Chemical Analysis of Counterfeit Hepatitis C Drug Found in Japan.

In January 2017, counterfeits of the hepatitis C drug 'HARVONI® Combination Tablets' (HARVONI®) were found at a pharmacy chain through unlicensed suppliers in Japan. A total of five lots of counterfeit HARVONI® (samples 1-5) bottles were found, and the ingredients of the bottles were all in tablet form. Among them, two differently shaped tablets were present in two of the bottles (categorized as samples 2A, 2B, 4A, and 4B). We analyzed the total of seven samples by high-resolution LC-MS, GC-MS and NMR. In samples 2A, 3 and 4B, sofosbuvir, the active component of another hepatitis C drug, SOVALDI® Tablets 400 mg (SOVALDI®), was detected. In sample 4A, sofosbuvir and ledipasvir, the active components of HARVONI®, were found. A direct comparison of the four samples and genuine products showed that three samples (2A, 3, 4B) are apparently SOVALDI® and that sample 2A is HARVONI®. In samples 1 and 5, several vitamins but none of the active compounds usually found in HARVONI® (i.e., sofosbuvir and ledipasvir) were detected. Our additional investigation indicates that these two samples are likely to be a commercial vitamin supplement distributed in Japan. Sample 2B, looked entirely different from HARVONI® and contained several herbal constitutents (such as ephedrine and glycyrrhizin) that are used in Japanese Kampo formulations. A further analysis indicated that sample 2B is likely to be a Kampo extract tablet of Shoseiryuto which is distributed in Japan. Considering this case, it is important to be vigilant to prevent a recurrence of distribution of counterfeit drugs.

Efficiently prepared ephedrine alkaloids-free Ephedra Herb extract: a putative marker and antiproliferative effects.

Ephedrine alkaloids (EAs) have been considered the main pharmacologically active substances in Ephedra Herb (, Mao; EH) since they were first identified by Prof. N. Nagai, and are known to induce palpitation, hypertension, insomnia, and dysuria as side effects. Therefore, the administration of drugs containing EH to patients with cardiovascular-related diseases is severely contraindicated. While our previous studies suggest that some of the effects of EH may not be due to EAs, considering their side effects would be expedient to develop a new EAs-free EH extract (EFE). Here, we established a preparation method for EFE and revealed its chemical composition, including the content of herbacetin, a flavonoid aglycon present in EH and a potential putative marker for EFE quality control. In addition, we showed the antiproliferative effects of EFE against the H1975 non-small cell lung cancer (NSCLC) cell line. EFE was prepared from EH extract using the ion exchange resin SK-1B. LC/Orbitrap MS analysis revealed the removal of EAs, 6-methoxykynurenic acid, and 6-hydroxykynurenic acid from the original extract. Quantitative analysis of herbacetin using LC/MS in acid-hydrolyzed EFE showed that its content was 0.104 %. Although several alkaloidal constituents were removed from EH extract, the antiproliferative effect of EFE against H1975 cells was comparable to that of EH extract. These results indicate that EFE retained the anticancer effect of EH and demonstrated its potential for future development as a new herbal medicine with reduced side effects.

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.

Identification of marker compounds for Japanese Pharmacopoeia non-conforming jujube seeds from Myanmar.

Jujube seed is a crude drug defined as the seed of Ziziphus jujuba Miller var. spinosa Hu ex H.F. Chou (Rhamnaceae) in the Japanese Pharmacopoeia (JP). Most of the jujube seed in the Japanese markets is imported from China, with the rest obtained from other Asian countries. Here we confirmed the botanical origins of jujube seeds from both China and Myanmar by a DNA sequencing analysis. We found that the botanical origins of the crude drugs from China and Myanmar were Z. jujuba and Z. mauritiana, respectively. Although the jujube seed from China conforms to the JP, that from Myanmar does not. A method for discriminating jujube seeds from China and Myanmar using a chemical approach is thus desirable, and here we sought to identify a compound specific to Z. jujuba. Jujuboside A (1) was identified as a compound specific to Z. jujuba. To establish a purity test of Jujube Seed in the JP against Z. mauritiana, we fractionated the extract of Z. mauritiana seeds and identified frangufoline (2) and oleanolic acid (4) as the marker compounds specific to Z. mauritiana. Thin-layer chromatography (TLC) and gas chromatography-mass spectrometry analyses revealed that the latter compound was useful for testing by TLC analysis. The established TLC conditions were as follows: chromatographic support, silica gel; developing solvent, n-hexane:EtOAc:HCOOH = 10:5:1; developing length, 7 cm; visualization, diluted sulfuric acid; R f value, 0.43 (oleanolic acid).

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.

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.

Identification of mutaprodenafil in a dietary supplement and its subsequent synthesis.

We isolated a new illegal sildenafil analogue named mutaprodenafil from a dietary supplement for erectile dysfunction (ED) and proposed that it is an aildenafil derivative containing an imidazole moiety. We subsequently synthesized mutaprodenafil from a thioaildenafil and authenticated its structure.

[Analytical data of designated substances (Shitei-Yakubutsu) controlled by the Pharmaceutical Affairs Law in Japan, part I: GC-MS and LC-MS].

In the last 10 years, many analogs of narcotic substances have been widely distributed in Japan as easily available psychotropic substances and this has become a serious problem. They have been sold as video cleaners, incense and reagents via the Internet or in video shops. They are not controlled under the Narcotics and Psychotropics Control Law because their pharmacological effects have not yet been proved scientifically. As a countermeasure to prevent the abuse of these substances, the Ministry of Health, Labor and Welfare amended the Pharmaceutical Affairs Law in 2006 so that 31 non-controlled psychotropic substances (11 tryptamines, 11 phenethylamines, 6 alkyl nitrites, 2 piperazines and salvinorin A) and 1 plant (Salvia divinorum) are now controlled as "Designated Substances (Shitei-Yakubutsu)" as of April 2007. Five other compounds (4 phenethylamines and 1 piperazine) were also added to this category in January 2008. In this study, we developed simultaneous analytical methods for these designated substances using gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) and present retention times, UV spectra, electron ionization (EI), GC-MS, and electrospray ionization (ESI) LC-MS data.

[Analytical data of designated substances (Shitei-Yakubutsu) controlled by the Pharmaceutical Affairs Law in Japan, part II: Color test and TLC].

Many psychotropic substances are readily available in Japan via the Internet. To avoid the spread of drug abuse, some drugs have been controlled as designated substances (Shitei-Yakubutsu) in Japan since 2007 by the Pharmaceutical Affairs Law. Twenty-nine designated substances (classified as tryptamine, phenethylamine and piperazine types) were analyzed using color tests and TLC. The color tests were examined with the Marquis, Ehrlich, Simon's, Liebermann-Burehard's, and Mandelin reagents. The color of beta-carbonyl-methylenedioxyphenetylamines produced by the Marquis reagent was yellow, and 4-halo-2,5-dimethoxy phenethylamines reacted with the Marquis reagent to a give deep yellow-green and/or a deep green color. Although all designated substances of the tryptamine type reacted with the Ehrlich reagent to give a brown color, only 1-(2,4,6-trimethoxyphenyl) propan-2-amine (TMA-6) among the phenethylamines showed a red color on treatment with the reagent. However, 3,4,5-trimethoxy and 2,4,5-trimethoxy isomers of TMA-6 were not colored with the reagent. Thus, TMA-6 could be distinguished from isomers using the Ehrlich reagent. We also analyzed the designated substances with thin-layer chromatography developed with two different solvent conditions. All substances were detected by UV(254 nm) and an iodoplatinate reagent. These results suggest that color tests and TLC, followed by GC-MS and LC-MS analyses, can be used for preliminary identification of designated substances.

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.

Detection of 1-O-malylglucose: pelargonidin 3-O-glucose-6''-O-malyltransferase activity in carnation (Dianthus caryophyllus).

Carnations have anthocyanins acylated with malate. Although anthocyanin acyltransferases have been reported in several plant species, anthocyanin malyltransferase (AMalT) activity in carnation has not been identified. Here, an acyl donor substance of AMalT, 1-O-beta-D-malylglucose, was extracted and partially purified from the petals of carnation. This was synthesized chemically to analyze AMalT activity in a crude extract from carnation. Changes in the AMalT activity showed close correlation to the accumulation of pelargonidin 3-malylglucoside (Pel 3-malGlc) during the development of red petals of carnation, but neither AMalT activity nor Pel 3-malGlc accumulation was detectable in roots, stems and leaves.

[Authentication and ultra performance liquid chromatography (UPLC)/MS analysis of magic mint, Salvia divinorum and its related plants].

Ultra performance liquid chromatography (UPLC)/mass spectrometry (MS) analysis was performed to investigate whether commercial Salvia cultivars available in the Japanese market contain salvinorin A (1), which is an hallucinogen present in magic mint (Salvia divinorum) prior to the regulation of S. divinorum by the Japanese Pharmaceutical Affairs Law. In addition, a previously reported method to authenticate S. divinorum, utilizing an amplification refractory mutation system (ARMS) was applied to the same samples to estimate the method's accuracy. As a result of the UPLC/MS analysis, it was clear that none of the tested cultivars possessed 1 while S. divinorum leaves and its processed products "concentrated salvia" contained 1 in the range from 0.19% to 0.58%. Furthermore, the ARMS method could clearly distinguish S. divinorum from the tested cultivars. In conclusion, the authentication method is considered to be useful for the practical regulation of S. divinorum due to its simplicity and accuracy.

Authentication and chemical study of isodonis herba and isodonis extracts.

Isodonis Herba is used as a Japanese dietary supplement and folk medicine. The extract of the herb (Isodonis extract) is also used as a food additive whose major compound is enmein (1). Here we compared internal transcribed spacer sequences of nuclear ribosomal DNA from Isodonis Herba available on the Japanese and Chinese crude drug markets, and found that the former derived from Isodon japonicus and Isodon trichocarpus, while the latter derived from distinct species such as Isodon eriocalyx. The liquid chromatography/mass spectrometry profiles of Isodonis Herba were classified into four chemotypes (A to D) according to the ratio of the major constituents. Types B and C contained 1 and oridonin (2) as major components, respectively. An intermediate (or mixed) form of types B and C in various ratios was designed type A. Type D contained eriocalyxin B (3) as its major component. Japanese herba were types A-C, while Chinese herba were types C and D. The commercial Isodonis extract products tested were classified as type D, suggesting that they originated from Chinese Herba. Understanding the relationship between extract constituents and DNA profiles is important for the official specification of dietary supplements and food additives of plant origin.