KAKI CALYX is defined in the Japanese standards for non-Pharmacopoeial crude drugs 2018 (non-JPS 2018) as the persistent calyx of mature fruit of Diospyros kaki Thunberg　(Ebenaceae), and is used for Kampo formulae such as Shiteito and Choukoushiteito that are prescribed for continuous hiccups. In order to help better understand and facilitate quality control of KAKI CALYX, HPLC methods for quantitative analyses of three triterpenoids, i.e. betulinic, ursolic and oleanolic acids in KAKI CALYX were elaborated. The calyces from various types of cultivated persimmon trees were collected and analyzed to clarify the difference affected by harvesting periods and sweet/astringent types of fruits. Market samples were also investigated and compared. As a result, betulinic acid content was larger in sweet persimmons than in astringent persimmons (p<0.05). Ursolic and oleanolic acids contents of cultivated persimmons were significantly higher than those of market samples (p<0.01).

We made an investigation into the current status of agrochemicals used for cultivation of Cornus fruit in four cultivation provinces in China: Anhui, Henan, Shaanxi and Sichuan. As a result, no agrochemicals except for aluminum phosphide were used in any province. Furthermore, we quantitatively determined the contents of residual pesticides on Cornus fruit which were available commercially in Japan. Although we were not able to obtain any Cornus fruit from Anhui in the Japanese market, seven and one pesticide residues were detected in the samples from Henan and Shaanxi, respectively, and none of those were detected in the samples from Sichuan. The amounts of the six pesticides, other than isocarbophos and triazophos in the samples from Henan, were less than each upper residual limit for fruit stipulated in the Food Sanitation Act in Japan, so we retested on the other three samples from Henan. As a result, isocarbophos and triazophos were not detected in those samples from Henan.

Many monographs dealing with the identification of crude drugs and Kampo formulae listed in the Japanese Pharmacopoeia (JP) describe tests using thin-layer chromatography (TLC). TLC identification tests are simple and easy but may not be applicable to some crude drugs due to their low sensitivity, and the objective identification of crude drugs by TLC can be difficult due to poor resolution. In addition, analogue data are not suitable for archival purposes.

In this study, we attempted to develop an alternative identification test which addresses these shortcomings. We studied Yokukansan Extract as a model Kampo formula using high-performance liquid chromatography coupled with mass spectrometry (LC/MS). Ions were detected by electrospray ionization (ESI) in a single quadrupole mass spectrometer by selected-ion monitoring (SIM), allowing the identification of Yokukasan Extract components with high sensitivity. Furthermore, minor components in Poria, Angelica and Cnidium, which cannot be identified by TLC, were observed using this technique. We confirmed the specificity and robustness of the proposed method by validation tests.

Application of the proposed LC/MS (SIM) identification test to Kampo formulae may provide higher efficiency, objectivity and accuracy than conventional TLC methods.

Effects of nutrient solution concentration on the growth and leaf chemical constituents of Artemisia princeps (Ibuki-yomogi) were investigated by cultivating the plant hydroponically under four different nutrient solution concentrations (25%, 50%, 75%, and 100% of the full strength). Even when concentration of nutrient solution was reduced to 25%, no significant differences were observed regarding shoot growth compared to all other treatments. Furthermore, as the concentration of nutrient solution decreased, the leaf nitrate content decreased, and leaf vitamin C, chlorogenic acid and total polyphenol contents tended to increase. These results indicated that the quality of Ibuki-yomogi as a functional food material was improved without growth reduction by the reduced nutrient solution concentration. Therefore, we concluded that nutrient solution of about 25% strength is adequate for hydroponic cultivation of Ibuki-yomogi plants, although management of nutrient solution pH and concentration during hydroponics should be noted due to the risk for pH increase and reduction of some mineral contents.