Aconitum carmichaelii is an important medicinal herb used widely in China, Japan, India, Korea, and other Asian countries. While extensive research on the characterization of metabolic extracts of A. carmichaelii has shown accumulation of numerous bioactive metabolites including aconitine and aconitine-type diterpene alkaloids, its biosynthetic pathway remains largely unknown. Biosynthesis of these secondary metabolites is tightly controlled and mostly occurs in a tissue-specific manner; therefore, transcriptome analysis across multiple tissues is an attractive method to identify the molecular components involved for further functional characterization. In order to understand the biosynthesis of secondary metabolites, Illumina-based deep transcriptome profiling and analysis was performed for four tissues (flower, bud, leaf, and root) of A. carmichaelii, resulting in 5.5 Gbps clean RNA-seq reads assembled into 128,183 unigenes. Unigenes annotated as possible rate-determining steps of an aconitine-type biosynthetic pathway were highly expressed in the root, in accordance with previous reports describing the root as the accumulation site for these metabolites. We also identified 21 unigenes annotated as cytochrome P450s and highly expressed in roots, which represent candidate unigenes involved in the diversification of secondary metabolites. Comparative transcriptome analysis of A. carmichaelii with A. heterophyllum identified 20,232 orthogroups, representing 30,633 unigenes of A. carmichaelii, gene ontology enrichment analysis of which revealed essential biological process together with a secondary metabolic process to be highly enriched. Unigenes identified in this study are strong candidates for aconitine-type diterpene alkaloid biosynthesis, and will serve as useful resources for further validation studies.

The pharmacological properties of Eleutherococcus senticosus leaf have not been clarified although it is taken as a food item. In this study, the effects of water extract of Eleutherococcus senticosus leaves on memory function were investigated in normal mice. Oral administration of the extract for 17 days significantly enhanced object recognition memory. Compounds absorbed in blood and the brain after oral administration of the leaf extract were detected by LC-MS/MS analyses. Primarily detected compounds in plasma and the cerebral cortex were ciwujianoside C3, eleutheroside M, ciwujianoside B, and ciwujianoside A1. Pure compounds except for ciwujianoside A1 were administered orally for 17 days to normal mice. Ciwujianoside C3, eleutheroside M, and ciwujianoside B significantly enhanced object recognition memory. These results demonstrated that oral administration of the leaf extract of E. senticosus enhances memory function, and that active ingredients in the extract, such as ciwujianoside C3, eleutheroside M, and ciwujianoside B, were able to penetrate and work in the brain. Those three compounds as well as the leaf extract had dendrite extension activity against primary cultured cortical neurons. The effect might relate to memory enhancement.

Numerous Foods with Function Claims that contain the extract of Pueraria flower (kudzu) isoflavones (PFI) are available in the Japanese market. These are labelled with function claims of reducing visceral fat. However, these foods have not undergone proper safety assessment such as the evaluation of their oestrogenic activity and effects on drug-metabolising enzymes (cytochrome P-450: CYP) in the liver. This study evaluated the estrogenic effect and the hepatic CYP activity and mRNA expression in normal female mice as a safety assessment of PFI (Experiment 1). In addition, the bone mineral density and visceral fat weight in ovariectomised mice (OVX) compared to soy isoflavones (SI) was evaluated to assess the efficacy of PFI (Experiment 2). OVX control fed a control diet, OVX fed a PFI diet (the recommended human intake of PFI), OVX fed a PFI20 diet (20- times the recommended PFI), OVX fed an SI diet (the recommended human intake of SI), and OVX fed an SI20 diet (20 -times the recommended intake of SI) for 28 days in Experiment 2. Body, liver, and visceral fat weights were not affected by the PFI, PFI20, SI, or SI20 diets. The hepatic CYP1A and CYP3A activities were elevated by the SI20 treatment. Ovariectomy-induced bone loss was inhibited by the SI20 treatment, but not by the PFI20 treatment. These results suggest that (1) PFI intake in human doses had no oestrogenic properties and did not affect CYP activity in the liver; (2) there was no evidence that PFI affects the amount of visceral fat in OVX mice.

Glycyrrhiza plants are important resources for sweeteners and medicines, because underground parts of them contain glycyrrhizic acid (GL), which has sweet taste and various pharmacological activities (ex. anti-inflammatory, antiallergy, antiviral activity, etc.). Although such importance of them, their supply still depends principally on the collection of wild plants. Therefore, it is an important issue to develop stable and efficient production system of Glycyrrhiza plants. To overcome this problem, we established the hydroponic cultivation system of Glycyrrhiza uralensis and selected superior G. uralensis clones with high-GL contents in the containment greenhouse. In this study, we aimed to develop a method of selecting these superior G. uralensis clones by DNA sequence polymorphisms in biosynthetic genes. Among the DNA sequences of GL biosynthetic key enzyme gene (CYP88D6), we found Glycyrrhiza species and clone-specific polymorphisms in intronic regions. By using these polymorphisms, discrimination among Glycyrrhiza species and G. uralensis clones became possible. Furthermore, the appearance frequency of superior clone-specific alleles in cloned CYP88D6 sequences was correlated with GL contents in crude drugs collected from the Japanese market. We also observed the tendency that G. uralensis seedlings having superior clone-specific alleles of CYP88D6 gene showed higher secondary metabolite productivity than those without the alleles. These results indicated that superior clone-specific alleles of CYP88D6 gene could be applied as DNA markers for selecting G. uralensis clones accumulating high secondary metabolites.

Aralia cordata is called “Udo” and is one of the famous herbaceous perennial plants found in Korea, China, and Japan. In Japan, aerial parts of A. cordata have been consumed. Furthermore, its rhizome and root are utilized as crude drugs known as “dokukatsu” and “wakyoukatsu”, respectively. A. cordata is cultivated as a vegetable in many places in Japan, and one of the production areas is Tokyo. A. cordata made in Tokyo is known as a high-quality “Udo” product (aerial part) using a unique cultivation method, known as “Udo muro”. “Udo muro” blocks light and maintains optimal temperature and humidity throughout the year, facilitating the soft cultivation of udo. However, the roots of A. cordata cultivated in Tokyo are all discarded. Thus, the utilization of the A. cordata root cultivated in Tokyo might lead to sustainability and income improvement for farmers. In this study, we investigated the effect of “Udo muro” with or without cultivation and drying temperature on chlorogenic acid (CA) contents in A. cordata root and compared it with A. cordata produced in other areas (“wakyoukatsu”) by a quantitative analysis of the CA content using high-performance liquid chromatography. The results indicate that the CA content of the roots of A. cordata grown in Tokyo was higher than those grown in other areas. Furthermore, the usefulness of A. cordata root was evaluated using inhibitory activity tests such as nitric oxide production and melanin production using Raw264.7 and B16F10 cell lines, respectively.

Paeonia lactiflora, the roots of which are used as a crude drug, is one of the most widely used and important medicinal plants. The long cultivation period and low proliferation rate of P. lactiflora makes it difficult to propagate large numbers of plants within a short period. We developed a bio-nursery system using plant tissue culture techniques to contribute to the supply of P. lactiflora seeds and seedlings in Japan. Here, we report on the improved tissue culture and acclimation conditions for a more stable and efficient bio-nursery system. We investigated the effect of culture conditions on shoot proliferation and the effect of calcium concentration during root induction and acclimation of cultured plantlets. The results demonstrated that the number of shoots increased under the 15/5°C diurnal temperature changing treatment [15°C, 12 h light (fluorescent light, 80–130 µmol m-2 s-1) / 5°C, 12 h dark] compared to a constant temperature of 15°C. A higher calcium concentration (6 mM Ca2+) during root induction resulted in more vigorous growth after transplantation to the soil. In addition, it was found that planting in a closed greenhouse at a constant temperature of 20°C after cold treatment was suitable for acclimation of cultured plantlets. These findings are expected to contribute to the future seedling supply of P. lactiflora.