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Extrahepatic delivery of small interfering RNAs (siRNAs) may have applications in the development of novel therapeutic approaches. However, reports on such approaches are limited, and the scarcity of reports concerning the systemically targeted delivery of siRNAs with effective gene silencing activity presents a challenge. We herein report for the first time the targeted delivery of CD206-targetable chemically modified mannose–siRNA (CMM–siRNA) conjugates to macrophages and dendritic cells (DCs). CMM–siRNA exhibited a strong binding ability to CD206 and selectively delivered contents to CD206-expressing macrophages and DCs. Furthermore, the conjugates demonstrated strong gene silencing ability with long-lasting effects and protein downregulation in CD206-expressing cells in vivo. These findings could broaden the use of siRNA technology, provide additional therapeutic opportunities, and establish a basis for further innovative approaches for the targeted delivery of siRNAs to not only macrophages and DCs but also other cell types.

Extrahepatic targeted delivery of oligonucleotides, such as small interfering RNA (siRNA) and antisense oligonucleotides (ASOs), is an attractive technology for the development of nucleic acid-based medicines. To target CD22-expressing B cells, several drug platforms have shown promise, including antibodies, antibody-drug conjugates, and nanoparticles, but to date CD22-targeted delivery of oligonucleotide therapeutics has not been reported. Here we report the uptake and enhancement of siRNA gene expression knockdown in CD22-expressing B cells using a chemically stabilized and modified CD22 glycan ligand-conjugated siRNA. This finding has the potential to broaden the use of siRNA technology, opening up novel therapeutic opportunities, and presents an innovative approach for targeted delivery of siRNAs to B cell lymphomas.

Saposhnikoviae Radix (SR), derived from the dried root and rhizome of Saposhnikovia divaricata, is a popular crude drug used in traditional Chinese and Japanese medicine. To evaluate the metabolites of S. divaricata roots from Mongolia and to investigate their geographical variation, we developed the HPLC method, determined the contents of 9 chromones and 4 coumarins, and conducted multivariate statistical analysis. All Mongolian specimens contained prim-O-glucosylcimifugin (1) and 4′-O-β-d-glucosyl-5-O-methylvisamminol (3), and their total amount (5.04–25.06 mg/g) exceeded the criterion assigned in the Chinese Pharmacopoeia. Moreover, the content of 1 (3.98–20.79 mg/g) was significantly higher in the Mongolian specimens than in Chinese SR samples. The specimens from Norovlin showed the highest contents of 1 and 3. The total levels of dihydropyranochromones were higher in the specimens from Bayan-Uul. The orthogonal partial least squares-discriminant analysis revealed that the Mongolian specimens tended to be separated into three groups based on growing regions, in which several chromones contributed to each distribution. Furthermore, 1H NMR analysis revealed that Mongolian specimens had less amount of sucrose and a substantial amount of polyacetylenes. Thus, in this study, the chemical characteristics of Mongolian S. divaricata specimens were clarified and it was found that the specimens from the northeast part of Mongolia, including Norovlin, had the superior properties due to higher amounts of major chromones.

In the Kali Gandaki Valley in Central Nepal, Ephedra gerardiana and E. pachyclada show species specificity for physical and chemical characteristics of soils. Here, the relationship between soil characteristics and ephedrine and pseudoephedrine contents was examined. E. gerardiana grew in moist alpine scrub and upper alpine meadow from 3735 to 4156 m a.s.l., while E. pachyclada grew in the lower Caragana steppe and dry alpine scrub from 2629 to 3671 m a.s.l. The soil texture of E. gerardiana and E. pachyclada collection sites were classified as loam or sandy loam mainly composed of sand and silt. Loss on ignition (%) of soil in E. gerardiana habitats (28.4–35.0%) was markedly higher than for that in E. pachyclada habitats (14.2–17.2%). E. pachyclada soil (pH 8.4–9.2) was more alkaline than that for E. gerardiana (pH 8.5). The five ions (Cl−, SO42−, Ca2+, Mg2+, and Na+) in soil of E. pachyclada (Cl−, 0.01–18.97 mmol/100 g dry soil weight; SO42−, 1.95–83.33; Ca2+, 3.79–77.91; Mg2+, 1.28–27.9; Na+, 0.94–34.49) were markedly higher than those of E. gerardiana (Cl−, 0.18–0.29; SO42−, 0.07–0.08; Ca2+, 4.19–4.59; Mg2+, 0.22–0.58; Na+, 0.93–1.40). The main factor contributing to strongly alkali soils for each species was different between E. gerardiana and E. pachyclada: CaCO3 for E. gerardiana and CaSO4, MgSO4, NaCl, or a combination of these for E. pachyclada. The total ephedrine and pseudoephedrine content in E. gerardiana and E. pachyclada ranged from 1.67–1.88%DW and 1.95–4.80%DW, respectively. Both E. gerardiana and E. pachyclada were amenable for use a raw material source for extraction of ephedrine and pseudoephedrine, and the ephedrine content of both species showed a statistically significantly positive correlation with Mg2+ and Na+ contents of the soil.

Aconitum kiyomiense Kadota (Ranunculaceae) is endemic to Takayama city, Gifu Prefecture, central Japan. We collected specimens from marshes and flood plains at altitudes ranging from 852 to 1085 m and from a new habitat consisting of a mesic meadow in the subalpine belt (1681 m). Glabrous pedicels and flowering sequence of inflorescence were used for identification, but intra-species variations in the pilus of pedicels (glabrous, pilose, and chimeric types) were observed. Although the flowering sequence has been reported as both indeterminate and determinate, all specimens in the present study were determinate. No intra-species variation was detected via partial nuclear internal transcribed space, and sequences did not match another 17 East Eurasian continent subgenus Aconitum species. The chloroplast trnL-trnF intergenic spacer region (trnL-trnF) showed three different haplotypes. The trnL-trnF dominant haplotype sequence was identical to that of A. kusnezoffii growing on the Eurasian continent, suggesting that A. kiyomiense is more primitive than other Japanese aconitum and a relic species of the Eurasian continent. We report the first detection of aconitine alkaloids in the tuberous roots, which exhibited aconitine alkaloid contents varying from 0.32 to 4.05 mg/g dry weight (mg/g) for aconitine, 0.02 to 4.12 mg/g for hypaconitine, undetectable to 0.05 mg/g for jesaconitine, and 0.42 to 3.76 mg/g for mesaconitine. The variation of aconitine alkaloid components and contents appeared to be random and did not vary with inflorescence phenotype, trnL-trnF haplotype, environmental habitat conditions, or the geographic region of the collection sites. Since most populations showed no genetic intra-variation, it will be necessary to maintain the continuity of habitats and designate areas for conservation of genetic diversity at the population level.

Pinellia tuber (PTE, 半夏, 大半夏, 掌葉半夏, 天南星, 水半夏, 半夏瀉心湯, 半夏厚朴湯, 六君子湯) is derived from the tuber of Pinellia ternata Breitenbach (Araceae), which is a crude drug used in traditional Japanese Kampo medicine for the purpose of antiemesis and expectoration. Since the separation of ephedrine from PTE in 1978, it has been listed as a PTE component in textbooks and internet information. Therefore, there are harmful effects on appropriate use in clinical practice because PTE is dealt with as a crude drug for doping target, and traditional Japanese Kampo medicine containing PTE must be carefully administered to the elderly. However, since the 1978 published report, there has not been any report on the isolation of ephedrine from PTE and the interpretation of biosynthesis remains questionable. In the present study, we analyzed the PTE samples in market distribution products by LC-TOF/MS. From the analysis of the result of ephedrine’s m/z 148.113 [M + H-H2O]+, PTE was not detected (n = 55, detection limit: 0.5 ppb). Additionally, the tuber of P. tripartite (PTR, 大半夏), the tuber of P. pedatisecta (PPE, 掌葉半夏), Arisaema Tuber (ART, 天南星), and the tuber of Typhonium flagelliforme (TFI, 水半夏) that have a similar description to PTE were also not detected. Moreover, the genetic analysis of experimental samples showed that PTE is derived from P. ternata. Furthermore, our attempt to isolate ephedrine from PTE based on the past literature was unsuccessful. These results suggest that PTE in market distribution products may not contain ephedrine as a component.

柿蔕のヘキサン、メタノール、50 % アセトン抽出物の、組み替えhepatitis C virus (HCV)の NS3/4A 蛋白質分解酵素に対する阻害活性を検討した。50 % アセトン抽出物が最も阻害活性が強く、この抽出物をSephadex LH20カラムで分画を行なった。得た各画分のうち阻害活性の強かった70 %メタノール画分を、さらにシリカゲルカラム／薄層クロマトグラフィーで分画し、トリテルペンのベルリン酸、オレアノール酸、ウルソール酸を分離した。これらのトリテルペンはpolycyclic aromatic hydrocarbons (PAH) HPLCH-NMRスペクトルで同定した。これらトリテルペンは柿蔕の50 %アセトン抽出物の主たる阻害物質と考えられる。, We tested hexane, methanol and 50% aqueous acetone extracts of kaki calyx for their inhibitoryactivity on recombinant hepatitis C virus NS3/4A protease. The 50 % aqueous acetone extract had thehighest inhibitory activity, and was fractioned by Sephadex LH20 column chromatography. The 70 %methanol fraction of the column chromatography was further fractioned by silica gel column/thin layerchromatography, and triterpenes, betulinic acid, oleanolic acid, and ursolic acid, were isolated. They wereidentified by analyses using HPLC equipped with a polycyclic aromatic hydrocarbons (PAH) column and1H-NMR spectroscopy. The triterpenes were thought to be the main inhibitory compounds in the 50 %aqueous acetone extract of kaki calyx, but there may be inhibitory compounds other than thetriterpenes.

桂皮（ケイヒ）は日本薬局方に収載される生薬で，抗アレルギーや解熱鎮痛炎症薬とみなされる漢方処方に配剤される．我々はケイヒの抗アレルギー作用および抗炎症作用について調査し，活性成分の同定を行った．ケイヒを70%メタノールで抽出し（Cin70M），シリカゲルカラムクロマトグラフィー（CC）およびオクタデシルシリルCCを用いて成分を分離した．分離した画分はin vitroでの実験としてラット好塩基球性白血病細胞株（RBL-2H3）およびマウスマクロファージ細胞株（RAW264）を用い，それらが産生するサイトカインmRNAの発現率によって抗アレルギーおよび抗炎症活性を示す画分を明らかにした．また，抗アレルギー作用においては，RBL-2H3の脱顆粒率を測定するために細胞培養液中のβ - ヘキソサミニダーゼも測定した．Cin70Mとシンナムアルデヒド（CNMA）はRBL-2H3細胞における脱顆粒およびインターロイキンのmRNA発現を阻害した．CNMAは300μMで脱顆粒を抑制し，その抑制作用はポジティブコントロールとして用いたケトチフェンより僅かに強かった．同様にCin70MとCNMA はRAW264が産生した一酸化窒素（NO）および炎症性サイトカインmRNA発現を抑制した．特にNO産生についてはCNMA 100μMで10μMのデキサメタゾンと同等の抑制作用を示した．ケイヒの特徴である甘い味と芳香は明らかにした活性成分の一つであるCNMAに由来する．今回の結果から，アレルギー疾患や炎症性疾患の患者に対し，ケイヒを含む漢方薬が処方された際には甘い味と芳香が強いケイヒを選んだ方が良いことが示された．, Cinnamomi Cortex (cinnamon bark) is a major crude drug. In Japan, it is used in Kampo medicine to treatallergic and inflammatory diseases. In this study, we examined the anti-allergic and anti-inflammatoryeffects of Cinnamomi Cortex and attempted to identify its active components. Cinnamomi Cortex wasextracted with 70% methanol (Cin70M) and separated into fractions via silica gel column chromatography(CC) or octadecylsilan (ODS) CC. In order to select fractions that have anti-allergic and anti-inflammatoryactivities, we evaluated the ability of the fractions to inhibit the expression of cytokine genes in vitro byusing a rat basophilic leukemia (RBL-2H3) cell line and mouse macrophage (RAW264) cell line. Geneexpression levels were assessed in both cell lines by using semi-quantitative reverse transcriptionpolymerase chain reaction (RT-PCR), and the RBL-2H3 degranulation ratio was quantified via ???? -hexosaminidase assay. Cin70M and cinnamaldehyde (CNMA) inhibited degranulation and the mRNAexpression of interleukins in RBL-2H3 cells. CNMA inhibited degranulation at 300 ????M, and its inhibitoryeffects were slightly stronger than those of ketotifen which is a typical anti-allergic drug. In RAW264 cells,nitric oxide (NO) production and the mRNA expression of inflammatory cytokines were reduced byCin70M and CNMA. In the NO production assay, 100 ????M CNMA was found to significantly decrease NOproduction from activated RAW264 cells, and its activity was the same as that of 10 ????M dexamethasone.The taste and scent of Cinnamomi Cortex were both attributed to CNMA. The results of the presentstudy indicate that it is more beneficial for patients with allergic and/or inflammatory diseases to useCinnamomi Cortex with a strong taste and scent.

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.

Pioglitazone is a peroxisome proliferator-activated receptor gamma (PPARγ) full agonist and useful for the treatment of type 2 diabetes mellitus. Naringenin is a citrus flavonoid with anti-inflammatory actions, which has been shown to prevent obesity-related diseases and to activate PPARγ. The aim of this study was to investigate whether dietary naringenin affects the actions of pioglitazone. We administered naringenin (100 mg/kg) and pioglitazone (10 mg/kg) to Tsumura Suzuki Obese Diabetes (TSOD) mice for 4 weeks and then conducted an oral glucose tolerance test. We found that oral administration of naringenin attenuated the hypoglycemic action of pioglitazone in TSOD mice. However, pioglitazone and naringenin did not affect fasting blood glucose levels, epididymal fat pad weight and body weight changes in this administration period. Pioglitazone suppressed expression of obesity-related adipokines such as tissue inhibitor of metalloproteinases-1 in adipose tissue of TSOD mice, but this effect was attenuated by naringenin. However, naringenin did not affect the pharmacokinetics of pioglitazone after single or repeated administration. Naringenin exhibited weak partial agonist activity in time-resolved fluorescence resonance energy transfer assay, but naringenin interfered with pioglitazone agonism, consistent with partial agonism. Our results suggest that it is advisable to avoid administering a combination of naringenin and pioglitazone.

Elucidation of the function of glycolipids in natural products is very important because ecology of animals containing them is not only understood, but also their compounds will lead to development of new drugs. This research tried to synthesis of new glycosphingolipids (1, 2, and 3) that were isolated from Hirsutella rhossiliensis. They have neogala-series, β-D-Galp(1→6)-β-D-Galp(1→6)-β-D-Galp, as core structure. Compound 1 contains also in Neurospora crassa, we already reported about synthesis of it. We attempted to synthesize of compounds 2 and 3.

C型肝炎ウイルス（HCV）感染者は、世界に１億人以上いるとされている。先進国では先端的な医薬品の開発で、HCVを病原とする慢性及び急性C型肝炎の治癒率は飛躍的に高まった。一方、世界には経済的に、また医療体制の問題から、それら先端医薬品を手に入れ難い地域も多い。我々は安価で、また手に入れやすいC型肝炎治療薬を開発するために、各地の伝統薬物（生薬）に着目した。特にタンニンを多く含んだ13生薬を選び、メタノールエキスを作成した。HCVの感染に必須であるHCVタンパク質分解酵素を、大腸菌で生成し、この組み換え酵素に対する阻害効果をスクリーニングした。13のエキスのうち６のエキスの100 μg/mlに、90％以上のHCVタンパク質分解酵素阻害効果を認めた。ポリフェノール含量の高いエキス（800-1500 μgGAE/ml）が阻害効果も高い傾向であったが、阿仙薬エキスはポリフェノール含量が高い（1300 μgGAE/ml以上）にも係らず、ほとんど阻害効果がなかった。一方柿蔕エキスは、ポリフェノール含量は比較的低かったが（400 μgGAE/ml以下）、高い阻害活性が認められ、ポリフェノールの種類によっても、阻害活性が異なることが示唆された。今後柿蔕の活性成分について、さらに詳しく研究する。, Aiming at finding novel hepatits C virus (HCV) drugs having easier access and requiring less economical burden, we studied on crude drugs for anti HCV protease activity. Thirteen crude drugs which are known rich in tannins were selected, since we had found peel and seeds of a tropical fruit in Peru, Camu camu, and their tannic ingredients were effectively inhibited the recombinant HCV protease. Methanolic extracts of the crude drugs were tested for inhibitory activity on the enzyme reaction, and 6 of them at a concentration of 100 μg/ml inhibited the reaction more than 90 %. Five of these effective extracts contained as much as 800-1500 μgGAE/ml phenolics, but the extract of kaki calyx (KC) contained less than 400 μgGAE/ml. On the other hand, that of gambir (Ga), which contained more than 1300 μgGAE/ml phenolics, did not effectively inhibit the reaction. We now focus on the effective ingredients contained in the extract of KC.

Arboreous fruit of Schisandra chinensis Baillon, Schisandra Fruit (SF), is a crude drug used in Japanese traditional Kampo medicine. The marker compounds of SF for quality control are lignans, such as schizandrin (Sz) and gomisin A (GmA). Kampo formulation containing SF is usually prepared as decoctions in the dosage form of whole crude drug (W), as its size is small enough to measure using a spoon. However, in some traditional books, it has been described that SF must be used in the dosage form of crushed or cut pieces (Cr). In this study, we evaluated the transferring ratio of lignans from SF to the decoction, and the stability and taste of the decoctions of shoseiryuto (SST) and ninjin’yoeito (NYT) using each dosage form, i.e., Cr and W, of SF. The transferring ratio of Sz and GmA was significantly higher in the decoction prepared with the Cr form than that prepared using the W form in both SST and NYT. The concentration of Sz and GmA in the decoctions was stable when maintained at 4 °C for 35 days. The taste of SST decoction prepared using the Cr form was more acidic, harsher, and bitterer than SST decoction prepared using the W form, and the taste of NYT decoction prepared using the Cr form was harsher than NYT decoction prepared using the W form. In conclusion, when SF is used in Kampo prescription, crushing the fruits and seeds can increase its effectiveness.

When oligosaccharides are built by coupling of sugars, stereoselectivity of glycosidic bonds is important for the effective synthesis of the target compound. Stereoselectivity of glycosylation depend on neighbouring group, solvents, and temperature. We tried to synthesize a glycosphingolipid from Neurospora crassa.

The Japanese Pharmacopoeia defines byakujutsu (Atractylodes rhizome) as the rhizome of Atractylodes japonica or A. macrocephala and sojutsu (Atractylodes lancea rhizome) as the rhizome of A. lancea, A. chinensis, or their interspecific hybrids. Because their pharmaceutical uses differ in traditional Japanese Kampo medicine and traditional Chinese medicine, with less apparent scientific evidence, we compared the pharmacological properties between byakujutsu and sojutsu. Crude drug specimens of byakujutsu (n = 40) and sojutsu (n = 49) obtained in markets were identified by their species using DNA profiling. Their pharmacological properties were evaluated by the inhibitory effect of a MeOH extract of the samples on nitric oxide (NO) production by lipopolysaccharide-stimulated murine macrophage-like RAW264.7 cells and by the inducing effect of boiling water extract of the samples on granulocyte-colony stimulating factor (G-CSF) secretion from murine normal colonic epithelial MCE301 cells. We authenticated A. macrocephala (n = 8), A. japonica (n = 35), and the hybrid between A. macrocephala and A. japonica (n = 1), and they were used as byakujutsu. We authenticated A. chinensis (n = 25), A. lancea (n = 14), and the hybrid between A. chinensis and A. lancea (n = 6), and they were used as sojutsu. The inhibitory effects of byakujutsu on NO production were significantly higher than those of sojutsu (P

糖質に分類されるシアル酸は、様々な生体機能を有していることが知られている。従って、シアル酸を含む糖鎖を化学合成することは、生命現象の解明に寄与するものと考えられる。しかし、シアル酸を糖供与体とする糖鎖合成は困難とされており、近年多くの研究者らによって、新たな処方が開発されてきている。我々は、シアル酸が持つ生体機能の興味を持ち、これを有する糖鎖誘導体 (5) の合成を試みた。, The sialic acids are now defined as a family of carbohydrates, have very important biological functions. Thus, it considers that oligosaccharides containing them employ elucidation of biological phenomena. However, for synthesis of them glycosylations using donor of sialic acid derivative have been very hard to achieve both high yields and stereoselectivity of them. But recently, Synthesis methods of oligosaccharides containing sialic acids were developed by many researchers. Highly regioand α-stereoselective sialyl glycoside syntheses have been achieved by using, as glycosyl donors, the 2-thioglycoside of Neu5Ac. We were interested in biological functions of oligosaccharide containing sialic acid, and tried to synthesize sialyl lewis X and synthesized trisaccharide derivative which was precursor of sialyl lewis X, α-L-Neu5Ac(2 → 3)β-D-Gal(1 → 4)β-D-GlcNAc-MP.

光アフィニティーラベル法は、糖鎖- レクチンの相互関係を明らかにする最も効果的な手法である。従来法では、プローブ合成時に還元末端糖が開環してしまっていたが、我々は、完全な糖鎖構造 (D- ラクトース) からなるリガンドとフェニルジアジリン基を有する分子プローブを開発した。次の問題点として、糖鎖は複雑な構造を有していることから、本分子プローブの合成法の確立が重要であると考えた。我々は、オルソゴナルグリコシル化法により糖鎖部分の合成を目指したが、反応時に糖受容体と糖供与体の脱離基が置換されてしまい、目的化合物が得られなかった。そこでトリクロロアセトイミデート法を用い、糖鎖合成に着手したところ、D- ガラクトース-D- グルコサミンから成る二糖誘導体が得られ分子プローブ(1、2、3) へと導かれた。, Photoaffinity labeling technology is the most efficient method for elucidation of carbohydrate - lectin interactions. However, when carbohydrate probes are synthesized according to conventional this method, the reducing terminus of the sugar is opened to provide an acyclic structure. We were undertaken to solve this problem, and developed new molecular probe containing complete oligosaccharide structure (D-lactose) and phenyldiazirine group for elucidation of carbohydrate - lectin interactions. Next problem, establishment of synthesis method of molecular probe is important subject because carbohydrates are composed of various complex structures. We tried synthesis of oligosaccharides using orthogonal glycosylation strategy. But when glycosyl donor and glycosyl acceptor were coupled by promoter, leaveing group of glycosyl donor (-F group) was substituted to leaving group of glycosyl acceptor (-SPh group). We synthesized Galp-GlcpNAc disaccharide derivatives using glycosyl trichloroacetimidate strategy, and then were obtained 3 types of molecular probes containing ligand and photoreactive group (1, 2, 3).

New clerodane diterpene, 16-hydroxy-pentandralactone (1) and known diterpene acuminolide (2) were isolated from the methanol extract of Vitex cofassus leaves. The chemical structure and the absolute configuration of 1 were determined by MS, NMR and electron circular dichroism (ECD) experiments. The isolated compounds were evaluated for their antiproliferative activities against a panel of human tumor cell lines, including a multidrug-resistant (MDR) cell line. Both compounds showed potent antiproliferative activities against all the tested cell lines with IC

発行後1年より全文公開, Poria, a dried sclerotium of Wolfiporiacocos Ryvarden et Gilbertson (Polyporaceae) has been used as a crude drug in both Chinese and Japanese (Kampo) traditional medicines. Recently, cultivated products of Chinese Poria strains have accounted for most of the market, while the cultivation of Japanese Poria strains has not been successful. Aiming to determine the relationship between the differences in cultivation characteristics and genetic polymorphism, we conducted a field cultivation experiment, a rot test, and rapid amplification of polymorphic DNA (RAPD) analysis of Poria strains collected from China and Japan: 3 Chinese and 7 Japanese strains. In field cultivation, although there was no marked inferiority of Japanese strains to Chinese ones in either mycelium propagation or the rate of sclerotium formation, Chinese strains formed whiter sclerotia with a mean size more than twice that of Japanese ones. Representatives of Chinese and Japanese strains, Yunnan and Kaimondake, respectively, were tested for wood-rotting ability. More wood was utilized and the wood color was darker in trials of the Yunnan strain. Amplifications of total DNA of these 10 fungal strains with 2 primers, PC-6 and PC-11, in RAPD analysis showed a difference in the amplicon profile between Japanese and Chinese strains, suggesting differences in their genetic background. © 2011 The Japanese Society of Pharmacognosy and Springer.

A short and practical synthesis of the PDE4 inhibitor K-34 (1) was developed. This synthesis was achieved in four steps and with a 58% overall yield. The unique spiro acetal was created with except...

Five novel phenolic glycosides, adenophorasides A (1), B (2), C (3), D (4), and E (5), were isolated from commercial Adenophora roots, together with vanilloloside (6), 3,4-dimethoxybenzyl alcohol 7-O-beta-D: -glucopyranoside (7), and lobetyolin (8). The structures of the new compounds (1-5) were characterized as 4-hydroxy-3-methoxyphenylacetonitrile 4-O-beta-D: -glucopyranoside (1), 4-hydroxy-3-methoxyphenylacetonitrile 4-O-beta-D: -glucopyranosyl-(1--6)-beta-D: -glucopyranoside (2), 4-hydroxy-3-methoxyphenylacetonitrile 4-O-alpha-L: -rhamnopyranosyl-(1--6)-beta-D: -glucopyranoside (3), 4-hydroxyphenylacetonitrile 4-O-beta-D: -glucopyranosyl-(1--6)-beta-D: -glucopyranoside (4), and 4-hydroxy-3-methoxybenzyl alcohol 4-O-beta-D: -glucopyranosyl-(1--6)-beta-D: -glucopyranoside (5), respectively, by means of spectroscopic and chemical analyses.

金沢大学医薬保健研究域薬学系, The stem of the Akebia plant, "Mokutsu", is a crude diuretic and antiphlogistic drug. Japanese products prepared from wild Akebia plants cover most of the Mokutsu market. Two Akebia plants, Akebia quinata Decaisne (Aq) and A. trifoliata Koidzumi (At) of Lardizabalaceae, are standardized as Mokutsu in Japanese pharmacopoeia. These two Akebia plants along with A. × pentaphylla Makino (Ap), which is considered a hybrid with the morphology of Aq and At, can be distinguished by DNA sequence analysis of internal transcribed spacers 1 and 2 (ITS) of nuclear ribosome DNA. Here, we report the results of molecular genetic analysis of Akebia plants grown in various wild habitats in Japan. We found that each of three Akebia plants could be distinguished in terms of their locality according to their nucleotide sequence in ITS, specifically at positions 91, 128, 133, 134, and 221. Plants with a comparable habitat had similar nucleotide sequences at these five points. We also found Aq with ITS and nucleotide deletion at position 86 that was distributed only around Awajishima in Shikoku (A), Harimanada (B), and Kinki (C), including the chief production center of Akebia Caulis. The results of these ITS sequences enabled discrimination of plants originating from Akebia Caulis. © 2009 The Japanese Society of Pharmacognosy and Springer.

We have measured the magnetic torque of an MgB 2 single crystal in the various different fields below 10 kG by using a torque magnetometer and a 4 K closed cycle refrigerator. The MgB 2 single crystal was synthesized by the vapor transport method. The torque can be measured as an off-balance signal of the Wheatstone bridge of the four piezoresistors on a Si cantilever. The torque curves are analyzed by the Kogan model. The superconducting anisotropy γ is rather independent of temperature in 5 and 10 kG, but is dependent on field up to 60 kG. We consider that the field dependence of γ comes from the nature of the multiple superconducting gaps. The experimental results show that the π-band superconducting gaps have been deteriorated gradually up to a crossover field H * (π) ∼ 20 kG at 10 K when the magnetic field increases.

Obese adipose tissue is characterized by increased macrophage infiltration, which results in chronic inflammation in adipose tissue and leads to obesity-related diseases such as type 2 diabetes mellitus and atherosclerosis. The regulation of macrophage infiltration into adipose tissue is an important strategy for preventing and treating obesity-related diseases. In this study, we report that naringenin, a citrus flavonoid, suppressed macrophage infiltration into adipose tissue induced by short-term (14 days) feeding of a high-fat diet in mice; although naringenin did not show any differences in high-fat diet-induced changes of serum biochemical parameters in this short administration period. Naringenin suppressed monocyte chemoattractant protein-1 (MCP-1) in adipose tissue, and this effect was mediated in part through inhibition of c-Jun NH2-terminal kinase pathway. Naringenin also inhibited MCP-1 expression in adipocytes, macrophages, and a co-culture of adipocytes and macrophages. Our results suggest a mechanism by which daily consumption of naringenin may exhibit preventive effects on obesity-related diseases.

The glycosphingolipid neurosporaside (α-D-Glcp-(1 → 2)-β-D-Galp-(1 → 6)-β-D-Galp-(1 → 6)-β-D-Galp-(1 →)-Cer) occurs in Neurospora crassa. We attempted to synthesize neurosporaside by block synthesis (route A) and linear synthesis (route B). Oligosaccharide derivatives were synthesized using trimethylsilyltrifluoromethanesulfonate and N-iodosuccinimide/trifluoromethane sulfonic acid as promoters. The target tetrasaccharide could not be attained via route A, but route B showed potential: glycosidic bonds (β-D-Galp-(1 → 6)-β-D-Galp-(1 → 6)-β-D-Galp) were formed stereoselectively, leading to the synthesis of glycosphingolipid 2.

Photoaffinity labeling technology is a highly efficient method for cloning carbohydrate-binding proteins. When the carbohydrate probes are synthesized according to conventional methods, however, the reducing terminus of the sugar is opened to provide an acyclic structure. Our continued efforts to solve this problem led to the development of new molecular tools with an oligosaccharide structure that contains a phenyldiazirine group for the elucidation of carbohydrate–protein interactions. We investigated whether carbohydrate–lectin interactions are affected by differences in the glycosidic formation and synthesized three types of molecular tools containing Galp–GlcpNAc disaccharide ligands and a photoreactive group (1, 2, 3). Photoaffinity labeling validated the recognition of the new ligand by different glycosidic bonds. Photoaffinity labeling also demonstrated that both the reducing end sugar and non-reducing end sugar recognized the Erythrina cristagalli agglutinin.

Aconite tuber is a representative crude drug for warming the body internally in Japanese Kampo medicine and Chinese traditional medicine. The crude drug is used in major prescriptions for the aged. Varieties of Aconitum plants are distributed throughout the Japanese Islands, especially Hokkaido. With the aim of identifying the medicinal potential of Aconitum plants from Hokkaido, 107 specimens were collected from 36 sites in the summer of 2011 and 2012. Their nuclear DNA region, internal transcribed spacer (ITS), and aconitine alkaloid contents were analyzed. Phylogenic analysis of ITS by maximum parsimony analysis showed that the majority of the specimens were grouped into one cluster (cluster I), separated from the other cluster (cluster II) consisting of alpine specimens. The aconitine alkaloid content of the tuberous roots of 76 specimens showed 2 aspects—specimens from the same collection site showed similar aconitine alkaloid profiles, and cluster I specimens from different habitats showed various alkaloid profiles. Environmental pressure of each habitat is presumed to have caused the morphology and aconitine alkaloid profile of these genetically similar specimens to diversify.

An efficient route suitable for a large-scale preparation of KF31327 (1), a potent phosphodiesterase V inhibitor, has been developed. We selected 7-chloro-2,4(1H,3H)-quinazolinedione (15) as a starting material, which gave the desired 6-nitro compound with good selectivity. In the chlorination of 7-ethylamino-6-nitro-2,4(1H,3H)-quinazolinedione (17), reaction conditions were optimized to minimize the amount of phosphorus oxychloride, and 2,4-dichloro-7-ethylamino-6-nitroquinazoline (14) was obtained in excellent yield. After the selective substitution at C4 position, the chloro substituent at C2 position was successfully removed by hydrogenation concomitant with the reduction of nitro group. The construction of the imidazothione ring was achieved by using phenyl isothiocyanate as a thiocarbonyl donor instead of extremely flammable carbon disulfide. Multikilograms of drug substance have been successfully prepared by these procedures.

We propose a two-step effective medium theory to predict the optical properties of anodized alumina films based on the assumption that spherical aluminum particles are distributed in columnar structures within an anodized alumina film (distributed metallic column model). Using this model, theoretical parameters such as the volume ratio and distribution range of alumina particles were determined by fitting the experimental values and their dependencies on the anodization condition were evaluated. Using the above results, the optical constants of the alumina films were calculated. © 1998 Scripta Technica, Electron Comm Jpn Pt 2, 81(3): 23–28, 1998

The benzodienone 4 as a stable analogue of the bracken ultimate carcinogen (2) has been designed and synthesized. In the reaction with DNA the compound 4 was found to give only the N-7 alkylated product of guanine and no N-3 alkylated product of adenine and reveal guanine-selective cleavage in contrast to the natural dienone 2.

The enantioselective total synthesis of aplyronine A (1), a potent antitumor substance of marine origin, was achieved by a convergent approach. Three segments 4, 5, and 6, corresponding to the C5−C11, C21−C27, and C28−C34 portions of aplyronine A (1), were prepared using the Evans aldol reaction and the Sharpless epoxidation as key steps. The coupling reaction of 4 with iodide 7 followed by Julia olefination with sulfone 8 gave the C5−C20 segment 9, while the Julia coupling reaction between segments 5 and 6 provided the C21−C34 segment 10. Julia olefination between segments 9 and 10 and the subsequent four-carbon homologation reaction led to seco acid 83, which was converted into aplyronine A (1) by Yamaguchi lactonization followed by the introduction of two amino acids. The use of the [(3,4-dimethoxybenzyl)oxy]methyl group as a protecting group for the hydroxyl at C29 was crucial for this synthesis. The enantioselective synthesis of two natural congeners, aplyronines B (2) and C (3), was also carried out...

The hot water extract of adzuki (HWEA), which is produced as a byproduct in the adzuki bean boiling process, has anti-tumor, antioxidative, and anti-diabetic activities. In this study, we fractionated HWEA to 4 fractions using stepwise gradient column chromatography with water and ethanol, and demonstrated the effects of each fraction on antigen (Ag)-stimulated degranulation in rat basophilic leukemia RBL-2H3 cells. The 40% ethanol eluate extract (EtEx.40) showed the strongest inhibition level of these fractions. To reveal the inhibitory mechanisms underlying degranulation by EtEx.40, we investigated intracellular reactive oxygen species (ROS) production, intracellular free Ca²⁺ concentration ([Ca²⁺]i), and early intracellular signaling pathways. Treatment with EtEx.40 markedly inactivated Lyn following Ag stimulation, resulting in the suppressions of intracellular elevation of [Ca²⁺]i and production of ROS. To identify the active compound in EtEx.40, we isolated 7 flavonoids from EtEx.40 and calculated their inhibition levels on Ag-stimulated degranulation. These flavonoids inhibited degranulation by about 25-60%. We further examined the in vivo effects of HWEA or EtEx.40 using a passive cutaneous anaphylaxis (PCA) reaction. Both extracts strongly suppressed the PCA reaction. These findings suggest that HWEA and/or EtEx.40 are beneficial for alleviating type I allergic symptoms.

The enantioselective total synthesis of aplyronine A (1), a potent antitumor substance of marine origin, was achieved by a convergent approach. Three segments 4, 5, and 6, corresponding to the C5−C11, C21−C27, and C28−C34 portions of aplyronine A (1), were prepared using the Evans aldol reaction and the Sharpless epoxidation as key steps. The coupling reaction of 4 with iodide 7 followed by Julia olefination with sulfone 8 gave the C5−C20 segment 9, while the Julia coupling reaction between segments 5 and 6 provided the C21−C34 segment 10. Julia olefination between segments 9 and 10 and the subsequent four-carbon homologation reaction led to seco acid 83, which was converted into aplyronine A (1) by Yamaguchi lactonization followed by the introduction of two amino acids. The use of the [(3,4-dimethoxybenzyl)oxy]methyl group as a protecting group for the hydroxyl at C29 was crucial for this synthesis. The enantioselective synthesis of two natural congeners, aplyronines B (2) and C (3), was also carried out...

Processed root of aconite, Aconitum carmichaeli Debeaux--known as bushi in Japan--is indispensable for treating diseases among elderly persons in Japanese and Chinese traditional medicine. Its active component is bushi diester alkaloid (BDA), which consists of aconitine (ACO), mesaconitine (MES), hypaconitine (HYP), and jesaconitine (JES). Since an overdose of BDA results in severe side effects, the BDA content should be within safe limits. However, the BDA content of raw aconite root, even that produced by standard cultivation procedures, varies greatly. In this study, to clarify the cause of BDA variation, we examined the weight and BDA content of each part of cultivated A. carmichaeli: the aerial part, the mother tuberous root (MT), the daughter tuberous root (DT), and the rootlet (RL). We found the following positive relationships: between aerial part weight and DT weight, aerial part weight and BDA content in stem of apex, and BDA content in stem of apex and total BDA of DT attached to the plant. Furthermore, DT belonging to a higher weight group showed less BDA content variation. In addition, BDA of DT and those of MT and RL differ in both content and composition. In conclusion, it was suggested that the weight or the size of the aerial part was a good marker for monitoring BDA content and its variation in the tuberous root, and it was found to be desirable to prevent mixing MT and RL at harvest.

金沢大学大学院自然科学研究科生理活性物質科学, 金沢大学薬学部, We determined the DNA sequences of the internal transcribed spacer 1 and 2 (ITS 1 and 2), the 5.8S rRNA gene and most of the 28S rRNA gene of Poria cocos for the first time, and conducted analysis of 20 samples including cultured mycelias and crude drug materials obtained from various localities and markets. Direct sequencing of the ITS 1 and 2 regions of the samples, except for four wild samples, showed that they had identical DNA sequences for ITS 1 and 2 with nucleotide lengths of 997 bps and 460 bps, respectively. By cloning, the four wild samples were found to have combined sequences of common ITS sequences with 1 or 2-base-pair insertions. Altogether both ITS 1 and 2 sequences were substantially longer than those of other fungal crude drugs such as Ganoderma lucidum and Polyporus umbellatus. Thus, Poria cocos could be distinguished from these crude drugs and fakes by comparing the nucleotide length of PCR products of ITS 1 and 2. Contrary to the basic homogeneity in ITS 1 and 2, three types (Group 1, 2, 3) of the 28S rRNA gene with distinctive differences in length and sequence were found. Furthermore, Group 1 could be divided into three subgroups depending on differences at nucleotide position 690. Products with different types of 28S rRNA gene were found in crude drugs from Yunnan and Anhui Provinces as well as the Korean Peninsula, suggesting that the locality of the crude drugs does not guarantee genetic uniformity. The result of DNA typing of Poria cocos may help discrimination of the quality of the crude drug by genotype. © 2007 Pharmaceutical Society of Japan.

We respond to the Comment by Angst et al. and report the analysis that our assumption of the upper critical field $\ensuremath{\eta}{H}_{c2}$ is reasonable.

We examine the superconducting anisotropy γ c =(m c /m a b ) 1 / 2 of a metallic high-T c superconductor MgB 2 by measuring the magnetic torque of a single crystal. The anisotropy γ c does not depend sensitively on the applied magnetic field at 10 K. We obtain the anisotropy parameter y c =4.31′0.14. The torque curve shows the sharp hysteresis peak when the field is applied parallel to the boron layers. This comes from the intrinsic pinning and is experimental evidence for the occurrence of superconductivity in the boron layers.

We have investigated the magnetic torque of MgB 2 by using a torque magnetometer consisting of a 4 K closed cycle refrigerator and a variable field permanent magnet. Single crystals of MgB 2 have been synthesized by the vapor transport method. We examine the mass anisotropy γ =( m c / m ab ) 1/2 by analyzing the magnetic torque curves of MgB 2 . We discuss that γ is not sensitive to temperature in a constant field 10 kG.

Serum zinc concentration was measured by atomic absorption spectrophotometry in 30 normal males, 17 normal females, 20 patients with arteriosclerosis obliterans, 26 patients with thromboangiitis obliterans, 40 patients with Takayasu's disease. The mean serum zinc concentration was 93.9 +/- S.E.4.0 mug/100 ml in the normal male controls, 75.1 +/- 3.3 mug/100 ml in patients with arteriosclerosis obliterans without ulcers (P less than 0.05) and 79.2 +/- 6.5 mug/100 ml in patients with thromboangiitis obliterans without ulcers. Serum zinc concentration showed to be more decreased in patients with these diseases who had ulcers. The mean serum zinc concentration was 77.9 +/- 4.0 mug/100 ml in the normal female controls, 71.8 +/- 5.1 mug/100 ml in patients with Takayasu's disease who had never been treated with steroids and 59.1 +/- 2.7 mug/100 ml in patients with this disease who had been treated with this drug (P less than 0.01). The zinc level was significantly lower in CRP positive patients with Takayasu's disease than in CRP negative patients (P less than 0.05).

When an activity of respiratory center in brain is depressed by high O2 pressure in blood, CO2 pressure in blood increases and respiratory acidosis appears in some patients especially with chronic respiratory diseases. Resipratory acidosis induces disturance of consciousness and the phenomenon is called as CO2 narcosis. The authors reported 2 cases of CO2 narcosis under an inhalation with high concentration of oxygen in O2-tent. The 1st case: 60 years old male was suffered from chronic cor pulmonale during 8 years. He was taken in O2-tent because he had infection of respiratory tract and showed dyspnea. Under the inhalation with air containing 50% of oxygen, he showed sopor 6 hr later. At that time, his PaO2 was 72 mmHg, PaCO2 was 75 mmHg and pH of blood was 7.34. The 2nd case : 70 years old female was suffered from pulmonary emphysema during 6-7 years. She was taken in O2-tent because she had infection of respiratory tract and showed dyspnea. Under the inhalation with air containing 50% of oxygen, she showed somnolence 22 hr later. Her PaO2 was 68 mmHg, PaCO2 was 96 mmHg and pH of blood was 7.33. When both the cases were taken out from the O2-tent and were deeply respired in room air by their voluntary movement, their consciousness, blood gas content and pH improved rapidly. These results suggest a large influence of respiratory air content as a condition of environmental factors on homeostasis.

Gorrelations of the metabolic state of human heart to EGG and to Xray findings were studied and discussed.There was a significant decrease in myocardial citrate extraction and a tendency of disturbed lactate and pyruvate metabolism in the cases with ST-T changes. In many cases with ST-T abnormalities, myocardial oxygen extraction coefficient was increased.QTc correlated, in general, with oxygen extraction coefficient. The magni-tude of G in frontal plane showed slight but significant negative correlation with oxygen extraction coefficient.Oxygen extraction coefficient increased distinctly with the enlargement of the heart in cases with valvular disease, while it was not so apparent in patients with hypertensive and/or sclerotic heart disease without failure.In cases with cardiac enlargement due to valvular disease of the heart, ST-T abnormalities, increase in QTc and reduction of the magnitude of G were frequently observed.

Pyridinolcarbamate; Anginin (B23) is a venous bradykinin antagonist, which antagonizes competitively and reversibly the venoconstrictive effect of bradykinin and its active homologues. This compound has also been shown to antagonize the congestion, exudation and hemorrhage induced by brady-kinin and its active homologues and to remove the edematous change from experimental atheroma and to promote the normal fibrous healing of atheroma.This compound has also exhibited clinically a beneficial effect in the atherosclerotic diseases of the brain and the heart, however, the evaluation of the effectiveness of this kind of drug in these morbid conditions had to depend upon rather the indirect evidences. In order to see directly the effect of pyridinolcarbamate on the arterial lesion the atherosclerosis of extremities seems much more suitable. The authors have first attempted this compound in the treatment of atherosclerotic occlusion in lower extremities of 2 hos-pitalized patients; 33 years old and 57 years old men.The first patient aged 33 years suffered from a marked cyanosis of his right foot with a history of 6 months and the pulsations of right popliteal, posterior tibial and dorsal pedal arteries were completely absent and arterio-graphy revealed the complete obstruction of popliteal artery immediately above the knee joint. Pyridinolcarbamate was given 1Gm. daily and on the 3rd day of the medication the cyanosis disappeared dramatically and the intermittent claudication exhibited a slight improvement. On the 14th day of the medication the right dorsal pedal pulsation was recognized and on the 15th day the pulsation became visible through the skin and at the same time the pulsation of right popliteal artery began to be recognized. The volume of pulsation increased gradually but relatively rapidly and concomi-tantly with the remarkable improvement of intermittent claudication and on the 25th day of the treatment there was no differnce of skin temperature be-tween right and left foot and the pulsation exhibited almost normal volume in dorsal pedal artery. On the 26th day the pulsation of his right posterior tibial artery also reappeared. Under the medication of this compound no relapse and no untoward effects of the drug have been noted for 3 months period of the observation.The second case aged 57 years suffered from intermittent claudication and the absence of the pulsation of bilateral popliteal, dorsal pedal and pos-terior tibial arteries and the arteriography revealed the occlusion of the middle part of the right femoral artery.The treatment with pyridinolcarbamate for 2 weeks in a daily dose of one Gm. induced a slight improvement of intermittent claudication and on the end of third week a marked improvement was noted and at the same time the pulsation of his right dorsal pedal artery reappeared. His inter-mittent claudication exhibited a marked improvement concomitantly with the appearance of his dorsal pedal artery. A definite shortening in crest time in plethysmogram was noted in the bilateral second toes on the 130th day of the treatment. Thereafter his condition has been continuously good and he had no relapse and no untoward effects of the drug during the whole observation period of 5 months.The effect of pyridinolcarbamate seems to originate from the improve-ment of atheromatous changes by the compound in these patients.

The antiatherosclerotic effect of pyridinolcarbamate in man has been subjected to the present controlled clinical trial in 43 patients suffering from atherosclerosis obliterans with relatively stable signs and short claudication times (101 ± 8 sec.). To isolate individual factors such as age, sex, constitution, social condition, and spontaneous fluctuations of symptoms from the drug effect, a double-blind crossover technique (within-subject comparison) has been applied. After the initial 3 weeks of placebo wash out, a 10 week regimen of placebo or pyridinolcarbamate (1.5 Gm. daily) and another 10 weeks of alternative regimens were given successively. After drug regimen the prolonged crest time in plethysmography was shortened (p < 0.01) and claudication time prolonged with a statistically significant correlation (p < 0.05). The statistically significant preference for pyridinolcarbamate over placebo has been established by the effect on crest time alone, claudication time alone, and also major symptoms including ischemic ulcer, cyanosis, pain, and paresthesia of affected legs (p < 0.001). The relatively slow-acting and persistent effect of pyridinolcarbamate on the clinical signs observed in these patients encourages further histologic analysis of its effect on atheromatous lesions in man.