Notopterygium Incisum Extract Promotes Apoptosis by Preventing the Degradation of BIM in Colorectal Cancer

Objective Colorectal cancer(CRC),a prevalent malignancy worldwide,has prompted extensive research into anticancer drugs.Traditional Chinese medicinal materials offer promising avenues for cancer management due to their diverse pharmacological activities.This study investigated the effects of Notopterygium incisum,a traditional Chinese medicine named Qianghuo(QH),on CRC cells and the underlying mechanism.Methods The sulforhodamine B assay and colony formation assay were employed to assess the effect of QH extract on the proliferation of CRC cell lines HCT116 and Caco-2.Propidium iodide(PI)staining was utilized to detect cell cycle progression,and PE Annexin V staining to detect apoptosis.Western blotting was conducted to examine the levels of apoptotic proteins,including B-cell lymphoma 2-interacting mediator of cell death(BIM),B-cell lymphoma 2(Bcl-2),Bcl-2-associated X protein(BAX)and cleaved caspase-3,as well as BIM stability after treatment with the protein synthesis inhibitor cycloheximide.The expression of BAX was suppressed using lentivirus-mediated shRNA to validate the involvement of the BIM/BAX axis in QH-induced apoptosis.The in vivo effects of QH extract on tumor growth were observed using a xenograft model.Lastly,APC^(Min+)mice were used to study the effects of QH extract on primary intestinal tumors.Results QH extract exhibited significant in vitro anti-CRC activities evidenced by the inhibition of cell proliferation,perturbation of cell cycle progression,and induction of apoptosis.Mechanistically,QH extract significantly increased the stability of BIM proteins,which undergo rapid degradation under unstressed conditions.Knockdown of BAX,the downstream effector of BIM,significantly rescued QH-induced apoptosis.Furthermore,the in vitro effect of QH extract was recapitulated in vivo.QH extract significantly inhibited the tumor growth of HCT116 xenografts in nude mice and decreased the number of intestinal polyps in the APC^(Min+)mice.Conclusion QH extract promotes the apoptosis of CRC cells by preventing the degradation of BIM.

GC-MS Analysis of Essential Oil Constituents from Rhizome and Root of Notopterygium incisum

Aim To analyse the chemical constituents of the essential oil extracted from the rhizome and root of Notopterygium incisum Ting ex H.T. Chang, providing scientific basis for quality control. Methods The total essential oil was extracted by water-steam distillation and separated by capillary gas chromatography （GC）. The components were determined by normalization method, and identified by GC-MS. Results GC-MS exhibited 242 peaks and 83 compounds were identified, accounting for 75.77% of the total essential oil. Conclusion In the total essential oil of the rhizome and root of N. incisum, monoterpenes and sesquiterpenes accounted for 13.63% and 67.93%, respectively, in which （ 1S）-β-pinene （ 1.67% ）, 3-carene （ 1.05% ）, limonene （ 1.22% ）, and 1S-endo-bornyl acetate （ 1.68% ） as the monoterpenes and its derivatives, and （ ＋ ） -β-elemene （6.78%）, sativene （1.54%）, α-caryophyllene （2.64%）, germacrene D （1.67%）, eudesma-4 （ 14 ）, ll-diene （2.36%）, α-selinene （2.42%）, δ-cadinene （ 1.55% ）, 3,7,11-trimethyl-2,6,10-dodecatrien-1-ol （1.03%）, （ ＋ ）-elemol （5.18%）, （-）-spathulenol （1.40%）, guaiol （3.81%）, dehydroxy-isocalamendiol （ 1.06% ）, γ-eudesmol （ 1.05% ）, α-eudesmol （7.97%）, bulnesol （3.09%）, and carotol （2. 30% ） as the sesquiterpenes and its derivatives were main components. In addition, isopropyl transcinnamate was the maximum compound （ 11.3% ） of the total essential oil.

羌活(Notopterygium incisum)nrDNAITS和cpDNA rpl20-rps1序列分子进化特点的分析

应用PCR产物直接测序法分析了羌活居群间nrDNA(核糖体DNA)ITS序列和cpDNA(叶绿体DNA)rpl20-rps12的碱基差异,从而初步研究两套植物基因组的变异速率。采用改良的CTAB法从硅胶干燥的羌活叶片中提取总DNA,并对nrDNA ITS和cpDNA rpl20-rps12区域进行扩增、纯化、测序。nrDNAITS序列共有635 bp,有变异位点17处,变异位点百分率为2.68%,(G+C)含量为57.83%。cpDNA rpl20-rps12序列共有767 bp,有变异位点35处,变异位点百分率4.56%,(G+C)含量为33.06%。比较发现,羌活nrDNAITS区域较cpDNA rpl20-rps12序列保守,变异速率较慢。通过对ITS和rpl20-rps12序列单倍型(haplotype)进行分析发现,两者得出的结论一致,即现有分布范围经历了居群近期范围扩张。因此,羌活nrDNA ITS序列适合该种的谱系地理学研究。

Transcriptome analysis of the endangered Notopterygium incisum: Coldtolerance gene discovery and identification of EST-SSR and SNP markers

Notopterygium incisum C. C. Ting ex H. T. Chang(Apiaceae) is an endangered perennial herb in China. The lack of transcriptomic and genomic resources for N. incisum greatly hinders studies of its population genetics and conservation. In this study, we employed RNA-seq technology to characterize transcriptomes for the flowers, leaves, and stems of this endangered herb. A total of 56 million clean reads were assembled into 120,716 unigenes with an N50 length of 850 bp. Among these unigenes, 70,245(58.19%) were successfully annotated and 65,965(54.64%) were identified as coding sequences based on their similarities with sequences in public databases. We identified 21 unigenes that had significant relationships with cold tolerance in N. incisum according to gene ontology(GO) annotation analysis. In addition, 13,149 simple sequence repeats(SSRs) and 85,681 single nucleotide polymorphisms were detected as potential molecular genetic markers. Ninety-six primer pairs of SSRs were randomly selected to validate their amplification efficiency and polymorphism. Nineteen SSR loci exhibited polymorphism in three natural populations of N. incisum. These results provide valuable resources to facilitate future functional genomics and conservation genetics studies of N. incisum.

A new dimeric furanocoumarin from Notopterygium incisum

A new dimeric psoralen-type furanocoumarin, notopterol-（18-O-20＇）-notopol （1）, was isolated from Notopterygium incisum for the first time and its structure was elucidated by spectroscopic methods.

The chromosome-scale assembly of the Notopterygium incisum genome provides insight into the structural diversity of coumarins

Coumarins,derived from the phenylpropanoid pathway,represent one of the primary metabolites found in angiosperms.The alignment of the tetrahydropyran(THP)and tetrahydrofuran(THF)rings with the lactone structure results in the formation of at least four types of complex coumarins.However,the mechanisms underlying the structural diversity of coumarin remain poorly understood.Here,we report the chromosome-level genome assembly of Notopterygium incisum,spanning 1.64 Gb,with a contig N50 value of 22.7 Mb and 60,021 annotated protein-coding genes.Additionally,we identified the key enzymes responsible for shaping the structural diversity of coumarins,including two p-coumaroyl CoA 2′-hydroxylases crucial for simple coumarins basic skeleton architecture,two UbiA prenyltransferases responsible for angular or linear coumarins biosynthesis,and five CYP736 cyclases involved in THP and THF ring formation.Notably,two bifunctional enzymes capable of catalyzing both demethylsuberosin and osthenol were identified for the first time.Evolutionary analysis implies that tandem and ectopic duplications of the CYP736 subfamily,specifically arising in the Apiaceae,contributed to the structural diversity of coumarins in N.incisum.Conclusively,this study proposes a parallel evolution scenario for the complex coumarin biosynthetic pathway among different angiosperms and provides essential synthetic biology elements for the heterologous industrial production of coumarins.

Anti-rheumatoid arthritic effect of volatile components in notopterygium incisum in rats via anti-inflammatory and anti-angiogenic activities

Notopterygium incisum(QH) has been used for the treatment of rheumatoid arthritis(RA), and volatile oils may be its mainly bioactive constituents. The present study was designed to analyze the volatile compounds in QH and to determine the anti-arthritic capacity of Notopterygium volatile oils and the potential mechanism of action. The volatile compounds analysis was conducted by GC-MS. The anti-arthritic capacity test of the volatile oils was conducted on adjuvant-induced arthritis(AIA) rats. The anti-inflammatory property was tested in NO release model in RAW 264.7 cells. Endothelial cells were used to evaluate the anti-proliferative and anti-tube formative effects. 70 compounds were analyzed by GC-MS in the volatile oils. Notopterygium volatile oils weakened the rat AIA in a dose-dependent manner(2, 4, and 8 g crude drug/kg). The NO production by RAW 264.7 was decreased by more than 50% in Notopterygium volatile oils(5, 15, and 45 μg·mL^(-1)) pretreated groups. Notopterygium volatile oils also inhibited EAhy926 cell proliferation and further delayed EAhy926 cell capillary tube formation in a concentration-dependent manner. The anti-NO productive, anti-proliferative, and anti-tube formative effects of Notopterygium volatile oils strongly suggested that the therapeutic effect of QH in AIA might be related to the potent anti-inflammatory and anti-angiogenic capacities of the volatile oils.

Cytotoxic polyacetylenes isolated from the roots and rhizomes of Notopterygium incisum

Phytochemical investigation on the roots and rhizomes of Notopterygium incisum led to the isolation of a new polyacetylene, notopolyenol A (1), along with thirteen known analogues (2-14). Their structures were elucidated by extensive analyses of NMR and HRMS data, and the absolute configuration of 1 was unambiguously determined as 3 R by comparison of its retention time and ECD curve with those of synthetic enantiomers (-)-1 and (+)-1, whose absolute configurations were established by using the modified Mosher's method. Subsequent activity screening revealed that (3 S)-1 exhibited the most significant cytotoxicity against MCF-7, H1299, and HepG2 cancer cells with IC_(50) values of 1.3 μmol/L,0.6μmol/L and 1.4μmol/L, respectively.

基于聚类及PCA分析的羌活栽培区土壤质量评价

为了准确评价川产道地药材羌活栽培区耕作层土壤质量状况,分别采用聚类分析法(CA)和主成分分析法(PCA)构建栽培区耕作层土壤质量最小数据集(minimum data set,MDS),利用最小数据集土壤质量指数(soil quality index-CA,SQI-CA和SQI-PCA)和全量数据集土壤质量指数(SQI-T)评价川西北羌活栽培区耕作层土壤质量。结果表明:(1)羌活栽培区土壤有机质含量为(19.14±6.75)g·kg^(−1),处于中度贫瘠化水平;土壤速效氮、速效磷和速效钾含量较高,分别为(129.78±47.78)mg·kg^(−1)、(22.89±14.78)g·kg^(−1)和(159.87±97.87)mg·kg^(−1);土壤为中性土壤,pH均值为7.20±1.68。(2)基于不同数据集的土壤质量指数均值排序为SQI-T>SQI-PCA>SQI-CA,而SQI-PCA与SQI-T之间的Nash有效系数高于SQI-CA,相对偏差系数低于SQI-CA,说明基于主成分分析的最小数据集(MDS-PCA)评价效果更优,该数据集包括土壤容重、抗剪强度、有机质含量、饱和导水率、黏粒含量、pH、速效氮和砂粒含量共8个指标。(3)川西北羌活栽培区土壤质量指数SQI-PCA<0.33,表明该研究区耕作层土壤质量总体水平较差,主要体现在土壤紧实、有机质含量低,需要通过合理耕作、施肥和土壤改良等方式对耕作层土壤质量进行有效调控。研究结果可为川西北高原羌活栽培区土壤质量改良和生产适宜性调控提供参考,有利于川西北高原区中药材产区土壤可持续利用。

家种羌活主要化合物生物合成途径的转录组学研究

羌活是我国传统常用大宗药材,以根茎及根入药,前期研究发现其根茎中以羌活醇和异欧前胡素为代表的次生代谢产物含量显著高于根。为探究其生物合成途径及调控机制,本研究利用RNA-seq和转录组学分析手段,以根为对照,对比分析家种羌活根茎中基因的差异表达。结果表明,1222个差异表达基因(DEGs)显著富集于苯丙烷生物合成、植物病原体相互作用、植物MAPK信号通路及植物激素信号转导途径等通路;苯丙烷生物合成通路中莽草酸O-羟基肉桂酰转移酶(HCT)、肉桂醇脱氢酶(CAD)和过氧化物还原酶6(PRDX6)等关键酶编码基因转录本在根茎中显著上调表达;AP2/ERF、WRKY、C2C2及NAC等转录因子家族的多个转录因子在根茎及根中显著差异表达,这些因子可能是影响羌活次生代谢产物生物合成的关键转录因子;基因的差异表达与羌活根茎及根中羌活醇和异欧前胡素含量差异相符。本研究结果可为进一步研究羌活药用部位有效成分积累机理及定向育种提供理论基础。

HPLC法同时测定羌活药材中甲基托布津和辛硫磷农药的残留

目的建立同时测定羌活中甲基托布津和辛硫磷2种农药残留的HPLC法。方法羌活药材样品加NaCl粉末,以0.1%甲酸乙腈为提取溶剂,通过无水硫酸钠粉末净化,所得净化液以反相C_(18)色谱柱为分离柱,以乙腈∶0.1%甲酸(含10 mmol/L甲酸铵)(70∶30)为流动相,流速0.8 mL/min,在检测波长280 nm处测定2种农药残留的量。结果甲基托布津和辛硫磷配制浓度与峰面积在0.02~0.2μg/mL范围内有良好的线性关系,相关系数为0.9986和0.9993,该方法的检测限分别为4 ng/mL和3 ng/mL。甲基托布津的加标回收率为92.14%~112.56%,辛硫磷的加标回收率为87.85%~103.45%。结论该HPLC检测方法的操作简单、准确而快速,重复性和稳定性均良好,可满足羌活药材中甲基托布津和辛硫磷农药残留检测的要求。

羌活种子不同休眠状态内生菌群落结构组成和差异性分析

为了探寻羌活种子不同休眠阶段内生菌的群落结构组成和差异性,利用Illumina Miseq平台分别对羌活种子不同休眠状态(形态生理双重休眠状态(GSA)、形态休眠解除状态(GSB)、生理休眠解除状态(GSC))细菌微生物16S V5+V7区域和真菌微生物ITS1-IF区域的扩增片段进行高通量测序,结合生物信息学进行分析。内生细菌的多样性较内生真菌丰富。内生菌群落种类在门水平上类似;在属水平上,从GSA到GSB阶段,内生菌群落组成发生变化,从GSB到GSC阶段,内生菌群落种类相似。通过Alpha多样性分析发现,从GSA到GSB再到GSC阶段,内生细菌群落多样性显著增大后直到种子生理休眠解除变化不变,而内生真菌群落多样性在种子整个休眠解除过程中变化不大。内生细菌群落结构从GSA到GSB再到GSC阶段均发生了显著性变化,而内生真菌群落结构从GSA到GSB发生显著变化后直到种子生理休眠解除,群落结构变化不大。LEfSe分析发现内生细菌共有37个差异丰富的分类学分支(a=0.01,LDA≥2.0),内生真菌则无。本研究明确了羌活种子不同休眠状态内生菌群落结构组成和差异性,丰富了羌活种子内生菌资源信息,对进一步研究利用微生物方法促进羌活种子休眠解除技术具有一定的指导意义。

羌活种子发育过程中形态及生理生化指标动态变化特征

【目的】探讨羌活种子发育过程中形态及生理生化指标变化规律,为羌活引种驯化、优质种质资源培育提供理论依据。【方法】采用定期观测采样及冷冻切片法,对不同发育阶段的羌活种子形态及生理生化指标进行测定。【结果】羌活种子发育过程中,种子大小、果翅厚度先升后降,果翅宽度升高,外种皮颜色由明亮的蓝绿色转为黄褐色;贮藏物质淀粉、可溶性糖、可溶性蛋白含量升高;淀粉酶活性小范围波动,琥珀酸脱氢酶活性升高,葡萄糖-6-磷酸脱氢酶活性交替升降,丙酮酸激酶活性呈“M”形变化,POD活性降低,而SOD活性先降后升;GA3、IAA和ZR含量降低,ABA含量升高;种子形态指标与SOD活性极显著负相关,贮藏物质含量与种子厚度极显著负相关,而与果翅宽度极显著正相关。【结论】在羌活种胚发育至心形胚前期过程中,外种皮由蓝绿色转为黄褐色,贮藏物质含量升高,关键酶活性波动变化,促生长激素含量波动降低,而胁迫激素含量升高。

羌活和宽叶羌活的环境土壤学研究

目的对羌活和宽叶羌活进行环境生态学比较,探讨环境植物学因素对野生羌活的影响,为羌活的野生抚育和引种驯化提供理论依据。方法对四川甘孜、阿坝、青海互助和甘肃临潭等主产区进行了长期的野生环境考察研究,并对各主产地的土壤养分状况进行了初步分析。结果野生羌活的生长、繁殖和分布主要受到3个方面的制约,即(1)土壤发育程度和养分状况,(2)土壤水分状况,(3)生境的植被覆盖状况。前二者受到植被覆盖状况的制约和影响,这些基础研究工作的欠缺是药用羌活植物引种长期难以取得成功的重要制约因素。结论羌活原生生境土壤有机质、氮素和有效磷与宽叶羌活有较大差异,对羌活和宽叶羌活的分布影响很大;土壤中有机质、水解性氮、有效磷对羌活和宽叶羌活的药材质量的影响极大,但其定量关系有待进一步研究。

羌活光合作用日变化及其与生理生态因子的关系

以大田引种栽培的羌活植株为材料,用Li-6400便携式光合作用测定系统原位测定自然条件下生长的羌活孕蕾期叶片的光合速率光响应、净光合速率及生理生态因子日变化,并通过相关分析、通径分析和逐步回归分析探讨净光合速率与生理生态因子的关系。结果表明:(1)羌活净光合速率(Pn)、蒸腾速率(Tr)、气孔导度(Gs)日变化均呈双峰曲线,其净光合速率具有典型的"午休"现象,并主要由非气孔限制因素造成;(2)影响羌活叶片Pn日变化的主要决定生理因子是Gs,主要限制因子是胞间CO2浓度(Ci);主要决定生态因子是空气相对湿度(RH),限制因子是气温(Ta);Gs是影响净光合速率最重要的生理生态因子。研究发现,羌活有较强的适应弱光环境的能力,属于阳性耐荫植物,宜选择海拔和荫蔽度均较高的环境进行引种驯化栽培,以利于其生长和存活。

羌活种子发芽及实生苗生长发育的研究

目的:研究打破休眠后的羌活种子的萌发过程、适宜培养基以及羌活实生苗生长规律和优化措施。方法:使用光照培养箱,设置25,20,15,10℃4个发芽温度条件,光周期为12h/12h光暗反应,计算发芽始期和发芽持续时间,以及发芽势和发芽率;9种基质、4种播种深度下定期对种子苗进行生长发育的观测。结果:羌活种子的发芽适温为15℃,基质A_6(锯末、蛭石、草炭灰、珍珠岩混合基质)是9种基质中对羌活实生苗生长最有利的基质,覆土深度1～1.5cm较好;实生苗子叶出土需要约10d,出苗约15d,第1片真叶展开约25d,第2片真叶展开2个月,倒苗时间12月份,返苗时间3月份。结论:解除休眠的羌活种子能长出高质量的实生苗,且适合生长在土质疏松、有机质丰富、土层深厚的土壤上。

中药羌活中的香豆素

从中药羌活ＮｏｔｏｐｔｅｒｇｉｕｍｉｎｃｉｓｕｍＴｉｎｇ．中分离鉴定了１５个香豆素，其中３个新化合物，分别为乙基羌话醇（ｅｔｈｙｌｎｏｔｏｐｔｅｒｏｌ，ＸＩＩ），羌活酚缩醛（ｎｏｔｏｐｔｏｌｉｄｅ，ＸＩＩＩ）和环氧脱水羌活酚（ａｎｈｙｄｒｏｎｏｔｏｐ－ｔｏｌｏｘｉｄｅ，ＸＩＶ）。１２个已知化合物（１～ＸＩ，ＸＶ）分别为佛手内酯、异欧芹素乙、去甲呋喃羽叶云香素、佛手酚、羌活醇、羌活酚、脱水羌活酚、花椒毒酚、佛手柑亭、７－ｉｓｏｐｅｎｔｅｎｙｌｏｘｙ－６－ｍｅｔｈｏｘｙ－ｃｏｕｍａｒｉｎ和７－（３，７－ｄｉｍｅｔｈｙｌ－２，６－ｏｃｔａｄｉｅｎｙｌｏｘｙ）－６－ｍｅｔｈｏｘｙ－ｃｏｕｍａｒｉｎ以及紫花前胡甙（ＸＶ）。

羌活的化学成分

目的：研究四川理县羌族地区产我国重要中药材羌活的化学成分,并比较与其他产地该药材之间的成分差别。方法：运用各种分离材料和方法分离羌活根的化学成分,采用波谱技术鉴定其结构。结果：从羌活根的70%丙酮提取物中分离得到12个化合物,经波谱解析分别鉴定为：notoptol（Ⅰ）、羌活醇（Ⅱ）、紫花前胡苷（Ⅲ）、镰叶芹二醇（Ⅳ）、（10E）1,10-heptadecadiene-4,6-diyne-3,8,9-triol（Ⅴ）、10-hydroxyverbenon（Ⅵ）、（＋）-bornylferulate（Ⅶ）、孕甾烯酮醇（Ⅷ）、香草酸（Ⅸ）、香豆酸（Ⅹ）、反式阿魏酸（Ⅺ）和9,12-octadecadienoic acid（Z,Z）-（2,2-dimethyl-1,3-dioxolan-4-yl）methyl ester（Ⅻ）,化合物Ⅰ-Ⅳ、Ⅶ和Ⅷ是其主要成分,化合物Ⅴ-Ⅶ和Ⅹ、Ⅻ为首次从该植物中分离得到。结论：四川理县产羌活的化学成分与其他产地的有所区别。

不同商品等级羌活中有机酸和香豆素类化合物的测定

目的比较同一产地不同商品等级羌活中主要有机酸和香豆素类化合物的含有量。方法采用HPLC-DAD法同时测定不同商品等级羌活中绿原酸、阿魏酸、紫花前胡苷、补骨脂素、佛手柑内酯、欧前胡素、羌活醇和异欧前胡素。色谱柱为C18TSK gel ODS-80Ts QA(250 mm×4.6 mm,5μm)。流动相为甲醇(A)-0.3%的冰醋酸水溶液(B),梯度洗脱;体积流量0.6 mL/min,进样量10μL,柱温30℃。结果 8种活性成分在80 min内达基线分离,线性关系良好(R2≥0.9992);平均加样回收率为96.27%～102.44%,RSD≤4.15%。绿原酸和阿魏酸的含有量与传统等级划分方法基本一致,但紫花前胡苷、佛手柑内酯、羌活醇和异欧前胡素的含有量与传统的以外观形态论品质的判断标准有一定的差异。补骨脂素和欧前胡素在所测样品中均未检测出。结论该方法灵敏、准确、可靠、重复性好,可用于羌活药材的质量评价。

药对羌活与独活及其单味药治疗佐剂关节炎初步研究

目的:探讨常用药对羌活与独活及其单味药治疗佐剂关节炎的作用,为临床羌活、独活配伍用药方式提供实验依据。方法:建立完全弗氏佐剂关节炎模型;造模成功后随机分空白组、模型组、阳性组、羌活组、独活组、羌独活组;观察大鼠机械疼痛阈值、足趾容积变化;酶联免疫吸附试验检测足趾组织研磨液IL-1β、IL-6、TNF-α、PGE2水平。结果:给药第14天后,给药组与模型组相较,大鼠对机械压力刺激的痛阈值显著增大(P<0.05),大鼠的足趾容积显著减小(P<0.05),各给药组均能够显著抑制IL-6、TNF-α、PGE2的水平(P<0.05),但是,羌活、独活组对IL-1β水平无影响,羌独活组能够显著减少IL-1β的产生(P<0.05)。结论:药对羌活与独活及其单味药能够在不同程度上显著性的减轻关节炎大鼠的足趾肿胀,提高机械压力刺激的痛阈值,其抑制IL-1β、IL-6、TNF-α、PGE2的异常分泌可能是治疗佐剂关节炎的机制之一。