Resultant compound from sublimation test for Gentianae Radix in Japanese Pharmacopoeia was 5-(hydroxymethyl)furfural.

Gentianae Radix, an herbal medicine, has been used as a gastrointestinal drug in Japan. In the Japanese Pharmacopoeia 18th Revision, the sublimation test is specified as an identification test for Gentianae Radix. The compound obtained in this sublimation test was believed to be gentisin, a xanthone family compound. However, the compound we identified using liquid chromatography-high-resolution mass spectrometry (LC-HRMS) and 1H- and 13C-NMR was 5-(hydroxymethyl)furfural (5-HMF). The same compound was found to be a sublimate of Gentianae Scabrae Radix and Gentianae Macrophyllae Radix, belonging to the same genus as Gentianae Radix. These results indicate the necessity to revise the identification test for Gentianae Radix to a more unique method.

Comparison of ephedrine and pseudoephedrine contents in 34 Kampo extracts containing Ephedrae Herba used clinically in Japan.

Ephedrae Herba is among the important crude drugs prescribed in Kampo medicine for the treatment of cold, flue, rhinitis, nasal congestion, cough, and asthma. The active ingredients of Ephedrae Herba, ephedrine (E) and pseudoephedrine (PE), are potent sympathomimetic compounds that stimulate α-, ß1-, and ß2-adrenoceptors resulting in dilatation and alleviation of nasal mucosal hyperemia. Hypertension, palpitations, insomnia, and dysuria are the main adverse effects of E and PE, which can be avoided by determining the actual contents of these alkaloids in Kampo extracts containing Ephedrae Herba. However, the extraction efficiencies of E and PE from Ephedrae Herba contained in Kampo formulas in combination with other crude drugs remain unknown. Therefore, we comprehensively determined the E and PE contents of 34 Kampo extracts containing Ephedrae Herba used clinically in Japan. The E and PE contents per daily dosage in Kampo extracts were generally proportional to the compounding amount of Ephedrae Herba. In contrast, the extraction efficiencies of E or PE were not constant and not influenced by the pH of the extracts. We assume that the extraction efficiencies of E and PE may be independently affected by other constituent crude drugs. Thus, it is necessary to investigate the cause and mechanism in the future. In conclusion, these results show that the E and PE content of each Kampo formulation can be estimated from the compounding amount of Ephedrae Herba. Therefore, the amount of Ephedrae Herba should be carefully considered to ensure the safe use of Kampo formulations containing Ephedrae Herba.

Miroestrol Quantification in Pueraria mirifica Crude Drugs and Products by Single-Reference UPLC/PDA/MS Using Relative Molar Sensitivities to Kwakhurin.

Owing to occasional health damages caused by health food products derived from Pueraria mirifica (PM), the Japanese government has designated PM as an "ingredient calling for special attention." Miroestrol is a specific isoflavone isolated from PM and possesses very strong estrogenic activity enough to induce side effects in small amount. Therefore, routine analyses for miroestrol quantification is recommended to control the safety and quality of PM products. However, miroestrol content in PM is quite low, and commercial reagent for its detection is rarely available. In this study, we developed a quantitative analysis method for miroestrol in PM without using its analytical standard by using the relative molar sensitivity (RMS) of miroestrol to kwakhurin, another PM-specific isoflavone, as a reference standard. The RMS value was obtained by an offline combination of 1H-quantitative NMR spectroscopy and a LC/photo diode array (PDA) and miroestrol content was determined by single-reference LC/PDA using RMS. Furthermore, we investigated miroestrol content in commercially available PM crude drugs and products, and the RMS method was compared with the conventional calibration curve method in terms of performance. The rate of concordance of miroestrol contents determined by two method was 89-101%. The results revealed that our developed LC/PDA/MS method with RMS using kwakhurin as a reference standard was accurate for routine monitoring of miroestrol content in PM crude drugs and products to control their quality.

Facile Synthesis of Kwakhurin, a Marker Compound of Pueraria mirifica and Its Quantitative NMR Analysis for Standardization as a Reagent.

The side effects of kwao keur dietary supplements (obtained from the tuberous root of Pueraria mirifica) have recently been reported by the Ministry of Health, Labour and Welfare, Japan. To control the quality of kwao keur products, its ingredients need to be maintained by characteristic marker compounds, such as miroestrol, deoxymiroestrol, and kwakhurin (KWA). In this study, we described the facile synthesis of KWA, a marker compound of P. mirifica. Our revised synthetic method produced KWA with shorter steps and higher yield than the reported method. Furthermore, the absolute purity of KWA was determined by quantitative NMR analysis for standardization as a reagent, and its purity was 92.62 ± 0.12%.

Quality Assessment of Medicinal Product and Dietary Supplements Containing Vitex agnus-castus by HPLC Fingerprint and Quantitative Analyses.

In this study, we aimed to evaluate the quality of 11 products sold in Japan (one medicinal product and 10 dietary supplements) containing/claiming to contain chasteberry extract (fruit of Vitex agnus-castus L.) using HPLC fingerprint (15 characteristic peaks), quantitative determination of chemical marker compounds, and a disintegration test. The HPLC profile of the medicinal product was similar to that of the reference standard of V. agnus-castus fruit dry extract obtained from European Directive for the Quality of Medicines (EDQM), whereas the profiles of some dietary supplements showed great variability, such as different proportions of peaks or lack of peaks. Results of the principal component analysis of the fingerprint data were consistent with those of the HPLC profile analysis. The contents of two markers, agnuside and casticin, in dietary supplements showed wide variability; this result was similar to that achieved with the HPLC fingerprint. In particular, agnuside and/or casticin was not detected in two dietary supplements. Furthermore, one dietary supplement was suspected to be contaminated with V. negundo, as evidenced from the results of agnuside to casticin ratio and assay of negundoside, a characteristic marker of V. negundo. Results of the disintegration test showed poor formulation quality of two dietary supplements. These results call attention to the quality problems of many dietary supplements, such as incorrect or poor-quality origin, different contents of the active ingredient, and/or unauthorized manufacturing procedures.

Effect of Schisandrae Fructus on glycyrrhizin content in Kampo extracts containing Glycyrrhizae Radix used clinically in Japan.

Glycyrrhizae Radix is an important crude drug in Japan and is the most frequently prescribed drug in Kampo medicines for the treatment of a wide range of diseases. Glycyrrhizin (GL), the major active ingredient of Glycyrrhizae Radix, has various pharmacological actions but causes adverse effects such as pseudoaldosteronism. In a previous study, the GL content of shoseiryuto was found to be unexpectedly low, and Schisandrae Fructus in shoseiryuto reduced the pH value of the decoction and drastically decreased the extraction efficiency of GL from Glycyrrhizae Radix. In the present study, we investigated the extraction efficiency of GL from Glycyrrhizae Radix in decoctions comprising Glycyrrhizae Radix and five different fruit-derived crude drugs. Among the five fruit-derived crude drugs tested, Schisandrae Fructus markedly decreased both the pH value of the decoction and the extraction efficiency of GL. A comparison of the pH value of the decoction and the GL content of 12 Kampo prescriptions (containing at least Glycyrrhizae Radix and Schisandrae Fructus) showed that the GL content per daily dose was proportional to the compounding amount of Glycyrrhizae Radix, and that the extraction efficiency of GL from Glycyrrhizae Radix was strongly correlated with the pH value of the decoction. In addition, the pH value of the decoction was similar to the pH value documented in interview forms provided by pharmaceutical companies. These results suggested that the GL content in Glycyrrhizae Radix-containing Kampo products can be estimated from both the compounding amounts of Glycyrrhizae Radix and the pH value documented in their interview forms. Knowledge of GL content will help avoid adverse reactions due to Glycyrrhizae Radix.

Correction to: Comparison of glycyrrhizin content in 25 major kinds of Kampo extracts containing Glycyrrhizae Radix used clinically in Japan.

The article Comparison of glycyrrhizin content in 25 major kinds of Kampo extracts containing Glycyrrhizae Radix used clinically in Japan, written by Mitsuhiko Nose, Momoka Tada, Rika Kojima, Kumiko Nagata, Shinsuke Hisaka, Sayaka Masada, Masato Homma and Takashi Hakamatsuka, was originally published Online First without open access. After publication in volume 71, issue 4, page 711-722 the author decided to opt for Open Choice and to make the article an open access publication. Therefore, the copyright of the article has been changed to

Survey on the Original Plant Species of Crude Drugs Distributed as Cynanchi Wilfordii Radix and Its Related Crude Drugs in the Korean and Chinese Markets.

Cynanchi Wilfordii Radix (CWR) is used in Korea as a substitute for Polygoni Multiflori Radix (PMR), which is a crude drug traditionally used in East Asian countries. Recently, the use of Cynanchi Auriculati Radix (CAR) in place of PMR and CWR has emerged a major concern in the Korean market. In Japan, PMR is permitted to be distributed as a pharmaceutical regulated by the Japanese Pharmacopoeia 17th edition (JP17). Although CWR and CAR have not traditionally been used as medicines, CWR was recently introduced as a health food. The distribution of unfamiliar CWR-containing products could lead to the misuse of original species for PMR and CWR like in Korea. To prevent this situation, the original species of plant products distributed as PMR, CWR, and CAR in the Korean and Chinese markets were surveyed and identified by their genes and components. The results revealed that all two PMR in the Korean market were misapplied as CAR, and that CAR was incorrectly used in eight of thirteen products distributed as CWR in both markets. As PMR is strictly controlled by JP17, the risk of mistaking PMR for CWR and CAR would be low in Japan. In contrast, the less stringent regulation of health food products and the present situation of misidentification of CWR in the Korean and Chinese markets could lead to unexpected health hazards. To ensure the quality and safety of crude drugs, it is important to use the information about the genes and components of these crude drugs.

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.

Comparison of glycyrrhizin content in 25 major kinds of Kampo extracts containing Glycyrrhizae Radix used clinically in Japan.

Glycyrrhizae Radix is the most frequently used crude drug in Japan and is prescribed in Kampo medicine for the treatment of a wide range of diseases. The major active ingredient of Glycyrrhizae Radix, glycyrrhizin (GL), has been shown to possess various pharmacological actions, but is also known to cause adverse effects such as pseudoaldosteronism. To avoid the adverse effects of GL, precautions have been indicated on the package inserts of Glycyrrhizae Radix-containing formulas depending on the amount of Glycyrrhizae Radix they contain. However, it remains unknown whether the extraction efficiency of GL from Glycyrrhizae Radix is constant throughout the different combinations of crude drugs in Glycyrrhizae Radix-containing formulas. To confirm the basis of the safety regulation, in this study we comprehensively determined the GL content of 25 major kinds of Kampo extracts compounding Glycyrrhizae Radix. We found that the GL content per daily dosage in all Kampo extracts are generally proportional to the compounding amount of Glycyrrhizae Radix, except in the case of shoseiryuto (Sho-seiryu-To). We also found that Schisandrae Fructus in Sho-seiryu-To decoction caused a lowered pH condition and drastically decreased the extraction efficacy of GL from Glycyrrhizae Radix. Moreover, we were able to confirm that the extraction efficiency of GL from Glycyrrhizae Radix is dependent on the pH value of the extraction solvent. The extraction efficiency of GL in the 25 kinds of Kampo extracts was not constant but it correlates significantly with the pH value of the decoction. Furthermore, the GL contents are well correlated with pseudoaldosteronism incidence data obtained from the Japanese Adverse Drug Event Report (JADER) database on the 25 kinds of Kampo extracts. This suggests that the GL content is a better index to consider to avoid the adverse effects of Glycyrrhizae Radix-containing Kampo formulas.

Qualitative and Quantitative Evaluation of Drug and Health Food Products Containing Red Vine Leaf Extracts on the Japanese Market.

Red vine leaf extracts (RVLEs) have traditionally been used for leg wellness and are now standardized to be used as OTC drugs in Europe. In Japan, one brand of RVLE products was recently approved as a direct OTC drug, and RVLEs are still used as ingredients in health food products. Since there is no mandated criterion for the quality of health food products in Japan, the consistent quality and composition of these products are not assured. Here we analyzed OTC drug and health food products containing RVLEs with different lot numbers by LC/MS. Subsequent multivariate analyses clearly indicated that the quality of the health food products was highly variable compared to that of the drug products. Surprisingly, the component contents in the health foods were different even within a same lot in a same brand. The quantitative analyses of flavonols and stilbene derivatives in the drugs and health foods indicated that the concentration of each substance was kept constant in the drugs but not in the health foods. These results strongly indicated that the quality of RVLEs as a whole was not properly controlled in the manufacturing process of health foods. Since RVLE is an active ingredient with pharmaceutical evidences and is used for drugs, the proper regulation for ensuring the consistent quality of RVLEs from product to product would be recommended even in the health foods.

Authentication of the botanical origin of Western herbal products using Cimicifuga and Vitex products as examples.

Various herbal medicines have been developed and used in various parts of the world for thousands of years. Although locally grown indigenous plants were originally used for traditional herbal preparations, Western herbal products are now becoming popular in Japan with the increasing interest in health. At the same time, there are growing concerns about the substitution of ingredients and adulteration of herbal products, highlighting the need for the authentication of the origin of plants used in herbal products. This review describes studies on Cimicifuga and Vitex products developed in Europe and Japan, focusing on establishing analytical methods to evaluate the origins of material plants and finished products. These methods include a polymerase chain reaction-restriction fragment length polymorphism method and a multiplex amplification refractory mutation system method. A genome-based authentication method and liquid chromatography-mass spectrometry-based authentication for black cohosh products, and the identification of two characteristic diterpenes of agnus castus fruit and a shrub chaste tree fruit-specific triterpene derivative are also described.

Identification of New Diterpenes as Putative Marker Compounds Distinguishing Agnus Castus Fruit (Chaste Tree) from Shrub Chaste Tree Fruit (Viticis Fructus).

Agnus Castus Fruit is defined in the European Pharmacopoeia as the dried ripe fruit of Vitex agnus-castus. In Europe it is used as a medicine targeting premenstrual syndrome and climacteric disorder. In Japan, Agnus Castus Fruit is becoming popular as a raw material for over-the-counter drugs and health food products, though its congenic species, Vitex rotundifolia and Vitex trifolia, have been used as Shrub Chaste Tree Fruit in traditional medicines. Therefore, it is important to discriminate these Vitex plants from the viewpoint of regulatory science. Here we tried to identify putative marker compounds that distinguish between Agnus Castus Fruit and Shrub Chaste Tree Fruit. We analyzed extracts of each crude drug by liquid chromatography-mass spectrometry, and performed differential analysis by comparison of each chromatogram to find one or more peaks characteristic of Agnus Castus Fruit. A peak was isolated and identified as an equilibrium mixture of new compounds named chastol (1) and epichastol (1a). The planar structures of 1 and 1a were determined spectroscopically. Their relative configurations were revealed by nuclear Overhauser effect spectroscopy and differential nuclear Overhauser effect-NMR data. Since avoiding contamination from closely related species is needed for the quality control of natural pharmaceuticals, this information will be valuable to establish a method for the quality control of both, Agnus Castus Fruit and Shrub Chaste Tree Fruit products.