Bamboo Leaf, the leaf of Phyllostachys nigra Munro var. henonis Stapf ex Rendle, P. bambusoides Siebold et Zuccarini, Bambusa textilis McClure or B. emeiensis L. C. Chia et H. L. Fung, is used as a crude drug in the Kampo medicine Chikuyo-sekko-to. Lophatherum Herb, which is derived from the leaves of Lophatherum gracile Brongniart, is a crude drug similar to bamboo leaf. There has been a concern over the misuse of these crude drugs. Recently, because Chikuyo-sekko-to has been approved by the Ministry of Health, Labour and Welfare as an OTC Kampo formula, we investigated the standardization of bamboo leaf and decided to list it in the non-JP crude drug standards 2015. In the process of the standardization, we designed identification and purity tests for the crude drug by using TLC. In addition, each indicator spot of the tests was isolated from bamboo leaf and Lophatherum Herb using repeated column chromatography and HPLC. The chemical structures of these spots were elucidated as p-coumaric acid (1) and trans-aconitic acid (2) based on m.p. and spectroscopic data including 1D- and 2D-NMR and MS.

The established TLC conditions were chromatographic support, silica gel; developing solvent, EtOAc/hexane/acetic acid (20/20/1) for the identification test, EtOAc/H₂O/formic acid (10/1/1) for the purity test; developing length, 7 cm; visualization, 4-methoxybenzaldehyde-sulphuric acid reagent for the identification test, UV (254 nm) for the purity test; R_f values, 0.4 (p-coumaric acid; 1) for the identification test, 0.6 (trans-aconitic acid; 2) for the purity test.

Coptis Rhizome is known to have a relatively high heavy metal content, and in Japanese Pharmacopoeia Supplement Two of the 16th edition, the Heavy Metal Limit Test is added for the purity test of Coptis Rhizome and Powdered Coptis Rhizome. In this Heavy Metal Limit Test, it states that when decision is difficult by the conventional colorimetric method, one should perform lead analysis by flame type Atomic absorption spectrophotometry (ASS) using the colorimetric test solution. However, the concentration of this colorimetric test solution is sometimes insufficient for the AAS test solution, so a new preparation method for the AAS test solution was investigated. The solvent extraction method, dry ashing method, and dry ashing with sulfuric acid method were examined, and the lead recovery rates of all three methods were 70% or more. Among these, the dry ashing method had the largest lead content amounts and lead recovery rate, and was also safe and easy.

As a result of lead validation of analytical procedures by AAS using the dry ashing method, no problem was identified in terms of linearity, detection limit, or quantitation limit. In addition, a lead recovery test using Coptis Rhizome, Orengedokuto Extract, which is a mixture of Coptis Rhizome, and Saikokeishito Extract, in which a slightly higher lead content in the actual condition survey was confirmed, was performed; The lead recovery rate was 70% or more at ashing temperatures of 450-550°C. Furthermore for cadmium, as a result of validation of the analytical procedures by AAS using the dry ashing method, no issues were identified in terms of linearity, detection limit, or quantitation limit. In addition, in a cadmium recovery test using Shoseiryuto Extract, which was confirmed to have a slightly higher cadmium content in the actual condition survey, it was confirmed that the cadmium recovery rate was 70% or more at an ashing temperature of 450°C. From the above, by using the dry ashing method, lead analysis of Coptis Rhizome, Orengedokuto Extract, and Saikokeishito Extract, and cadmium analysis of Shoseiryuto Extract by AAS have become possible.

Extracts of Kampo formulations have been listed in the Japanese Pharmacopoeia since its 15th edition and their importance as pharmaceuticals is increasing, as is the recognition of the need to ensure the safety of them. The purity test for extracts of Kampo formulations includes tests for heavy metals and arsenic, but since the Heavy Metals Limit Test uses a colorimetric method, there is no specificity for individual metals. Meanwhile, with the development of highly sensitive instrumental analytical methods, it is now a trend worldwide to set standards for individual elements based on degree of hazard. Accordingly, the JKMA have investigated the amount of arsenic, cadmium, lead and mercury, which are significantly toxic, in the target 22 extracts of Kampo formulations manufactured by the member companies and listed in the 16th Edition of the Japanese Pharmacopoeia in 2012 and 2013. As a result, data were provided for 613 samples for arsenic and 524 samples for cadmium, lead and mercury, and all of them were confirmed to be below the criteria values indicated in the Japanese Pharmacopoeia and Hong Kong Chinese Materia Medica Standards.

The Japan Kampo Medicines Manufacturers Association (JKMA) set up “self-imposed limits of residual pesticides for Kampo products, conventional crude drug products and crude drugs” in 1996, in order to guarantee the safety of Kampo products and conventional crude drug products. These self-imposed limits have been modified twice, and now organochlorine, organophosphorus, and pyrethroid classes are covered. The JKMA carried out a survey of the actual implementation of these self-regulations in member companies for a one-year target period from April 1, 2012 to March 31, 2013. As a result, the implementation rate for these limits was 89.3%, and the measured number of samples was at least 21,358 lots. The implementation rate and the measured number of samples have increased compared to the previous survey. In addition, no cases displayed results where the detected residual pesticides were beyond the levels imposed by self-regulation.

Seven compounds (1-7) were isolated from the root of Odontioda Marie Noel ‘Velano’. These were identified as phenanthrene and stilbene derivatives with spectroscopic analysis. 1, 3 and 4 reported here were isolated for the first time from this plant.