)、木犀草素-7-O-β-D-葡萄糖苷(10)、蒙花苷(11)、山柰酚-3-O-β-芸香糖苷(12)、柚皮苷(13)、橙皮苷(14)。除5~7外的所有化合物均从该植物中首次分离。对分离所得的化合物进行了体外细胞毒和抗氧化活性测试,结果显示化合物6、9和10表现出一定的细胞毒活性,所有化合物均具有不同程度的抗氧化活性。     

香樟叶化学成分的研究

采用硅胶色谱柱层析、聚酰胺色谱柱层析、制备薄层色谱及其它分离手段从香樟叶(Cinnamomum camphora Leaves)70%乙醇提取中分离得到9个化合物,根据化合物理化性质和波谱数据分别鉴定为:邻苯二甲酸二丁酯(1)、l-细辛脂素(2)、(8R,8'R)-3,3',4,4'-四甲氧基-9-氧代-8-8',9-O-9'-木脂素(3)、槲皮素-3-Ο-β-D-葡萄糖苷(4)、山奈酚-3-O-β-芸香糖苷(5)、山柰酚-3-O-α-L-吡喃鼠李糖苷(6)、槲皮素3-Ο-α-L-鼠李糖苷(7)、芦丁(8)、异鼠李素-3-O-β-芸香糖苷(9)。其中化合物1,2,6和9为首次从该种植物叶中分离得到。     

幌伞枫叶的化学成分研究

为了解幌伞枫(Heteropanax fragrans)的药理活性化学基础,从其叶的乙醇提取物中分离得到8个化合物,经波谱分析分别鉴定为:(7S,8R)-蛇菰脂醛素-4-O-β-D-吡喃葡萄糖苷(1)、4β,10α-香木兰烷二醇(2)、原儿茶酸(3)、3′-甲氧基-槲皮素-3-O-β-D-吡喃葡萄糖苷(4)、山柰酚-3-O-β-D-吡喃葡萄糖苷(5)、槲皮素-3-O-β-D-吡喃葡萄糖苷(6)、槲皮素-3-O-β-D-芸香糖苷(7)和山柰酚-3-O-β-D-芸香糖苷(8)。这8个化合物均为首次从幌伞枫中分离得到。     

矮生胡枝子的化学成分研究CSCD

研究植物矮生胡枝子(Lespedeza forrestii)全株的化学成分。采用Sephadex LH-20和反相制备型HPLC等方法分离与纯化,运用NMR、MS等波谱技术鉴定化合物结构。从矮生胡枝子的甲醇提取物中分离得到10个化合物,分别鉴定为山柰酚-3-O-α-L-鼠李糖苷(1)、槲皮素-3-O-α-L-鼠李糖苷(2)、山柰酚-3-O-芸香糖苷(3)、槲皮素-3-O-芸香糖苷(4)、山柰酚-3-O-洋槐糖苷(5)、牡荆素(6)、荭草素(7)、维采宁-2(8)、染料木素(9)和dalbergioidin(10),均为首次从矮生胡枝子中分离得到。对化合物1~10在小鼠巨噬细胞RAW 264.7细胞株上进行抗炎活性的筛选显示,在20μmol/L浓度下化合物8和10分别对IL-6和IL-1β的mRNA表达水平具有抑制活性,显示一定的抗炎作用。     

钝裂银莲花主要色素成分及适应机制的研究

为确定调控花色深浅的主要色素成分及相关合成酶,本文以钝裂银莲花(Anemone obtusiloba)为材料,用紫外-可见光谱扫描法、高效液相色谱法和液质联用法(HPLC-MS)对不同花色花色素进行研究。结果显示:钝裂银莲花含类胡萝卜素和类黄酮两种色素,两者总吸光值均随花色深浅规律变化,但类黄酮随花色变浅吸光度变化更明显,通过HPLC-MS分析,确定了7种黄酮类化合物:木犀草素-3-7-O-葡萄糖苷、槲皮素-3-O-芸香糖苷、山奈酚-3-O-槐糖苷、杨梅黄素-3-O-鼠李糖、槲皮素-3-O-半乳糖苷、槲皮素-3-O-葡萄糖苷、槲皮素-3-O-鼠李糖苷。槲皮素的衍生物为引起花色差异的主要色素,催化其合成的类黄酮3’-羟化酶为关键酶。推测钝裂银莲花花色适应环境的机制是通过类黄酮3’-羟化酶来调控花色深浅。     

五种金花茶组植物类黄酮成分及其与花色关系

以金花茶、小果金花茶、扶绥金花茶、龙州金花茶和陇瑞金花茶等五种金花茶组植物为试验材料,按照CIE L*a*b*表色系法测量其花色,利用超高效液相色谱-四极杆-飞行时间质谱(UPLC-Q-TOF-MS)联用技术定性定量分析其花中类黄酮成分与含量,运用多元线性回归方法研究花色与类黄酮成分之间的关系。结果表明:5种金花茶组植物花中共检测到8种类黄酮成分,其中天竺葵素-3-O-葡萄糖苷(Pg3G)、木犀草素-7-O-芸香糖苷(Lu7R)、芸香柚皮苷和圣草素为金花茶组植物中首次发现;槲皮素-3-O-葡萄糖苷(Qu3G)、槲皮素-7-O-葡萄糖苷(Qu7G)、槲皮素-3-O-芸香糖苷(Qu3R)和山柰酚-3-O-葡萄糖苷(Km3G)为扶绥金花茶和小果金花茶中首次发现;金花茶花中类黄酮成分总量最高,其次是扶绥金花茶和小果金花茶,陇瑞金花茶和龙州金花茶较低;金花茶和小果金花茶主要类黄酮成分为Qu3G、Qu3R和Pg3G,扶绥金花茶为Qu3G和Qu7G,陇瑞金花茶和龙州金花茶为圣草素和芸香柚皮苷; Qu3G和Qu3R是决定金花茶组植物花瓣呈现黄色的主要成分,圣草素与花瓣红晕显著正相关,Pg3G影响花色鲜艳程度。     

长春花萜类吲哚生物碱含量测定及相关基因的表达分析

采用反相高效液相色谱法(RP-HPLC)对23个长春花品种中的重要萜类吲哚生物碱文多灵、长春质碱和长春碱含量进行了测定,发现这3种生物碱的含量在不同品种中存在较大差异。相关性分析表明,文多灵、长春质碱和长春碱这3种生物碱中,文多灵含量和长春碱含量有极显著的相关性(P0.01)。利用RT-PCR分析了香叶醇10-脱羧酶基因(g10h)和异胡豆苷合成酶基因(str)在萜类吲哚生物碱含量有显著差异的品种之间的表达水平差异,并结合生物碱含量数据结果,发现g10h和str的表达水平和文多灵和长春质碱的总含量有显著的正相关性(P0.05),说明g10h和str基因可以作为长春花中文多灵和长春质碱含量的参考基因标记。该研究对为选育高萜类吲哚生物碱含量长春花品种及长春花萜类吲哚生物碱代谢工程具有重要意义。     

樟树叶化学成分的研究

本文研究了樟树叶的化学成分。主要采用硅胶、Sephadex LH-20凝胶和聚酰胺等柱层析方法对樟树叶的化学成分进行分离纯化,并根据理化性质、波谱数据(1H NMR,13C NMR)鉴定化合物的结构。从樟树叶的甲醇提取物中分离鉴定出9个化合物,分别为山柰酚-3-O-β-D-葡萄糖苷(1)、槲皮素-3-O-β-D-鼠李糖苷(2)、槲皮素-3-O-β-D-葡萄糖苷(3)、异鼠李素-3-O-β-D-葡萄糖苷(4)、黑色五味子单体苷(5)、山奈酚-3-O-β-D-芸香糖苷(6)、异鼠李素-3-O-β-D-芸香糖苷(7)、新芝麻脂素(8)和maculatin(9)。化合物1为首次从樟属中分离得到,化合物4、5、8为首次从樟科植物中分离得到。     

C-prolinylquercetins from the yellow cocoon shell of the silkworm, Bombyx mori

Two flavonoids containing the l-proline moiety, 6-C-[(2S,5S)-prolin-5-yl] quercetin (prolinalin A) and 6-C-[(2S,5R)-prolin-5-yl] quercetin (prolinalin B), were isolated from the cocoon shell of the silkworm, Bombyx mori. Their structural elucidation was achieved by application of acid hydrolysis and spectroscopic methods. These compounds were not found in the leaves of mulberry (Morus alba L.), the host plant of the silkworm, suggesting that the flavonoids are metabolites of the insect. This is the first time that flavonoids with an amino acid moiety have been found as naturally occurring compounds.     

Purification and Identification of Flavonoids from the Yellow Green Cocoon Shell (Sasamayu) of the Silkworm,Bombyx mori

Three quercetin glycosides, quercetin 5-O-β-D-glucoside, quercetin 7-O-β-D-glucoside, and quercetin 4′-O-β-D-glucoside, and two kaempferol glycosides, kaempferol 5-O-β-D-glucoside and kaempferol 7-O-β-D-glucoside, along with their aglycones, quercetin and kaempferol, were isolated from an ethanolic extract of Sasamayu cocoon shells. The chemical structures were characterized by chemical and spectroscopic methods including UV spectrometry and HPLC-ESI-MS. The five flavonol glycosides of the shell are different structurally from those of the leaves of mulberry (Morus alba). It was suggested that potent antioxidative activity in the cocoon is mainly due to flavonoid compounds since free radical scavenging activity was found in the cocoon flavonoids identified here.     

Microsatellite markers application on domesticated silkworm and wild silkworm

Abstract Twenty-seven sets of simple sequence repeat (SSR) primers were developed through hybridizing of (CA)n, (CT)n and (GT)n and sequencing the positive clones in libraries constructed by using p50 silkworm strain. Of those primer pairs, 26 sets of SSR primers amplified well in two regional wild silkworm strains. Ten domesticated silkworm strains and two regional wild silkworm strains were used for comparing the polymorphisms and for constructing a phylogenetic tree employing the UPGAM method. The result showed that the genetic distances within Japanese strains are closer than those of Chinese strains. And this result also implies that Japanese strains diverged from domesticated silkworm later than Chinese strains. According to the clustering result, the domesticated silkworm is firstly clustered in one class, but could be classified into two groups. Within a strain, the individual polymorphism of wild silkworm was significantly higher in abundance than those of domesticated silkworm. The S SR primers of domesticated silkworm could be used in genetic studies for wild silkworm.     

Flavonoid 5-glucosides from the cocoon shell of the silkworm, Bombyx mori

The flavonoid 5-glucosides, quercetin 5,4'-di-O-beta-D-glucopyranoside and quercetin 5,7,4'-tri-O-beta-D-glucopyranoside, together with the known quercetin 5-O-beta-D-glucopyranoside, were isolated from the cocoon shell of the silkworm, Bombyx mori. The structures were identified by spectroscopic analysis. These flavonoid glucosides were not present in mulberry leaves, the silkworm's only food, and they are considered to be metabolites produced by the silkworm.     

Purification and identification of flavonoids from the yellow green cocoon shell (Sasamayu) of the silkworm,Bombyx mori

Three quercetin glycosides, quercetin 5-O-beta-D-glucoside, quercetin 7-O-beta-D-glucoside, and quercetin 4'-O-beta-D-glucoside, and two kaempferol glycosides, kaempferol 5-O-beta-D-glucoside and kaempferol 7-O-beta-D-glucoside, along with their aglycones, quercetin and kaempferol, were isolated from an ethanolic extract of Sasamayu cocoon shells. The chemical structures were characterized by chemical and spectroscopic methods including UV spectrometry and HPLC-ESI-MS. The five flavonol glycosides of the shell are different structurally from those of the leaves of mulberry (Morus alba). It was suggested that potent antioxidative activity in the cocoon is mainly due to flavonoid compounds since free radical scavenging activity was found in the cocoon flavonoids identified here.     

An integrated database for the enhanced identification of silkworm gene resources

The National Academy of Agricultural Science (NAAS) has developed a web-based database to provide characterization information in silkworm. The silkworm database has four major function menus: variety searching, characterization viewing, general information and photo gallery. It provides 321 silkworm varieties characterization information for six different regions namely, Korean, Japanese, Chinese, European, Tropical and non-classified group. Additionally, the database provides 1,132 photo images regarding life cycle of various silkworm varieties. A specific characterization information table provides accession number, variety, strain and larval marking, blood color, cocoon color, cocoon shape, egg colors, remarks and image table provides photos which consist of shape and color in the different stages of larval, egg and cocoon stages. AVAILABILITY: The database is available for free at http://www.naas.go.kr/silkworm/english/     

Purification and characterization of cocoonase from the silkworm Bombyx mori

Cocoonase (CCN) which facilitates the degradation of a cocoon is recognized as a trypsin-like serine protease. In this study, CCN from the silkworm Bombyx mori was purified and comprehensively characterized. Its activity was maximal at about pH 9.8. It was stable above pH 3.4 at 4?°C and below 50?°C at pH 7.5. CuSO₄, FeSO₄, and ZnSO₄ showed inhibitory effects on CCN, but other salts improved activity. Typical trypsin inhibitors inhibited CCN, but the relative inhibitory activities were much lower than those against bovine trypsin. An extract of cocoon shells inhibited trypsin, but it was only slightly inhibitory against CCN. There were significant differences in catalytic efficiencies and substrate specificities as between CCN and bovine trypsin.     

SSR based linkage and mapping analysis of C,a yellow cocoon gene in the silkworm,Bombyx mori

The yellow color of the cocoon of the silkworm Bombyx mori is controlled by three genes, Y (Yellow haemolymph), I (Yellow inhibitor) and C (Outer‐layer yellow cocoon), which are located on linkage groups 2, 9 and 12, respectively. Taking advantage of a lack of crossing over in females, reciprocal backcrossed F₁ (BC₁) progeny were used for linkage analysis and mapping of the C gene using silkworm strains C108 and KY, which spin white and yellow cocoons, respectively. DNA was extracted from individual pupae and analyzed for simple sequence repeat (SSR) markers. The C gene was found to be linked to seven SSR markers. All the yellow cocoon individuals from a female heterozygous backcross (BC₁ F) showed a heterozygous profile for SSR markers on linkage group 12, whereas individuals with light yellow cocoons showed the homozygous profile of the strain C108. Using a reciprocal heterozygous male backcross (BC₁ M), we constructed a linkage map of 36.4 cM with the C gene located at the distal end, and the closest SSR marker at a distance of 13.9 cM.     

Ecdysteroid as a mediator in the regulation of silk protein synthesis and its influence on silkworm (Bom., Lepidoptera) genome

Abstract:  Ecdysteroid is one of the two most important insect hormones, which controls growth and developmental activities of insect. In silkworm, Bombyx mori L., basically ecdysteroid induces molting and metamorphosis in larvae and exogenous administration hastens the silk synthetic activity and cocoon spinning process but it strictly depends on the time of administration. In the present study, we administered phytogenous ecdysteroid to the 48-h-old 5th instar silkworm, B. mori at a single dose of 2  μ g per larva to study its effects on the larval growth, cocoon and silk variables. The possible role of ecdysteroid in altering the developmental sequence of silkworm and thereby its effect on cocoon and silk production at molecular level have been tried to get elucidated. The genomic DNA was isolated from the posterior silk gland on day 5 and 7 of the 5th instar from the ecdysteroid treated and the control larvae and was randomly amplified with arbitrary primers. The result presented notable variation in the amplified product suggesting the participation of ecdysteroid in regulating the silk gene. The feeding period of treated larvae was unaffected while the cocoon characters exhibited considerable improvement. The filament traits also were improved notably in the treated larvae. The participation of ecdysteroid in the silk biosynthetic process with its physiological and molecular implications was discussed.     

Isolation, purification and characterization of silk protein sericin from cocoon peduncles of tropical tasar silkworm, Antheraea mylitta

A high molecular weight water-soluble glue protein, sericin was identified in the cocoon peduncle (a strong thread connecting the cocoons to the branches of the tree with a ring) of the tropical tasar silkworm, Antheraea mylitta. The sericin was isolated by 8 M urea containing 1% sodium dodecyl sulfate and β-mercaptoethenol (2%) or by 1% sodium chloride. The protein was purified by gel filtration chromatography. In SDS-PAGE, a single band of approximately 200 kDa was detected both in non-reducing and reducing conditions. Amino acid analysis showed that the protein is enriched in glycine and serine. There is a slight difference observed in amino acid composition between the sericin from cocoon peduncle and cocoon of A. mylitta. Secondary structure estimation by circular dichroism spectrometry showed 36.7% β-sheets, 52.7% random coils, 10.6% turns and no helices.