Hirotoshi Fushimi

Development of a DNA Microarray for Authentication of Ginseng Drugs Based on 18S rRNA Gene Sequence

Ginseng drugs, derived from underground parts of Panax species (Araliaceae), are the most important group of herbal medicines in the Orient. Previously, the nucleotide sequences of the nuclear 18S rRNA gene of 13 Panax taxa were determined, as were the specific polymorphic nucleotides for identification of each species. On the basis of the nucleotide difference, a DNA microarray (PNX array) was developed for the identification of various Panax plants and drugs. Thirty-five kinds of specific oligonucleotide were designed and synthesized as probes spotting on a decorated glass slide, which included 33 probes corresponding to the species-specific nucleotide substitutions and 2 probes as positive and negative controls. The species-specific probes were of 23-26 bp in length, in which the substitution nucleotide was located at the central part. Triplicate probes were spotted to warrant accuracy by correcting variation of fluorescent intensity. Partial 18S rRNA gene sequences amplified from Panax plants and drugs as well as their derived health foods were fluorescently labeled as targets to hybridize to the PNX array. After hybridization under optimal condition, specific fluorescent patterns were detected for each Panax species, and the analyzed results could be indicated as barcode patterns for quick distinction. The developed PNX array provided an objective and reliable method for the authentication of Panax plants and drugs as well as their derived health foods.

Inhibitory effects of Cordia spinescens extracts and their constituents on reverse transcriptase and protease from human immunodeficiency virus

By bioactive‐guided fractionation of a water extract of Cordia spinescens, magnesium lithospermate (1), calcium rosmarinate (2) and magnesium rosmarinate (3) were isolated as potent inhibitory substances against HIV‐1 reverse transcriptase (RT) with IC50 values of 0.8, 5.8 and 3.1 μM, respectively. However, they were weak HIV‐1 protease (PR) inhibitors with IC50>100 μM. The RT inhibition by these compounds was noncompetitive with respect to dTTP substrate.

Effects of goshajinkigan, hachimijiogan, and rokumigan on mechanical allodynia induced by Paclitaxel in mice.

Peripheral neuropathy is a major dose-limiting side effect of the chemotherapeutic agent paclitaxel. This study examined whether the three related traditional herbal formulations, goshajinkigan (GJG; 牛車腎氣丸 Niú Chē Shèn Qì Wán), hachimijiogan (HJG; 八味地黃丸 Bā Wèi Dì Huáng Wán), and rokumigan (RMG; 六味丸 Liù Wèi Wán), would relieve paclitaxel-induced mechanical allodynia in mice. A single intraperitoneal injection of paclitaxel (5 mg/kg) induced mechanical allodynia, which peaked on day 14 after injection. On day 14 after paclitaxel injection, oral administration of GJG (0.1-1.0 g/kg) produced a significant inhibition of established allodynia, but HJG and RMG did not affect the allodynia. Repeated oral administration of GJG (0.1-1.0 g/kg) starting from the day after paclitaxel injection did not affect allodynia development, but significantly inhibited allodynia exacerbation. Repeated oral administration of HJG produced a slight inhibition of allodynia exacerbation, but that of RMG did not. These results suggest that prophylactic administration of GJG is effective in preventing the exacerbation of paclitaxel-induced allodynia. The herbal medicines Plantaginis Semen (車前子 Chē Qián Zǐ) and Achyranthis Radix (牛膝 Niú Xī), which are present in GJG but not in HJG, may contribute to the inhibitory action of GJG on the exacerbation of paclitaxel-induced allodynia.

Effect of Curcuma Herbs on Vasomotion and Hemorheology in Spontaneously Hypertensive Rat

Curcuma herbs have a vasodilator effect. The effects of C. longa, which induces only endothelium-independent vasodilatation, and C. zedoaria, which induces both endothelium-dependent and -independent vasodilatation, were studied on vasomotion and hemorheology in spontaneously hypertensive rats. Spontaneously hypertensive eight-week-old male rats were assigned to five groups. For 12 weeks, the control group received standard chow. The 3%CL (C. longa) group received standard chow containing 3% (wt/wt) C. longa. The 1%CZ and 3%CZ (C. zedoaria) groups received standard chow containing 1% and 3% (wt/wt) C. zedoaria, respectively. The captoril group received standard chow and 100 mg/kg/day of captoril in drinking water. Blood pressure, vasomotion, hemorheology, etc. were examined. Systolic blood pressure of the 3%CZ and captoril groups decreased significantly as compared to the control group. Acetylcholine-induced endothelium-dependent relaxations of the 3%CZ and captoril groups were increased to a greater degree, significantly, than the control group. When testing xanthine oxidase-induced contraction, the 3%CZ group was significantly decreased as compared to the control group. Low shear stress of whole blood viscosity showed the 3%CL and 3%CZ groups to be decreased significantly compared to the control group. Thus, Curcuma herbs have hypotensive and protective effect on the endothelium in spontaneously hypertensive rats. Especially, C. zedoaria is more effective than C. longa, and its mechanism is thought to be related to a radical scavenging effect and improvement of hemorheology.

Morphological, genetic, and chemical polymorphism of Curcuma kwangsiensis

Previously, Chinese Gajutsu available in Japan was identified, from the chloroplast trnK gene sequence, to be the rhizomes of Curcuma phaeocaulis and two genotypes of C. kwangsiensis. Although we defined the two genotypes, the pl and gl types, on the basis of the nucleotide difference, their external features did not correspond to the two phenotypes described in the literature. In this paper, to investigate the relationship between genotype and phenotype of C. kwangsiensis, a field investigation was carried out in its main cultivation areas of Guangxi Zhuangzu Autonomous Region and Guangdong Province, China, and sequence analysis of the trnK gene and single-nucleotide polymorphism (SNP) analysis of the nuclear 18S rRNA gene were performed on the collected specimens. Four genotypes of C. kwangsiensis were recognized from the combined 18S rRNA gene–trnK gene sequences: homozygote-K(gl)Wtk type, homozygote-K(pl)Ztk type, heterozygote-K(gl)Wtk type, and heterozygote-Ltk type. Among the four genotypes, C. kwangsiensis in a field used for cultivation of Gajutsu was of heterozygote-K(gl)Wtk type. Formation of a heterozygote in the 18S rRNA gene might be a result of crossbreeding of C. kwangsiensis with several Curcuma species which had cytosine at nucleotide position 234. GC analysis of the rhizomes revealed that C. kwangsiensis was characterized by camphor and β-elemene, and by detecting additional components such as curdione and curcumenol Curcuma species involved in the formation of the heterozygote might be speculated upon.

Comprehensive quality evaluation and comparison of Angelica sinensis radix and Angelica acutiloba radix by integrated metabolomics and glycomics

Angelica radix (Danggui in Chinese) used in China and Japan is derived from two species of Angelica, namely Angelica sinensis and Angelica acutiloba, respectively. The differences in quality between A. sinensis radix (ASR) and A. acutiloba radix (AAR) should be therefore investigated to guide the medicinal and dietary applications of these two species. Secondary metabolites and carbohydrates have been demonstrated to be the two major kinds of bioactive components of Danggui. However, previously, quality comparison between ASR and AAR intensively concerned secondary metabolites but largely overlooked carbohydrates, thus failing to include or take into consideration an important aspect of the holistic quality of Danggui. In this study, untargeted/targeted metabolomics and glycomics were integrated by multiple chromatography-based analytical techniques for qualitative and quantitative characterization of secondary metabolites and carbohydrates in Danggui so as to comprehensively evaluate and compare the quality of ASR and AAR. The results revealed that not only secondary metabolites but also carbohydrates in ASR and AAR were different in type and amount, which should collectively contribute to their quality difference. By providing more comprehensive chemical information, the research results highlighted the need to assess characteristics of both carbohydrates and secondary metabolites for overall quality evaluation and comparison of ASR and AAR.