Takuro Maruyama

Simultaneous analysis of mitragynine, 7-hydroxymitragynine, and other alkaloids in the psychotropic plant “kratom” (Mitragyna speciosa) by LC-ESI-MS

The leaves of Mitragyna speciosa, a tropical plant known as “kratom,” have been traditionally used as a substitute for opium in Thailand and Malaysia. Mitragynine, a major constituent of M. speciosa, has an opioid agonistic activity, and its derivative 7-hydroxymitragynine (7-OH-mitragynine) (a minor constituent) is much more potent than mitragynine or morphine. Recently, many products containing this plant have been distributed as “incense” on the drug market in Japan for their expected narcotic effects. Despite their potency and their wide distribution for abuse, there are no reports on the quantitative analysis of mitragynine and 7-OH-mitragynine in the raw materials or in the commercial products of kratom. In this study, a method for simultaneous analysis of mitragynine, 7-OH-mitragynine, and other indole alkaloids (speciogynine, speciociliatine, and paynantheine), present in the raw materials and commercial products of kratom, was developed using liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS). By this method, mitragynine, 7-OH-mitragynine, and the other alkaloids were detected in 11 of the 13 products. The content of mitragynine in the products ranged from 1% to 6%, and that of 7-OH-mitragynine from 0.01% to 0.04%. Because 7-OH-mitragynine is much more potent than morphine, M. speciosa abuse is a matter of major concern. The present analytical method is considered useful for the screening of M. speciosa products in the drug market.

Paradisin C: A new CYP3A4 inhibitor from grapefruit juice

A new furanocoumarin derivative, paradisin C, was isolated from grapefruit juice as an inhibitor of cytochrome P450 (CYP) 3A4. Its stereochemistry was elucidated by spectroscopic methods. The stereochemistries of 17,18-dihydroxybergamottin and 17-epoxybergamottin were also elucidated.

The botanical origin of kratom ( Mitragyna speciosa ; Rubiaceae) available as abused drugs in the Japanese markets

Kratom is the leaves of Mitragyna speciosa (Rubiaceae). Recently, kratom has been sold in street shops or on the Internet in Japan for the purpose of abuse due to its opium-like effects. In this study, we investigated the botanical origin of the commercial kratom products using the internal transcribed spacer (ITS) sequence analysis of rDNA in preparation for future regulation of this product. In addition, a previously reported method to authenticate the plant, utilizing polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was applied to the same products in order to estimate the method’s accuracy and utility. The ITS sequence analysis of the commercial kratoms revealed that most of them were derived from M. speciosa or closely related plants, while the others were made from the same tribe plant as M. speciosa. The reported PCR-RFLP method could clearly distinguish kratoms from the other psychoactive plants available in the Japanese markets and also from related plants. The authentication method is considered to be useful for the practical regulation of the plant due to its wide range of application, high accuracy and simplicity.

Authentication of the Traditional Medicinal Plant Eleutherococcus senticosus by DNA and Chemical Analyses

Shigoka (SGK), the rhizome of Eleutherococcus senticosus, is a traditional medicine used as a tonic in northeastern Asia and far eastern Russia. We analyzed the nuclear ribosomal DNA internal transcribed spacer (ITS) sequence of the medicine available on the Japanese and Chinese markets and found that at least 3 species were used as the source plant of the commercial SGKs and that only 70% of all samples was made from the correct species. Furthermore, we performed the quantitative determination of 3 marker compounds, eleutheroside B (EB), syringaresinol diglucoside (Syr), and isofraxidin (Iso) by ultraperformance liquid chromatography (UPLC)/mass spectrometry (MS). We found that EB and Iso are specific to the correct source plant of SGK. Of them, EB is thought to be the best marker compound for quality assurance of the SGK from the viewpoint of its pharmacological activity.

Ephedrine alkaloids-free Ephedra Herb extract: a safer alternative to ephedra with comparable analgesic, anticancer, and anti-influenza activities

It is generally accepted that the primary pharmacological activities and adverse effects of Ephedra Herb are caused by ephedrine alkaloids. Interestingly, our research shows that Ephedra Herb also has ephedrine alkaloid-independent pharmacological actions, such as c-MET inhibitory activity. This study describes the preparation of an ephedrine alkaloids-free Ephedra Herb extract (EFE) by ion-exchange column chromatography, as well as in vitro and in vivo evaluation of its pharmacological actions and toxicity. We confirmed that EFE suppressed hepatocyte growth factor (HGF)-induced cancer cell motility by preventing both HGF-induced phosphorylation of c-Met and its tyrosine kinase activity. We also investigated the analgesic effect of EFE. Although the analgesic effect of Ephedra Herb has traditionally been attributed to pseudoephedrine, oral administration of EFE reduced formalin-induced pain in a dose-dependent manner in mice. Furthermore, we confirmed the anti-influenza virus activity of EFE by showing inhibition of MDCK cell infection in a concentration-dependent manner. All assessments of toxicity, even after repeated oral administration, suggest that EFE would be a safer alternative to Ephedra Herb. The findings described here suggest that EFE has c-Met inhibitory action, analgesic effect, and anti-influenza activity, and that it is safer than Ephedra Herb extract itself. Therefore, EFE could be a useful pharmacological agent.

Biological Activities of Indian Celery, Seseli diffusum (Roxb. ex Sm.) Sant. & Wagh

In continuation of our work on Indian celery (Seseli diffusum (Roxb. ex Sm.) Santapau & Wagh; Umbelliferae), the fractionation of the 80% MeOH–H2O extract of the seeds was performed to identify the principles responsible for its folk use as an antispasmodic and diuretic. Several compounds were isolated as active components: seselin (1) and anthriscinol methyl ether (4) showed a selective cytotoxicity to some yeast strains. Compound 1 also showed spasmolytic activity. On the other hand, isopimpinellin (3) and isorutarin (5) exhibited a spasmogenic effect on the smooth muscle preparations. Compound 5 was also found to have antioxidant activity. Among them, compound 4 was isolated for the first time from this plant. Copyright

Chemical analysis reveals the botanical origin of shatavari products and confirms the absence of alkaloid asparagamine A in Asparagus racemosus

Shatavari—a famous Ayurveda materia medica used mainly as a tonic for women—is distributed in health food products all over the world. The Ayurvedic Pharmacopoeia of India identifies the botanical origin of shatavari as the tuberous root of Asparagus racemosus. We recently investigated by DNA analysis the botanical origin of shatavari products on the Japanese market. The results suggested that their botanical origin was Asparagus; however, species identification was difficult. In this study, we analyzed steroidal saponins, including those specific to this plant, in these products and confirmed their origin as A. racemosus. Next, alkaloid analyses of an authentic A. racemosus plant and these products were performed, because several papers have reported the isolation of a pyrrolo[1,2-a]azepine alkaloid, asparagamine A, from this plant. Our results suggest that neither plant material nor products contained asparagamine A. It has been pointed out that Stemona plants are sometimes mistaken for shatavari, because their tuberous roots have a similar shape to that of A. racemosus, and pyrrolo[1,2-a]azepine alkaloids are thought to be Stemona-specific. These data strongly suggest that A. racemosus does not contain asparagamine A, and that previous isolation of asparagamine A from materials claimed as originating from A. racemosus was likely caused by misidentification of Stemona plants as A. racemosus.

Botanical origin of dietary supplements labeled as “Kwao Keur”, a folk medicine from Thailand

In the course of our study on the quality of dietary supplements in Japan, both the internal transcribed spacer (ITS) sequence of nrDNA and the rps16 intron sequence of cpDNA of products labeled as “Kwao Keur” were investigated. As a result, the DNA sequence of Pueraria candollei var. mirifica, which is the source plant of Kwao Keur, was observed in only about half of the products. Inferred from the determined sequences, source plants in the other products included Medicago sativa, Glycyrrhiza uralensis, Pachyrhizus erosus, and Ipomoea batatas, etc. These inferior products are estimated to lack the efficacy implied by their labeling. In order to guarantee the quality of dietary supplements, it is important to identify the source materials exactly; in addition, an infrastructure that can exclude these inferior products from the market is needed for the protection of consumers from potential damage to their health and finances. The DNA analysis performed in this study is useful for this purpose.

Isoheleproline: a new amino acid-sesquiterpene adduct from Inula helenium

A new amino acid–sesquiterpene adduct, isoheleproline (1), was isolated from the roots of Inula helenium (elecampane), together with four known sesquiterpene lactones (2–5). The planar configuration of 1 was elucidated on the basis of spectroscopic data analysis, and the relative configuration of 1 was determined by performing a detailed analysis of NOESY correlations and comparing its physicochemical data with the d- and l-proline adducts of 2 obtained by Michael addition. This is the first report of a new amino acid–sesquiterpene adduct from Inula plants.

Determination and identification of a specific marker compound for discriminating Shrub Chaste Tree Fruit from Agnus Castus Fruit based on LC/MS metabolic analysis

Shrub Chaste Tree Fruit (SCTF) is defined as the fruits of Vitex rotundifolia L. f. and V. trifolia L. and has been used as a component of some traditional Japanese medicines (Kampo formulations). Agnus Castus Fruit (ACF) is defined as the dried ripe fruits of V. agnus-castus L.; it is used in traditional European medicines, but is becoming popular in Japan as both an over-the-counter drug and as an ingredient in health foods for treating premenstrual syndrome (PMS). To ensure the efficacy and safety of both SCTF and ACF products, it is important to precisely authenticate their botanical origins and to clearly distinguish between SCTF and ACF. Therefore, we tried to identify SCTF-specific marker compounds based on LC/MS metabolic analysis. The multivariate analysis of LC/MS data from SCTF and ACF samples furnished candidate marker compounds of SCTF. An SCTF-specific marker was isolated from SCTF crude drugs and identified as 3-O-trans-feruloyl tormentic acid on the basis of spectroscopic data from NMR and MS. Since avoiding contamination from closely related species is a significant requirement for pharmaceuticals of natural origin, this information will be valuable for the quality control of both SCTF and ACF products from the viewpoint of regulatory science.