Ryota Tsuchihashi

Microbial transformation and bioactivation of isoflavones from Pueraria flowers by human intestinal bacterial strains

The flowers from Pueraria, which are called Puerariae Flos, have been used since ancient times for recovery from alcohol intoxication. We elucidated the microbial transformation of the main isoflavones (1, 1a and 2) by using 29 commercially available human intestinal bacterial strains together with the bioactivation of the hepatoprotective activity of their metabolites. Tectoridin (1a), which contains only one glucosyl moiety, was metabolized to its aglycone 1b by various bacterial strains. On the other hand, the metabolism of 1 and 2, which both have disaccharide groups, was limited to specific bacterial strains. The metabolites 1c and 2c obtained from the Peptostreptococcus productus strain were completely different from those produced by the other strains. These metabolites were identified as 6-hydroxygenistein and 6-hydroxybiochanin A, respectively. The glycosides 1, 1a and 2 did not show any hepatoprotective activity, whereas aglycones 1b and 2b showed moderate activity. Furthermore, the hepatoprotective activity of the demethylated metabolites 1c and 2c was extremely potent. Although not all people have P. productus in their gastrointestinal tract, the O-demethylated compounds might become one of the bioactivated metabolites when Puerariae Flos is administered orally.

Microbial metabolism of soy isoflavones by human intestinal bacterial strains

Intestinal bacteria play an important role for the metabolism of soy isoflavonoids. When soy foods are consumed, the soy isoflavone glucosides are metabolized into their aglycones and the related isoflavonoids by intestinal bacteria. We designed an in vitro microbial metabolic system using 29 commercially available human intestinal bacterial strains and elucidated the metabolism of soy isoflavone glucosides. The strains were classified into three categories, which were 14 facultative anaerobes, 13 obligate anaerobes, and 2 aerobes. Almost all facultative anaerobe strains metabolized soy isoflavone glucosides to their aglycones. The ratio of metabolism from glucoside to aglycone was different in each strain. Contrary to the facultative anaerobes, some of the obligate anaerobes did not metabolize soy isoflavone glucosides at all. Both the aerobic bacteria hardly metabolized soy isoflavone glucosides. The bacterial growth speed might show good correlation to the metabolizing speed of both glucosides. Therefore, the speed of metabolism would be different in each bacterial strain, too.

Enzyme-linked immunosorbent assay for total isoflavonoids in Pueraria candollei using anti-puerarin and anti-daidzin polyclonal antibodies.

Pueraria candollei (White Kwao Khuer) is a medicinal plant containing puerarin, daidzin, genistin, daidzein, and genistein as major isoflavonoids used for its rejuvenating and estrogenic effects. In order to analyze these compounds, a single enzyme-linked immunosorbent assay (ELISA) for total isoflavonoids was developed using anti-puerarin and anti-daidzin polyclonal antibodies (PAbs). The range for calibration of isoflavonoids by ELISA was 0.05-6.25 microg/mL. Total isoflavonoid concentrations in P. candollei samples determined by the newly developed assay system showed good agreement with those analyzed by HPLC. Based on validation analysis, this analytical method by ELISA is a precise, accurate, and sensitive method for the determination of total isoflavonoids in P. candollei.

Simultaneous analysis of isoflavones and saponins in Pueraria flowers using HPLC coupled to an evaporative light scattering detector and isolation of a new isoflavone diglucoside

The Pueraria flowers, Puerariae Flos [Puerariae Lobatae Flos (the flowers of P. lobata) and Puerariae Thomsonii Flos (the flowers of P. thomsonii)], have been used as crude drugs to counteract the overconsumption of alcohol in Japan and China. Both types of Puerariae Flos contain a large amount of isoflavones and saponins. Simultaneous analysis of the total saponin and isoflavone fraction within the flowers has proven difficult thus far; however, profile analysis of saponin and isoflavone levels was attempted in this study by using HPLC coupled to an evaporative light scattering detector (ELSD). A characteristic peak of kakkalide was common in the chromatograms of extracts originating from the flowers of P. lobata, though original habitats were different. Tiny peaks of saponins were also observed in chromatograms of all specimens derived from P. lobata. In the chromatograms of six out of eight extracts of Puerariae Thomsonii Flos originating from Guangdong, China, characteristic twin peaks corresponding to tectorigenin 7-O-xylosylglucoside and tectoridin were observed. The distinctive twin peaks were not found in the chromatograms of the extracts produced in Hunan and Sichuan. Although the amounts of total saponins and isoflavones obtained from Puerariae Lobatae Flos were almost the same, those of Puerariae Thomsonii Flos varied remarkably and were not related to habitat. A good yield of a new isoflavone glycoside was obtained from some specimens of Puerariae Thomsonii Flos; the structure was determined to be 6-hydroxygenistein 6,7-di-O-glucoside.

Expression, purification, and characterization of anti-plumbagin single-chain variable fragment antibody in Sf9 insect cell.

Plumbagin (PL; 5-hydroxy-2-methyl-1, 4-naphthoquinone) is an important secondary metabolite, mainly produced in the Plumbago zeylanica L. (Plumbaginaceae). A single-chain variable fragment (scFv) antibody, fusion of the variable regions of the heavy chain and light chain of immunoglobulin against PL (PL-scFv) was expressed by Bac-to-Bac Baculovirus Expression System using Spodoptera frugiperda (Sf9) insect cells and characterized to investigate potential use of PL-scFv as a tool for plant immunomodulation. Functional PL-scFv expressed in the Sf9 insect cells were purified using cation exchange chromatography followed by immobilized metal ion affinity chromatography (IMAC). The yields of the purified PL-scFv in the culture supernatant and Sf9 insect cells were 2.0 mg and 5.2 mg per 1 liter of Sf9 culture medium, respectively. Recombinant purified PL-scFv was then characterized by the indirect competitive enzyme-linked immunosorbent assay (ELISA). The cross-reactivity and sensitivity of PL-scFv expressed in Sf9 insect cells were compared with PL-scFv expressed in Escherichia coli and its parental anti-plumbagin monoclonal antibody (MAb 3A3) secreted from hybridoma cells. Intriguingly, the specificity of the PL-scFv expressed in Sf9 insect cells was found to be different from that expressed in E. coli and parental MAb 3A3, although the detectable level (0.2-25 μg/mL) was the same in ELISA using each antibody. Even more interestingly, the characteristics of PL-scFv, which have wide cross-reactivity against 1,4-napththoquinone, suggest its potential use as a tool for plant immunomodulation not only for breeding Plumbaginacea family containing PL but also for breeding other medicinal plants containing bioactive naphthoquinones.

Screening of promising chemotherapeutic candidates against human adult T-cell leukemia/lymphoma from plants: active principles from Physalis pruinosa and structure–activity relationships with withanolides

Adult T-cell leukemia/lymphoma (ATL) is a peripheral T-cell malignancy caused by human T-cell lymphotropic virus type I (HTLV-1). Clinical manifestations of ATL range from smoldering to chronic, lymphoma and acute subtypes. Patients with acute and lymphoma-type ATL require therapeutic intervention. Conventional chemotherapeutic regimens used against other malignant lymphoma have been administered to ATL patients, but the therapeutic outcomes of acute and lymphoma-type ATL remain very poor. In this study, 214 extracts from 162 plants belonging to 65 families were screened for the purpose of elucidating the anti-proliferative effect against HTLV-1-infected T-cell lines. Extracts from aerial parts of Physalis pruinosa showed potent inhibitory effect. We isolated five withanolides from the extracts by activity-guided fractionation and examined the structure–activity relationships. The presence of a 5β,6β-epoxy function is suggested to be essential for the activity, and the most active principle showed selective toxicity to HTLV-1-infected T-cell lines.

Screening of promising chemotherapeutic candidates from plants against human adult T-cell leukemia/lymphoma (II): apoptosis of antiproliferactive principle (24,25-dihydrowithanolide D) against ATL cell lines and structure–activity relationships with withanolides isolated from solanaceous plants

Adult T-cell leukemia/lymphoma (ATL) is an incurable peripheral T-cell malignancy caused by human T-cell lymphotropic virus type I. In our preceding paper, 214 extracts from 162 plants were screened to elucidate the antiproliferative principles against ATL cell lines. Several withanolides were isolated and the structure–activity relationships (SAR) examined. To extend the search for SAR, 31 further withanolides, previously isolated from solanaceous plants, were tested against ATL cell lines. The presence of a 4β-hydroxy group as well as a 5β,6β-epoxy group appeared to be essential for the activity. In contrast, the presence of a sugar moiety at either the 3- or the 27-position led to a reduction in the activity. Furthermore, 24,25-dihydrowithanolide D (13) was identified as the most potent inhibitor, showing selective toxicity against ATL cell lines by inducing apoptotic cell death.

Screening of promising chemotherapeutic candidates from plants against human adult T-cell leukemia/lymphoma (V): coumarins and alkaloids from Boenninghausenia japonica and Ruta graveolens

During the course of our studies towards the identification of promising chemotherapeutic candidates from plants against two human T-cell lymphotropic virus type I-infected T-cell lines (MT-1 and MT-2), we screened 17 extracts from 9 rutaceous plants against MT-1 and MT-2 cells. The extracts from the aerial parts and roots of Boenninghausenia japonica, as well as the leaves and roots of Ruta graveolens showed potent antiproliferative effects. After activity-guided fractionation, we isolated 44 compounds from two rutaceous plants, including three new compounds (1–3), which were classified into 26 coumarin analogs (13 coumarins, 8 furanocoumarins, 4 dihydrofuranocoumarins and one dihydropyranocoumarin), 15 alkaloid analogs (7 quinolone alkaloids, 4 acridone alkaloids, 3 furanoquinoline alkaloids and one tetrahydroacridone alkaloid) and 3 flavonoid glycosides. Structure–activity relationship studies were also evaluated. The coumarin compounds (2, 3 and 7–9) bearing a 3-dimethylallyl moiety showed potent activity. Similarly, of all the furanocoumarins evaluated in the current study, compound 17 bearing a 3-dimethylallyl group also showed potent activity. A dihydrofuranocoumarin (27) bearing a 3-dimethylallyl moiety showed the most potent activity. Following 27, compound 28 showed potent activity. These results therefore suggested that the presence of a 3-dimethylallyl moiety was important to the antiproliferative activity of these coumarin analogs.