Molecular analysis of medicinally-used Chinese and Japanese Curcuma based on 18S rRNA gene and trnK gene sequences.

Curcuma drugs have been used discriminatingly for invigorating blood circulation, promoting digestion, and as a cholagogic in China. However, there is confusion about the drug's botanical origins and clinical uses because of morphological similarity of Curcuma plants and drugs. In order to develop an ultimate identification, molecular analysis based on 18S rRNA gene and trnK gene sequences were performed on 6 Curcuma species used medicinally in China and Japan. The 18S rRNA gene sequences were found to be of 1810 bps in length. In comparison with the common sequence of C. longa, C. phaeocaulis, C. wenyujin and C. aromatica, that of C. kwangsiensis had one base substitution, and the same base difference was observed between the Chinese and the Japanese populations of C. zedoaria. The trnK gene sequences were found to span 2698-2705 bps. There were base substitutions, small deletions or insertions at some sites between the trnK coding region and matK region among each species. Based on the base substitutions, C. zedoaria and C. kwangsiensis specimens were divided into two groups, respectively. An identical sequence was detected in C. phaeocaulis and in the Chinese population of C. zedoaria, as well as in the Japanese population of C. zedoaria and in one group of C. kwangsiensis with a purple-colored band in leaves. New taxonomic information to be used for authenticating Curcuma drugs was obtained.     

Application of single-nucleotide polymorphism analysis of the trnK gene to the identification of Curcuma plants

We previously found that Curcuma plants and drugs derived from Curcuma longa, C. phaeocaulis, C. zedoaria, and C. aromatica could be identified by the nucleotide differences at two sites and the existence of a 4-base indel on trnK gene. In this paper, based on species-specific nucleotide sequences, the application of a new method, single-nucleotide polymorphism (SNP) analysis was investigated to identify Curcuma plants more conveniently. First, three types of reverse primer were synthesized in different lengths, 34 mer, 26 mer, and 30 mer, to anneal the template DNAs from each species at sites immediately upstream from substitution positions 177 and 645, and at the site including the 4-base insertion from 728 to 731, respectively. After single-base extension reaction of these primers using fluorescent-labeled ddNTPs and PCR products of the trnK gene region as template, the resulting products were detected using an ABI PRISM 310 Genetic Analyzer. The electrophoretogram showed three or two peaks at different positions depending on the 27 mer, 31 mer, and 35 mer product lengths. Each peak was derived from the incorporated fluorescent-labeled ddNMPs complementary to template nucleotides at positions 645, 724, and 177, respectively. C. phaeocaulis showed three peaks of ddCMP, ddAMP, and ddAMP. The other three species showed two peaks derived from 27 mer and 35 mer products: peaks of ddCMP and ddAMP in C. longa, those of ddCMP and ddTMP in C. zedoaria, and those of ddTMP and ddAMP in C. aromatica. Thus SNP analysis to identify four Curcuma plants was newly developed.     

Authentication of Curcuma species (Zingiberaceae) based on nuclear 18S rDNA and plastid trnK sequences

Curcuma drugs have been used discriminatingly for invigorating blood circulation, promoting digestion, and as a cholagogic in China. However, there is confusion about the drug's botanical origins and clinical uses because of morphological similarity of Curcuma plants and drugs. Comparative sequencing of the 18S rRNA gene in nuclear ribosomal DNA (rDNA) and trnK gene in chloroplast DNA (cpDNA) was carried out in order to examine interspecies phylogeny and to identify ultimately Curcuma species. A total of a hundred of accessions of eighteen species were analyzed. This resulted in an aligned matrix of 1810 bp for 18S rDNA and 2 800 bp for trnK. 18S rDNA sequence divergence within the ingroup ranged from 0-0.05%, trnK ranged from 0-0.19%. One base transversion-substituted site (from cytosine to thymine) was observed from the upstream of 18S rDNA at nucleotide position 234 in C. kwangsiensis and Japanese population of C. zedoaria which have separated genetic distance to other Curcuma taxa. Two noncoding regions embedded in trnK intron showed higher variability, including nucleotide substitutions, repeat insertion and deletions. Based on consensus of relationship, eighteen major lineages within Curcuma are recognized at the species level. The results suggest that Curcuma is monophyletic with 100% bootstrap support and sister to the genera Hedychium and Zingiber. The trnK sequences showed considerable variations between Curcuma species and thus were revealed as a promising candidate for barcoding of Curcuma species, which provide valuable characters for inferring relationship within species but are insufficient to resolve relationships among closely related taxa.     

6种川产姜黄属药用植物叶绿体trnK基因序列变异分析及其分子鉴定

目的建立6种川产姜黄属(Curcuma)药用植物快速简单的分子鉴定方法.方法采用叶绿体赖氨酸tRNA基因(trnK)测序与序列变异分析方法.结果 6种姜黄属药用植物(包括姜黄C. longa、莪术C. phaeocaulis、川郁金C. sichuanensis、川郁金C. chuanyujin、川黄姜C. chuanhuangjiang、川莪术C. chuanezhu)完整trnK基因长度在2699～2705 bp.序列可变区包括matK基因编码区和trnK外显子与matK内含子之间区域,共有6个单核苷酸多态性(SNPs)位点、1个9-bp的缺失重复序列和2个4-bp、14-bp插入重复序列.结论 trnK基因序列可变位点可以作为6种川产姜黄属药用植物快速简单的分子鉴定标记,并为它们之间种的归并提供了分子依据.     

6种川产姜黄属药用植物叶绿体trnK基因序列变异分析及其分子鉴定

目的建立6种川产姜黄属（Curcuma）药用植物快速简单的分子鉴定方法。方法采用叶绿体赖氨酸tRNA基因（trnK）测序与序列变异分析方法。结果6种姜黄属药用植物（包括姜黄C. longa、莪术C. phaeocaulis、川郁金C. sichuanensis、川郁金C. chuanyujin、川黄姜C. chuanhuangjiang、川莪术C. chuanezhu）完整trnK基因长度在2699～2705 bp。序列可变区包括matK基因编码区和trnK外显子与matK内含子之间区域，共有6个单核苷酸多态性（SNPs）位点、1个9-bp的缺失重复序列和2个4-bp、14-bp插入重复序列。结论trnK基因序列可变位点可以作为6种川产姜黄属药用植物快速简单的分子鉴定标记，并为它们之间种的归并提供了分子依据。     

Identification and quantitation of eleven sesquiterpenes in three species of Curcuma rhizomes by pressurized liquid extraction and gas chromatography-mass spectrometry

In this paper, GC-MS and pressurized liquid extraction (PLE) was developed for identification and quantitative determination/estimation 11 sesquiterpenes including germacrene D, curzerene, gamma-elemene, furanodienone, curcumol, isocurcumenol, furanodiene, germacrone, curdione, curcumenol and neocurdione in Ezhu which are derived from three species of Curcuma, i.e., Curcuma phaeocaulis, Curcuma wenyujin and Curcuma kwangsiensis by using an analogue as standard. The results showed the methodology could quantitatively compare the quality of three species of Curcuma. The contents of investigated sesquiterpenes in three species of Curcuma were high variant. Hierarchical clustering analysis based on characteristics of 11 identified peaks in GC profiles showed that 18 samples were divided into two main clusters, C. phaeocaulis and C. wenyujin, respectively. C. kwangsiensis showed the characters closed to C. phaeocaulis or C. wenyujin based on its location. Five components such as furanodienone, germacrone, curdione, curcumenol and neocurdione were optimized as markers for quality control of Ezhu.     

Rapid identification of Curcuma longa and C. aromatica by LAMP

We have developed a novel method for the identification of Curcuma longa and C. aromatica called "loop-mediated isothermal amplification (LAMP)," based on trnK gene sequences. LAMP employs four primers that recognize six regions on the target DNA. Cycling elongation was initiated when the four primers were annealed to the target DNA. Amplifications were detected by measuring turbidity due to the formation of magnesium pyrophosphate. We designed allele-specific primer sets for C. longa and C. aromatica, respectively. LAMP using a primer set for C. longa and total DNA extracted from C. longa rhizome (0.5-10.0 ng) as template was detected up to 70 min. On the other hand, in the reaction using a primer set for C. longa and total DNA from C. aromatica as template, no amplifications were detected. The same tendency could be seen in the reactions using a set of primers for C. aromatica. LAMP enabled not only identification but also detection with high specificity. This rapid, specific, sensitive, and convenient method is expected to be applicable to the identification of the botanical origin of commercially available herbal products.     

Effects of curcuma drugs on vasomotion in isolated rat aorta

The effectiveness of Curcuma drugs against "Oketsu" and the differences in their efficacy were evaluated by examining their vasomotional effects as one index. Since nitric oxide (NO) is the relaxation factor of vascular smooth muscle and also an inhibitor of platelet aggregation in blood vessels, substances showing NO-dependent relaxation are thought to be effective in improving Oketsu. In this study, five Curcuma drugs derived from Curcuma longa, C. kwangsiensis, C. phaeocaulis, C. wenyujin, and C. zedoaria were used. Methanol extracts exhibited intense effects on relaxation in rings precontracted by prostaglandin F(2alpha) (PGF(2alpha)) despite pretreatment with and without N(G)-nitro-l-arginine methyl ester (L-NAME) as an inhibitor of NO synthesis. The maximal activities were approximately 80% at 10(-3) g/ml. From these methanol extracts, curcumin and eight sesquiterpenes were isolated. Since all these compounds showed NO-independent relaxation effects with almost the same intensities, the relaxation effects of Curcuma drugs can be estimated by the total amounts of curcumin and sesquiterpenes. Polysaccharides, the main constituents of methanol-insoluble compounds of water extracts, in contrast, showed contraction effects; only polysaccharides in C. zedoaria showed NO-dependent relaxation as well as contraction. All water extracts showed relaxation effects as sum of the methanol-soluble compounds-induced relaxation and polysaccharides-induced contraction. Therefore, all Curcuma drugs tested in the present study can be effective for vasodilation. Moreover, the drug derived from C. zedoaria has potential to cure Oketsu with its various acting points.     

Qualitative and quantitative analysis of four species of Curcuma rhizomes using twice development thin layer chromatography

The rhizomes of Curcuma phaeocaulis, Curcuma kwangsiensis, Curcuma wenyujin and Curcuma longa are used as Ezhu or Jianghuang in traditional Chinese medicine for a long time. Due to their similar morphological characters, it is difficult to distinguish their origins of raw materials used in clinic. In this study, a simple, rapid and reliable twice development TLC method was developed for qualitative and quantitative analysis of the four species of Curcuma rhizomes. The chromatography was performed on silica gel 60F(254) plate with chloroform-methanol-formic acid (80:4:0.8, v/v/v) and petroleum ether-ethyl acetate (90:10, v/v) as mobile phase for twice development. The TLC markers were colorized with 1% vanillin-H(2)SO(4) solution. The four species of Curcuma were easily discriminated based on their characteristic TLC profiles, and simultaneous quantification of eight compounds, including bisdemethoxycurcumin, demethoxycurcumin, curcumine, curcumenol, curcumol, curdione, furanodienone and curzerene, in Curcuma were also performed densitometrically at lambda(scan)=518nm and lambda(reference)=800 nm. The investigated compounds had good linearity (r(2)>0.9905) within test ranges. Therefore, the developed TLC method can be used for quality control of Curcuma rhizomes.     

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。     

不同产地莪术挥发油的研究

目的：研究不同产地莪术挥发油的化学成分，了解同产地莪术挥发性成分的差别，为控制莪术挥发油的质量提供理论依据。方法：采用水蒸气蒸馏法提取不同产地莪术挥发油，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：不同产地莪术水蒸气蒸馏提取物得率在在0．26％－0．35％之间，分别鉴定出29－42个化学成份。结论：不同品种，不同产地莪术挥发性成分有较大差别，主要为萜类及倍半萜衍生物。     

Possible inhibitory mechanism of Curcuma drugs on CYP3A4 in 1alpha,25 dihydroxyvitamin D3 treated Caco-2 cells

Curcuma longa and C. zedoaria, belonging to genus Curcuma, have become prevalent as supplements in East Asia. Curcumin is the most well-studied bioactive component isolated from rhizomes of C. longa and other Curcuma species except C. zedoaria. In this study, we investigated the affects of C. longa, C. zedoaria from Japan and curcumin on CYP3A4. Caco-2 cells, in which CYP3A4 expression was induced by 1alpha,25-(OH)(2)-D(3), were used to mimic the metabolism of small intestine. Caco-2 cells were treated with methanol extracts from two Curcuma rhizomes (0.1mg/ml) or curcumin (30 microM) for 72 h. Both extracts significantly decreased the activity of CYP3A4 by about 85-98%. The 50% inhibitory concentrations of C. longa and C. zedoaria extracts were 0.019 and 0.014 mg/ml, respectively. They caused a 60-70% decrease in CYP3A4 protein. Otherwise, curcumin treatment caused a 30-40% decrease in CYP3A4 catalytic activity and a 38% decrease in CYP3A4 protein expression. Moreover, it was found that both Curcuma extracts and curcumin treatment had no influence on CYP3A4 mRNA expression. Our results suggested that administration of Curcuma drugs might inhibit the catalytic activity of intestinal CYP3A4. However, curcumin was not the major compound responsible for this inhibitory effect.     

Phylogenetic relationship in the genus Panax: inferred from chloroplast trnK gene and nuclear 18S rRNA gene sequences

Chloroplast trnK gene and nuclear 18S rRNA gene sequences of 13 Panax taxa, collected mainly from Sino-Japanese floristic region, were investigated in order to construct phylogenetic relationship and to assist taxonomic delimitation within this genus. The length of trnK gene sequence varied from 2537 bp to 2573 bp according to the taxa, whereas matK gene sequences, embedded in the intron of trnK gene, were of 1512 bp in all taxa. Species-specific trnK/ matK sequence provided much insight into phylogeny and taxonomy of this genus. 18S rRNA gene sequences were of 1808 or 1809 bps in length, only 9 types of 18S rRNA sequences were observed among 13 taxa. Parsimony and neighbor-joining analyses of the combined data sets of trnK-18S rRNA gene sequences yielded a well-resolved phylogeny within genus Panax, where three main clades were indicated. P. pseudoginseng and P. stipuleanatus formed a sister group located at a basal position in the phylogenetic tree, which suggested the relatively primitive position of these two species. Monophyly of P. ginseng, P. japonicus (Japan) and P. quinquefolius, which are distributed in northern parts of Asia or America, was well supported (Northern Clade). The remaining taxa distributed in southern parts of Asia formed a relatively large clade (Southern Clade). The taxonomic debated taxa traditionally treated as subspecies or varieties of P. japonicus or P. pseudoginseng showed various nucleotide sequences, but all fell into one cluster. It might suggest these taxa are differentiated from a common ancestor and are in a period of high variation, which is revealed not only on morphological appearance, but also on molecular divergence. By comparing trnK and 18S rRNA gene sequences among 13 Panax taxa, a set of valuable molecular evidences for identification of Ginseng drugs was obtained.     

Molecular identification of "Chuanxiong" by nucleotide sequence and multiplex single base extension analysis on chloroplast trnK gene

Chloroplast trnK gene sequences of Cnidium officinale and Ligusticum chuanxiong were determined to establish an effective method for identifying Japanese Senkyu and Chinese Chuanxiong, the two which have the same drug name in Chinese characters, similar external feature, but different botanical origins. Three sites of nucleotide differences were found between these 2 species at positions 767,924 and 964 from upstream in trnK gene sequence, allowing molecular identification of the two plants and crude drugs. Further, three kinds of specific primers of 14 mer, 23 mer and 30 mer long were designed to detect these 3 sites of marker nucleotides. By using multiplex single base extension (MSBE) analysis with the 3 specific primers, C. officinale and L. chuanxiong could be distinguished clearly by the electrophoretograms, where 3 peaks with different color of ddTMP, ddCMP and ddTMP were observed in case of C. officinale and those of ddGMP, ddAMP and ddGMP in L. chuanxiong. Moreover, trnK gene sequence of "Dongxiong," a kind of Chuanxiong cultivated in Northeast China, suggested that its botanical origin was C. officinale.     

土壤—温郁金体系中硒的形态分析

目的:研究土壤-温郁金体系内硒(Se)的形态分布及其温郁金对土壤中硒的吸收富集情况,旨在为温郁金药材的进一步开发研究提供理论和实验依据。方法:采用盆栽方法,在温郁金种植的土壤中施以不同浓度的硒,用连续浸提法提取各形态硒,通过氢化物发生—原子吸收法光谱法检测硒的含量。结果:根系土壤中,可利用态硒含量所占比值最高,易于硒的迁移,当土壤中硒浓度低时,温郁金块根和叶子中有机硒含量大于无机硒,且以蛋白硒为主,而当土壤中硒浓度增加到0.5 mg.kg-1时,对温郁金产生毒害作用,块根中各有机态硒的含量呈现下降趋势。结论:温郁金药材中硒含量与其在土壤中含量呈较强的正相关性(P<0.05)。     

Species identification from Ginseng drugs by multiplex amplification refractory mutation system (MARMS)

The multiplex amplification refractory mutation system (MARMS) was applied to the identification of 5 Panax species ( P. ginseng, P. japonicus, P. quinquefolius, P. notoginseng and P. vietnamensis). A set of specific primers, including 2-pair primers on chloroplast trnK gene and nuclear 18S rRNA gene regions, respectively, was designed and synthesized for each species on the basis of species-specific sequences of the 2 genes. By using 5 sets of specific primers, in turn, PCR amplifications were performed with total DNA extracted from 5 Panax species as template under appropriate condition, and each resulting product was detected by agarose gel electrophoresis. The results showed that two expected fragments, one from trnK gene and another from 18S rRNA gene regions, were observed simultaneously only when the set of species-specific primers encountered template DNA of the corresponding species. This assay could give more reliable results for identification of not only 5 Panax species but also corresponding Ginseng drugs by simultaneous detection of 4-site nucleotide differences on 2 completely different genes.     

Authentication of Panax notoginseng by 5S-rRNA spacer domain and random amplified polymorphic DNA (RAPD) analysis

The great majority of Panax species are well-known herbal medicines in the Orient, and many of them share a close resemblance in appearance and chemical composition. Among these Panax species, the root of P. notoginseng (Sanqi) is a unique herb that has distinct clinical usage. Here, the 5S-rRNA spacer domains were isolated from P. notoginseng, P. japonicus var. major, P. stipuleanatus, P. quinquefolius, P. ginseng, P. zingiberensis, and P. wangianus, and four common adulterants of P. notoginseng including Curcuma wenyujin, Curcuma longa, Bletilla striata and Gynura segetum. The spacer domains were sequenced and compared, which showed over 75 % DNA identity among all Panax species, but not for the adulterants. In addition, random amplification of polymorphic DNA (RAPD) analysis was used to distinguish different members of Panax genus as well as the morphological variants of P. notoginseng. These molecular methods could be used in the authentic identification of P. notoginseng from other Panax species.     

猪基源的肝素粗品及混淆品的AS-PCR及ARMS分子检测

本文建立了一种简便、准确的鉴别猪基源的肝素粗品及其混淆品的DNA分子鉴定方法。在对家猪、野猪及其他7种动物(均用于加工猪肝素粗品的混淆品)的mtDNA D-loop区进行序列分析的基础上，设计了专门用于鉴别猪基源肝素钠的AS-PCR(allele-specific PCR)引物及ARMS(amplification refractory mutation system)引物，对家猪、野猪及其他7种动物来源的肝素、肌肉或血液共49个样品的基源进行了分子检测。结果表明：AS-PCR及ARMS方法均可用于猪基源肝素粗品的快速鉴别。AS-PCR鉴别时的复性温度为54～56 ℃，ARMS引物鉴别时的复性温度更宽，为52～58 ℃。在用两种引物对肝素样品进行鉴别时，仅猪来源的肝素DNA模板能扩增得到约170 bp的扩增条带，而其他动物来源的肝素DNA模板在同样条件下无扩增产物。     

我国姜黄属植物的研究——Ⅱ.五种姜黄属药用植物根茎挥发油化学成分的比较

用GC,GC-MS和TLC分析了国产5种姜黄属植物(姜黄,温郁金,莪术,桂莪术和川郁金)根茎挥发油的化学成分。在OV-17柱上分离出30多个色谱峰,鉴定了24种成分,其中4种(柠檬烯,α-松油烯,芳樟醇和丁香烯)以前未见报道。不同姜黄属植物有许多相同成分,但也有很高含量的不同成分,例如川郁金中的桉叶素,莪术中的莪术酮,姜黄中的姜黄酮和芳姜黄酮。因此根据5种挥发油中主要成分的差别,列出了区分姜黄属品种的表。     

Curcuma drugs and curcumin regulate the expression and function of P-gp in Caco-2 cells in completely opposite ways

Curcumin is a phenolic compound isolated from rhizomes of C. longa, C. aromatica and other Curcumas except C. zedoaria. Recently, both curcumin and Curcumas have become prevalent as supplement. P-gp has been reported as an important determinant for drug absorption in small intestine. In this study, Caco-2 cell monolayers were treated with methanol extracts of Curcumas (0.1 mg/ml) or curcumin (30 microM) for 72h to investigate the relationship between the potential affects of Curcumas and curcumin on P-gp. [(3)H]-digoxin and rhodamine 123 were used to evaluate P-gp activity. All Curcumas significantly increased the activity of P-gp by up-regulating the expressions of P-gp protein and MDR1 mRNA levels. Interestingly, contrary to Curcumas, curcumin treatment inhibited the activity of P-gp with a decrease in P-gp protein and MDR1 mRNA expression levels. Curcumas might alter the pharmacokinetics of co-administrated drugs by up-regulating the function and expression levels of intestinal P-gp. However, curcumin has no relationship with the inductive effect of Curcumas since curcumin showed an opposite effects. Caution should be exercised when Curcumas or curcumin are to be consumed with drugs that are P-gp substrates because Curcumas and curcumin might regulate the function of P-gp in completely opposite ways.