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Relative molar sensitivity (RMS) determined using quantitative 1H NMR and HPLC with a refractive index (RI) detector was applied as a specific value for quantifying the levels of heptaoxyethylene dodecyl ether (HOEDE), a typical non-ionic surfactant, in methanol solutions. RMS was robust against changes of the analytical conditions (i.e., RI cell temperature, acetonitrile content in the mobile phase, HPLC system). Furthermore, the obtained HOEDE concentrations using a previously evaluated RMS were comparable to those obtained using a reference method for over 1 year.

Perillaldehyde (PRL) is one of the essential oil components derived from perilla plants (Perilla frutescens Britton) and is a characteristic compound of the traditional medicine "perilla herb ()" listed in the The Japanese Pharmacopoeia, 17th edition (JP17). HPLC using an analytical standard of PRL has been used to quantitatively determine the PRL content in perilla herb. However, PRL reagents have been reported to decompose easily. In this study, we utilized an alternative quantitative method using on a single reference with relative molar sensitivity (RMS) based on the results of experiments performed in two laboratories. It was possible to calculate the exact RMS using an offline combination of 1H-quantitative NMR spectroscopy (1H-qNMR) and an HPLC/photodiode array (PDA) detector (or an HPLC/variable-wavelength detector [VWD]). Using the RMS of PRL to the single-reference compound diphenyl sulfone (DFS), which is an inexpensive and stable compound, the PRL content in the perilla herb could be determined using HPLC/PDA or HPLC/VWD without the need for the analytical standard of PRL. There was no significant difference between the PRL contents of perilla herb determined using the method employing the single-reference DFS with RMS and using the JP17 assay, the calibration curve of which was generated using the analytical standard of PRL with adjusted purity measured by 1H-qNMR. These results demonstrate that our proposed method using a single reference with RMS is suitable for quantitative assays of perilla herb and can be an alternative method for the current assay method defined in the JP17.

In general, carnations (Dianthus caryophyllus) have each of four kinds of anthocyanins acylated by malic acid. A few carnation cultivars are known to display a peculiar dusky color supposedly caused by anthocyanic vacuolar inclusions (AVIs). The hereditary pattern suggests that the peculiar color is controlled by a single recessive factor tightly linked with existence of AVIs containing non-acylated anthocyanins. To diversify the peculiar color carnation, we produced a bluish purple line displaying a highly novel metallic appearance by crossbreeding. By subjecting the line to ion-beam irradiation, we generated metallic reddish purple, metallic crimson and metallic red lines. The major anthocyanin of the metallic bluish purple and reddish purple lines was pelargonidin 3,5-diglucoside, whereas that of the metallic crimson and red lines was pelargonidin 3-glucoside. All four metallic lines did not have transcripts for anthocyanin malyltransferase. Metallic crimson and red lines did not express the acyl-glucose-dependent anthocyanin 5-O-glucosyltransferase gene. In contrast to the dusky color types, metallic lines have highly condensed AVIs and water-clear vacuolar sap in the petal adaxial epidermal cells. Differences in the number of AVIs on the abaxial side were observed within mutants containing the same anthocyanin, thereby affecting their shade and hue. We demonstrated that (1) a factor generating the AVIs is inactivated anthocyanin malyltransferase gene, (2) AVIs in water-clear vacuolar sap in the adaxial epidermal cells generate the novel metallic appearance, and (3) ion beam breeding is a useful tool for increasing metallic colors by changing anthocyanin structure and the level of AVIs.

The pink, red and crimson petal colors of carnations (Dianthus caryophyllus) are produced by anthocyanins. The anthocyanins, pelargonidin and cyanidin can be modified by two glucoses at the 3 and 5 positions, and by a single malic acid. Petal color variation can result from failure of such modification, for example, the lack of a glucose at the 5 position is responsible for the color variants of some commercial varieties. With respect to this variation, modification by 5-O-glucosyltransferase plays the most important role in glucosylation at the 5 position. Recently, we identified a novel acyl-glucose-dependent anthocyanin 5-O-glucosyltransferase (AA5GT), that uses acyl-glucoses, but not UDP-glucose, as the glucose donor. Although we showed that loss of AA5GT expression was responsible for loss of glucosylation at the 5 position of anthocyanin in some varieties, the cause of this repression of AA5GT expression could not be determined. Here, we have succeeded in isolating the AA5GT gene and found that it consists of 12 exons and 11 introns. In carnation varieties lacking a glucose at the 5 position, we identified the insertion of two different retrotransposons, Ty1dic1 and Retdic1, into AA5GT. Ty1dic1, which belongs to the class I long terminal repeat (LTR)-retrotransposons of Ty1/copia families, was inserted into exon 10. Retdic1, which includes a long interspersed nuclear element (LINE)-like sequence, was inserted into intron 5. Thus, insertion of either Ty1dic1 or Retdic1 can disrupt AA5GT and result in the lack of glucosylation at the 5 position in anthocyanins.