Development of SCAR Markers to Identify Medicinal Cultivars of Paeonia lactiflora.

Paeoniae Radix, the dried root of Paeonia lactiflora, is one of the most important ingredients in Kampo medicine. It is known that Paeoniae Radix is derived from various P. lactiflora cultivars, including medicinal and horticultural cultivars, and that cultivar identification by DNA analysis has been unsuccessful. We attempted to develop sequence characterized amplified region (SCAR) markers as useful DNA markers for the identification and herbal medicine authentication of two cultivars developed in Japan, 'Bonten' and 'Kitasaisho,' which are two superior medicinal strains of P. lactiflora. Sequence-related amplified polymorphism (SRAP) analysis was conducted on fourteen P. lactiflora cultivars, and polymorphic fragments specific to 'Bonten' or 'Kitasaisho' were detected. Then, SCAR markers for 'Bonten' and 'Kitasaisho' were developed from the sequence information of these polymorphic fragments. Thirty cultivars of P. lactiflora and five herbal medicine samples were used to validate the specificity of the developed SCAR markers. As a result, we confirmed that our SCAR markers can identify 'Bonten' or 'Kitasaisho' from the plant samples and the herbal medicine samples. Thus, we have successfully designed two highly specific DNA markers and established an easy, rapid, and cost-efficient method to identify specific cultivars of P. lactiflora. Our SCAR markers are expected to contribute to the maintenance of P. lactiflora cultivars such as 'Bonten' as superior medicinal strains, the development of more elite cultivars in the future, and the deterrence of outflow of original cultivars to foreign countries.

Scopolia mild mottle virus: a new tobamovirus isolated from a Scopolia japonica plant in Japan.

A tobamovirus was isolated from leaves of a Scopolia japonica plant showing mild yellowing. Back-inoculation of healthy Scopolia japonica with the isolated virus induced mild mottle on upper leaves. Phylogenetic analysis based on coat protein and replicase protein sequences revealed that the newly isolated tobamovirus was most closely related to yellow tailflower mild mottle virus (YTMMV). The newly isolated tobamovirus shared the highest nucleotide sequence identity (71%) with YTMMV, which is lower than the cutoff (90%) set for species demarcation in the genus Tobamovirus. Thus, our result suggested that scopolia mild mottle virus (SMMoV) is a new tobamovirus that infects Scopolia japonica plants in Japan.

Efficient fertilization in the cultivation of Angelica acutiloba (Siebold & Zucc.) Kitag. in Hokkaido: Effect of amount of supplied nitrogen on growth, yield, and quality of A. acutiloba.

Angelica acutiloba (Siebold & Zucc.) Kitag., the source plant of the crude drug Angelicae acutilobae radix, is traditionally cultivated in western regions of Japan. Since A. acutiloba is now also grown in Hokkaido, the northernmost prefecture of the country, the cultivation method, especially the quantity of fertilizer, needs to be adapted because of the cooler climate and shorter growing period. In this study, we compared plant growth and harvest yield of A. acutiloba cultivated with different amounts of nitrogen (N) fertilizer. When plants were fertilized with 24 kg N/10 a, the aerial part was lush, and the diameter at the top of the root was about 1.3 times thicker than that in plants treated with 12 kg N/10 a. On the other hand, the weight of the harvested root grown with 24 kg N/10 a was slightly, although not significantly, less than that in plants grown with 12 kg N/10 a. In addition, we found that the content of (Z)-ligustilide, a major essential oil contained in A. acutiloba root, is affected by nitrogen application. Interestingly, it increased with increasing amounts of supplied nitrogen. However, the importance of Angelicae acutilobae radix as the crude drug is not limited to its (Z)-ligustilide content, and there are also other crucial quality features, such as having thick lateral roots, which were generated in this study with a moderate nitrogen application. We conclude that about 12 kg N/10 a is the optimal amount of nitrogen for healthy growth of A. acutiloba in Hokkaido.

Polymorphism analysis of TCP gene region to intraspecific analysis of Paeonia lactiflora, and application for authentication of Paeoniae Radix.

We attempted to conduct an intraspecific analysis of 30 peony cultivars in Japan and to authenticate five herbal medicine samples derived from Paeoniae Radix by polymorphism analysis of the TEOSINTE BRANCHED1, CYCLOIDEA, and PCF (TCP) gene region. We focused on cultivar-dependent differences in leaf margin undulation and analyzed the sequence of the related TCP gene region. As a result, we found that the nucleotide sequences of 29 cultivars of Paeonia lactiflora except 'America' exhibit interspecific variations compared with the nucleotide sequences of Paeonia suffruticosa and Paeonia tenuifolia. Therefore, in the dendrogram constructed on the basis of the sequence similarity in the TCP gene region, the 29 cultivars of P. lactiflora were separated from P. suffruticosa, P. tenuifolia, and 'America', and clustered into three subgroups. There were 16 variations containing heterogenous DNA sequences within P. lactiflora species, and two characteristic variations in subgroup I. Some P. lactiflora cultivars showed the same nucleotide sequence in the TCP gene region, whereas the five herbal medicine samples showed different sequences, although all of them could be authenticated. The results suggest that Paeoniae Radix in the Japanese crude drug market can be authenticated by analysis of the TCP gene region.

Simultaneous UHPLC/MS quantitative analysis and comparison of Saposhnikoviae radix constituents in cultivated, wild and commercial products.

Differences in the components of the crude drug Saposhnikoviae radix, both wild and cultivated, and the cultivation duration were examined by UHPLC/MS. As a result, there was no significant difference in composition depending on the region in China where the drug was produced. The most abundant components in all samples were prim-O-glucosylcimifugin, 4'-O-glucosyl-5-O-methylvisamminol, 3'-O-acetylhamaudol and cimifugin. The 1 year-old Saposhnikoviae radix cultivated in Japan had a low component content overall. A comparison of components according to root thickness revealed that glycosides, such as prim-O-glucosylcimifugin and 4'-O-glucosyl-5-O-methylvisamminol, were accumulated in thin roots. In a comparison of the components according to the drying temperature, a large difference was observed in the content of glycosides, and a difference was observed depending on the sugar-binding position. According to a metabolome analysis in domestic commercial products by LC/MS, a characteristic component in the cultivated product was found and its content was low in the 1 year-old sample and high in the 2 year-old sample. If the cultivation duration is prolonged up to about 6 years, the contents of the ingredients are close to those of wild products.

New isoflavone glucosides in yabumame (Amphicarpaea bracteata (L.) Fernald subsp. edgeworthii (Benth.) H.Ohashi var. japonica (Oliv.) H.Ohashi) and their effect on leukotriene B4 production in mast cells.

Yabumame (Amphicarpaea bracteata (L.) Fernald subsp. edgeworthii (Benth.) H.Ohashi var. japonica (Oliv.) H.Ohashi) is a legume plant that the Ainu people eat as a traditional food, although the bioactive ingredients other than vitamins have not been studied. In this study, the structures of yabumame isoflavone glucosides were determined and their effect on leukotriene (LT) B4, a chemical mediator of type I allergy, produced in mast cells, was investigated in vitro. Seven compounds were isolated from yabumame. Their structures were determined by spectroscopic and spectrometric analyses, which were genistein-7-O-ß-D-glucoside (1), formononetin-7-O-(2″-O-ß-D-glucosyl)-ß-D-glucoside (2), formononetin-7-O-ß-D-glucoside (3), biochanin A-7-O-(2″-O-ß-D-glucosyl)-ß-D-glucoside (4), formononetin-7-O-(6″-O-malonyl)-ß-D-glucoside (5), biochanin A-7-O-(2″-O-ß-D-glucosyl-6″-O-ß-D-glucosyl)-ß-D-glucoside (6), and biochanin A-7-O-(6″-O-malonyl)-ß-D-glucoside (7). Compounds 2, 4, and 6 were determined as new compounds. Compound 3 showed statistically significant suppressive effect on LTB4 production in mast cells, although the activity was not strong. On the other hand, biochanin A, an aglycone common to compounds 4, 6, and 7, strongly inhibited the LTB4 production. The results suggest that some of yabumame isoflavone glucosides might contribute to mitigate type I allergy. Seven isoflavone glucosides including 3 new compounds were found in yabumame and their anti-allergic effect was evaluated.

Evaluation of the safety and efficacy of Glycyrrhiza uralensis root extracts produced using artificial hydroponic and artificial hydroponic-field hybrid cultivation systems III: anti-allergic effects of hot water extracts on IgE-mediated immediate hypersensitivity in mice.

To evaluate the safety and efficacy of Glycyrrhiza uralensis root extracts produced using artificial hydroponic and artificial hydroponic-field hybrid cultivation systems, we investigated anti-allergic action in mice using IgE-mediated immediate hypersensitivity. Hot water extracts obtained from the roots of Glycyrrhiza uralensis cultivated using two systems were orally administered at a dose of 100 mg/kg as glycyrrhizin (GL) and compared with the commercial crude drug, Glycyrrhizae Radix. Both the artificial hydroponic and artificial hydroponic-field hybrid cultivated root extracts showed anti-allergic effects on IgE-mediated immediate hypersensitivity in mice, as did the commercial crude drugs. These results highlight the potential for artificially cultivated roots of Glycyrrhiza uralensis to be used as an alternative medicinal source.

Evaluation of the safety and efficacy of Glycyrrhiza uralensis root extracts produced using artificial hydroponic-field hybrid cultivation systems II: comparison of serum concentration of glycyrrhetinic acid serum concentration in mice.

To evaluate the safety and efficacy of Glycyrrhiza uralensis root produced using artificial hydroponic and artificial hydroponic-field hybrid cultivation systems, we investigated the pharmacokinetics of a major metabolite of glycyrrhizin (GL), glycyrrhetinic acid (GA). Hot water extracts obtained from the roots of the artificial hydroponic-field hybrid cultivated Glycyrrhiza uralensis were orally administered at a dose of 100 mg/kg as GL in mice and, compared with a commercial crude drug, Glycyrrhizae Radix. The temporal changes in serum GA concentration was found to depend on the GL concentration of the hot-water extracts. When hot-water extracts containing relatively high GL were administered, bimodal peaks appeared. In contrast, a broad single peak was detected when a hot-water extract containing relatively low GL content was administered. These tendencies in the serum GA concentration time course were observed for all samples, regardless of their derivation. Moreover, we compared the pharmacokinetic parameters and found that the Cmax and AUC0-48 values after oral administration of the extracts from Glycyrrhiza uralensis roots produced by the artificial cultivation system are within the range of variation for the commercial crude drugs. These results suggest the possibility that roots of Glycyrrhiza uralensis cultivated by the artificial hydroponic-field hybrid cultivation system can be used in addition to currently available commercial crude drugs produced from wild plant resources.

Determination of (E)-ferulic acid content in the root of Angelica acutiloba: a simple chemical evaluation method for crude drug quality control.

The root of Angelica acutiloba Kitagawa is an important crude drug in Kampo medicines (traditional Japanese medicine). Chemical evaluation of crude drugs is crucial to ensuring the safety and efficacy of herbal medicine; however, there is currently no chemical standard for the A. acutiloba crude drug in Japanese pharmacopoeia. (E)-ferulic acid (FA) is an important active ingredient of Angelica spp., including A. sinensis (Oliv.) Diels, and has been suggested as a marker for quality evaluation of those crude drugs. However, it has been controversial whether FA is a reliable marker constituent of A. acutiloba. To achieve effective extraction of FA from A. acutiloba, we compared three different extraction methods: alkaline hydrolysis, ethanol extraction, and hexane extraction. FA levels in these extracts were assessed using high performance liquid chromatography (HPLC), and alkaline hydrolysis was found to be the most effective. Furthermore, in the hydrolysate, FA was distinctly identified by thin layer chromatography (TLC) analysis. These results provide useful information for the quality control of the A. acutiloba crude drug.

Prenylated benzophenones from Triadenum japonicum.

Six new prenylated benzophenones, (-)-nemorosonol (1) and trijapins A-E (2-6), were isolated from the aerial parts of Triadenum japonicum. (-)-Nemorosonol (1) and trijapins A-C (2-4) have a common tricyclo[4.3.1.0(3,7)]decane skeleton, while 1 is an enantiomer of (+)-nemorosonol previously isolated from Clusia nemorosa. The absolute configuration of (-)-nemorosonol (1) was assigned by ECD spectroscopy. Trijapins A-C (2-4) are analogues of 1 possessing an additional tetrahydrofuran ring. Trijapins D (5) and E (6) are prenylated benzophenones with a 1,2-dioxane moiety and a hydroperoxy group, respectively. (-)-Nemorosonol (1) exhibited antimicrobial activity against Escherichia coli (MIC, 8 µg/mL), Staphylococcus aureus (MIC, 16 µg/mL), Bacillus subtilis (MIC, 16 µg/mL), Micrococcus luteus (MIC, 32 µg/mL), Aspergillus niger (IC50, 16 µg/mL), Trichophyton mentagrophytes (IC50, 8 µg/mL), and Candida albicans (IC50, 32 µg/mL), while trijapin D (5) showed antimicrobial activity against C. albicans (IC50, 8 µg/mL).

Diversity in aconitine alkaloid profile of Aconitum plants in Hokkaido contrasts with their genetic similarity.

Aconite tuber is a representative crude drug for warming the body internally in Japanese Kampo medicine and Chinese traditional medicine. The crude drug is used in major prescriptions for the aged. Varieties of Aconitum plants are distributed throughout the Japanese Islands, especially Hokkaido. With the aim of identifying the medicinal potential of Aconitum plants from Hokkaido, 107 specimens were collected from 36 sites in the summer of 2011 and 2012. Their nuclear DNA region, internal transcribed spacer (ITS), and aconitine alkaloid contents were analyzed. Phylogenic analysis of ITS by maximum parsimony analysis showed that the majority of the specimens were grouped into one cluster (cluster I), separated from the other cluster (cluster II) consisting of alpine specimens. The aconitine alkaloid content of the tuberous roots of 76 specimens showed 2 aspects-specimens from the same collection site showed similar aconitine alkaloid profiles, and cluster I specimens from different habitats showed various alkaloid profiles. Environmental pressure of each habitat is presumed to have caused the morphology and aconitine alkaloid profile of these genetically similar specimens to diversify.

Highly oxygenated monoterpene acylglucosides from Spiraea cantoniensis.

Six new monoterpene acylglucosides named kodemariosides A-F (1-6) were isolated from the leaves and flowers of Spiraea cantoniensis. Their absolute structures including a highly oxygenated monoterpene aglycon part were determined by NMR experiments and chemical derivatization.

Flavonol Caffeoylglycosides as alpha-Glucosidase Inhibitors from Spiraea cantoniensis Flower.

In the screening experiments for rat intestinal alpha-glucosidase inhibitors in 218 plants cultivated in the Japanese temperate region, potent maltase-inhibiting activity was found in the extract of flowers of Spiraea cantoniensis. The enzyme assay guided fractionation of the extract led to the isolation of three flavonol caffeoylglycosides, quercetin 3- O-(6- O-caffeoyl)-beta-galactoside ( 1), kaempferol 3- O-(6- O-caffeoyl)-beta-galactoside ( 2), and kaempferol 3- O-(6- O-caffeoyl)-beta-glucoside ( 3), as rat intestinal maltase inhibitors. This is the first report on the alpha-glucosidase-inhibitory activity of those flavonol caffeoylglycosides. Comparison in the activity of the isolates indicated the importance of caffeoyl substructures in the molecule for the alpha-glucosidase-inhibiting activity. The relatively high contents of the active isolates in the plant suggest that S. cantoniensis could be physiologically useful for treatment of diabetes.

Comparison of the major flavonoid content of S. baicalensis, S. lateriflora, and their commercial products.

According to the notification for definition of pharmaceuticals from the Director-General of the Pharmaceutical and Food Safety Bureau, Ministry of Health Labour and Welfare of Japan, the roots of Scutellaria baicalensis (Chinese skullcap) and S. lateriflora (skullcap) are classified as "the raw materials exclusively used as pharmaceuticals", but their aerial parts are classified as "non-pharmaceuticals" so, in principle, there are no health claims for these materials and no descriptions of drug-like dosages or administration directions. Dried root of S. baicalensis is also registered in Japanese Pharmacopoeia XV as scutellaria root. Scutellaria root is considered to have the adverse drug reactions of interstitial pneumonia and drug-induced hepatopathy in kampo medicines (Japanese traditional herbal formulations), and baicalin, its major constituent, is considered to be the cause of the adverse reaction. This study was conducted to evaluate the validity of this borderline between pharmaceuticals and non-pharmaceuticals by analyzing the amounts of four flavonoids, including baicalin, in the roots, stems, and leaves of S. baicalensis and S. lateriflora, and in the commercial products herbal tea and dietary supplements prepared from S. lateriflora. These flavonoids were found in the root of S. baicalensis; its aerial parts, however, did not contain them. On the other hand, the amounts of those flavonoids in the aerial parts of S. lateriflora were larger than in the root. Herbal tea and dietary supplements of S. lateriflora obtained commercially also contained those flavonoids, and the dietary supplements contained amounts of them comparable with that in kampo medicine. These results suggest that classification that the aerial parts of S. lateriflora as non-pharmaceuticals in Japan needs reconsideration.

Screening of indigenous plants from Japan for modulating effects on transformation of the aryl hydrocarbon receptor.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor with which halogenated and polycyclic aromatic hydrocarbons such as dioxins and benzo[a]pyrene interact as ligands. Since such compounds cause various toxicological effects, including cancer, through the transformation of AhR, it is important to determine influence of modulating factors. It has been reported that certain plant components such as flavonoids and indoles can affect AhR transformation. In this study, to obtain clues to novel ligands of AhR, 191 species of indigenous plants were collected in Japan, and their 50% methanolic extracts (total 368 plant parts) were tested for modulating effects on AhR transformation in a cell-free system using a rat hepatic cytosolic fraction. Among tested extracts at a concentration of 1 mg dry weight of plant/mL, 174 of 368 extracts suppressed 1 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced AhR transformation to 50% or less, while 9 extracts per se induced AhR transformation equivalent to more than 20% of that induced by 1 nM TCDD. Mallotus japonicus (Thunb.) Muell. (leaf) and Trichosanthes rostrata Kitamura (fruit and fruit skin) strongly suppressed 1 nM TCDD-induced AhR transformation, while Phellodendron amurense Ruprecht (seed) per se strongly induced AhR transformation. These results suggest that a large variety of plants in Japan contain various compounds modulating, mainly suppressing, AhR transformation.

The sequences of the spacer region between the atpF and atpA genes in the plastid genome allows discrimination among three varieties of medicinal Angelica.

The dried roots of Angelica acutiloba Kitagawa var. acutiloba Kitagawa, A. acutiloba Kitagawa var. iwatensis Hikino and A. acutiloba Kitagawa var. sugiyamae Hikino have been used as the herbal medicine known in Japan as Japanese Angelica Root. The respective morphological features of, in particular, A. acutiloba var. sugiyamae and A. acutiloba var. iwatensis are similar, and they are not easy to distinguish morphologically from each other. In an attempt to find a method for discriminating among these three varieties, we compared the nucleotide sequence of the spacer region between the atpF and atpA genes among the respective plastid genomes. Comparison of these sequences allowed us to identify each of the three varieties unequivocally.

The sequences of the plastid gene rpl16 and the rpl16-rpl14 spacer region allow discrimination among six species of Scutellaria.

Dried aerial parts of Scutellaria galericulata L. (Labiatae; marsh skullcap) and Scutellaria lateriflora L. (Labiatae; mad dog skullcap) are mainly used as skullcap, a medicinal herb, in Europe and the United States. The respective dried aerial parts of the two species are difficult to distinguish morphologically from each other. We attempted to discriminate among six species (Scutellaria altissima L. (Labiatae), Scutellaria baicalensis Georgi (Labiatae), Scutellaria galericulata, Scutellaria incana Spreng. (Labiatae), Scutellaria indica L. (Labiatae) and Scutellaria lateriflora) of Scutellaria, which include three medicinal species (Scutellaria galericulata, Scutellaria lateriflora and Scutellaria baicalensis), by comparing the respective nucleotide sequences of the plastid rpl16 gene and the rpl16-rpl14 spacer region. Comparisons of these sequences allowed us to identify each of the six species unequivocally.

Discrimination among species of Papaver based on the plastid rpl16 gene and the rpl16-rpl14 spacer sequence.

An attempt was made to discriminate among six species of Papaver (P. bracteatum, P. orientale, P. pseudo-orientale, P. rhoeas, P. setigerum and P. somniferum) by comparing the nucleotide sequences of the plastid rpl16 gene and the rpl16-rpl14 spacer region. Comparison of sequences allowed us to distinguish five species, namely P. bracteatum, P. orientale, P. pseudo-orientale, P. rhoeas and P. setigerum plus P. somniferum from one another, but sequences from P. setigerum and P. somniferum were identical. It is difficult to distinguish between P. bracteatum, P. orientale and P. pseudo-orientale at the vegetative stage of growth. However, our method allowed us to distinguish between these three species and the others using nucleotide sequences and should allow identification of P. bracteatum that has been cultivated illegally in the garden in Japan. Furthermore, P. rhoeas was clearly discriminated from P. setigerum and P. somniferum by reference to the sequence of the rpl16 exon using young seedlings.