The Clausena lansium genome provides new insights into alkaloid diversity and the evolution of the methyltransferase family

Wampee(Clausena lansium)is an important evergreen fruit tree native to southern China that has a long history of use for medicinal purposes.Here,a chromosome-level genome of C.lansium was constructed with a genome size of 282.9 Mb and scaffold N50 of 30.75 Mb.The assembled genome contains 48.70%repetitive elements and 24,381 protein-coding genes.Comparative genomic analysis showed that C.lansium diverged from Aurantioideae 15.91-24.95 million years ago.Additionally,some expansive and specific gene families related to methyltransferase activity and S-adenosylmethionine-dependent methyltransferase activity were also identified.Further analysis indicated that N-methyltransferase(NMT)is mainly involved in alkaloid biosynthesis and O-methyltransferase(OMT)participates in the regulation of coumarin accumulation in wampee.This suggested that wampee's richness in alkaloids and coumarins might be due to the gene expansions of NMT and OMT.The tandem repeat event was one of the major reasons for the NMT expansion.Hence,the reference genome of C.lansium will facilitate the identification of some useful medicinal compounds from wampee resources and reveal their biosynthetic pathways.

STUDIES ON THE CHEMICAL CONSTITUENTS OF CLAUSENA LANSIUM(LOUR.)SKEELS Ⅲ.The structural elucidation of homo-and zeta-clausenamide(1)

The structures of two novel lactams isolated from Clausena lansium were elucidated mainly on the bases of their spectral data. They are homoclausenamide(1),a -lactam,and zetaclausenamide(4), an eight-membered ring lactam.

Prenylated Coumarins from Heracleum stenopterum,Peucedanum praeruptorum,Clausena lansium,and Murraya paniculata

Four hitherto unknown prenylated coumarins,namely 600-O-b-D-apiofuranosylapterin(1),40-O-isobutyroylpeguangxienin(2),6-(3-methyl-2-oxobutyroyl)-7-methoxycoumarin(3),and 6-hydroxycoumurrayin(4),were isolated from the ethanol extract of Heracleum stenopterum,Peucedanum praeruptorum,Clausena lansium,and Murraya paniculata,respectively.Their chemical structures were established on the basis of extensive spectroscopic analysis.Compound 2 exhibited in vitro cytotoxic activity against five human cancer cell lines(HL-60,A-549,SMMC-7721,MCF-7,and SW-480)with IC50 values ranging from 15.9 to 23.2μM.

STUDIES ON THE CHEMICAL CONSTITUENTS OF CLAUSENA LANSIUM (LOUR.) SKEELS Ⅳ THE STRUCTURAL ELUCIDATION OF SECO-AND SECODEMETHYL-CLAUSENAMIDE

The spectral data and chemical properties of two new amides isolated from Clausena lansium led to the assignment of acyclic structures of C-6 and C-7. The absolute configurations of the two chiral carbons in C-6 were determined by degradation to be 3S4R.

Clausenlanins A and B,Two Leucine-Rich Cyclic Nonapeptides from Clausena lansium

Two new cyclic nonapeptides,named clausenlanins A(1)and B(2),were isolated from the roots and rhizomes of Clausena lansium.Their structures were elucidated as cyclo-(Gly^(1)-L-Leu^(2)-L-Ile^(3)-L-Leu^(4)-L-Leu^(5)-L-Leu^(6)-L-Leu^(7)-L-Leu^(8)-L-Leu^(9))(1)and cyclo-(Gly^(1)-L-Leu^(2)-L-Val^(3)-L-Leu^(4)-L-Leu^(5)-L-Leu^(6)-L-Leu^(7)-L-Leu^(8)-L-Leu^(9))(2)respectively on the basis of extensive spectroscopic analysis,particularly 2D NMR spectra taken at the temperature of 338 or 303 K and MS.

黑果黄皮Clausena dunniana Levl.中一个新的咔唑类生物碱(英文)

从黑果黄皮ClusenadunnianaLevl.中分到一个新的咔唑类生物碱 1,并通过谱学手段确定了 1的化学结构为 3

Chemical composition and larvicidal activity of leaf essential oil from Clausena anisata (Willd.) Hook.f.ex Benth (Rutaceae) against three mosquito species

Objective:To determine the mosquito larvicidal activity of leaf essential oil and their chemical constituents from Clausena anisata（C.anisata）（Willd.） Hook.f.ex Benth.against Culex quinquefasciatus,Aedes aegypti and Anopheles Stephensi.Methods:Essential oil was obtained by hydro-distillation and the chemical composition of the leaf essential oil was analyzed using gas chromatography—mass spectrometry.The mosquitoes were reared in the vector control laboratory and twenty lateⅢinstar larvae of three mosquito species were exposed to based on the wide range and narrow range tests,essential oil was tested at 50.100.150,200 and 250 ppm and each compound was tested at various concentration（5-75 ppm） and were assayed in the laboratory by using the protocol of WHO 2005:the 24 h LC50 values of the C.anisata leaf essential oil and their major compounds were determined following Probit analysis.Results:The oil contained were mainlyβ-pinene（32.8%）,sabinene（28.3%）,germacrene-D（12.7%）,estragole （6.4%） and linalool（5.9%）.The essential oil from the leaves of C.anisata exhibited significant larvicidal activity,with 24 h LC50 values of 140.96,130.19 and 119.59 ppm.respectively.The five pure constituents extracted from the C.anisata leaf essential oil were also tested individually against three mosquito larvae.The LC50 values ofβ-pinene,sabinene,germacrene-D,estragole and linalool appeared to be most effective against Anopheles stephensi(LC50-23.17,19.67,16.95. 11.01,35.17 ppm) followed by Aedes aegypti(LC50-27.69,21.20,18.76,12.70,38.64 ppm) and Culex quinquefasciatus(LC50-32.23,25.01.21.28,14.01,42.28).Conclusions:The essential oil of C. anisata contains five major compounds and has remarkable larvicidal properties,which may be considered as a potent source for the production of natural larvicides.

Two New O-terpenoidal Coumarins, Excavacoumarin A and B from Clausena excavata

Two new O-terpenoidal coumarins named excavacoumarin A (2): B (3), and a known one 1 were isolated from the leaves of Clausena excavata Burm. f. (Rutaceae) collected in Xishuangbanna, Yunnan, Structure elucidation and unambiguous NMR assignments for the title compounds were carried out on the basis of 1D and 2D NMR experiments.

A Novel Dimeric Coumarin from Clausena lenis

A novel dimeric coumarin (1), a dimeric of seselin named diseselin A, was isolated from the aerial part of Clausena lenis. The structure was elucidated based on the MS, 1D and 2D NMR data.

Effect of clausenamide on hippocampal neuron apoptosis induced by sodium nitroprusside

BACKGROUND： Aggregation of β-amyloid peptide （A β ）, excitatory intoxication, oxidation injury and inflammation reaction are generally regarded as the main pathogenesis for Alzheimer disease （AD）. （ - ） clausenamide is characterized by promoting intelligent development, resisting oxidation, cleaning free radicals, resisting A β neurotoxicity and nerve cell apoptosis, inhibiting over phosphorylation of tau protein, and improving central cholinergic system. However, whether （-） clausenamide has an effect on hippocampal neuron apoptosis or not need further study. OBJECTIVE： To observe the effect of （ - ） clausenamide on survival rate of hippocampal neurons due to sodium nitroprusside （SNP） and analyze the possible pathways. DESIGN： Contrast observation. SETTING： Institute of Biochemistry and Molecular Biology, Guangdong Medical College. MATERIALS： A total of 12 male SD rats of 24 hours old were provided by the Experimental Animal Center of Guangdong Medical College. The primer was synthesized by Beijing Huada Genetic Engineering Company and （-） clausenamide （99.6%） was provided by Pharmacological Department of Chinese Academy of Medical Sciences. SNP was provided by Sigma Company. METHODS： Bilateral hippocampus was collected from newborn rats to establish single cell suspension. On the 12th day, hippocampal neurons were pretreated with 0.2, 0.4, 0.8 and 1.6 la mol/L （ - ） clausenamide for 6 hours; the culture medium was gotten rid of and neurons were washed with non-serum DMEM solution for three times. In addition, non-serum DMEM solution was added with the above mentioned volume of （ - ） clausenamide and 50 μ mol/L SNP to culture neurons for 24 hours and the collected cells were prepared for the experiment. Neurons were equally divided into control group （culture medium control）, model group （SNP treatment） and experimental group [（ - ） clausenamide ＋ SNP]. Experiment of each group was done for three times at least. Survival rate of cells was measured with MTT chromatometry; levels of mRNA of hippocampal neuron bcl-2 and bax gene were detected with reverse transcription polymerase chain reaction （RT-PCR）; expression of hippocampal neuron Bcl-2 and Bax protein was measured with Western blot technique. MAIN OUTCOME MEASURES： ① Effect of （-） clausenamide on survival rate of SNP-induced hippocampal neuron apoptosis; ②bcl-2 and bax mRNA and protein expression ofhippocampal neurons. RESULTS： ①Survival rate of hippocampal neurons： Survival rate of hippocampal neurons affected by 0.4 - 1.6 μ mol/L （ - ） clausenamide was higher in the experimental group than the model group （P 〈 0.01）, and the survival rate was increased with the larger volume of （ - ） clausenamide. Survival rate was the highest when hippocampal neurons were induced by 1.6 μ mol/L, and it had obvious dosage dependence （P 〈 0.01）. ②Expression of bcl-2 and bax mRNA： Hippocampal neurons were pretreated with 0.2 - 1.6 μ mol/L （ - ） clausenamide for 6 hours in the experimental group and strap of PCR product of bcl-2 gene was brightened gradually. This suggested that, with the increase of concentration, expression of bcl-2 mRNA was increased simultaneously. However, when strap of PCR product of bax gene was darkened, expression of bax was decreased with the increase of concentration. ③Expression of Bcl-2 and Bax protein： Hippocampal neurons were pretreated with 0.2 - 1.6 μ mol/L （ - ） clausenamide for 6 hours in the experimental group and strap of PCR product of Bcl-2 protein was thickened gradually. This suggested that, with the increase of concentration, expression of Bcl-2 protein was increased simultaneously. However, when strap of PCR product of Bax protein was thinned, expression of Bax protein was decreased with the increase of concentration. CONCLUSION： （ - ） clausenamide can resist neurotoxic effect of SNP through dosage dependence, and the mechanism may be related to promoting expression of anti-apoptotic bcl-2 gene and inhibiting expression of pro-apoptotic bax gene.

Antiplasmodial and analgesic activities of Clausena anisata

Objective:Antiplasmodial and analgesic activities of the leaf extract and fractions of Clausena anisata(C.anisata) were evaluated for antimalarial and analgesic activities.Methods:The crude leaf extract(39-117mg/kg) and fractions(chloroform and acqeous;78mg/kg) of C. anisata were investigated for antiplasmodial activity against chloroquine-sensitive Plasmodium berghei(P.berghei) infections in mice using suppressive,prophylactic and curative models and analgesic activity against acetic acid,formalin and heat-induced pains.Artesunate,5 mg/kg and pyrimethamine,1.2mg/kg were used as positive controls.Thin films made from tail blood of each mouse were used to assess the level of parasitaemia of the mice.Results:The extract and its fractions dose-dependently reduced parasitaemia induced by chloroquine-sensitive P.berghei in prophylactic,suppressive and curative models in mice.These reductions were statistically significant(P【0.001).They also improved the mean survival time(MST) from 17 to 21 days relative to control(P【0.01-0.001).On chemically and thermally-induced pains,the extract inhibited acetic acid and formalin-induced inflammation as well as hot plate-induced pain in mice.These inhibitions were statistically significant(P【0.001) and in a dose-dependent fashion. Conclusions:The antiplasmodial and analgesic effects of this plant may in part be mediated through its chemical constituents and it can be concluded that the C.anisata possess significant antimalarial and analgesic properties.

20份黄皮种质资源果肉游离氨基酸对风味品质和药用价值的影响及其评价

为研究黄皮果实中游离氨基酸的组分特征、科学评价不同黄皮种质游离氨基酸对果实风味和药用价值的影响,利用高效液相色谱法,测定20份黄皮种质果肉的20种常见游离氨基酸,采用味觉活度值、主成分分析和聚类分析等方法对各种质进行综合评价。结果表明:在20份黄皮种质果肉中均含有除胱氨酸和精氨酸以外的18种游离氨基酸,种类丰富,且多数种质间含量差异显著;各种质的总游离氨基酸质量浓度在1309.7~9832.0 mg/kg之间,其中非必需氨基酸含量最高,必需氨基酸含量次之,半必需氨基酸含量最低;药用氨基酸占总游离氨基酸的6.3%~18.6%,以天冬氨酸和谷氨酸为主;根据氨基酸呈味特性的不同,将18种氨基酸分为3大类呈味氨基酸,发现种质M1的甜味氨基酸含量最高,G2的鲜味氨基酸和苦味氨基酸含量最高;仅16份种质中的丙氨酸与少数种质中的天冬酰胺、天冬氨酸的味觉活度值(taste activity values,TAV)>1,对黄皮果实风味有贡献;大多数游离氨基酸呈显著或极显著正相关关系;主成分分析提取到4个主成分,累计贡献率达到89.766%,能较好地反映出黄皮果肉游离氨基酸的综合特征;各黄皮种质的综合得分在–3.18~3.51之间,M1的综合得分最高;聚类分析将20份黄皮种质聚为3大类,其中第Ⅲ类群的X2、Y-1、G2、M1的总游离氨基酸、药用氨基酸、呈味氨基酸含量高,氨基酸营养品质好,风味品质佳。研究结果可为黄皮良种选育和品质评价提供参考依据。

Methanolic extract of Clausena excavata promotes wound healing via antiinflammatory and anti-apoptotic activities

Objective: To investigate the anti-inflammatory properties of methanolic extract of Clausena excavata in lipopolysaccharide(LPS)-activated macrophages(J774 A.1) and the effect on skin wound in a rat model through determining cytokine levels and gene expressions. Methods: The effects of methanolic extract of Clausena excavata on in vitro viability and TNF-α, IL-6, IL-10, and nitric oxide release by LPS-activated J774 A.1 cells were determined. In addition, relative expressions of BAX, BCL-2 and COX-2 genes were examined in healed wounds of rats. Results: The methanolic extract of Clausena excavata was not toxic to J774 A.1 cells at the highest dose of 400 μg/m L. It decreased levels of TNF-α and IL-6, while increasing IL-10 level in LPSactivated J774 A.1 cells and in the healed wounds of rats. The methanolic extract of Clausena excavata also inhibited nitric oxide production in LPS-activated J774 A.1 cells. The BAX and COX-2 genes were downregulated while the BCL-2 gene was upregulated in the healed wound of rats. Conclusions: The methanolic extract of Clausena excavata promotes wound healing via its anti-inflammatory and anti-apoptotic activities.

戊糖片球菌协同蜂蜜接合酵母LGL-1混合发酵对黄皮果酒品质的影响

目的探究戊糖片球菌协同蜂蜜结合酵母LGL-1(Zygosacharomyces mellis LGL-1,LGL-1)混合发酵对黄皮果酒品质的影响。方法从米香型白酒酒厂的小曲中筛选乳酸菌;进行形态学、分子生物学鉴定及生长测定;以乳酸菌协同LGL-1进行果酒酿造;采用顶空固相微萃取-气相色谱-质谱法(headspace solid phase microextraction-gas chromatography-mass spectrometry,HS-SPME-GC-MS)和气相色谱法(gas chromatography,GC)对风味物质进行分析。结果所筛菌株BJQ-1、CZZX-2和LJQ-1为戊糖片球菌(Pediococcus pentosaceus);3株菌皆能利用葡萄糖和果糖进行生长且葡萄糖/果糖质量比1:1复配糖耐受性约300 g/L,BJQ-1、LJQ-1乙醇耐受性达14%vol,CZZX-2乙醇耐受性达16%vol,即所筛菌株适合复配协同酵母菌进行果酒酿造;当CZZX-2、BJQ-1和LJQ-1分别协同LGL-1进行黄皮果酒酿造时,与对照组相比,戊糖片球菌CZZX-2的强化发酵提升黄皮果酒品质的效果最佳,果酒中挥发性物质的种类由32种增加至44种,果酒中2-苯乙醇、异丁醇及异戊醇含量与对照组相比,分别降低24.72%、48.89%和51.57%,乙酸乙酯含量显著提升614.70%。结论戊糖片球菌CZZX-2能协同LGL-1进行黄皮果酒酿造,达到增酯降高级醇的目的,提升其品质。本研究可为黄皮果酒的工业化生产提供菌种及一定的理论基础。

儋州本地黄皮核型分析及同源多倍体诱导

海南省儋州市本地黄皮是著名的热带水果,但存在核大、可食率低、品质不一等问题,为选育无(少)核黄皮新品种,开展儋州本地黄皮核型分析和多倍体诱导试验,对供试黄皮露白种子分别采用0.25%、0.5%、1%秋水仙素和浸泡24、36、48 h 2个因素3个水平共9个处理诱导多倍体,取幼苗根尖进行染色体计数,剥取叶片下表面观测气孔大小和密度,取幼嫩叶片采用流氏细胞仪鉴定倍性,筛选较优的秋水仙素和时间处理后,添加0.05%、0.10%、0.20%二甲基亚砜(DMSO)和5、10、15 mg/L萘乙酸(NAA)优化多倍体诱导方式。结果表明,儋州本地黄皮体细胞染色体数量2n=18,核型公式是2n=2x=18=12m+6sm,相对长度组成是2n=1s+4M1+4M2,2B型,核型不对称性高,进化程度高。0.50%秋水仙碱浸泡36 h处理的下胚轴膨大率最高,为35.53%,多倍体和同源四倍体诱导率最高,分别为4.82%和2.07%,表型变异株中多倍体和同源四倍体占比分别为13.62%和5.83%,为多倍体诱导较优处理。该处理优化以0.10%DMSO+10 mg/L NAA处理最优,多倍体和同源四倍体诱导率最高,分别为18.18%和8.74%,除了0.20%DMSO+10 mg/L NAA外,极显著高于其他处理(p<0.01)。

鸡皮果-百香果复合饮料的研制及抗氧化性研究

为了探究鸡皮果-百香果复合饮料的制作工艺及其抗氧化性,以鸡皮果和百香果为原料,通过单因素和响应面试验,优化复合饮料的制作工艺。结果表明,复合饮料的最佳工艺为百香果汁添加量45%、鸡皮果汁添加量55%、罗汉果苷添加量0.018%、果葡糖浆添加量2%,得到的饮料酸甜适中、风味独特。在此工艺条件下,通过单因素和正交试验,确定最佳稳定剂复配条件为CMC-Na添加量0.08%、黄原胶添加量0.2%;最佳护色剂为茶多酚添加量0.012%、D-异抗坏血酸添加量0.01%复配。评价复合饮料体外抗氧化能力,得到ABTS自由基清除率为96.7%、羟自由基清除率为90.5%。

胡椒、芒果和黄皮的化感作用潜力

以胡椒、芒果和黄皮作为供体,玉米、黄豆、南瓜、花生、萝卜、稗草、马唐、柱花草为受体,通过种子萌发法和根生长法测定胡椒、芒果和黄皮的化感作用潜力.结果表明,玉米、黄豆、南瓜、稗草和马唐的萌发和根生长都受到3种供体水浸提液的影响,3种供体水浸提液在高浓度下抑制而在低浓度下则促进受体的萌发和根生长;胡椒和芒果根系周围的土壤对玉米萌发和根生长有促进作用而对花生的萌发和根生长则有抑制作用.3种供体的水浸提液通过乙酸乙酯、正丁醇萃取分离成不同极性的三相后分别对玉米、萝卜和柱花草进行处理.结果表明,胡椒和芒果浸提液的水相与正丁醇相对受体的抑制作用明显强于乙酸乙酯相,表明胡椒和芒果的化感物质具有较强的极性.

黄皮种子的保湿贮藏及胚轴的超低温保存

以典型的顽拗性种子黄皮（Ｃｌａｕｓｅｎａｌａｎｓｉｕｍ）为材料，研究了其种子的保湿贮藏及胚轴的超低温保存．结果表明：部分脱水的生理成熟期种子，每１００ｇ种子混入４～６ｇ百菌清贮于１５℃，８００多ｄ后仍保持较高的发芽率及种子活力．经梯度蔗糖、梯度蔗糖加脱落酸（Ａｂｓｃｉｓｉｃａｃｉｄ，ＡＢＡ）及１１０ｇ／Ｌ蔗糖（Ｓｕｃｒｏｓｅ，Ｓｕｃ蔗糖）＋２０μｍｏｌ／ＬＡＢＡ预培养的生理成熟期胚轴，脱水８～１０ｈ后加入预冷的冰冻保护剂直接入液氮（ＬｉｑｕｉｄＮｉｔｒｏｇｅｎ，ＬＮ），２４ｈ后取出，３７℃水浴化冻并转移至３０ｇ／ＬＳｕｃ的木本植物培养基（ＷｏｏｄｙＰｌａｎｔＭｅｄｉｕｍ，ＷＰＭ）中恢复生长，两个月后再转移至含不同组合的萘乙酸（αｎａｐｈｔｈａｌｅｎｅａｃｅｔｉｃａｃｉｄ，ＮＡＡ）和６－基腺嘌呤（６－Ｂｅｎｚｙ－ｌａｄｅｎｉｎｅ，６－ＢＡ）的３０ｇ／ＬＳｕｃ的ＷＰＭ培养基中生长两个月，发现：梯度蔗糖预培养的胚轴生根率为５０％，而经梯度蔗糖加ＡＢＡ及１１０ｇ／ＬＳｕｃ＋２０μｍｏｌ／ＬＡＢＡ预培养的胚轴则分别保持３０％及１０％的生根率，并能诱导产生少量的愈伤组织；用新鲜的胚轴及子叶为外植体，能诱导产生愈伤组?

黄皮叶黄酮类化合物的抗氧化性研究

【目的】研究黄皮叶黄酮类化合物的抗氧化性,为黄皮叶的开发利用提供参考依据。【方法】采用邻苯三酚自氧化法、水杨酸法和硫代硫酸钠滴定法,分别测定黄皮叶黄酮类化合物对超氧阴离子自由基、羟自由基的清除率以及油脂的过氧化值,探讨黄皮叶黄酮类化合物的抗氧化性。【结果】黄皮叶黄酮类化合物对超氧阴离子自由基和羟自由基的清除作用均呈正相关,当其浓度达0.12 mg/mL时,对超氧阴离子自由基和羟自由基的清除率均超过50.0%;黄皮叶黄酮类化合物对油脂的抗氧化性随着黄酮浓度的增加而增强。【结论】黄皮叶黄酮类化合物具有清除超氧阴离子自由基、羟自由基的作用,同时具有良好的抗油脂氧化活性,开发利用市场前景广阔。

黄皮种子提取物对斜纹夜蛾幼虫的杀虫活性及有效成分鉴定

【目的】测定黄皮Clausena lansium种子提取物对斜纹夜蛾Spodoptera litura幼虫的拒食活性,对有效成分进行分离、鉴定,并进一步测定其拒食活性、生长发育抑制活性及细胞毒性,为筛选斜纹夜蛾生态防治植物源杀虫剂提供依据。【方法】采用叶蝶法测定黄皮种子甲醇提取物及其石油醚、乙酸乙酯、正丁醇和水4种溶剂萃取物对斜纹夜蛾3龄幼虫的非选择性拒食活性和生长发育抑制活性;用硅胶柱层析进行石油醚萃取物和乙酸乙酯萃取物拒食活性成分分离;用核磁共振谱(NMR)进行拒食有效成分结构鉴定;用MTT法测定拒食活性成分对SL细胞毒性。【结果】黄皮种子甲醇提取物对斜纹夜蛾3龄幼虫具有较强的拒食活性,活性成分主要分布于石油醚相和乙酸乙酯相中;石油醚相和乙酸乙酯相经硅胶柱层析分离得10个馏分,以黄色晶体馏分2的拒食活性最强;经分离纯化及核磁共振氢谱和碳谱鉴定,黄皮新肉桂酰胺B(lansiumamide B)为主要的拒食活性成分,对斜纹夜蛾3龄幼虫48 h拒食中浓度为214.95μg/m L,同时具有较强的细胞毒性,对SL细胞48 h抑制中浓度为10.09μg/m L。【结论】黄皮种子提取物对斜纹夜蛾幼虫具有较强的拒食活性及细胞毒性,黄皮新肉桂酰胺B是主要活性成分之一,作为植物源杀虫剂开发具有较好的发展前景。