Essential oil of Curcuma wenyujin induces apoptosis in human hepatoma cells

AIM: To investigate the effects of the essential oil of Curcuma wenyujin (CWO) on growth inhibition and on the induction of apoptosis in human HepG2 cancer cells. METHODS: The cytotoxic effect of drugs on HepG2 cells was measured by 3-(4,5-dimethylthiazol-2- yl)-2,5-diphenyltetra-zolium bromide (MTT) assay. DNA fragmentation was visualized by agarose gel electrophoresis. Cell cycle and mitochondrial transmembrane potential (△Ψm) were determined by flow cytometry (FCM). Cytochrome C immunostaining was evaluated by fluorescence microscopy. Caspase-3 enzymatic activity was assayed by the cleavage of Ac-DEVD-R110. Cleaved PARP and active caspase-3 protein levels were measured by FCM using BD? CBA Human Apoptosis Kit. RESULTS: Treatment with CWO inhibited the growth of HepG2 cells in a dose-dependent manner, and the IC50 of CWO was approximately 70 μg/mL. CWO was found to inhibit the growth of HepG2 cells by inducing a cell cycle arrest at S/G2. DNA fragmentation was evidentlyobserved at 70 μg/mL after 72 h of treatment. During the process, cytosolic HepG2 cytochrome C staining showed a markedly stronger green fluorescence than in control cells in a dose-dependent fashion, and CWO also caused mitochondrial transmembrane depolarization. Furthermore, the results clearly demonstrated that both, activity of caspase-3 enzyme and protein levels of cleaved PARP, significantly increased in a dose- dependent manner after treatment with CWO. CONCLUSION: CWO exhibits an antiproliferative effect in HepG2 cells by inducing apoptosis. This growth inhibition is associated with cell cycle arrest, cytochrome C translocation, caspase 3 activation, Poly- ADP-ribose polymerase (PARP) degradation, and loss of mitochondrial membrane potential. This process involves a mitochondria-caspase dependent apoptosis pathway. As apoptosis is an important anti-cancer therapeutic target, these results suggest a potential of CWO as a chemotherapeutic agent.

Quantitative determination of polysaccharide in Curcuma wenyujin, a traditional Chinese medicine, by a phenol-sulfuric acid method

Curcuma wenyujin has been widely used as a traditional medicine in China. In this paper a strategy for the quantitative determination of the polysaccharide by a phenol-sulfuric acid method was described. Involved in three factors, 5% phenol volume, H2SO4 volume, and temperature of water bath, we adopted the L9（3）3 orthogonal array design to gain the optimal colorimetric method. 3.0 ml of polysaccharide solution, 1.0 ml, 5% phenol and 7.0 ml H2SO4 were mixed with constant stirring in a glass vessel, and then kept in a water bath at 40 ℃. After cooling to room temperature for 20 min, the absorbance values were recorded by the UV-2501 PC spectrometer at the wavelength range of 485 nm. The polysaccharide content in Curcuma wenyujin were 3.21%, 3.23%, 3.20%, 3.18~/0, 3.22% and 2.38%, respectively. All results showed that this method was adequate, valid and applicable, may be applied to the determination of other bacterial polysaccharide as well.

不同配方基肥对温郁金产量及其营养元素含量的影响

本试验研究了基肥氮磷钾不同比例配施对温郁金产量与品质的影响,为提高温郁金产量及品质提供科学依据。本研究采用大田试验,设置4个不同的施肥配方,各处理基肥用量均为750 kg·hm^(-2)。结果表明,在氮磷钾总用量相等的前提下,基施配方肥2的温郁金产量最高,与常规施肥比较,温郁金地下部分根茎、块根分别增产11.3%、17.9%。配方肥1和3相较常规施肥也提高了温郁金的产量,但统计上无显著差异。与常规施肥相比,配方肥处理提高了温郁金中氮和磷含量。同时,基施配方肥还可以提高根茎、块根中Ca、Mg及微量元素Zn、B等含量。综上,基肥施用配方肥2既能提高温郁金产量,又能提高根茎、块根中的大量元素及中微量元素含量,具有较好的推广应用价值。

温郁金根茎腐烂病的病原鉴定

在浙江省瑞安市陶山镇温郁金主产区,部分田块温郁金发生大规模倒伏、地下根茎严重腐烂。为明确引起该病害的病原物,利用LB培养基和TTC(2,3,5-氯化三苯基四氮唑)培养基从温郁金腐烂根茎中分离获得细菌菌株JF1,采用灌根法、芽侵染法和根茎侵染法确定该菌株对温郁金的致病性。通过形态学特征观察、生理生化测定、多基因序列(16S rDNA、rpoB、gyrB)系统发育分析,明确引起田间温郁金根茎腐烂的病原菌为桑肠杆菌(Enterobacter mori)。

温郁金连作和熏蒸处理土壤微生物种群数量分析

【目的】明确温郁金连作和熏蒸土壤中微生物种群结构,为缓解温郁金连作障碍奠定基础。【方法】选择温郁金连作3年的土壤,在播种前1个月,用棉隆进行土壤熏蒸处理,在温郁金种植7个月后,分别取连作、棉隆熏蒸及未种植温郁金地块等3种土壤,提取土壤总DNA,通过PacBio Sequel微生物3代测序手段,分析土壤真菌及细菌种群数量及多样性。【结果】土壤经熏蒸处理种植的温郁金植株长势好,病害发生少,而连作种植的温郁金植株长势差,死苗较多。细菌群落属水平的分析结果表明,与未种植土壤比较,连作根际土壤中的AD3、TK10和WPS-2的丰度降低。熏蒸土壤中,芽孢杆菌属(Bacillus)和鞘脂单胞菌属(Sphingomonas)等一些具有生防功能的细菌丰度增加。在3种土壤中,有5个真菌门类丰度较高,分别为子囊菌门(Ascomycota)、担子菌门(Basidiomycota)、壶菌门(Chytridiomycota)、被孢菌门(Mortierellomycota)以及未分类真菌门(unclassified_k_Fungi)。其中子囊菌门真菌在未种植土壤中丰度最低,连作使子囊菌门真菌的丰度显著升高、担子菌门真菌的丰度显著降低。大量有益的真菌属,如毛壳菌属(Chaetomium)、青霉属(Penicillium)、曲霉属(Aspergillus)、漆斑菌属(Myrothecium)、梭孢壳霉属(Thielavia)、腐质霉属(Humicola)、Rhizophlyctis、Microdochium、Ramicandelaber、Albifimbria、Curvularia、巨孢囊霉属(Gigaspora)、黑孢菌属(Nigrospora)、Neocosmospora、Talaromyces、Roussoella、Rhytidhysteron、Boothiomyces、Apiotrichum、Ochroconis、Saitozyma、丝孢酵母属(Trichosporon)、茎点霉属(Phoma)等在熏蒸土壤中的丰度明显高于连作土壤。而在连作土壤中则表现为较多的致病微生物的累积。【结论】温郁金连作可使土壤有害微生物的积累,造成温郁金连作障碍,施用棉隆熏蒸处理土壤后,可减少土壤中病原菌的数量,增加有益微生物种群数量,削减温郁金的连作障碍。

Comparative Analysis of the Volatile Components in the Fresh Roots and Rhizomes of Curcuma wenyujin by Static Headspace Gas Chromatography Mass Spectrometry

Static headspace GC-MS method coupled with H/D exchange was firstly developed to determine and identify the volatile components in the fresh root and rhizome of Curcuma wenyujin. The TIC chromatograms of 3 batches of fresh roots harvested at different time showed significant difference in the volatile components： the constitution was the same but the content of them was different. More than 60 volatile components in fresh roots （Root of C. wenyujin） and rhizomes （Rhizome of C. wenyujin） of C. wenyujin were detected, of which 51 and 48 volatile components were identified respectively. The fresh roots and rhizomes of C. wenyujin were found to have the similar volatile components. The contents of these components were calibrated by the response of β-elemene. In addition, the principal active component, β-elemene, was further confirmed and relatively quantified by its standard. γ-terpinene showed obvious allylic hydrogen/deuterium exchange using deuterium oxide which gave a new method to identify some compounds containing allylic hydrogen. At the same time, the active hydrogen compounds were also further confirmed. The results show that HS-GC-MS of volatile components from medical plants. method is a fast, simple and efficient way for the analysis

Sesquiterpenoids from Curcuma wenyujin dreg and their biological activities

Four new sesquiterpenoids, 4,8-dioxo-6βhydroxyl-7β,l 1-epoxycarabrane （1）, 4,8-dioxo-6βhydroxyl- 7β,11-epoxycarabrane （2）, wenyujinins Q and R （3-4）, and nine known sesquiterpenoids （5-13） were isolated from the Curcuma wenyujin （C wenyujin） dreg. Their structures and relative configurations wereelucidated using 1D, 2D NMR, and HR-ESI-MS data. All the compounds were isolated for the first time from the C wenyujin dreg and evaluated for their antibacterial and antifungal activities. Compounds 3, 5- 8 exhibited strong broad-spectrum antifungal activities against tested nine pathogenic fungi.

Gas Chromatography–Mass Spectrometry for Quantitative and Qualitative Analysis of Essential Oil from Curcuma wenyujin Rhizomes

Objectives:A rapid and sensitive gas chromatography–mass spectrometry(GC–MS)method for quantitative and qualitative analysis of essential oil from Curcuma wenyujin rhizomes was established.Methods:The essential oil of C.wenyujin rhizomes was extracted by supercritical CO2 extraction(SFE).Six main bioactive compounds(eucalyptol,β-elemene,curzerene,germacrone,curdione,and curcumol)were analyzed in selected ion monitoring mode(SIM).Results:Curzerene is not originally present in C.wenyujin rhizomes,but is a product of the transformation of furanodiene at high temperature.The six target components demonstrated good linearity(R2>0.9979)over a relatively wide concentration range.The interday and intraday variations had relative standard deviation values less than 5%and the average recovery ranged from 96.95%to 100.04%.The limit of quantitation ranged from 0.032 to 0.235μg/mL.The developed method was successfully used to analyze the six compounds in 17 samples collected from different origins.Significant variation was observed for the concentrations of the six compounds.In addition,51 constituents were identified in C.wenyujin rhizome essential oil,consisting of 87.66%of the total essential oil,including curdione,curzerene,dehydrocurdione,germacrone,1,4-bis(2-benzimidazoyl)benzene,neocurdione,curcumenone,andβ-elemene.Conclusions:The proposed method will be useful in the quality control of C.wenyujin rhizome essential oil production.

Ultra-performance Liquid Chromatography-Quadrupole/Time-of-Flight Mass Spectrometry Based Bile and Urine Metabonomics Study on the Ameliorative Effects of Curcuma wenyujin Rhizoma on Acute Blood Stasis in Rats

Background: Curcuma wenyujin rhizome(CWR) is a commonly used Chinese herbal medicine for treating blood stasis in China for 1000 of years. However, the underlying mechanism of CWR remains unclear. Aims and Objectives: The purpose of this study is to clarify the bioactive mechanism of CWR in treating blood stasis. Materials and Methods: In this study, pharmacological indexes, including hemorheology and four blood coagulation indexes were tested. Bile and urine metabolomics were engaged by UPLC-Q/TOF-MS. Multivariate statistical analysis were used to screen out differential endogenous metabolites. Results: The results indicated that CWR significantly ameliorated the hemorheology and coagulation functions of acute blood stasis(ABS) model rats. Moreover, 27 endogenous metabolites between the CWR group and the ABS group were screened, and the levels were all improved to certain degrees by CWR preadministration. Metabonomics results indicated that ABS was mainly related to linoleic acid metabolism, arachidonic acid metabolism, glycerophospholipid metabolism, sphingolipid metabolism, pentose and glucuronate intercereasonversions, steroid hormone biosynthesis, and primary bile acid biosynthesis. Conclusion: In a word, the metabolomics method is consistent with the holistic view of traditional Chinese medicine(TCM) that can be a powerful means to illustrate the biological activity mechanism of CWR in treating blood stasis and to offer research demonstration for further study on the effector mechanism of TCM.

温郁金抗肿瘤有效成分及相关机制进展

近年来,癌症的发病率逐渐上升,且呈年轻化发展趋势,以其难治性和高致死率的特征成为危害人类生命健康的主要原因。姜科姜黄属植物温郁金是一味具有潜在应用价值的抗癌中药,因其用药历史悠久,祛除瘀血佳,安全性高,在癌症的治疗中引起了广泛关注且发挥了至关重要的作用。随着现代分子生物技术与中药学的交叉渗透,温郁金的多种抗肿瘤有效成分被分离鉴定,其中挥发油(榄香烯、呋喃二烯、姜黄烯醇、吉马酮、二萜类化合物C、姜黄醇)和姜黄素类等有效成分,主要通过抑制癌细胞增殖、诱导凋亡、逆转多药耐药、抑制血管生成、增强放化疗敏感性和增强机体免疫功能等途径发挥抗肿瘤作用。目前,温郁金被批准为国家二类非细胞毒性抗肿瘤药物,在临床上已经被开发为榄香烯注射液及榄香烯口服乳剂等抗癌制剂广泛应用于不同肿瘤的治疗中,发挥增效减毒的作用。就温郁金的本草考证、抗肿瘤有效成分和相关机制做一综述,旨在为癌症的临床治疗和抗癌中药的开发提供借鉴。

防控温郁金细菌性枯萎病的生物有机肥研制与应用

茄科劳尔氏菌(Ralstonia solanaceraum)引起的温郁金(Curcuma wenyujin Y.H.Chen et C.Ling)细菌性枯萎病是目前温郁金种植中的毁灭性病害。本文利用2株高效拮抗菌Paenibacillus jamila ZJU-C612-3和Bacillus amyloliquefaciens ZJU-C612-4研制生物有机肥,并进行田间防治效果试验。温室盆栽试验结果显示,施生物有机肥的温郁金发病率下降了60百分点;田间试验显示,每667 m 2施200 kg生物有机肥作为基肥的田块中,健康根茎的发病率降低17.66百分点,且植株生长健壮。此外,施生物有机肥能缓解温郁金的连作障碍,在5 a连作田块施该肥后,在病害高发期的7月和8月,连作根茎的发病率分别下降9.43百分点和19.75百分点。研究结果可为今后防治温郁金细菌性枯萎病的专用生物有机肥的产业化开发提供依据。

海南产温郁金离体繁殖体系的优化

目的:对海南地区引种的温郁金进行离体培养,建立离体快繁体系,为工厂化育苗奠定基础。方法:对海南地区温郁金根茎进行预处理,以萌芽为外植体,进行温郁金的初代培养、继代增殖、组培苗的生根和移栽,并对各培养阶段的培养基进行优化。结果:已贮藏半年的温郁金根茎以10 mg/L的GA_(3)浸泡30 min,芽萌发速度明显加快、萌芽率提高;根茎萌芽初代培养污染率低于5%,芽诱导率为100%;以MS+4 mg/L ZT+0.4 mg/L IAA配比的培养基进行继代增殖,增殖系数为2.4;组培生根苗以轻基质网袋容器育苗,移栽成活率为93%以上。结论:以海南引种温郁金根茎萌芽为外植体,建立离体快繁体系,可应用于温郁金组培苗工厂化繁育,保证优质种苗的供应,促进海南温郁金产业的可持续发展。

温郁金地上部分的化学成分(英文)

目的:研究中药莪术来源植物之一温郁金地上部分的化学成分。方法:利用反复硅胶色谱柱进行分离纯化,通过理化性质和波谱数据分析鉴定化合物结构。结果:分离并鉴定出6种化合物:codonolactone(1),vole-neol(2),octacosanoic ac id(3),β-谷甾醇(4),m angdesisterol(5)和胡萝卜苷(6)。结论:两个倍半萜化合物1,2和一个甾醇5为首次从该植物中分得。

GC-MS法建立温莪术挥发油指纹图谱研究

目的 :建立温莪术挥发油的指纹图谱测定分析方法 ,为温莪术药材的质量控制提供参考 ,同时也为温莪术中药制剂制定指纹图谱奠定基础。方法 :采用水蒸气蒸馏法提取温莪术挥发油 ,用GC MS联用技术对其进行指纹图谱测定 ,并采用国家药品监督管理局推荐的中药指纹图谱计算软件进行计算 ,建立温莪术挥发油的共有指纹图谱。结果与结论 :建立的GC MS研究温莪术挥发油指纹图谱的方法有较好的重现性、精密度和稳定性 (RSD均小于 5 % ) ,再加上GC MS具有多成分同时定性的优势 ,所建立的温莪术挥发油的GC MS指纹图谱为其质量控制打下基础。

温郁金和川郁金的PAPD研究

目的：探讨用ＰＡＰＤ技术解决Ｃｕｒｃｕｍａ属植物和药物分类存在的问题和争议。方法：对温郁金Ｃ．ｗｅｎｙｕｊｉｎ和川郁金Ｃ．ｓｉｃｈｕａｎｅｎｓｉｓ进行了ＰＡＰＤ研究。结果：２种亲缘关系很近，聚类结果表明难以从ＤＮＡ水平上把二者划分为２个种。结论：结合化学和形态资料，我们认为２种应予以合并，学名应用Ｃ．ｗｅｎｙｕｊｉｎ，但温郁金和川郁金经长期栽培，化学品质有一些区别，可分为２个药材品种。同时也指出了用花葶中央出和侧生作为分类依据值得商榷。此外，还证明温郁金与毛郁金Ｃ．

中药莪术GC-MS指纹图谱研究

目的研究中药莪术的GC-MS指纹图谱。方法运用GC-MS分析3种基原的18个莪术样品,所得TIC图谱采用“中药色谱指纹图谱计算机相似性评价系统”软件进行模式分析及相似度计算。通过质谱数据与文献对照检识出主要特征峰成分,并根据主要特征成分对18个样品进行SPSS聚类分析。结果3种基原18个莪术样品的相似度(全谱)计算结果较低;通过聚类分析结果可将莪术分为两类,温郁金和蓬莪术各为一类,广西莪术样品则无法成类。因此基于聚类分析结果,建立了温郁金和蓬莪术的指纹图谱共有模式,并分别具有较高的相似度,而广西莪术6个样品因成分差异较大,暂未建立指纹图谱共有模式。结论共有指纹图谱是多基原中药质量控制的基础,但多基原中药莪术GC-MS指纹图谱无法建立,提示不同基原莪术化学成分差异与药理作用的关系值得研究。

温莪术环状含氧倍半萜类化学成分的研究

采用超临界流体CO2萃取(SFE-CO2)技术提取温莪术Curcuma wenyujinY.H.Chen et C.L ing挥发油,用多种色谱和波谱方法对油中主成分进行分离和结构鉴定。结果分离得到5个环状含氧倍半萜类化合物,分别鉴定为:蓬莪术环二烯(Ⅰ)(furanod iene)、莪术二酮(Ⅱ)(curd ione)、莪术双环烯酮(Ⅲ)(curcum enone)、(4S,5S)大=牛儿酮4,5环氧化物(Ⅳ)((4S,5S)-germ acrone-4,5-epoxide)、莪术烯(Ⅴ)(curzerene);其中蓬莪术环二烯含量约占莪术油30%,莪术双环烯酮系首次从温莪术中得到。并对上述5个化合物进行了THP-1细胞分泌TNFα炎症因子抑制作用的体外活性实验,实验结果表明:蓬莪术环二烯和莪术烯对THP-1细胞分泌TNFα炎症因子有明显的抑制作用。

温郁金生长规律研究

目的:研究温郁金生长发育规律,为获得优质、高产药材提供理论依据。方法:定期观察植株形态变化,并测定叶片、根茎、块根干物质及挥发油含量。结果:温郁金发育过程大体分为5个时期,分别为发根发芽期、苗期、叶丛期、根茎膨大期、干物质积累期。在根茎膨大期以前,地上部分干物质占全株干物质的70%以上,在叶丛期,其增长率达到最大值每天3.90 g/株;在干物质积累期,根茎干物质所占比例迅速增加,达到33%以上,增长率最大值每天3.83 g/株,期末根茎挥发油含量大值1.20 mL.100 g-1。结论:温郁金经历了2个生长中心,当地上部分干物质增长率曲线与根茎干物质增长率曲线相交时,可作为地上部分的生长向根茎部生长转移的依据。根茎挥发油含量随其干物质增长率的迅速上升而增加,12月中旬为最佳采收期。

姜科姜黄属植物有效成分的研究

研究不同产地姜科姜黄属植物挥发油的化学成分 ,了解不同产地姜科姜黄属植物挥发性成分的差别 ,为控制其药材质量提供理论依据。采用水蒸气蒸馏法提取不同产地姜科姜黄属植物的挥发油 ,用气相色谱 -质谱联用仪对其进行分离测定 ,结合计算机检索对分离的化合物进行结构鉴定 ,应用色谱峰面积归一化法测定各成分的相对百分含量。结果发现 ,不同产地姜科姜黄属植物的水蒸气蒸馏提取物得率在0.20%～2.13 %之间 ,分别鉴定出24～40个化学成分。不同品种、不同产地姜科姜黄属植物的挥发油含量和其所含成分有较大差别 ,文献报道的某些有效成分如莪术酮、莪术二酮、莪术醇和表莪术酮在某些供试品中未检测到。实验中10个供试品药材中挥发性成分主要为单萜类及倍半萜类化合物及其衍生物 ,倍半萜类化合物的百分含量 (44.57%～88.65 %)明显高于单萜类化合物的百分含量 (1.63 %～26.69%)。

温郁金内生真菌Chaetomium globosum L18对植物病原菌的抑菌谱及拮抗机理

通过体外培养法,研究了药用植物温郁金内生真菌Chaetomium globosum L18对几种常见的植物病原菌的抑菌谱及其拮抗机理。结果表明,Chaetomium globosum L18对多种植物病原真菌和细菌均有不同程度的抑制作用,具有较广的抑菌谱,但对不同植物病原菌的抑制作用具有显著性差异(P<0.05),抑制率最高可达到92.9%;抑菌机制结果显示,竞争作用和重寄生作用是其主要的拮抗机制之一;发酵产物抑制作用测定发现,内生真菌Chaetomium globosum L18能够分泌产生抗菌物质抑制病原菌菌丝的生长和孢子萌发,可引起病原菌菌丝菌丝膨大成串珠状,分枝增多,分枝顶端膨胀后细胞壁破裂,原生质外溢,产生溶菌作用;使分生孢子萌发畸形,萌发率降低。