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.

Development of a DNA microarray for authentication of ginseng drugs based on 18S rRNA gene sequence.

Ginseng drugs, derived from underground parts of Panax species (Araliaceae), are the most important group of herbal medicines in the Orient. Previously, the nucleotide sequences of the nuclear 18S rRNA gene of 13 Panax taxa were determined, as were the specific polymorphic nucleotides for identification of each species. On the basis of the nucleotide difference, a DNA microarray (PNX array) was developed for the identification of various Panax plants and drugs. Thirty-five kinds of specific oligonucleotide were designed and synthesized as probes spotting on a decorated glass slide, which included 33 probes corresponding to the species-specific nucleotide substitutions and 2 probes as positive and negative controls. The species-specific probes were of 23-26 bp in length, in which the substitution nucleotide was located at the central part. Triplicate probes were spotted to warrant accuracy by correcting variation of fluorescent intensity. Partial 18S rRNA gene sequences amplified from Panax plants and drugs as well as their derived health foods were fluorescently labeled as targets to hybridize to the PNX array. After hybridization under optimal condition, specific fluorescent patterns were detected for each Panax species, and the analyzed results could be indicated as barcode patterns for quick distinction. The developed PNX array provided an objective and reliable method for the authentication of Panax plants and drugs as well as their derived health foods.

Comprehensive quality evaluation and comparison of Angelica sinensis radix and Angelica acutiloba radix by integrated metabolomics and glycomics.

Angelica radix (Danggui in Chinese) used in China and Japan is derived from two species of Angelica, namely Angelica sinensis and Angelica acutiloba, respectively. The differences in quality between A. sinensis radix (ASR) and A. acutiloba radix (AAR) should be therefore investigated to guide the medicinal and dietary applications of these two species. Secondary metabolites and carbohydrates have been demonstrated to be the two major kinds of bioactive components of Danggui. However, previously, quality comparison between ASR and AAR intensively concerned secondary metabolites but largely overlooked carbohydrates, thus failing to include or take into consideration an important aspect of the holistic quality of Danggui. In this study, untargeted/targeted metabolomics and glycomics were integrated by multiple chromatography-based analytical techniques for qualitative and quantitative characterization of secondary metabolites and carbohydrates in Danggui so as to comprehensively evaluate and compare the quality of ASR and AAR. The results revealed that not only secondary metabolites but also carbohydrates in ASR and AAR were different in type and amount, which should collectively contribute to their quality difference. By providing more comprehensive chemical information, the research results highlighted the need to assess characteristics of both carbohydrates and secondary metabolites for overall quality evaluation and comparison of ASR and AAR.

Effect of curcuma herbs on vasomotion and hemorheology in spontaneously hypertensive rat.

Curcuma herbs have a vasodilator effect. The effects of C. longa, which induces only endothelium-independent vasodilatation, and C. zedoaria, which induces both endothelium-dependent and -independent vasodilatation, were studied on vasomotion and hemorheology in spontaneously hypertensive rats. Spontaneously hypertensive eight-week-old male rats were assigned to five groups. For 12 weeks, the control group received standard chow. The 3%CL (C. longa) group received standard chow containing 3% (wt/wt) C. longa. The 1%CZ and 3%CZ (C. zedoaria) groups received standard chow containing 1% and 3% (wt/wt) C. zedoaria, respectively. The captoril group received standard chow and 100 mg/kg/day of captoril in drinking water. Blood pressure, vasomotion, hemorheology, etc. were examined. Systolic blood pressure of the 3%CZ and captoril groups decreased significantly as compared to the control group. Acetylcholine-induced endothelium-dependent relaxations of the 3%CZ and captoril groups were increased to a greater degree, significantly, than the control group. When testing xanthine oxidase-induced contraction, the 3%CZ group was significantly decreased as compared to the control group. Low shear stress of whole blood viscosity showed the 3%CL and 3%CZ groups to be decreased significantly compared to the control group. Thus, Curcuma herbs have hypotensive and protective effect on the endothelium in spontaneously hypertensive rats. Especially, C. zedoaria is more effective than C. longa, and its mechanism is thought to be related to a radical scavenging effect and improvement of hemorheology.

Effects of goshajinkigan, hachimijiogan, and rokumigan on mechanical allodynia induced by Paclitaxel in mice.

Peripheral neuropathy is a major dose-limiting side effect of the chemotherapeutic agent paclitaxel. This study examined whether the three related traditional herbal formulations, goshajinkigan (GJG; Niú Che Shèn Qì Wán), hachimijiogan (HJG; Ba Wèi Dì Huáng Wán), and rokumigan (RMG; Liù Wèi Wán), would relieve paclitaxel-induced mechanical allodynia in mice. A single intraperitoneal injection of paclitaxel (5 mg/kg) induced mechanical allodynia, which peaked on day 14 after injection. On day 14 after paclitaxel injection, oral administration of GJG (0.1-1.0 g/kg) produced a significant inhibition of established allodynia, but HJG and RMG did not affect the allodynia. Repeated oral administration of GJG (0.1-1.0 g/kg) starting from the day after paclitaxel injection did not affect allodynia development, but significantly inhibited allodynia exacerbation. Repeated oral administration of HJG produced a slight inhibition of allodynia exacerbation, but that of RMG did not. These results suggest that prophylactic administration of GJG is effective in preventing the exacerbation of paclitaxel-induced allodynia. The herbal medicines Plantaginis Semen ( Che Qián Zǐ) and Achyranthis Radix ( Niú Xi), which are present in GJG but not in HJG, may contribute to the inhibitory action of GJG on the exacerbation of paclitaxel-induced allodynia.

Molecular analysis and chemical evaluation of ephedra plants in Mongolia.

Ephedrae herba has been used in traditional Chinese and Japanese (Kampo) medicine from ancient times, with the primary resource being in China. In the present study, a field survey as well as molecular and chemical assessments were conducted on Ephedra plants in Mongolia to clarify whether they could be an alternative resource of the Ephedrae herba used in Japanese Kampo medicine. Ephedra sinica, E. equisetina, E. przewalskii, E. regeliana, E. monosperma and an unknown taxon (ESP) collected in Mongolia were divided into 9 genotypes on the basis of nucleotide sequences of 18S ribosomal RNA (rRNA) gene and trnK gene. E. sinica, E. equisetina, and E. monosperma presented completely identical sequences to the corresponding species from China. The sequences of trnK gene and 18S rRNA gene provide a useful index for identification and taxonomic classification of Mongolian Ephedra plants. Quantitative analysis of 5 ephedrine alkaloids revealed that almost all Mongolian Ephedra plants contained high amounts of total ephedrine alkaloids (TAs, 1.86-4.90%) and a high percentage of pseudoephedrine in TAs differed obviously from the Chinese. E. sinica and E. equisetina found in eastern and central Mongolia, showing total contents of ephedrine and pseudoephedrine higher than 1.43%, were potential new resources of Japanese Pharmacopoeia grade Ephedrae herba.

Morphological, genetic, and chemical polymorphism of Curcuma kwangsiensis.

Previously, Chinese gajutsu available in Japan was identified, from the chloroplast trnK gene sequence, to be the rhizomes of Curcuma phaeocaulis and two genotypes of C. kwangsiensis. Although we defined the two genotypes, the pl and gl types, on the basis of the nucleotide difference, their external features did not correspond to the two phenotypes described in the literature. In this paper, to investigate the relationship between genotype and phenotype of C. kwangsiensis, a field investigation was carried out in its main cultivation areas of Guangxi Zhuangzu Autonomous Region and Guangdong Province, China, and sequence analysis of the trnK gene and single-nucleotide polymorphism (SNP) analysis of the nuclear 18S rRNA gene were performed on the collected specimens. Four genotypes of C. kwangsiensis were recognized from the combined 18S rRNA gene-trnK gene sequences: homozygote-K(gl)Wtk type, homozygote-K(pl)Ztk type, heterozygote-K(gl)Wtk type, and heterozygote-Ltk type. Among the four genotypes, C. kwangsiensis in a field used for cultivation of gajutsu was of heterozygote-K(gl)Wtk type. Formation of a heterozygote in the 18S rRNA gene might be a result of crossbreeding of C. kwangsiensis with several Curcuma species which had cytosine at nucleotide position 234. GC analysis of the rhizomes revealed that C. kwangsiensis was characterized by camphor and beta-elemene, and by detecting additional components such as curdione and curcumenol Curcuma species involved in the formation of the heterozygote might be speculated upon.

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.

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.

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.

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 analysis of Rheum species used as Rhei Rhizoma based on the chloroplast matK gene sequence and its application for identification.

Rhei Rhizoma (Dahuang in Chinese) is widely known as a purgative and antiinflammatory agent. In the Japanese Pharmacopoeia, Rhei Rhizoma is prescribed for four Rheum species, Rheum palmatum, R. tanguticum, R. officinale, and R. coreanum, while the first three species are prescribed for Dahuang in the Chinese Pharmacopoeia. Due to the morphologic similarity of the aerial parts and frequent occurrence of intermediate forms, the taxonomy of this genus and the correct identification of Rheum species and their derivative drugs are very difficult. To resolve taxonomic problems of the genus Rheum and develop an ultimate identification method for plants and drugs, molecular analysis of the chloroplast matK gene and nuclear 18S ribosomal RNA gene were performed on nine species. The sequence comparison of the matK gene revealed that most species had variable sequences not only inter- but also intraspecies. However, the specimens of the same species belonged to the same subclade in the phylogenetic tree constructed based on matK gene sequences, except for R. palmatum, in which specimens belonged to three subclades related to their production areas. The nucleotide differences at positions 587, 707, and 838 distinguished official species from others, while specific nucleotides at positions 367 and 937 became identification markers for R. palmatum, R. tanguticum, and R. officinale (or R. coreanum). Moreover, three groups of R. palmatum, each belonging to three subclades, were characterized by the nucleotides at positions 619, 769, 883, and 1061. By detecting marker nucleotides, the botanical origins of Rhei Rhizoma were determined.

Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and amplification refractory mutation system (ARMS) analyses of medicinally used Rheum species and their application for identification of Rhei Rhizoma.

Previously, we have determined marker nucleotides on the chloroplast matK gene to identify Rheum palmatum, R. tanguticum and R. officinale used as Rhei Rhizoma officially. In the present study, we further developed a convenient and efficient identification method on the basis of marker nucleotides with Amplification Refractory Mutation System analysis. On the basis of the nucleotide substitutions at positions 367 and 937 among the three species on the matK gene, at each position two kinds of reverse primers with complementary 3'-terminal nucleotides were designed. Upon PCR amplification using three sets of primers and template DNA from each species, one or two fragments (202 bp or/and 770 bp) were detected. As the resultant three fragment profiles were species-specific, the procedure enabled us to classify the botanic origins of 22 drug samples of Rhei Rhizoma.

Comparative study on triterpene saponins of Ginseng drugs.

A comparative study on the triterpene saponins of 47 samples of Ginseng drugs derived from 12 Panax taxa was conducted using a reverse-phase high-performance liquid chromatography (HPLC)method. Eleven ginsenosides, which represent 4 types of typical sapogenins, were chosen as standards for quantitative determination in order to characterize the chemical constituent pattern of each Ginseng drug and investigate the relationship between genetic varieties and chemical constituent pattern. The results showed that the ginsenoside compositions in Ginseng drugs of different origins were of considerable variability. Total saponin contents varied by 10-fold from the highest drug to the lowest one. Chikusetsu-ninjin derived from P. japonicus (Japan) was found to have the highest content (192.80 - 296.18 mg/g) and Ginseng from P. ginseng to be the lowest (5.78 - 15.63 mg/g). Two main groups (I and II) suggested by phytochemical data were clearly observed; group I mainly containing dammarane saponins consisted of P. ginseng, P. quinquefolius, P. notoginseng, P. vietnamensis and P. vietnamensis var. fuscidiscus; and group II containing a large amount of oleanolic acid saponins was com-posed of P.japonicus (apan), P. zingiberensis, P.japonicus (China),P. japonicus var. angustifolius, P. japonicus var. major, P. japonicus var. bipinnatifidus and P. stipuleanatus. The ratios of the subtotal of dammarane saponins to that of oleanolic acid saponins (D/0) were found to be > 1.9 and < 0.25 for groups I and II, respectively. The drug samples derived from the same botanical origin revealed similar constituent patterns, in other words, each Panaxtaxon showed its own characteristic chromatographic profile,which appeared in the specific shape of an 11-direction radar graph constructed on the basis of the result of quantitative analysis. Similarities of chemical constitution were seen among the closely phylogenetically-related taxa, including P. ginseng and P.quinquefolius, P. vietnamensis and P. vietnamensis var.fuscidiscus,P. japonicus (China) and its varieties were demonstrated, except P. japonicus (Japan) and P. zingiberensis.

Sequence analysis of Chinese and Japanese Curcuma drugs on the 18S rRNA gene and trnK gene and the application of amplification-refractory mutation system analysis for their authentication.

The botanical origins of Chinese and Japanese Curcuma drugs were determined to be Curcuma longa, C. phaeocaulis, the Japanese population of C. zedoaria, C. kwangsiensis, C. wenyujin, and C. aromatica based on a comparison of their 18S rRNA gene and trnK gene sequences with those of six Curcuma species reported previously. Moreover, to develop a more convenient identification method, amplification-refractory mutation system (ARMS) analysis of both gene regions was performed on plants. The ARMS method for the 18S rRNA gene was established using two types of forward primers designed based on the nucleotide difference at position 234. When DNAs of four Curcuma species were used as templates, PCR amplification with either of the two primers only generated a fragment of 912 base pairs (bp). However, when DNAs of the purple-cloud type of C. kwangsiensis and C. wenyujin were used, PCR amplifications with both primers unexpectedly generated the fragment, suggesting that these two were heterozygotes. The ARMS method for the trnK gene was also established using a mixture of four types of specific reverse primers designed on the basis of base substitutions and indels among six species, and common reverse and forward primers. C. phaeocaulis or the Chinese population of C. zedoaria, the Japanese population of C. zedoaria or the purple-cloud type of C. kwangsiensis, the pubescent type of C. kwangsiensis or C. wenyujin, and C. aromatica were found to show specific fragments of 730, 185, 527 or 528, and 641 or 642 bp, respectively. All species including C. longa also showed a common fragment of 897-904 bp. Using both ARMS methods, together with information on producing areas, the identification of Curcuma plants was achieved. Moreover, the ARMS method for the trnK gene was also useful for authentication of Curcuma drugs.