Chemical quality evaluation and characteristics of interspecific hybrid (G. uralensis × G. glabra) C-18 strain as a new Licorice cultivar.

The development of new cultivars is essential for establishing a method of producing licorice in Japan. A suitable new cultivar for domestic licorice production, known as the interspecific hybrid strain C-18, was developed by crossbreeding Glycyrrhiza uralensis Fisch. (as the seed parent, possessing a high glycyrrhizin (GL) content, strain OMP-28) and Glycyrrhiza glabra L. (as the pollen parent, known for vigorous growth, strain OMP-10). Both G. uralensis and G. glabra are specified in the Pharmacopoeia of Japan (18th edition) as the source plants for Glycyrrhizae Radix. After 2 years of cultivation, strain C-18 exhibited robust growth, with a fresh weight of 148.8 g and a stem diameter of 0.89 mm. The GL content in the dry weight was measured at 3.61%. Seedlings cultivated from rhizomes in the field for 2 years showed a tap root fresh weight per plant of 120 ± 21 g, with an average GL content relative to the dry weight of 2.68% ± 0.38%. Although glabridin, a characteristic compound of G. glabra, was not detected, glycycoumarin, a characteristic compound of G. uralensis, was detected via HPLC analysis. Strain C-18 contained glycycoumarin as a characteristic compound of G. uralensis but lacked glabridin, a compound characteristic of G. glabra. Additionally, 2,3-dehydrokievitone (1) and parvisoflavone A (2) were identified as distinctive components of the interspecific hybrid (G. uralensis × G. glabra) C-18.

Characterization of a glycyrrhizin-deficient strain of Glycyrrhiza uralensis.

A triterpene saponin, glucoglycyrrhizin, was isolated from a glycyrrhizin-deficient strain 83-555 of Glycyrrhiza uralensis (Leguminosae), and the structure was determined by chemical and spectral data to be 3-O-[ß-D-glucopyranosyl-(1→2)-ß-D-glucuronopyranosyl]-glycyrrhetinic acid. Since this saponin has a 2'-O-ß-D-glucopyranosyl moiety instead of the 2'-O-ß-D-glucuronopyranosyl moiety of glycyrrhizin, the glucuronidation of 3-O-ß-D-glucuronopyranosyl-glycyrrhetinic acid leading to glycyrrhizin is inhibited in this strain. All 4 offspring of the 83-555 strain produced glucoglycyrrhizin. Interestingly, 2 of the offspring produced both glycyrrhizin and glucoglycyrrhizin, and sequence analysis of the pkr gene suggested that these 2 offspring were hybrids of 83-555 strain and glycyrrhizin-producing strains.

Seasonal variation in the total saponin content of platycodon roots cultivated in Japan.

Roots of Platycodon grandiflorus A. De Candolle (Campanulaceae), with the bark removed, have been used as food and frequently employed as herbal medicines for inflammatory diseases such as tonsillitis, dermatitis, and cough. Platycodins are the bioactive saponin components of these crude medicines. Recently, P. grandiflorus have been cultivated in Japan and are harvested from October to December according to conventional practices. Seasonal fluctuations in the total saponin content of these roots were determined using LC/MS methods to recommend harvesting times when the saponin content is high. Platycodins A and C are monoacetylated forms of platycodin D; however, the acetyl form is unstable and deacetylates easily. Here, the contents of platycodin D, platycodin D2, and platyconic acid A were measured as the total saponin content using alkaline hydrolysis for monoacetylated platycodins D, D2, and platyconic acid A. The results demonstrated that the saponin content in the roots decreased in summer, increased in autumn, but decreased again in late autumn.

Determination of flavonoids in licorice using acid hydrolysis and reversed-phase HPLC and evaluation of the chemical quality of cultivated licorice.

Licorice contains flavonoids and triterpenoids as the major bioactive components. Most of the flavonoids are the glycosidic form of liquiritigenin (LIQ), isoliquiritigenin (ISO) and formononetin (FOR). A reversed-phase HPLC method for the quantification of LIQ, ISO and FOR in licorice was developed. This method does not measure each glycoside but measures the aglycones using acid hydrolysis. All calibration curves showed good linear regression (r > 0.9998). The method showed good precision for intraday (RSD < 2.14 %) and interday (RSD < 0.51 %) assays. The limit of detection was 0.031 microg for LIQ, 0.011 microg for ISO and 0.006 microg for FOR. The limit of quantification was 0.31 microg for LIQ, 0.11 microg for ISO and 0.06 microg for FOR. The flavonoid contents along with the glycyrrhizin content of cultivated licorice from seedling plants in Japan and commercial wild licorice were investigated. This new method could be extremely useful for evaluating the quality of licorice.

Anti-inflammatory activities of Ophiopogonis Radix on hydrogen peroxide-induced cellular senescence of normal human dermal fibroblasts.

Ophiopogonis Radix (Ophiopogon root), which nourishes the yin, has been used in clinical practice to promote fluid secretion and to moisturize the lungs and skin in traditional Chinese and Japanese (Kampo) medicine. To evaluate this traditional medicinal effect, we investigated the anti-chronic inflammatory effect of Radix Ophiopogonis on senescent cells. Conversely, although several phenotypes of Ophiopogon japonicus Ker-Gawler (Liliaceae) are prevalent in Japan and China, we used these Ophiopogon roots by considering them as one crude drug, Ophiopogonis Radix. In this study, it was revealed that there are two chemotypes (Types A and B) in the root of the original plant, O. japonicus. Methylophiopogonanone A (compound 1) and methylophiopogonanone B (compound 2) were isolated as index compounds from Type A and compound 1 and ophiopogonanone A (compound 3) from Type B. In addition, ophiopogonin B (compound 4) was isolated as the main steroidal saponin from both Type A and B. The results indicated that two different methanol extracts (from Types A and B) and the main constituents of O. japonicus (compound 1-4), significantly downregulated the expression of interleukin (IL)-6 and IL-8, which were enhanced by senescent normal human dermal fibroblasts. Moreover, the two different methanol extracts and compounds 1-4 decreased IL-6 production in a strong and concentration-dependent manner by the ELISA method.

Evaluation of the chemical quality of Sekkoku () in current Japanese commercial crude drugs: constituents of Flickingeria xantholeuca (Rchb. f.) A.D. Hawkes.

In our investigation, most Shihu (; Japanese name, Sekkoku) in current Japanese commercial crude drugs were from Flickingeria xantholeuca (Orchidaceae). As the index compounds, three new ent-pimarane-type diterpenes, flickinxthanthosides A-C (1-3), one known analogue (7), and three new ent-kaurane-type diterpenes, flickinxanthosides D (4) and E (5) and flickinxanthol A (6) were isolated from the stem of F. xantholeuca. The structures of the new compounds were elucidated on the basis of spectroscopic analyses and chemical methods. We attempted to detect these index compounds from the MeOH extracts of other Dendrobium or Flickingeria plants using TLC and LC/MS.

Rice Proteome Database based on two-dimensional polyacrylamide gel electrophoresis: its status in 2003.

The Rice Proteome Database is the first detailed database to describe the proteome of rice. The current release contains 21 reference maps based on two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) of proteins from rice tissues and subcellular compartments. These reference maps comprise 11 941 identified proteins showing tissue and subcellular localization, corresponding to 4180 separate protein entries in the database. The Rice Proteome Database contains the calculated properties of each protein such as molecular weight, isoelectric point and expression; experimentally determined properties such as amino acid sequences obtained using protein sequencers and mass spectrometry; and the results of database searches such as sequence homologies. The database is searchable by keyword, accession number, protein name, isoelectric point, molecular weight and amino acid sequence, or by selection of a spot on one of the 2D-PAGE reference maps. Cross-references are provided to tools for proteomics and to other 2D-PAGE databases, which in turn provide many links to other molecular databases. The information in the Rice Proteome Database is updated weekly, and is available on the World Wide Web at http://gene64.dna.affrc.go.jp/RPD/.

[Pharmaceutical botanical studies on some Glycyrrhiza species].

Some Glycyrrhiza species grown in several domestic research gardens of medicinal plants were collected by the Osaka University of Pharmaceutical Sciences and were cultivated to compare their morphological properties. HPLC profile analysis was performed and index compounds of MeOH extracts of aerial parts and EtOAc extracts of subterranean parts were determined. Glycyrrhizin contents and growth rates of the underground parts of some types of Glycyrrhiza uralensis and Glycyrrhiza glabra were compared and four excellent types were selected as candidates for cultivation. One of them was due to Kanzo-Yashiki (Enzan, Yamanashi prefecture), where G. uralensis was cultivated in the Edo period. Alkaloidal constituents of G. uralensis and G. glabra were also investigated and anabasine (an insecticide) and a new tricyclic alkaloid were obtained.

Aim for production of Glycyrrhizae Radix in Japan (3): development of a new licorice cultivar.

The development of cultivars is indispensable for the establishment of a method aimed at producing licorice in Japan. The cultivar should have the following attributes: (1) the underground parts should grow vigorously; (2) the glycyrrhizin (GL) content must be 2.5 % or greater; and (3) the architecture of the aerial parts should be erect. A new cultivar suitable for the domestic production of licorice was developed by crossbreeding between strain A-19 (with a high GL content) as the mother and strain G-6 (with vigorous growth) as the father. After 2 years of cultivation, strain C-2 exhibited vigorous growth; the fresh weight and stem diameter were 148.8 g and 0.89 mm, respectively. Moreover, the dry-weight GL and total flavonoid contents of the new cultivar (strain C-2) from cultured plants were 3.61 and 1.365 %, respectively.

Comparative analysis of ten strains of Glycyrrhiza uralensis cultivated in Japan.

Comparative analysis of 10 strains of Glycyrrhiza uralensis cultivated in Kyoto, Japan, was undertaken to characterize their variations. Based on the chemical characteristics of their leaves and underground parts, the 10 strains were divided into two chemotypes, the China type and Kazakhstan type. The contents of licoleafol in the leaves of the China type (0-0.03% of dry weight) were lower than those of the Kazakhstan type (0.05-1.16% of dry weight). In addition, a China type-specific unidentified compound was also detected in the leaves of China-type plants. Glycyrrhizin contents in the underground parts of the China type (2.08-5.12% of dry weight) were relatively higher than those of the Kazakhstan type (0.75-2.55% of dry weight). Contents of glycycoumarin, a species-specific flavonoid of G. uralensis, in the underground parts of China-type plants (0.07-0.28% of dry weight) were higher than those of Kazakhstan-type plants (0.01-0.08% of dry weight). These 10 strains were also divided into two genotypes, the GA type and AT type, based on their chloroplast ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit gene (rbcL) sequences, although there was no correlation between the chemotype and the rbcL genotype.