Design and application of ascorbic acid derivatives, Evaluation and identification of food factors for promoting health and healing, Analysis of structure-function relationship of bioactive compounds with chemical modification
Akihiro Tai and 山本 格 : ビタミン総合事典 (日本ビタミン学会 編) 9. ビタミンC, 9.5 薬理作用, 朝倉書店, Tokyo, Nov. 2010.
4.
Jun Takebayashi, Jianbin Chen and Akihiro Tai : Advanced Protocols in Oxidative Stress II (Donald Armstrong ed.) 20. A method for evaluation of antioxidant activity based on inhibition of free radical-induced erythrocyte hemolysis, The Humana Press, Totowa, New Jersey, Dec. 2009.
(Summary)
There are many in vitro methods for evaluating antioxidant activity. In this chapter, we describe an operationally simple cell-based assay, oxidative hemolysis inhibition assay (OxHLIA). OxHLIA is based on inhibition of free radical-induced membrane damage in erythrocytes by antioxidants. The advantage of this method is that it uses peroxyl radicals as pro-oxidants and erythrocytes as oxidizable targets so that the results obtained reflect biologically relevant radical-scavenging activity and microlocalization of antioxidants. We also present here a comparison of OxHLIA with other common methods (DPPH, ABTS(*+), and ORAC assays).
Rie Mukai, Okuyama Hitomi, Uchimura Miku, Sakao Kozue, Matsuhiro Miyu, Ikeda-Imafuku Mayumi, Yu Ishima, Nishikawa Miyu, Ikushiro Shinichi and Akihiro Tai : The binding selectivity of quercetin and its structure-related polyphenols to human serum albumin using a fluorescent dye cocktail for multiplex drug-site mapping., Bioorganic Chemistry, Vol.145, 107184, 2024.
2.
Takeru Koga, Kawahara Naoaki, Aburada Mei, Ono Asako, Mae Shiori, Yoshida Aina, Iwaoka Yuji, Ito Hideyuki and Akihiro Tai : Antiallergic activity of 3-O-dodecyl-L-ascorbic acid, Molecules, Vol.29, No.1, 69, 2024.
3.
Takeru Koga, Hideyuki Ito, Yuji Iwaoka, Toshiro Noshita and Akihiro Tai : Neurite outgrowth-promoting compounds from the petals of Paeonia lactiflora in PC12 cells, Molecules, Vol.27, No.22, 7670, 2022.
Takeru Koga, Nanako Shiki, Hideyuki Ito, Yuji Iwaoka and Akihiro Tai : Degranulation inhibitors from petals of Coreopsis grandiflora, Records of Natural Products, Vol.16, No.6, 645-650, 2022.
Yuji Iwaoka, Misaki Fukushima, Hideyuki Ito, Takeru Koga, Naoaki Kawahara and Akihiro Tai : Synthesis of ascorbic acid derivatives with different types of C8 straight acyl chain and their neurite outgrowth-enhancing activities, Journal of Nutritional Science and Vitaminology, Vol.68, No.3, 236-239, 2022.
(Summary)
We previously reported that 2-O-α-D-glucopyranosyl-6-O-octanoyl-L-ascorbic acid, having a C straight acyl chain, at a concentration of 100 μM remarkably enhanced nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells after being metabolized to L-ascorbic acid (AA) as an active form by esterase and α-glucosidase. In this study, to examine the structure-activity relationship of 6-O-substituted AA derivatives with a C straight acyl chain for neurite outgrowth-promoting activity, we synthesized AA derivatives 1-4 and compared their activities for promoting NGF-induced neurite outgrowth in PC12 cells. AA derivatives 1-4 showed neurite outgrowth-enhancing activity at 100 μM, while AA derivative 2 also showed the enhancing activity at 3 μM. Furthermore, AA derivative 2 as well as AA enhanced NGF-induced neurite outgrowth after being incorporated into PC12 cells via sodium-dependent vitamin C transporter as an anion transporter. The results suggested that AA derivative 2 has neurite outgrowth-enhancing activity in its intact form at a low concentration (3 μM) and that AA derivatives 1-4 showed their activities in the form of AA, a metabolite of these derivatives, at a higher concentration (100 μM).
Takeru Koga and Akihiro Tai : Neurite outgrowth-promoting compounds from cockscomb hydrolysate, Nutrients, Vol.14, No.7, 1422, 2022.
(Summary)
Cockscomb hydrolysate was found to have neurite outgrowth-promoting activity in PC12 cells. To investigate the neurite outgrowth-promoting compounds derived from cockscomb hydrolysate, bioassay-guided purification was carried out. Purified active fractions were obtained by liquid-liquid partition, followed by column chromatography. High-performance liquid chromatography and proton nuclear magnetic resonance analyses of the purified active fractions clarified that the main compounds are threonine, alanine, valine, and methionine. By screening for 20 kinds of amino acids, it was shown that valine and methionine, but not threonine and alanine, have neurite outgrowth-promoting activity. The results of activity evaluation of the mixture of amino acids indicated that alanine enhanced the activity of valine and that the mixture of valine and methionine showed a higher ratio of neurite formation than did each of them alone. On the other hand, dipeptides formed by valine and methionine showed weak neurite outgrowth-promoting activity. A mixture of threonine, alanine, valine, and methionine at the same concentrations as those in cockscomb hydrolysate showed neurite outgrowth-promoting activity comparable to that of cockscomb hydrolysate although threonine, alanine, valine, and methionine alone did not show activity at their concentrations in cockscomb hydrolysate. Therefore, the strong neurite outgrowth-promoting activity of cockscomb hydrolysate was considered to be due to the synergistic effect of threonine, alanine, valine, and methionine.
Iwaoka Yuji, Fukushima Misaki, Ito Hideyuki and Akihiro Tai : Highly efficient and low-cost process for synthesis of 2-O-α-D-glucopyranosyl-6-O-(2-propylpentanoyl)-L-ascorbic acid, Process Biochemistry, Vol.111, No.Part1, 71-77, 2021.
Kaori Miura, Hiroaki Matsuno, Yuji Iwaoka, Hideyuki Ito and Akihiro Tai : Antiallergic activity of 6-deoxy-2-O-methyl-6-(N-hexadecanoyl)amino-L-ascorbic acid, Molecules, Vol.26, No.15, 4684, 2021.
(Summary)
Allergy is an excessive immune response to a specific antigen. Type I allergies, such as hay fever and food allergies, have increased significantly in recent years and have become a worldwide problem. We previously reported that an ascorbic acid derivative having palmitoyl and glucosyl groups, 2--α-d-glucopyranosyl-6--hexadecanoyl-l-ascorbic acid (6-sPalm-AA-2G), showed inhibitory effects on degranulation in vitro and on the passive cutaneous anaphylaxis (PCA) reaction in mice. In this study, several palmitoyl derivatives of ascorbic acid were synthesized and a structure-activity relationship study was performed to discover more potent ascorbic acid derivatives with degranulation inhibitory activity. 6-Deoxy-2--methyl-6-(-hexadecanoyl)amino-l-ascorbic acid (2-Me-6--Palm-AA), in which a methyl group was introduced into the hydroxyl group at the C-2 position of ascorbic acid and in which the hydroxyl group at the C-6 position was substituted with an -palmitoyl group, exhibited much higher inhibitory activity for degranulation in vitro than did 6-sPalm-AA-2G. 2-Me-6--Palm-AA strongly inhibit the PCA reaction in mice at lower doses than those of 6-sPalm-AA-2G. These findings suggest that 2-Me-6--Palm-AA may be a promising therapeutic candidate for allergic diseases.
Doi Natsumi, Togari Hiro, Minagi Kenji, Iwaoka Yuji, Akihiro Tai, Nakaoji Koichi, Hamada Kazuhiko and Tatsuka Masaaki : 2-O-Octadecylascorbic acid represses RhoGDIβ expression and ameliorates DNA damage-induced abnormal spindle orientations, Journal of Cellular Biochemistry, Vol.122, No.7, 739-751, 2021.
(Summary)
The appropriate regulation of spindle orientation maintains proper tissue homeostasis and avoids aberrant tissue repair or regeneration. Spindle misorientation due to imbalance or improper functioning leads to a loss of tissue integrity and aberrant growth, such as tissue loss or overgrowth. Pharmacological manipulation to prevent spindle misorientation will enable a better understanding of how spindle orientation is involved in physiological and pathological conditions and will provide therapeutic possibilities to treat patients associated with abnormal tissue function caused by spindle misorientation. N-terminal-deleted Rho guanine nucleotide dissociation inhibitor β (RhoGDIβ/RhoGDI2/LyGDI) produced by caspase-3 activation perturbs spindle orientation in surviving cells following exposure to either ionizing radiation or UVC. Thus, presumably, RhoGDIβ cleaved by caspase-3 activation acts as a determinant of radiation-induced spindle misorientation that promote aberrant tissue repair due to deregulation of directional organization of cell population and therefore becomes a potential target of drugs to prevent such response. The objective of this study was to screen and identify chemicals that suppress RhoGDIβ expression. We focused our attention on ascorbic acid (AA) derivatives because of their impact on the maintenance of skin tissue homeostasis. Here, we screened for AA derivatives that suppress RhoGDIβ expression in HeLa cells and identified a lipophilic derivative, 2-O-octadecylascorbic acid (2-OctadecylAA), as a novel RhoGDIβ inhibitor that ameliorated ionizing radiation-induced abnormal spindle orientations. Among all examined AA derivatives, which were also antioxidative, the inhibition activity was specific to 2-OctadecylAA. Therefore, this activity was not due to simple antioxidant properties. 2-OctadecylAA was previously shown to prevent hepatocellular carcinoma development. Our findings suggest that the anticarcinogenic effects of 2-OctadecylAA are partly due to RhoGDIβ inhibition mechanisms by which spindle orientation perturbations are attenuated. Thus, the molecular targeting features of RhoGDIβ warrant its further development for the treatment or control of spindle orientation abnormalities that affect epithelial homeostasis.
Toshiro Noshita, Kentaro Fujita, Takeru Koga, Hidekazu Ouchi and Akihiro Tai : Synthesis and biological activity of (±)-7,3',4'-trihydroxyhomoisoflavan and its analogs, Bioorganic & Medicinal Chemistry Letters, Vol.31, 127674, 2021.
(Summary)
Acetylcholinesterase (AChE) inhibitors and neurite outgrowth promoters are thought to alleviate the symptoms of degenerative brain disorders, such as Alzheimer's disease. We designed and synthesized a series of homoisoflavonoids based on the structure of natural homoisoflavan isolated from Dracaena cambodiana dragon's blood. The homoisoflavonoids were then evaluated as AChE inhibitors and neurite outgrowth promoters. The catechol structure of the homoisoflavan B rings was important for AChE inhibition, and some of the homoisoflavonoids significantly promoted neurite outgrowth induced by nerve growth factor (NGF).
Takeru Koga, Takaiku Sakamoto, Eiji Sakuradani and Akihiro Tai : Neurite Outgrowth-Promoting Activity of Compounds in PC12 Cells from Sunflower Seeds, Molecules, Vol.25, No.20, 4748, 2020.
(Summary)
In the current super-aging society, the establishment of methods for prevention and treatment of Alzheimer's disease (AD) is an urgent task. One of the causes of AD is thought to be a decrease in the revel of nerve growth factor (NGF) in the brain. Compounds showing NGF-mimicking activity and NGF-enhancing activity have been examined as possible agents for improving symptoms. In the present study, sunflower seed extract was found to have neurite outgrowth-promoting activity, which is an NGF-enhancing activity, in PC12 cells. To investigate neurite outgrowth-promoting compounds from sunflower seed extract, bioassay-guided purification was carried out. The purified active fraction was obtained by liquid-liquid partition followed by some column chromatographies. Proton nuclear magnetic resonance and gas chromatography-mass spectrometry analyses of the purified active fraction indicated that the fraction was a mixture of β-sitosterol, stigmasterol and campesterol, with β-sitosterol being the main component. Neurite outgrowth-promoting activities of β-sitosterol, stigmasterol, campesterol and cholesterol were evaluated in PC12 cells. β-Sitosterol and stigmasterol showed the strongest activity of the four sterol compounds (β-sitosterol stigmasterol > campesterol > cholesterol), and cholesterol did not show any activity. The results indicated that β-sitosterol was the major component responsible for the neurite outgrowth-promoting activity of sunflower seeds. Results of immunostaining also showed that promotion by β-sitosterol of neurite formation induced by NGF was accompanied by neurofilament expression. β-Sitosterol, which showed NGF-enhancing activity, might be a candidate ingredient in food for prevention of AD.
Yuji Iwaoka, Nao Ikeda, Asako Ohno, Hideyuki Ito and Akihiro Tai : Antioxidant activity and Neurite outgrowth-enhancing activity of scorbamic acid and a red pigment derived from ascorbic acid., Natural Product Research, Vol.34, No.6, 838-842, 2020.
(Summary)
L-Ascorbic acid (AA), known as vitamin C, can form browning products by a non-enzymatic process during storage and the browning products cause deterioration of agricultural products. In the browning reaction, a red pigment, 2,2´-nitrilodi-2(2´)-deoxy-L-ascorbic acid ammonium salt (NDA), is generated from AA via L-scorbamic acid (SCA) as an intermediate. However, the biological activities of SCA and NDA have not yet been clarified. In this study, we assayed the antioxidant activities of SCA and NDA using ABTS radical cation and their neurite outgrowth-enhancing activities in PC12 cells. SCA showed stronger radical-scavenging activity than that of AA, while NDA hardly showed any activity. SCA and NDA enhanced the neurite outgrowth induced by dibutyryl cyclic AMP after their incorporation into cells in the same manner as that of AA. The results indicated that SCA has antioxidant activity and that SCA and NDA have neurite outgrowth-enhancing activity.
Yuta Morishita, Kaoru Ikeda, Hiroaki Matsuno, Hideyuki Ito and Akihiro Tai : Identification of degranulation inhibitors from rooibos (Aspalathus linearis) tea in rat basophilic leukaemia cells, Natural Product Research, Vol.33, No.10, 1472-1476, 2019.
(Summary)
Quercetin, luteolin and chrysoeriol were identified from rooibos tea as degranulation inhibitors in rat basophilic leukaemia cells. The degranulation inhibitory activity of chrysoeriol was first discovered in the present study. When quercetin, luteolin and chrysoeriol were mixed in the ratio that occurs in rooibos tea extract, the mixture inhibited antigen- and calcium ionophore-stimulated degranulation to the same degree as that by the whole rooibos tea extract. These findings indicate that these three flavonoids are the key factors underlying the degranulation inhibitory activity of rooibos tea.
Toshiro Noshita, Ryoya Onishi, Kaori Miura, Yoshitomo Hamada, Yuki Nishino, Hidekazu Ouchi and Akihiro Tai : Synthetic and in vitro studies to indicate that the structure of the PTP1B inhibitor isolated from Acanthopanax senticosus requires reinvestigation, Phytochemistry Letters, Vol.27, 214-218, 2018.
(Summary)
We herein describe the syntheses of 3,5-dimethoxy-4-{2-methoxy-4-[(1E)-3-oxoprop-1-en-1-yl]phenoxy}benzaldehyde (1) (the proposed structure for the naturally occurring protein tyrosine phosphatase 1B (PTP1B) inhibitor isolated from Acanthopanax senticosus) and its three positional isomers 3, 4, and 5. The inhibitory activities of these four compounds against PTP1B were also investigated. Comparison of the spectral data for 1, 3, 4, and 5 with the data obtained for the PTP1B inhibitor isolated from A. senticosus revealed that the original structural assignment as compound 1 was incorrect. In addition, none of the above four compounds exhibited inhibitory activity against PTP1B. Based on these results, it is apparent the structure of the PTP1B inhibitor isolated from Acanthopanax senticosus requires reinvestigation.
Toshiro Noshita, Kaori Miura, Kaoru Ikeda, Hidekazu Ouchi, Takuya Matsumoto and Akihiro Tai : Structure-activity relationships of flavanones, flavanone glycosides, and flavones in anti-degranulation activity in rat basophilic leukemia RBL-2H3 cells, Journal of Natural Medicines, Vol.72, No.2, 551-556, 2018.
(Summary)
The incidence of type I allergies, which are associated with mast cell degranulation and local inflammation, is increasing, and new treatments are needed. To date, structure-activity relationships of flavonoids in their degranulation-inhibiting activity have not been systematically characterized. In the current study, the degranulation-inhibiting activity of a series of flavonoids was evaluated. The following three observations were made: (1) the activity disappears when a sugar moiety is introduced into the A ring of the flavanone; (2) the activity depends on the number of hydroxyl groups on the B ring; (3) the activity is markedly enhanced when a double bond is introduced into the C ring. The information obtained in the current study may guide the development of a therapy for type I allergies.
Yuji Iwaoka, Kohei Nishino, Takahiro Ishikawa, Hideyuki Ito, Yoshihiro Sawa and Akihiro Tai : Affinity resins as new tools for identifying target proteins of ascorbic acid, Analyst, Vol.143, No.4, 874-882, 2018.
(Summary)
l-Ascorbic acid (AA) has diverse physiological functions, but little is known about the functional mechanisms of AA. In this study, we synthesized two types of affinity resin on which AA is immobilized in a stable form to identify new AA-targeted proteins, which can provide important clues for elucidating unknown functional mechanisms of AA. To our knowledge, an affinity resin on which AA as a ligand is immobilized has not been prepared, because AA is very unstable and rapidly degraded in an aqueous solution. By using the affinity resins, cytochrome c (cyt c) was identified as an AA-targeted protein, and we showed that oxidized cyt c exhibits specific affinity for AA. These results suggest that two kinds of AA-affinity resin can be powerful tools to identify new target proteins of AA.
Kaori Miura, Misaki Haraguchi, Hideyuki Ito and Akihiro Tai : Potential Antitumor Activity of 2-O-α-d-Glucopyranosyl-6-O-(2-Pentylheptanoyl)-l-Ascorbic Acid, International Journal of Molecular Sciences, Vol.19, No.2, E535, 2018.
(Summary)
Intravenous administration of high-dose ascorbic acid (AA) has been reported as a treatment for cancer patients. However, cancer patients with renal failure cannot receive this therapy because high-dose AA infusion can have side effects. To solve this problem, we evaluated the antitumor activity of a lipophilic stable AA derivative, 2-O-α-d-glucopyranosyl-6-O-(2-pentylheptanoyl)-l-ascorbic acid (6-bOcta-AA-2G). Intravenous administration of 6-bOcta-AA-2G suppressed tumor growth in colon-26 tumor-bearing mice more strongly than did AA, even at 1/10 of the molar amount of AA. Experiments on the biodistribution and clearance of 6-bOcta-AA-2G and its metabolites in tumor-bearing mice showed that 6-bOcta-AA-2G was hydrolyzed to 6-O-(2-propylpentanoyl)-l-ascorbic acid (6-bOcta-AA) slowly to yield AA, and the results suggested that this characteristic metabolic pattern is responsible for making the antitumor activity of 6-bOcta-AA-2G stronger than that of AA and that the active form of 6-bOcta-AA-2G showing antitumor activity is 6-bOcta-AA. In in vitro experiments, the oxidized form of 6-bOcta-AA as well as 6-bOcta-AA showed significant cytotoxicity, while the oxidized forms of ascorbic acid showed no cytotoxicity at all, suggesting that the antitumor activity mechanism of 6-bOcta-AA-2G is different from that of AA and that the antitumor activity is due to the reduced and oxidized form of 6-bOcta-AA. The findings suggest that 6-bOcta-AA-2G is a potent candidate as an alternative drug to intravenous high-dose AA.
Kaori Miura, Yuta Morishita, Hiroaki Matsuno, Yusuke Aota, Hideyuki Ito and Akihiro Tai : Anti-Allergic Activity of Monoacylated Ascorbic Acid 2-Glucosides, Molecules, Vol.22, No.12, E2202, 2017.
(Summary)
2-O-α-d-Glucopyranosyl-l-ascorbic acid (AA-2G) is one of the stable ascorbic acid (AA) derivatives known as provitamin C agents. We have previously synthesized two types of monoacylated derivatives of AA-2G, 6-O-acyl-2-O-α-d-glucopyranosyl-l-ascorbic acids having a straight-acyl chain of varying length from C to C18 (6-sAcyl-AA-2G) and a branched-acyl chain of varying length from C to C16 (6-bAcyl-AA-2G) in order to improve the bioavailability of AA-2G. In this study, 6-sAcyl-AA-2G and 6-bAcyl-AA-2G per se showed the inhibitory effects on hyaluronidase activity and degranulation. 6-sAcyl-AA-2G exhibited strong inhibitory effects on hyaluronidase activity and degranulation in a concentration-dependent manner, and the inhibitory effects tended to become stronger with increasing length of the acyl chain. 2-O-α-d-Glucopyranosyl-6-O-hexadecanoyl-l-ascorbic acid (6-sPalm-AA-2G), which has a straight C16 acyl chain, was the most potent effective for inhibition of hyaluronidase activity and for inhibition of degranulation among the 6-sAcyl-AA-2G derivatives and the two isomers of 6-sPalm-AA-2G. Furthermore, percutaneous administration of 6-sPalm-AA-2G significantly inhibited IgE-mediated passive cutaneous anaphylaxis reaction in mice. These findings suggest that 6-sPalm-AA-2G will be useful for treatment of allergies.
Akihiro Tai, Atsuko Iomori and Hideyuki Ito : Structural evidence for the DPPH radical-scavenging mechanism of 2-O-α-d-glucopyranosyl-l-ascorbic acid, Bioorganic & Medicinal Chemistry, Vol.25, No.20, 5303-5310, 2017.
(Summary)
2-O-α-d-Glucopyranosyl-l-ascorbic acid (AA-2G) exhibits biological activities after enzymatic hydrolysis to ascorbic acid (AA) by α-glucosidase. We have found that AA-2G per se exerted radical-scavenging activity toward 1,1-diphenyl-2-picrylhydrazyl radical (DPPH radical). The radical-scavenging property of AA-2G was greatly different from that of AA; that is, the reaction rate with DPPH radical of AA-2G was far slower than that of AA, but the long-lasting radical-scavenging ability per one molecule of AA-2G was superior to that of AA. We purified key intermediates for the characteristic radical-scavenging reaction of AA-2G and carried out time-course studies of the radical-scavenging reactions of the intermediates, AA-2G and AA to determine both the reaction rate and stoichiometry of AA-2G with DPPH radical. One mole of AA-2G quenched 2.7mol of DPPH radical over a period of 120min, while one mole of AA quenched 1.9mol of the radical. The high reaction stoichiometry of AA-2G against DPPH radical was associated with adduct formation of AA-2G with DPPH radical. The radical-scavenging reaction mechanism of AA-2G consists of the following three steps: (1) At an early stage of the reaction, AA-2G scavenged DPPH radical to generate AA-2G radical, (2) AA-2G radical immediately reacted with an additional DPPH radical to give two types of AA-2G-DPPH adducts and (3) AA-2G-DPPH adducts slowly quenched the other DPPH radical to generate several reaction products. Our results suggest the practical value of AA-2G, even before being converted into AA, as a beneficial antioxidant in food and cosmetic applications.
Toshiro Noshita, Akihiro Tai, Takuya Matsumoto, Kaori Miura, Kaoru Ikeda and Yoshitomo Hamada : Structure-activity relationship of flavanone. Anti-degranulation activity of 7-O-substituted hesperetin, Natural Product Research, Vol.31, No.18, 2137-2142, 2017.
(Summary)
A series of 7-O-substituted hesperetins was evaluated for degranulation-inhibiting activity in rat basophil leukaemia cells. 7-O-Methyl and 7-O-ethyl hesperetin exhibited potent anti-degranulation activity compared with the original hesperetin.
(Keyword)
Animals / Cell Line / Drug Evaluation, Preclinical / Flavanones / Hesperidin / Rats / structureactivity relationship
Kaori Miura and Akihiro Tai : 2-O-α-D-Glucopyranosyl-l-ascorbic acid as an antitumor agent for infusion therapy, Biochemistry and Biophysics Reports, Vol.10, 232-236, 2017.
(Summary)
Ascorbic acid (AA) has been reported as a treatment for cancer patients. Intravenous (iv) administration of high-dose AA increases plasma AA levels to pharmacologic concentrations and generates reactive oxygen species (ROS) to exert anti-tumor activity via enhancement of oxidative stress. However, AA is very unstable in aqueous solutions and it is impossible to preserve AA for a long period in a solution. 2-O-α-D-Glucopyranosyl-l-ascorbic acid (AA-2G), which is a glucoside derivative of AA, has been found to exhibit much higher stability than AA in aqueous solutions and it shows vitamin C activity after enzymatic hydrolysis to AA. To evaluate the effectiveness of AA-2G for cancer treatment, we examined the antitumor activity of AA-2G to murine colon carcinoma (colon-26) cells and in tumor-bearing mice. AA-2G did not show cytotoxicity to colon-26 cells, whereas AA exhibited a significant cytotoxic effect in a concentration-dependent manner. In colon-26 tumor-bearing mice, iv administration of high-dose AA-2G as well as that of AA significantly inhibited tumor growth. Experiments on the biodistribution and clearance of AA-2G in tumor-bearing mice showed that AA-2G was rapidly hydrolyzed to AA and exhibited significant antitumor activity. Treatment of tumor-bearing mice with AA-2G tended to increase plasma malondialdehyde level. These results indicated that the antitumor activity of AA-2G was caused by ROS generated by AA released by rapid hydrolysis of AA-2G.
Akihiro Tai, Asako Ohno and Hideyuki Ito : Isolation and Characterization of the 2,2'-Azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) Radical Cation-Scavenging Reaction Products of Arbutin, Journal of Agricultural and Food Chemistry, Vol.64, No.38, 7285-7290, 2016.
(Summary)
Arbutin, a glucoside of hydroquinone, has shown strong 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical cation-scavenging activity, especially in reaction stoichiometry. This study investigated the reaction mechanism of arbutin against ABTS radical cation that caused high stoichiometry of arbutin in an ABTS radical cation-scavenging assay. HPLC analysis of the reaction mixture of arbutin and ABTS radical cation indicated the existence of two reaction products. The two reaction products were purified and identified to be a covalent adduct of arbutin with an ABTS degradation fragment and 3-ethyl-6-sulfonate benzothiazolone. A time-course study of the radical-scavenging reactions of arbutin and the two reaction products suggested that one molecule of arbutin scavenges three ABTS radical cation molecules to generate an arbutin-ABTS fragment adduct as a final reaction product. The results suggest that one molecule of arbutin reduced two ABTS radical cation molecules to ABTS and then cleaved the third ABTS radical cation molecule to generate two products, an arbutin-ABTS fragment adduct and 3-ethyl-6-sulfonate benzothiazolone.
Nao Ishimata, Hideyuki Ito and Akihiro Tai : Structure-activity relationships of vanillic acid ester analogs in inhibitory effect of antigen-mediated degranulation in rat basophilic leukemia RBL-2H3 cells, Bioorganic & Medicinal Chemistry Letters, Vol.26, No.15, 3533-3536, 2016.
(Summary)
Methyl vanillate (1) showed strong degranulation inhibitory activity among vanillin derivatives tested. In order to find structure-activity relationships for developing anti-allergic agents with simple structures and potent activity, we synthesized several vanillic acid (VA) ester derivatives with C1-C4 and C8 alkyl chains and evaluated their degranulation inhibitory activities. The most active compound of VA ester derivatives was derivative 5 with a C4 straight alkyl chain, and derivative 5 exhibited approximately three-fold greater inhibitory activity than that of 1. Moreover, we designed 8 types of analogs based on 5, and we found that the minimum structure for potent degranulation inhibitory activity requires direct connection of the butyl ester moiety on the benzene ring and at least one hydroxyl group on the benzene ring. Butyl meta or para hydroxyl benzoate (10 or 11) has a simpler structure than that of 5 and exhibited more potent degranulation inhibitory activity than that of 5.
Ai Morita, Akihiro Tai, Hideyuki Ito, Natsuki Ganeko and Shin-Ichi Aizawa : Proanthocyanidins in an astringent persimmon inhibit Salmonella pathogenicity island 1 (SPI1) secretion, Journal of the Science of Food and Agriculture, Vol.96, No.5, 1798-1802, 2016.
(Summary)
Proanthocyanidins suppress the secretion of Salmonella pathogenicity island 1 virulence proteins.
Toshiro Noshita, Akihiro Tai, Hikaru Nishikawa, Kaoru Ikeda, Hidekazu Ouchi, Yoshitomo Hamada, Akiko Saito and Teiko Yamada : Synthesis and degranulation-inhibiting activities of the proposed apteniols B, C, and G, Bioscience, Biotechnology, and Biochemistry, Vol.79, No.11, 1743-1749, 2015.
(Summary)
The synthesis of compounds with the structures proposed for the oxyneolignan apteniols B, C, and G is described. The diphenyl ether skeletons of the proposed apteniols were formed via Ullmann ether synthesis. In particular, the spectral data for the synthesized apteniols B, C, and G did not agree with those previously reported for the isolated compounds. Furthermore, the synthesized proposed apteniol B did not show degranulation-inhibiting activity, while the prepared proposed apteniols C and G exhibited activities considerably weaker than that of the methyl ester of proposed apteniol A.
Kaori Miura, Futoshi Yazama and Akihiro Tai : Oxidative stress-mediated antitumor activity of erythorbic acid in high doses, Biochemistry and Biophysics Reports, Vol.3, 117-122, 2015.
(Summary)
Intravenous (iv) infusion of high-dose ascorbic acid (AA) has been used as a treatment for cancer patients. The tumoricidal action of AA occurs due to its prooxidant effect. Erythorbic acid (EA), one of the AA epimers, has reduced vitamin C activity, while the antioxidant activity of EA is similar to that of AA. Currently, other physiological and pharmacological functions of EA are not well known. We examined the cytotoxicity of EA to murine colon carcinoma (colon-26) cells and the antitumor activity of EA in tumor-bearing mice. Cytotoxic activity of EA to colon-26 cells was evaluated by using the calcein-AM assay. EA showed the same cytotoxic activity to colon-26 cells as that of AA. The cytotoxicity of EA was shown to be caused by oxidative stress. Next, colon-26 tumor-bearing mice were iv administered EA and AA on alternate days for 4 times, and tumor growth rates were measured. Tumor growth was significantly inhibited by administration of high-dose EA in vivo as well as AA. Finally, the in vivo biodistribution and clearance of EA and AA were investigated in tumor-bearing mice. Endogenous AA in the tumor was consumed to resist oxidative stress caused by reactive oxygen species that was generated by administered EA. These results indicated that the oxidative stress-mediated antitumor activity is one of the pharmacological functions of high-dose iv EA.
Tairo Ogura, Takeshi Bamba, Akihiro Tai and Eiichiro Fukusaki : Method for the Compound Annotation of Conjugates in Nontargeted Metabolomics Using Accurate Mass Spectrometry, Multistage Product Ion Spectra and Compound Database Searching, Mass Spectrometry, Vol.4, No.1, A0036, 2015.
(Summary)
Owing to biotransformation, xenobiotics are often found in conjugated form in biological samples such as urine and plasma. Liquid chromatography coupled with accurate mass spectrometry with multistage collision-induced dissociation provides spectral information concerning these metabolites in complex materials. Unfortunately, compound databases typically do not contain a sufficient number of records for such conjugates. We report here on the development of a novel protocol, referred to as ChemProphet, to annotate compounds, including conjugates, using compound databases such as PubChem and ChemSpider. The annotation of conjugates involves three steps: 1. Recognition of the type and number of conjugates in the sample; 2. Compound search and annotation of the deconjugated form; and 3. In silico evaluation of the candidate conjugate. ChemProphet assigns a spectrum to each candidate by automatically exploring the substructures corresponding to the observed product ion spectrum. When finished, it annotates the candidates assigning a rank for each candidate based on the calculated score that ranks its relative likelihood. We assessed our protocol by annotating a benchmark dataset by including the product ion spectra for 102 compounds, annotating the commercially available standard for quercetin 3-glucuronide, and by conducting a model experiment using urine from mice that had been administered a green tea extract. The results show that by using the ChemProphet approach, it is possible to annotate not only the deconjugated molecules but also the conjugated molecules using an automatic interpretation method based on deconjugation that involves multistage collision-induced dissociation and in silico calculated conjugation.
Kohei Kawata, Ken-ichi Morishita, Mariko Nakayama, Shoya Yamada, Toshiki Kobayashi, Yuki Furusawa, Sakae Arimoto-Kobayashi, Toshitaka Oohashi, Makoto Makishima, Hirotaka Naitou, Erika Ishitsubo, Hiroaki Tokiwa, Akihiro Tai and Hiroki Kakuta : RXR partial agonist produced by side chain repositioning of alkoxy RXR full agonist retains antitype 2 diabetes activity without the adverse effects, Journal of Medicinal Chemistry, Vol.58, No.2, 912-926, 2015.
(Summary)
We previously reported RXR partial agonist CBt-PMN (1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)-1H-benzotriazole-5-carboxylic acid: 5, EC50 = 143 nM, Emax = 75%), which showed a potent glucose-lowering effect without causing serious adverse effects. However, it remains important to elucidate the structural requirements for RXR efficacy and the glucose-lowering effect because RXR-permissive heterodimers such as PPAR/RXR or LXR/RXR are reported to be activated differently depending upon the chemical structure of RXR agonists. In this work, we show that an RXR partial agonist, NEt-4IB (6-[ethyl-(4-isobutoxy-3-isopropylphenyl)amino]pyridine-3-carboxylic acid: 8b, EC50 = 169 nM, Emax = 55%), can be obtained simply by repositioning the side chains (interchanging the isobutoxy and isopropoxy groups) at the hydrophobic moiety of the RXR full agonist NEt-3IB (6-[ethyl-(3-isobutoxy-4-isopropylphenyl)amino]pyridine-3-carboxylic acid: 7b, EC50 = 19 nM). NEt-4IB (8b) showed antitype 2 diabetes activity without the above side effects upon repeated oral administration to mice at 10 mg/kg/day, similarly to 5.
(Keyword)
Animals / COS Cells / Cercopithecus aethiops / Diabetes Mellitus, Type 2 / Female / Hypoglycemic Agents / Male / Mice / Mice, Inbred ICR / Rats / Rats, Sprague-Dawley / Retinoid X Receptors
Toshiki Kobayashi, Yuki Furusawa, Shoya Yamada, Masaru Akehi, Fumiaki Takenaka, Takanori Sasaki, Akiya Akahoshi, Takahisa Hanada, Eiji Matsuura, Hiroyuki Hirano, Akihiro Tai and Hiroki Kakuta : Positron emission tomography to elucidate pharmacokinetic differences of regioisomeric retinoid X receptor agonists, ACS Medicinal Chemistry Letters, Vol.6, No.3, 334-338, 2015.
(Summary)
RXR partial agonist NEt-4IB (2a, 6-[ethyl-(4-isobutoxy-3-isopropylphenyl)amino]pyridine-3-carboxylic acid: EC50 = 169 nM, E max = 55%) showed a blood concentration higher than its E max after single oral administration at 30 mg/kg to mice, and repeated oral administration at 10 mg/kg/day to KK-A(y) mice afforded antitype 2 diabetes activity without the side effects caused by RXR full agonists. However, RXR full agonist NEt-3IB (1a), in which the isobutoxy and isopropyl groups of 2a are interchanged, gave a much lower blood concentration than 2a. Here we used positron emission tomography (PET) with tracers [(11)C]1a, [(11)C]2a and fluorinated derivatives [(18)F]1b, [(18)F]2b, which have longer half-lives, to examine the reason why 1a and 2a exhibited significantly different blood concentrations. As a result, the reason for the high blood concentration of 2a after oral administration was found to be linked to higher intestinal absorbability together with lower biliary excretion, compared with 1a.
Akihiro Tai, Mei Aburada and Hideyuki Ito : A simple efficient synthesis and biological evaluation of 3-O-ethylascorbic acid, Bioscience, Biotechnology, and Biochemistry, Vol.78, No.12, 1984-1987, 2014.
(Summary)
A single-step synthesis of 3-O-ethyl-l-ascorbic acid was performed without the induction of protecting groups. Sodium l-ascorbate reacted with ethyl bromide in DMSO to give 3-O-ethylascorbic acid in a yield of 51.0%. 3-O-Ethylascorbic acid enhanced dibutyryl cyclic AMP-induced neurite outgrowth in PC12 cells.
Ryuji Asano, Amon Nagami, Yuki Fukumoto, Kaori Miura, Futoshi Yazama, Hideyuki Ito, Isao Sakata and Akihiro Tai : Synthesis and biological evaluation of new BSH-conjugated chlorin derivatives as agents for both photodynamic therapy and boron neutron capture therapy of cancer, Journal of Photochemistry and Photobiology B: Biology, Vol.140, 140-149, 2014.
(Summary)
New disodium mercaptoundecahydro-closo-dodecaborate (BSH)-conjugated chlorin derivatives 11, 12, 16 and 20 as agents for both photodynamic therapy (PDT) and boron neutron capture therapy (BNCT) of cancer were synthesized. The in vivo biodistribution and clearance of 11, 12, 16 and 20 were investigated in tumor-bearing mice. Compounds 12 and 16 showed good tumor-selective accumulation among the four derivatives. The time to maximum accumulation of compound 16 in tumor tissue was one-fourth of that of compound 12, and clearance from normal tissues of compound 16 was similar to that of compound 12. The in vivo therapeutic efficacy of PDT using 16, which has twice as many boron atoms as 12, was evaluated by measuring tumor growth rates in tumor-bearing mice with 660 nm light-emitting diode irradiation at 6h after injection of 16. Tumor growth was significantly inhibited by PDT using 16. These results suggested that 16 is a good candidate for both PDT and BNCT of cancer.
Akihiro Tai, Kanako Fukunaga, Asako Ohno and Hideyuki Ito : Antioxidative properties of ascorbigen in using multiple antioxidant assays, Bioscience, Biotechnology, and Biochemistry, Vol.78, No.10, 1723-1730, 2014.
(Summary)
The antioxidative properties of ascorbigen, one of the major indole-derived compounds of Brassica vegetables, were systematically evaluated using multiple assay systems with comparison to the well-known antioxidants ascorbic acid and Trolox. We first performed assays using model radicals, DPPH radical, galvinoxyl radical, and ABTS radical cation (ABTS(•+)). Ascorbigen showed stronger activity than that of ascorbic acid in the ABTS(•+)-scavenging assay but showed no activity in the DPPH radical- and galvinoxyl radical-scavenging assays. In the ABTS(•+)-scavenging assay, the indole moiety of ascorbigen contributed to scavenging of the radicals to produce indole-3-aldehyde as one of the final reaction products. The activity of ascorbigen was then evaluated by an oxygen radical absorbance capacity assay and an oxidative hemolysis inhibition assay using physiologically relevant peroxyl radicals, AAPH-derived radicals. Ascorbigen showed much stronger antioxidant activity than did ascorbic acid and Trolox. Therefore, antioxidant activity of ascorbigen might be more beneficial than has been thought for daily health care.
Hikaru Nishikawa, Toshiro Noshita, Akihiro Tai, Hidekazu Ouchi, Taisuke Okamoto, Akiko Saito, Teiko Yamada, Atsuko Iomori and Nao Ishimata : Syntheses and biological activities of the proposed structure of apteniol A and its derivatives, Bioscience, Biotechnology, and Biochemistry, Vol.78, No.9, 1485-1489, 2014.
(Summary)
We describe the syntheses of the proposed structure of diphenyl ether oxyneolignan, apteniol A and its derivatives. The diphenyl ether moiety of proposed apteniol A was formed via Ullmann ether synthesis, but the spectral data of the synthesized apteniol A did not agree with that in previous studies. The dimethyl ester derivative of the proposed apteniol A was found to enhance neurite outgrowth in PC12 cells and inhibit antigen-induced degranulation in RBL-2H3 cells.
Ryuji Asano, Amon Nagami, Yuki Fukumoto, Kaori Miura, Futoshi Yazama, Hideyuki Ito, Isao Sakata and Akihiro Tai : Synthesis and biological evaluation of new boron-containing chlorin derivatives as agents for both photodynamic therapy and boron neutron capture therapy of cancer, Bioorganic & Medicinal Chemistry Letters, Vol.24, No.5, 1339-1343, 2014.
(Summary)
New boron-containing chlorin derivatives 9 and 13 as agents for both photodynamic therapy (PDT) and boron neutron capture therapy (BNCT) of cancer were synthesized from photoprotoporphyrin IX dimethyl ester (2) and L-4-boronophenylalanine-related compounds. The in vivo biodistribution and clearance of 9 and 13 were investigated in tumor-bearing mice. The time to maximum accumulation of compound 13 in tumor tissue was one-fourth of that of compound 9, and compound 13 showed rapid clearance from normal tissues within 24h after injection. The in vivo therapeutic efficacy of PDT using 13 was evaluated by measuring tumor growth rates in tumor-bearing mice with 660 nm light-emitting diode irradiation at 3h after injection of 13. Tumor growth was significantly inhibited by PDT using 13. These results suggested that 13 might be a good candidate for both PDT and BNCT of cancer.
Yukako Hanada, Atsuko Iomori, Rie Ishii, Eiichi Gohda and Akihiro Tai : Protection of free radical-induced cytotoxicity by 2-O-α-D-glucopyranosyl-L-ascorbic acid in human dermal fibroblasts, Bioscience, Biotechnology, and Biochemistry, Vol.78, No.2, 301-306, 2014.
(Summary)
The stable ascorbic acid (AA) derivative, 2-O-α-D-glucopyranosyl-L-ascorbic acid (AA-2G), exhibits vitamin C activity after enzymatic hydrolysis to AA. The biological activity of AA-2G per se has not been studied in detail, although AA-2G has been noted as a stable source for AA supply. The protective effect of AA-2G against the oxidative cell death of human dermal fibroblasts induced by incubating with 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) for 24 h was investigated in this study. AA-2G showed a significant protective effect against the oxidative stress in a concentration-dependent manner. AA-2G did not exert a protective effect during the initial 12 h of incubation, but had a significant protective effect in the later part of the incubation period. Experiments using a α-glucosidase inhibitor and comparative experiments using a stereoisomer of AA-2G confirmed that AA-2G had a protective effect against AAPH-induced cytotoxicity without being converted to AA. Our results provide an insight into the efficacy of AA-2G as a biologically interesting antioxidant and suggest the practical use of AA-2G even before being converted into AA as a beneficial antioxidant.
Akihiro Tai, Yuji Iwaoka and Hideyuki Ito : Highly efficient and regioselective production of an erythorbic acid glucoside using cyclodextrin glucanotransferase from Thermoanaerobacter sp. and amyloglucosidase, Journal of Molecular Catalysis B: Enzymatic, Vol.92, 19-23, 2013.
(Summary)
In order to continuously supply erythorbic acid (EA) for long-term cell cultures, we synthesized a stable EA derivative, 2-O-α-d-glucopyranosyl-d-erythorbic acid (EA-2G), as a useful tool for analyzing the biological function of EA. The specific and efficient production process of EA-2G consisted of two steps: transglycosylation by cyclodextrin glucanotransferase (CGTase) from Thermoanaerobacter sp. and hydrolysis by amyloglucosidase from Aspergillus niger. EA-2G was regioselectively formed by CGTase using EA and γ-cyclodextrin in pH 4.0 acetate buffer at 40 °C for 24 h. It seemed that several EA-2-oligoglucosides were also formed in this reaction mixture. Additional hydrolysis at 60 °C for 2 h of the reaction mixture by glucoamylase resulted in efficient production of EA-2G. EA-2G was obtained in two steps in 49.1% overall yield from EA.
Yoshimi Watanabe, Kazuyasu Murakami, Koji Sakamoto, Tomoko Fujiwara, Akihiro Tai and Norio Muto : Production of γ-Aminobutyric Acid in Pumpkin Tissue Treated with Freeze-thaw Infusion of L-Glutamic Acid Monosodium Salt, Food Science and Technology Research, Vol.19, No.4, 641-646, 2013.
(Summary)
We studied a method for producing high concentrations of γ-aminobutyric acid (GABA) in pumpkin tissue using freeze-thaw infusion. We introduced L-glutamic acid monosodium salt (MSG) into pumpkins by freeze thaw infusion, and observed that high concentrations of GABA were produced due to the action of the glutamate decarboxylase present in the pumpkins. For an enzyme reaction at 3°C, the amount of GABA peaked when the MSG concentration was 1% (w/w) and enzyme reaction time was about 48 h. When a softening enzyme (Macerozyme 2A) was added together with MSG and infusion was performed, a soft pumpkin that contained GABA at high concentrations was obtained. By freeze-thawing with MSG, it is now possible to manufacture functional foods that have two added values, i.e., in addition to enrichment of GABA, their hardness can also be adjusted.
Ryuji Asano, Amon Nagami, Yuki Fukumoto, Futoshi Yazama, Hideyuki Ito, Isao Sakata and Akihiro Tai : Synthesis and biological evaluation of new chlorin derivatives as potential photosensitizers for photodynamic therapy, Bioorganic & Medicinal Chemistry, Vol.21, No.8, 2298-2304, 2013.
(Summary)
Three new water-soluble chlorin derivatives 3, 5 and 8 for potential use as photosensitizers in photodynamic therapy (PDT) for cancer were synthesized from photoprotoporphyrin IX dimethyl ester (1). The in vivo biodistribution and clearance of chlorin derivatives 3, 5 and 8 were investigated in tumor-bearing mice. Iminodiacetic acid derivative 8 showed the greatest tumor-selective accumulation among the new chlorin derivatives with maximum accumulation in tumor tissue at 3h after intravenous injection and rapid clearance from normal tissues within 24h after injection. The in vivo therapeutic efficacy of PDT using 8 was evaluated by measuring tumor growth rates in tumor-bearing mice with 660 nm light-emitting diode irradiation at 3h after injection of 8. Tumor growth was significantly inhibited by PDT using 8. These results indicate that iminodiacetic acid derivative 8 is useful as a new photosensitizer to overcome the disadvantages of photosensitizers that are currently in clinical use.
Fuminori Ohsawa, Shoya Yamada, Nobumasa Yakushiji, Ryosuke Shinozaki, Mariko Nakayama, Kohei Kawata, Manabu Hagaya, Toshiki Kobayashi, Kazutaka Kohara, Yuuki Furusawa, Chisa Fujiwara, Yui Ohta, Makoto Makishima, Hirotaka Naitou, Akihiro Tai, Yutaka Yoshikawa, Hiroyuki Yasui and Hiroki Kakuta : Mechanism of retinoid X receptor partial agonistic action of 1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)-1H-benzotriazole-5-carboxylic acid and structural development to increase potency, Journal of Medicinal Chemistry, Vol.56, No.5, 1865-1877, 2013.
(Summary)
We have reported that retinoid X receptor (RXR) partial agonist 1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)-1H-benzotriazole-5-carboxylic acid (CBt-PMN, 4a) shows a significant antidiabetes effect in the KK-A(y) type 2 diabetes model mice, with reduced side effects compared to RXR full agonists. To elucidate the mechanism of the RXR partial agonist activity of 4a, we synthesized derivatives of 4a, evaluated their RXR agonist activity, and performed structure-activity relationship analysis. Reporter gene assay revealed that though 6b, which possesses an amino group at the 2-position of 5-carboxybenzimidazole, showed RXR full-agonist activity, compounds 6d and 6e, which possess an oxygen atom and a sulfur atom at the corresponding position, respectively, showed weak RXR agonist activity. On the other hand, 6c, which has a trifluoromethyl group at the corresponding position, acts as an RXR partial agonist, having similar Emax (67 ± 2%) and lower EC50 (15 ± 0 nM) compared to those of 4a (Emax = 75 ± 4%, EC50 = 143 ± 2 nM). A fluorescence polarization assay of cofactor recruitment confirmed that fluorescein-labeled D22 coactivator peptide was less efficiently recruited to RXR by 4a and 6c than by LGD1069 (1), a known RXR full agonist. Electrostatic potential field calculations and computational docking studies suggested that full agonists show an electrostatic attraction, which stabilizes the holo structure and favors coactivator recruitment, between the side chain at the benzimidazole 2-position and the α-carbonyl oxygen of asparagine-306 in helix 4 (H4) of the RXR receptor. However, RXR partial agonists 4a and 6c lack this interaction. Like 4a, 6c showed a significant antidiabetes effect in KK-A(y) type 2 diabetes model mice with reduced levels of the side effects associated with RXR full agonists. These findings should aid the design of new RXR partial agonists as antitype 2 diabetes drug candidates.
Jun Takebayashi, Noriko Iwahashi, Yoshiko Ishimi and Akihiro Tai : Development of a simple 96-well plate method for evaluation of antioxidant activity based on the oxidative hemolysis inhibition assay (OxHLIA), Food Chemistry, Vol.134, No.1, 606-610, 2012.
(Summary)
OxHLIA is a cell-based antioxidant assay for evaluating inhibition of free radical-induced haemolysis in sheep erythrocytes by antioxidants. The original OxHLIA was performed in a test-tube format, and the degree of haemolysis was determined by absorbance of the supernatant after centrifugation. This made it difficult to evaluate large numbers of samples at the same time. Thus, we first showed here that the degree of haemolysis can be determined by turbidity of the erythrocyte suspension without centrifugation. We then developed OxHLIA in a 96-well plate format by adapting the turbidity method. There have already been some methodological studies on cell-based antioxidant assays using erythrocytes, and the method described here was comprised of the best mix of these earlier works with ingenious modifications. This plate method enables evaluation of large numbers of samples in small quantity at the same time with satisfactory precision and reproducibility.
Hiroki Kakuta, Nobumasa Yakushiji, Ryosuke Shinozaki, Fuminori Ohsawa, Shoya Yamada, Yui Ohta, Kohei Kawata, Mariko Nakayama, Manabu Hagaya, Chisa Fujiwara, Makoto Makishima, Shigeyuki Uno, Akihiro Tai, Ami Maehara, Masaru Nakayama, Toshitaka Oohashi, Hiroyuki Yasui and Yutaka Yoshikawa : RXR Partial Agonist CBt-PMN Exerts Therapeutic Effects on Type 2 Diabetes without the Side Effects of RXR Full Agonists, ACS Medicinal Chemistry Letters, Vol.3, No.5, 427-432, 2012.
(Summary)
Treating insulin resistance and type 2 diabetes in rodents, currently known retinoid X receptor (RXR) agonists induce significant adverse effects. Here we introduce a novel RXR partial agonist CBt-PMN (11b), which shows a potent glucose-lowering effect and improvements of insulin secretion and glucose tolerance without the serious adverse effects caused by RXR full agonists. We suggest that RXR partial agonists may be a new class of antitype 2 diabetes drug candidates.
Akihiro Tai, Sayoko Ikeda, Futoshi Yazama and Hideyuki Ito : Intramolecular acyl migration and enzymatic hydrolysis of 2-O-α-D-glucopyranosyl-6-O-(2-pentylheptanoyl)-L-ascorbic acid, Journal of Bioscience and Bioengineering, Vol.113, No.4, 545-548, 2012.
(Summary)
2-O-α-D-Glucopyranosyl-6-O-(2-pentylheptanoyl)-L-ascorbic acid (6-bDode-AA-2G) underwent an intramolecular acyl migration to yield approximately 12% of 2-O-α-D-glucopyranosyl-5-O-(2-pentylheptanoyl)-L-ascorbic acid (5-bDode-AA-2G) in neutral solutions for 3 days. In small intestine homogenate from guinea pigs for 12h, 6-bDode-AA-2G, which hardly underwent acyl migration to give 5-bDode-AA-2G, was predominantly hydrolyzed with α-glucosidase and then with esterase to ascorbic acid.
Hiroki Kakuta, Fuminori Ohsawa, Shoya Yamada, Makoto Makishima, Akihiro Tai, Hiroyuki Yasui and Yutaka Yoshikawa : Feasibility of structural modification of retinoid X receptor agonists to separate blood glucose-lowering action from adverse effects: studies in KKA(y) type 2 diabetes model mice, Biological & Pharmaceutical Bulletin, Vol.35, No.4, 629-633, 2012.
(Summary)
Retinoid X receptor (RXR) agonists are reported to exhibit blood glucose-lowering action owing to peroxisome proliferator-activated receptor (PPAR)/RXR or liver X receptor (LXR)/RXR activation, but may also cause adverse effects such as blood triglyceride elevation. In order to examine the feasibility of separating the glucose-lowering action from the adverse effects, we examined the effects of RXR agonists (NEt-TMN), NEt-3IB, and NEt-3IP, which have different heterodimer-activating patterns, in KKA(y) type 2 diabetes model mice. We found that NEt-3IB induced lower degrees of hepatomegaly and blood triglyceride (TG) elevation than the other RXR agonists, even though all of them showed similar blood glucose-lowering action on repeated administration. These findings indicate that structural modification of RXR agonists is a potentially effective strategy to reduce adverse effects while retaining desired activities.
(Keyword)
Animals / Blood Glucose / COS Cells / Cercopithecus aethiops / cholesterol / Diabetes Mellitus, Type 2 / Disease Models, Animal / Hypoglycemic Agents / Liver / Liver X Receptors / Male / Mice / Organ Size / Orphan Nuclear Receptors / PPAR gamma / Retinoid X Receptors / Triglycerides
Akihiro Tai, Takeshi Sawano and Hideyuki Ito : Antioxidative properties of vanillic acid esters in multiple antioxidant assays, Bioscience, Biotechnology, and Biochemistry, Vol.76, No.2, 314-318, 2012.
(Summary)
The antioxidative properties of vanillic acid esters were systematically evaluated by multiple assays to compare with the well-known antioxidants, vanillic acid and Trolox. We first performed assays with the model radicals, DPPH, galvinoxyl and ABTS cation (ABTS(•+)) types. Methyl vanillate, ethyl vanillate and butyl vanillate showed stronger activity than Trolox in the ABTS(•+)-scavenging assay, but showed no activity in the DPPH radical- and galvinoxyl radical-scavenging assays. In contrast, vanillic acid could quench the three radicals. We then evaluated their antioxidative activities by an ORAC assay and an oxidative hemolysis inhibition assay (OxHLIA), using physiologically relevant peroxyl radicals. Vanillic acid esters and vanillic acid exerted much stronger activity than Trolox in the ORAC assay and OxHLIA. The antioxidative activity by OxHLIA was strongly correlated to the lipophilicity of vanillic acid and its esters. These results indicate that the protective effect of vanillic acid esters against free radical-induced biomembrane damage increased with increasing lipophilicity.
Hidekazu Ishimoto, Akihiro Tai, Morio Yoshimura, Yoshiaki Amakura, Takashi Yoshida, Tsutomu Hatano and Hideyuki Ito : Antioxidative properties of functional polyphenols and their metabolites assessed by an ORAC assay, Bioscience, Biotechnology, and Biochemistry, Vol.76, No.2, 395-399, 2012.
(Summary)
We compared the antioxidative activities of polyphenol metabolites with those of intact functional polyphenols by an assay of the oxygen radical absorbance capacity (ORAC). The metabolites of ellagitannin geraniin, chlorogenic acid, and (-)-epigallocatechin gallate displayed more potent antioxidative activity than their respective original compounds. Our findings suggest that these metabolites may play important roles as biological antioxidants after their consumption.
Akihiro Tai, Takeshi Sawano and Futoshi Yazama : Antioxidant properties of ethyl vanillin in vitro and in vivo, Bioscience, Biotechnology, and Biochemistry, Vol.75, No.12, 2346-2350, 2011.
(Summary)
We systematically evaluated the antioxidant activity of ethyl vanillin, a vanillin analog, as compared with the activities of vanillin and other vanillin analogs using multiple assay systems. Ethyl vanillin and vanillin exerted stronger antioxidant effects than did vanillyl alcohol or vanillic acid in the oxygen radical absorbance capacity (ORAC) assay, although the antioxidant activities of vanillyl alcohol and vanillic acid were clearly superior to those of ethyl vanillin and vanillin in the three model radical assays. The antioxidant activity of ethyl vanillin was much stronger than that of vanillin in the oxidative hemolysis inhibition assay, but was the same as that of vanillin in the ORAC assay. Oral administration of ethyl vanillin to mice increased the concentration of ethyl vanillic acid, and effectively raised antioxidant activity in the plasma as compared to the effect of vanillin. These data suggest that the antioxidant activity of ethyl vanillin might be more beneficial than has been thought in daily health practice.
(Keyword)
Administration, Oral / Animals / Benzaldehydes / Free Radical Scavengers / Hemolysis / Male / Mice / Mice, Inbred ICR / oxidation and reduction / reactive oxygen species
Akihiro Tai, Yuji Iwaoka and Hideyuki Ito : Regioselective monoacylation of 2-O-α-D-glucopyranosyl-L-ascorbic acid by a polymer catalyst in N,N-dimethylformamide, Carbohydrate Research, Vol.346, No.15, 2511-2514, 2011.
(Summary)
6-O-Dodecanoyl-2-O-α-D-glucopyranosyl-L-ascorbic acid (6-sDode-AA-2G) was synthesized from 2-O-α-D-glucopyranosyl-L-ascorbic acid and lauric anhydride with a polymer catalyst, poly(4-vinylpyridine), in N,N-dimethylformamide without the introduction of protecting groups. The optimum reaction conditions enabled 6-sDode-AA-2G to be synthesized in a yield of 49.7%. The yield and the regioselectivity in this method were far superior to those in our previous method by using an enzyme. The polymer catalyst could be recycled more than five times without any significant activity loss.
Hidekazu Ishimoto, Mari Shibata, Yuki Myojin, Hideyuki Ito, Yukio Sugimoto, Akihiro Tai and Tsutomu Hatano : In vivo anti-inflammatory and antioxidant properties of ellagitannin metabolite urolithin A, Bioorganic & Medicinal Chemistry Letters, Vol.21, No.19, 5901-5904, 2011.
(Summary)
Urolithin A is a major metabolite produced by rats and humans after consumption of pomegranate juice or pure ellagitannin geraniin. In this study, we investigated the anti-inflammatory effect of urolithin A on carrageenan-induced paw edema in mice. The volume of paw edema was reduced at 1h after oral administration of urolithin A. In addition, plasma in treated mice exhibited significant oxygen radical antioxidant capacity (ORAC) scores with high plasma levels of the unconjugated form at 1h after oral administration of urolithin A. These results indicate strong associations among plasma urolithin A levels, the plasma ORAC scores, and anti-inflammatory effects and may help explain a mechanism by which ellagitannins confer protection against inflammatory diseases.
Futoshi Yazama and Akihiro Tai : Unexpected role of α-fetoprotein in spermatogenesis, PLoS ONE, Vol.6, No.5, e19387, 2011.
(Summary)
AFP is a well-known cancer-specific marker, but AFP has no known function in healthy human beings. Our findings indicate that AFP expressed under EC conditions plays a role as a regulatory factor in spermatogenesis and in hepatic generation.
(Keyword)
Animals / Cryptorchidism / Electrophoresis, Polyacrylamide Gel / Hepatectomy / Humans / immunohistochemistry / Male / Mice / Molecular Sequence Data / Seminiferous Tubules / Spermatogenesis / Testis / alpha-Fetoproteins
Ryosuke Fukai, Xiaoxia Zheng, Kazunori Motoshima, Akihiro Tai, Futoshi Yazama and Hiroki Kakuta : Design and synthesis of novel cyclooxygenase-1 inhibitors as analgesics: 5-amino-2-ethoxy-N-(substituted-phenyl)benzamides, ChemMedChem, Vol.6, No.3, 550-560, 2011.
(Summary)
We previously found that N-(4-aminophenyl)-4-trifluoromethylbenzamide (TFAP), a COX-1 inhibitor, exhibits an analgesic effect without causing gastric damage. Unfortunately, TFAP causes reddish purple coloration of urine, and its analgesic effect is less potent than that of indomethacin. Herein we describe our study focusing on the development of 4- and 5-amino-2-alkoxy-N-phenylbenzamide scaffolds, designed on the basis of the structures of TFAP and parsalmide, another known COX-1 inhibitory analgesic agent. 5-Amino-2-ethoxy-N-(2- or 3-substituted phenyl)benzamide derivatives exhibited analgesic activity in a murine acetic acid induced writhing test. Among these compounds, 5-amino-2-ethoxy-N-(2-methoxyphenyl)benzamide (9 v) possesses potent COX-1 inhibitory and analgesic activities, similar to those of indomethacin. In addition, 5-amino-2-ethoxy-N-(3-trifluoromethylphenyl)benzamide (9 g) showed a more potent analgesic effect than indomethacin or 9 v without causing apparent gastric damage or coloration of urine, although its COX-1 inhibitory activity was weaker than that of indomethacin or 9 v. Thus, 9 g and 9 v appear to be promising candidates for analgesic agents and are attractive lead compounds for further development of COX-1 inhibitors.
Akihiro Tai, Takeshi Sawano, Futoshi Yazama and Hideyuki Ito : Evaluation of antioxidant activity of vanillin by using multiple antioxidant assays, Biochimica et Biophysica Acta (BBA) - General Subjects, Vol.1810, No.2, 170-177, 2011.
Tuty Anggraini, Akihiro Tai, Tomoyuki Yoshino and Tomio Itani : Antioxidative activity and catechin content of four kinds of Uncaria gambir extracts from West Sumatra, Indonesia, African Journal of Biochemistry Research, Vol.5, No.1, 33-38, 2011.
54.
Akihiro Tai, Yuji Iwaoka, Tasuku Mori and Hideyuki Ito : Protease-catalyzed monoacylation of 2-O-α-D-glucopyranosyl-L-ascorbic acid in three solvent systems, Bioscience, Biotechnology, and Biochemistry, Vol.74, No.9, 1969-1971, 2010.
(Summary)
6-O-dodecanoyl-2-O-α-D-glucopyranosyl-L-ascorbic acid (6-sDode-AA-2G) was synthesized from 2-O-α-D-glucopyranosyl-L-ascorbic acid and vinyl laurate with a protease from Bacillus subtilis in 30% dimethylformamide (DMF)/dioxane with a low water content. The addition of 3% (v/v) water to DMF/dioxane dramatically enhanced the 6-sDode-AA-2G synthesis. The optimum reaction conditions enabled 6-sDode-AA-2G to be synthesized in a yield of 38.1%.
Shuji Fujii, Fuminori Ohsawa, Shoya Yamada, Ryosuke Shinozaki, Ryosuke Fukai, Makoto Makishima, Shuichi Enomoto, Akihiro Tai and Hiroki Kakuta : Modification at the acidic domain of RXR agonists has little effect on permissive RXR-heterodimer activation, Bioorganic & Medicinal Chemistry Letters, Vol.20, No.17, 5139-5142, 2010.
(Summary)
Retinoid X receptors (RXRs) function as homo- or heterodimers with other nuclear receptors, such as peroxisome proliferator-activated receptors (PPARs), which are targets for treatment of hyperlipidemia and type 2 diabetes, or liver X receptors (LXRs), which are involved in glucose/lipid metabolism. PPAR/RXR or LXR/RXR are known as permissive RXR-heterodimers because they are activated by RXR agonists alone. Interestingly, the pattern of RXR-heterodimer activation is different depending on the RXR agonist structure, but the structure-activity relationship has not been reported. Here we show that modification or replacement of the carboxyl group in the acidic domain of RXR agonists has little or no effect on permissive RXR-heterodimer activation. Phosphonic acid (9), tetrazole (10), and hydroxamic acid (12) analogues were synthesized from the common bromo intermediate 7. Except for 9, these compounds showed RXR full-agonistic activities in the concentration range of 1-10 microM. The order of agonistic activity toward both PPARgamma/RXRalpha and LXRalpha/RXRalpha was the same as it was for RXR, that is, 11>10>12. These results should be useful for the development of RXR agonists with improved bioavailability.
(Keyword)
Dimerization / Dose-Response Relationship, Drug / Models, Molecular / Retinoid X Receptors / Transcriptional Activation
Shoya Yamada, Fuminori Ohsawa, Shuji Fujii, Ryosuke Shinozaki, Makoto Makishima, Hirotaka Naitou, Shuichi Enomoto, Akihiro Tai and Hiroki Kakuta : Fluorescent retinoid X receptor ligands for fluorescence polarization assay, Bioorganic & Medicinal Chemistry Letters, Vol.20, No.17, 5143-5146, 2010.
(Summary)
Retinoid X receptor (RXR) agonists are candidate agents for the treatment of metabolic syndrome and type 2 diabetes via activation of peroxisome proliferator-activated receptor (PPAR)/RXR or liver X receptor (LXR)/RXR-heterodimers, which control lipid and glucose metabolism. Reporter gene assays or binding assays with radiolabeled compounds are available for RXR ligand screening, but are unsuitable for high-throughput screening. Therefore, as a first step towards stabilizing a fluorescence polarization (FP) assay system for high-throughput RXR ligand screening, we synthesized fluorescent RXR ligands by modification of the lipophilic domain of RXR ligands with a carbostyril fluorophore, and selected the fluorescent RXR agonist 6-[ethyl(1-isobutyl-2-oxo-4-trifluoromethyl-1,2-dihydroquinolin-7-yl)amino]nicotinic acid 8d for further characterization. Compound 8d showed FP in the presence of RXR and the FP was decreased in the presence of the RXR agonist LGD1069 (2). This compound should be a lead compound for use in high-throughput assay systems for screening RXR ligands.
(Keyword)
Dimerization / Fluorescence Polarization / Ligands / Retinoid X Receptors
Akihiro Tai, Tasuku Mori, Yuka Kimura and Hideyuki Ito : Monoacylation of 2-O-alpha-D-glucopyranosyl-L-ascorbic acid by protease in N,N-dimethylformamide with low water content, Carbohydrate Research, Vol.345, No.12, 1658-1662, 2010.
(Summary)
2-O-alpha-D-Glucopyranosyl-L-ascorbic acid (AA-2G) laurate was synthesized from AA-2G and vinyl laurate with a protease from Bacillus subtilis in N,N-dimethylformamide (DMF) with low water content. Addition of water to DMF dramatically enhanced monoacyl AA-2G synthesis. Maximum synthetic activity was observed when 3% (v/v) water was added to the reaction medium. Under the optimal reaction conditions, 5-O-dodecanoyl-2-O-alpha-D-glucopyranosyl-L-ascorbic acid, 2-O-(6'-O-dodecanoyl-alpha-D-glucopyranosyl)-L-ascorbic acid, and 6-O-dodecanoyl-2-O-alpha-D-glucopyranosyl-L-ascorbic acid were synthesized in yields of 5.5%, 3.2%, and 20.4%, respectively.
Akihiro Tai, Tasuku Mori, Masaya Urushihara, Hideyuki Ito, Daisuke Kawasaki and Itaru Yamamoto : Intramolecular acyl migration and enzymatic hydrolysis of a novel monoacylated ascorbic acid derivative, 6-O-dodecanoyl-2-O-alpha-d-glucopyranosyl-L-ascorbic acid, Bioorganic & Medicinal Chemistry, Vol.18, No.16, 6179-6183, 2010.
(Summary)
A stable ascorbic acid derivative, 2-O-alpha-D-glucopyranosyl-L-ascorbic acid (AA-2G), exhibits vitamin C activity in vitro and in vivo after enzymatic hydrolysis to ascorbic acid. AA-2G has been approved by the Japanese Government as a quasi-drug principal ingredient in skin care and as a food additive. In order to achieve efficient action as an ascorbic acid source, a pro-vitamin C agent, on a variety of cells or tissues, we have synthesized a series of monoacyl AA-2G derivatives. Our previous studies indicate that a series of the derivatives is a readily available source of AA activity in vitro and in vivo, and suggested that intramolecular acyl migration of the derivatives might have occurred in a neutral aqueous solution. In this study, intramolecular acyl migration and enzymatic hydrolysis of a monoacyl AA-2G derivative, 6-O-dodecanoyl-2-O-alpha-D-glucopyranosyl-L-ascorbic acid (6-sDode-AA-2G), were investigated. 6-sDode-AA-2G underwent an intramolecular acyl migration to yield ca. 10% of an isomer in neutral aqueous solutions, and the acyl-migrated isomer was isolated and characterized as 5-O-dodecanoyl-2-O-alpha-D-glucopyranosyl-L-ascorbic acid (5-sDode-AA-2G). In some tissue homogenates from guinea pigs as well as in neutral aqueous solutions, 6-sDode-AA-2G underwent partial acyl migration to give 5-sDode-AA-2G. 6-sDode-AA-2G and the resulting 5-sDode-AA-2G were predominantly hydrolyzed with esterase to AA-2G and then with alpha-glucosidase to ascorbic acid in the tissue homogenates. The results will provide a further basis for its use as an ingredient in skin care, as an effective pharmacological agent and as a promising food additive.
Jun Takebayashi, Rie Ishii, Jianbin Chen, Teruki Matsumoto, Yoshiko Ishimi and Akihiro Tai : Reassessment of antioxidant activity of arbutin: multifaceted evaluation using five antioxidant assay systems, Free Radical Research, Vol.44, No.4, 473-478, 2010.
(Summary)
Arbutin, a practically used skin-lightening agent, has been reported to possess a weak antioxidant activity compared to that of its precursor, hydroquinone. However, its antioxidant activity has not been systematically evaluated. Hence, this study reassessed its activity using five assay systems. Assays were first performed using model radicals, DPPH radical and ABTS(*+). Arbutin showed weak DPPH radical-scavenging activity compared to that of hydroquinone, but showed strong ABTS(*+)-scavenging activity. Its activity by ORAC assay was then evaluated using a physiologically relevant peroxyl radical. Arbutin exerted weak but long-lasting radical-scavenging activity and showed totally the same antioxidant activity as that of hydroquinone. Finally, it was shown that, in two cell-based antioxidant assays using erythrocytes and skin fibroblasts, arbutin exerted strong antioxidant activity comparable or even superior to that of hydroquinone. These findings indicate that the antioxidant activity of arbutin may have been under-estimated and suggest that it acts as a potent antioxidant in the skin.
Hiroki Kakuta, Ryosuke Fukai, Zheng Xiaoxia, Fuminori Ohsawa, Takeshi Bamba, Kazumasa Hirata and Akihiro Tai : Identification of urine metabolites of TFAP, a cyclooxygenase-1 inhibitor, Bioorganic & Medicinal Chemistry Letters, Vol.20, No.6, 1840-1843, 2010.
(Summary)
Only a few COX-1-selective inhibitors are currently available, and the research on COX-1 selective inhibitors is not fully developed. The authors have produced several COX-1 selective inhibitors including N-(5-amino-2-pyridinyl)-4-trifluoromethylbenzamide: TFAP (3). Although 3 shows potent analgesic effect without gastric damage, the urine after administration of 3 becomes red-purple. Since the colored-urine should be avoided for clinical use, in this research we examined the cause of the colored-urine. UV-vis spectra and LC-MS/MS analyses of urine samples and metabolite candidates of 3 were performed to afford information that the main reason of the colored urine is a diaminopyridine (4), produced by metabolization of 3. This information is useful to design new COX-1 selective inhibitors without colored urine based on the chemical structure of 3.
(Keyword)
Aminopyridines / Benzamides / Chromatography, Liquid / Cyclooxygenase 1 / Cyclooxygenase Inhibitors / Spectrophotometry, Ultraviolet / Tandem Mass Spectrometry
Kenji Ichiyama, Hitoshi Mitsuzumi, Ming Zhong, Akihiro Tai, Akihiro Tsuchioka, Saeko Kawai, Itaru Yamamoto and Eiichi Gohda : Promotion of IL-4- and IL-5-dependent differentiation of anti-mu-primed B cells by ascorbic acid 2-glucoside, Immunology Letters, Vol.122, No.2, 219-226, 2009.
(Summary)
The stable ascorbic acid derivative 2-O-alpha-D-glucopyranosyl-L-ascorbic acid (AA-2G) was used to investigate the role of ascorbic acid (AA) in B cell differentiation in vitro. AA-2G is stable in a solution unlike AA but is hydrolyzed by cellular alpha-glucosidase to release AA. Mouse spleen B cells were primed for 2 days with an anti-mu antibody in the presence of interleukin (IL)-4 and IL-5 and then washed and recultured with AA-2G in the presence of IL-4 and IL-5. AA-2G, but not AA, dose-dependently increased IgM production, the greatest enhancement being 150% at concentrations of more than 0.5mM. In the absence of IL-4 and IL-5, primed B cells produced a negligible amount of IgM, and AA-2G had no effect. AA-2G-induced IgM production in the presence of IL-4 and IL-5 was inhibited by the alpha-glucosidase inhibitor castanospermine. Intracellular AA content, depleted during the priming period, increased by adding AA-2G at the start of reculture. Treatment of B cells with AA-2G resulted in an increase in the number of IgM-secreting cells, CD138-positive cells and CD45R/B220-negative cells. The number of viable cells in untreated cultures decreased gradually, but the decrease was significantly attenuated by AA-2G, resulting in about 70% more viable cells in AA-2G-treated cultures. AA-2G caused a slight but reproducible enhancement of DNA synthesis and a slight decrease in the number of cells with a sub-G1 DNA content. These results demonstrated that AA released from AA-2G enhanced cytokine-dependent IgM production in anti-mu-primed B cells and suggest that its effect is caused through promoting the differentiation of B cells to plasma cells and attenuating the gradual decrease in the number of viable cells.
Ken-ichi Morishita, Nobumasa Yakushiji, Fuminori Ohsawa, Kayo Takamatsu, Nobuyasu Matsuura, Makoto Makishima, Masatoshi Kawahata, Kentaro Yamaguchi, Akihiro Tai, Kenji Sasaki and Hiroki Kakuta : Replacing alkyl sulfonamide with aromatic sulfonamide in sulfonamide-type RXR agonists favors switch towards antagonist activity, Bioorganic & Medicinal Chemistry Letters, Vol.19, No.3, 1001-1003, 2009.
(Summary)
Retinoid X receptor (RXR) ligands are attractive candidates for clinical application because of their activity against tamoxifen-resistant breast cancer, taxol-resistant lung cancer, metabolic syndrome, and allergy. Though several RXR ligands, especially RXR antagonists, have been reported, the rational molecular design of such compounds is not well advanced. 4-[N-Methanesulfonyl-N-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-2-naphthyl)amino]nicotinic acid (5a) is a moderately RXRalpha-preferential agonist, and we examined the feasibility of replacing the methyl group on the sulfonamide with a longer alkyl chain or an aromatic ring as an approach to produce new RXR antagonists. Several of the resulting benzenesulfonanilide-type compounds showed RXR antagonist activity. This design strategy should be a useful approach for addressing the lack of structure diversity of RXR antagonists.
Xiaoxia Zheng, Hiroyuki Oda, Shun Harada, Yukio Sugimoto, Akihiro Tai, Kenji Sasaki and Hiroki Kakuta : Effect of the oral absorption of benzenesulfonanilide-type cyclooxygenase-1 inhibitors on analgesic action and gastric ulcer formation, Journal of Pharmaceutical Sciences, Vol.97, No.12, 5446-5452, 2008.
(Summary)
A benzensulfonanilide-type cyclooxygenase-1 (COX-1)-selective inhibitor, ZXX2-77: 4-amino-4'-chloro-N-methylbenzenesulfonanilide (4a), has been reported as a novel analgesic that does not cause gastric damage. This compound has a weak analgesic effect but has potent in vitro COX-1 inhibitory activity. Since the reason for the weak analgesic effect in vivo was thought to be the low rate of oral absorption, the blood concentration of ZXX2-77 (4a) was measured in rats. It was found that the C(max) value (1.2 microM) of ZXX2-77 (4a) at a dose of 30 mg/kg did not reach the COX-1 IC(50) value (3.2 microM). On the other hand, ZXX2-79 (4b) (SO(2)NH derivative of ZXX2-77 (4a); 4-amino-4'-chlorobenzenesulfonanilide), which shows less potent COX inhibitory activities (COX-1 IC(50) = 12 microM, COX-2 IC(50) = 150 microM) than those of ZXX2-77 (4a) in vitro, was found to be more absorbable (C(max) = 16 microM at a dose of 30 mg/kg in rats) than ZXX2-77 (4a). Furthermore, ZXX2-79 (4b) not only showed a potent analgesic effect in a formalin test but also caused little gastric damage. These findings indicate that demethylated sulfonamide compounds are more easily absorbed than are N-methylated sulfonamide compounds and suggest that COX-1-selective inhibitors will be useful as analgesics that do not cause gastric damage.
Jun Takebayashi, Yasuyuki Yagi, Rie Ishii, Shigeki Abe, Kazuhiko Yamada and Akihiro Tai : Antioxidant properties of 2-O-beta-D-glucopyranosyl-L-ascorbic acid, Bioscience, Biotechnology, and Biochemistry, Vol.72, No.6, 1558-1563, 2008.
(Summary)
The antioxidant activity of a provitamin C agent, 2-O-beta-D-glucopyranosyl-L-ascorbic acid (AA-2betaG), was compared to that of 2-O-alpha-D-glucopyranosyl-L-ascorbic acid (AA-2G) and ascorbic acid (AA) using four in vitro methods, 1,1-diphenyl-picrylhydrazyl (DPPH) radical-scavenging assay, 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation (ABTS(*+))-scavenging assay, oxygen radical absorbance capacity (ORAC) assay, and 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH)-induced erythrocyte hemolysis inhibition assay. AA-2betaG slowly and continuously scavenged DPPH radicals and ABTS(*+) in roughly the same reaction profiles as AA-2G, whereas AA quenched these radicals immediately. In the ORAC assay and the hemolysis inhibition assay, AA-2betaG showed similar overall activities to AA-2G and to AA, although the reactivity of AA-2betaG against the peroxyl radical generated in both assays was lower than that of AA-2G and AA. These data indicate that AA-2betaG had roughly the same radical-scavenging properties as AA-2G, and a comprehensive in vitro antioxidant activity of AA-2betaG appeared to be comparable not only to that of AA-2G but also to that of AA.
Kensuke Namba, Xiaoxia Zheng, Kazunori Motoshima, Hidetomo Kobayashi, Akihiro Tai, Eizo Takahashi, Kenji Sasaki, Keinosuke Okamoto and Hiroki Kakuta : Design and synthesis of benzenesulfonanilides active against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus, Bioorganic & Medicinal Chemistry, Vol.16, No.11, 6131-6144, 2008.
(Summary)
Vancomycin is mainly used as an antibacterial agent of last resort, but recently vancomycin-resistant bacterial strains have been emerging. Although new antimicrobials have been developed in order to overcome drug-resistant bacteria, many are structurally complex beta-lactams or quinolones. In this study, we aimed to create new anti-drug-resistance antibacterials which can be synthesized in a few steps from inexpensive starting materials. Since sulfa drugs function as p-aminobenzoic acid mimics and inhibit dihydropteroate synthase (DHPS) in the folate pathway, we hypothesized that sulfa derivatives would act as folate metabolite-mimics and inhibit bacterial folate metabolism. Screening of our sulfonanilide libraries, including benzenesulfonanilide-type cyclooxygenase-1-selective inhibitors, led us to discover benzenesulfonanilides with potent anti-methicillin-resistant Staphylococcus aureus (MRSA)/vancomycin-resistant Enterococcus (VRE) activity, that is, N-3,5-bis(trifluoromethyl)phenyl-3,5-dichlorobenzenesulfonanilide (16b) [MIC=0.5microg/mL (MRSA), 1.0microg/mL (VRE)], and 3,5-bis(trifluoromethyl)-N-(3,5-dichlorophenyl)benzenesulfonanilide (16c) [MIC=0.5microg/mL (MRSA), 1.0microg/mL (VRE)]. These compounds are more active than vancomycin [MIC=2.0microg/mL (MRSA), 125microg/mL (VRE)], but do not possess an amino group, which is essential for DHPS inhibition by sulfa drugs. These results suggested that the mechanism of antibacterial action of compounds 16b and 16c is different from that of sulfa drugs. We also confirmed the activity of these compounds against clinical isolates of Gram-positive bacteria.
Mai Yoshikawa, Kazunori Motoshima, Kanji Fujimoto, Akihiro Tai, Hiroki Kakuta and Kenji Sasaki : Pyridinium cationic-dimer antimalarials, unlike chloroquine, act selectively between the schizont stage and the ring stage of Plasmodium falciparum, Bioorganic & Medicinal Chemistry, Vol.16, No.11, 6027-6033, 2008.
(Summary)
Malaria is a leading cause of death in developing countries, and the emergence of strains resistant to the main therapeutic agent, chloroquine, has become a serious problem. We have developed cationic-dimer type antimalarials, MAP-610 and PMAP-H10, which are structurally different from chloroquine. In this study, we introduced several substituents on the terminal phenyl rings of PMAP-H10. The electronic and hydrophobic properties of the substituents were correlated with the antimalarial activity and cytotoxicity of the compounds, respectively. Studies with synchronized cultures of malarial plasmodia showed that our cationic-dimers act selectively between the schizont stage and the ring stage of the parasitic cycle, unlike chloroquine, which has a stage-independent action. Thus, the mechanism of action of our antimalarials appears to be different from that of chloroquine, and our compounds may be effective against chloroquine-resistant strains.
Kayo Takamatsu, Atsushi Takano, Nobumasa Yakushiji, Kazunori Morohashi, Ken-ichi Morishita, Nobuyasu Matsuura, Makoto Makishima, Akihiro Tai, Kenji Sasaki and Hiroki Kakuta : The first potent subtype-selective retinoid X receptor (RXR) agonist possessing a 3-isopropoxy-4-isopropylphenylamino moiety, NEt-3IP (RXRalpha/beta-dual agonist), ChemMedChem, Vol.3, No.5, 780-787, 2008.
(Summary)
Retinoid X receptor (RXR) agonists (rexinoids) are attracting much attention for their use in treatment of cancers, including tamoxifen-resistant breast cancer and taxol-resistant lung cancer, and metabolic disease. However, known RXR agonists have a highly lipophilic character. In addition, no subtype-selective RXR agonists have been found. We previously reported an RXRalpha-preferential agonist 4-[N-methanesulfonyl-N-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-2-naphthyl)amino]benzoic acid (6 a). The RXR agonistic activity is much less than that of well-known RXR agonists. To develop potent, less-lipophilic, and subtype-selective RXR agonists, we created new RXR agonists possessing alkoxy and isopropyl groups as a lipophilic domain of the common structure of well-known RXR agonists. As a result, compounds possessing branched alkoxy groups, 6-[N-ethyl-N-(3-isopropoxy-4-isopropylphenyl)amino]nicotinic acid (NEt-3IP: 7 a) and 6-[N-ethyl-N-(3-isobutoxy-4-isopropylphenyl)amino]nicotinic acid (NEt-3IB: 7 c), showed RXR agonistic activity as potent as, or more potent than, the activities of representative RXR agonists. Moreover, NEt-3IP (7 a) was found to be the first RXRalpha/beta-selective (or RXRalpha/beta-dual) agonist. Being potent, less lipophilic, and having RXR subtype-selective activity, NEt-3IP (7 a) is expected to become a new drug candidate and to be a useful biological tool for clarifying each RXR subtype function.
Hiroki Kakuta, Xiaoxia Zheng, Hiroyuki Oda, Shun Harada, Yukio Sugimoto, Kenji Sasaki and Akihiro Tai : Cyclooxygenase-1-selective inhibitors are attractive candidates for analgesics that do not cause gastric damage. design and in vitro/in vivo evaluation of a benzamide-type cyclooxygenase-1 selective inhibitor, Journal of Medicinal Chemistry, Vol.51, No.8, 2400-2411, 2008.
(Summary)
Although cyclooxygenase-1 (COX-1) inhibition is thought to be a major mechanism of gastric damage by nonsteroidal anti-inflammatory drugs (NSAIDs), some COX-1-selective inhibitors exhibit strong analgesic effects without causing gastric damage. However, it is not clear whether their analgesic effects are attributable to COX-1-inhibitory activity or other bioactivities. Here, we report that N-(5-amino-2-pyridinyl)-4-(trifluoromethyl)benzamide ( 18f, TFAP), which has a structure clearly different from those of currently available COX-1-selective inhibitors, is a potent COX-1-selective inhibitor (COX-1 IC 50 = 0.80 +/- 0.05 microM, COX-2 IC 50 = 210 +/- 10 microM). This compound causes little gastric damage in rats even at an oral dose of 300 mg/kg, though it has an analgesic effect at as low a dose as 10 mg/kg. Our results show that COX-1-selective inhibitors can be analgesic agents without causing gastric damage.
(Keyword)
Aminopyridines / Animals / Benzamides / Cyclooxygenase 1 / Cyclooxygenase Inhibitors / Drug Evaluation, Preclinical / Magnetic Resonance Spectroscopy / Male / Mice / Mice, Inbred ICR / molecular structure / Rats / Rats, Sprague-Dawley / Spectrometry, Mass, Fast Atom Bombardment / Spectrophotometry, Infrared / Stomach
Kayo Takamatsu, Atsushi Takano, Nobumasa Yakushiji, Ken-ichi Morishita, Nobuyasu Matsuura, Makoto Makishima, Ismail Hamed Ali, Eiichi Akaho, Akihiro Tai, Kenji Sasaki and Hiroki Kakuta : Reduction of lipophilicity at the lipophilic domain of RXR agonists enables production of subtype preference: RXRalpha-preferential agonist possessing a sulfonamide moiety, ChemMedChem, Vol.3, No.3, 454-460, 2008.
(Summary)
Retinoid X receptor agonists (RXR agonists, rexinoids) are interesting candidates for the treatment of cancers such as tamoxifen-resistant breast cancer and taxol-resistant lung cancer. However, well-known RXR agonists possess a strong lipophilic character. In addition, although RXR has three subtypes, no subtype-selective RXR agonists are known. Thus we aimed to produce less-lipophilic and subtype-selective RXR agonists. By designing sulfonamide-type RXR agonists, 4-[N-methanesulfonyl-N-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-2-naphthyl)amino]benzoic acid (8 a) was found to prefer RXRalpha over RXRbeta and RXRgamma, although the potency is less than the potencies of well-known RXR pan-agonists. Moreover, our results suggest that the reduction of lipophilicity at the hydrophobic interaction region of RXR agonists enables production of RXR subtype preference. Our finding will be useful for the creation of more potent and less-lipophilic subtype-selective RXR agonists aimed at the reduction of undesirable side effects.
(Keyword)
Animals / Anti-Obesity Agents / Antineoplastic Agents / COS Cells / Cell Differentiation / Cercopithecus aethiops / Drug Synergism / Hydrophobic and Hydrophilic Interactions / Models, Chemical / Nitroblue Tetrazolium / Oxidation-Reduction / Retinoid X Receptor alpha / Retinoid X Receptor beta / Retinoid X Receptor gamma / Retinoid X Receptors / Sulfonamides
Jun Takebayashi, Hiroaki Kaji, Kenji Ichiyama, Kazutaka Makino, Eiichi Gohda, Itaru Yamamoto and Akihiro Tai : Inhibition of free radical-induced erythrocyte hemolysis by 2-O-substituted ascorbic acid derivatives, Free Radical Biology and Medicine, Vol.43, No.8, 1156-1164, 2007.
(Summary)
Inhibitory effects of 2-O-substituted ascorbic acid derivatives, ascorbic acid 2-glucoside (AA-2G), ascorbic acid 2-phosphate (AA-2P), and ascorbic acid 2-sulfate (AA-2S), on 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH)-induced oxidative hemolysis of sheep erythrocytes were studied and were compared with those of ascorbic acid (AA) and other antioxidants. The order of the inhibition efficiency was AA-2S> or =Trolox=uric acid> or =AA-2P> or =AA-2G=AA>glutathione. Although the reactivity of the AA derivatives against AAPH-derived peroxyl radical (ROO(*)) was much lower than that of AA, the derivatives exerted equal or more potent protective effects on AAPH-induced hemolysis and membrane protein oxidation. In addition, the AA derivatives were found to react per se with ROO(*), not via AA as an intermediate. These findings suggest that secondary reactions between the AA derivative radical and ROO(*) play a part in hemolysis inhibition. Delayed addition of the AA derivatives after AAPH-induced oxidation of erythrocytes had already proceeded showed weaker inhibition of hemolysis compared to that of AA. These results suggest that the AA derivatives per se act as biologically effective antioxidants under moderate oxidative stress and that AA-2G and AA-2P may be able to act under severe oxidative stress after enzymatic conversion to AA in vivo.
Kazunori Motoshima, Yoshiko Hiwasa, Mai Yoshikawa, Kanji Fujimoto, Akihiro Tai, Hiroki Kakuta and Kenji Sasaki : Antimalarial cation-dimers synthesized in two steps from an inexpensive starting material, isonicotinic acid, ChemMedChem, Vol.2, No.10, 1527-1532, 2007.
(Summary)
Malaria is one of the three major serious infectious diseases in the world. As the area affected by malaria includes a large proportion of developing countries, there is a need for new antimalarials that can be synthesized and supplied inexpensively. To generate low-cost antimalarials, the MAP series 6-10, bis-cation dimers, synthesized by amidating the carboxyl group of isonicotinic acid (11) with various amines and by cationizing the nitrogen atoms of the pyridine ring with the corresponding alkyl bromides, were designed. This design enabled expansion of the structural variations of bis-cation-type antimalarial compounds. The compounds bearing alkyl or phenyl groups in the amide moieties showed remarkable antimalarial activities in vitro. Moreover, 1,1'-(1,12-dodecanediyl)bis[4-[(buthylamino)carbonyl]pyridinium bromide], MAP-412 (6 d), exhibited a potent antimalarial activity (ED(50)=8.2 mg kg(-1)). Being prepared at low cost, our bis-cation-type antimalarial compounds may be useful as lead compounds for inexpensive antimalarials.
Akihiro Tai and Eiichi Gohda : Determination of ascorbic acid and its related compounds in foods and beverages by hydrophilic interaction liquid chromatography, Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences, Vol.853, No.1-2, 214-220, 2007.
(Summary)
A new hydrophilic interaction liquid chromatography method for the simultaneous determination of ascorbic acid (AA), erythorbic acid (EA), 2-O-alpha-D-glucopyranosyl-L-ascorbic acid (AA-2G) and 2-O-beta-D-glucopyranosyl-L-ascorbic acid (AA-2betaG) was developed using a diol column with an isocratic solution of acetonitrile-66.7 mM ammonium acetate solution (85:15, v/v) at a detection wavelength of 260 nm. The calibration curves were found to be linear in the range of 1-50 microg/ml for AA and EA and in the range of 2.5-100 microg/ml for AA-2G and AA-2betaG. Detection limits of AA, EA, AA-2G and AA-2betaG were 0.3, 0.3, 0.03 and 0.03 microg/ml, respectively. This method was satisfactorily applied to the determination of AA, EA, AA-2G and AA-2betaG in a fruit, a food and beverages. The results show that the procedure is simple and sensitive and that it can be employed for the simultaneous determination of AA and its related compounds in foods and beverages.
Yuka Kamata, Hiromi Shiraga, Akihiro Tai, Yuko Kawamoto and Eiichi Gohda : Induction of neurite outgrowth in PC12 cells by the medium-chain fatty acid octanoic acid, Neuroscience, Vol.146, No.3, 1073-1081, 2007.
(Summary)
It has been shown that polyunsaturated fatty acids such as arachinonic and docosahexanoic acids but not monounsaturated and saturated long-chain fatty acids promote basal and nerve growth factor (NGF)-induced neurite extension of PC12 cells, a line derived from a rat pheochromocytoma. On the other hand, short-chain fatty acids and valproic acid (2-propylpentanoic acid) enhance the growth of neurite processes of the cells only in the presence of inducers. In this study, we demonstrated that straight medium-chain fatty acids (MCFAs) at millimolar concentrations alone potently induced neuronal differentiation of PC12 cells. Hexanoic, heptanoic and octanoic acids dose-dependently induced neurite outgrowth of the cells: their maximal effects determined 2 days after addition to the culture medium were more marked than the effect of NGF. PC12 cells exposed to octanoic acid expressed increased levels of the neuronal marker beta-tubulin isotype III. Nonanoic, decanoic, and dodecanoic acids also induced growth of neurite processes, but their maximal effects were less marked than that of octanoic acid. In contrast, the polyunsaturated fatty acid linoleic acid and short-chain fatty acids had only slight or almost no effects on neurite formation in the absence of NGF. The effect of octanoic acid was synergistic with or additive to the effects of NGF and dibutyryl cyclic AMP. Octanoic acid upregulated phosphorylation of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK), critical signaling molecules in neuronal differentiation, but not phosphorylation of Akt, a signaling molecule downstream of phosphatidylinositol 3-kinase (PI3K). Moreover, growth of neurites induced by octanoic acid was potently inhibited by treatment of cells with the p38 MAPK inhibitor SB203580 and the ERK kinase inhibitor PD98059 but not inhibited and only slightly inhibited by the JNK inhibitor SP600125 and the PI3K inhibitor wortmannin, respectively. Taken together, our results indicate that MCFAs, including octanoic acid, induced neurite outgrowth of PC12 cells in the absence of NGF and suggest that the activation of p38 MAPK and ERK pathways is involved in this process.
Jun Takebayashi, Ryuji Asano, Yoshinori Nakae, Morio Saito, Eiichi Gohda, Itaru Yamamoto and Akihiro Tai : 2-O-alpha-D-glucopyranosyl-L-ascorbic acid scavenges 1,1-diphenyl-2-picrylhydrazyl radicals via a covalent adduct formation, Bioscience, Biotechnology, and Biochemistry, Vol.71, No.3, 754-760, 2007.
(Summary)
The 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging mechanism of 2-O-alpha-D-glucopyranosyl-L-ascorbic acid (AA-2G) was studied. We found two undefined products, named X and Y, in the reaction mixture of AA-2G and the DPPH radical under acidic conditions by HPLC analysis. The reaction mixture was further subjected to LC-MS analysis. X was found to be a covalent adduct of AA-2G and the DPPH radical. On the other hand, Y could not be identified, probably because it was a mixture. A time-course study of the radical-scavenging reaction revealed that one molecule of AA-2G scavenged one molecule of DPPH radical to generate an AA-2G radical, which readily reacted with another molecule of the DPPH radical to form a covalent adduct (X). Subsequently, this adduct slowly quenched a third molecule of the DPPH radical, resulting in reaction products (Y). Therefore, one molecule of AA-2G has only one oxidizable -OH group, but can scavenge three molecules of the DPPH radical. The radical-scavenging mechanism of AA-2G elucidated in this study should be useful in understanding the biological roles of AA-2G per se in the food and cosmetic fields.
Kenji Ichiyama, Akihiro Tai and Itaru Yamamoto : Augmentation of antigen-specific antibody production and IL-10 generation with a fraction from Rooibos (Aspalathus linearis) tea, Bioscience, Biotechnology, and Biochemistry, Vol.71, No.2, 598-602, 2007.
(Summary)
Rooibos tea was extracted with boiling water. The aqueous extract was chromatographed in a Diaion HP20 column eluted stepwise with water, 25%, 50% and 75% (v/v) aqueous methanol, and 100% methanol. The water eluate (fraction A) showed an augmenting effect on anti-ovalbumin (anti-OVA) immunoglobulin M (IgM) production in OVA-stimulated murine splenocytes in vitro. Fraction A also showed a strong augmenting effect on interleukin-10 generation in murine splenocytes. Furthermore, continuous ingestion of fraction A was found to increase the anti-OVA IgM level in the sera of OVA-immunized mice.
Xiaoxia Zheng, Hiroyuki Oda, Kayo Takamatsu, Yukio Sugimoto, Akihiro Tai, Eiichi Akaho, Ismail Hamed Ali, Toshiyuki Oshiki, Hiroki Kakuta and Kenji Sasaki : Analgesic agents without gastric damage: design and synthesis of structurally simple benzenesulfonanilide-type cyclooxygenase-1-selective inhibitors, Bioorganic & Medicinal Chemistry, Vol.15, No.2, 1014-1021, 2007.
(Summary)
In order to create novel analgesic agents without gastric disturbance, structurally simple cyclooxygenase-1 (COX-1) inhibitors with a benzenesulfonanilide skeleton were designed and synthesized. As a result, compounds 11f and 15a, which possess a p-amino group on the benzenesulfonyl moiety and p-chloro group on the anilino moiety, showed COX-1-selective inhibition. Moreover compound 11f, which is the most potent compound in this study showed more potent analgesic activity than that of aspirin at 30 mg/kg by po. The anti-inflammatory activity and gastric damage, however, were very weak or not detectably different from aspirin. Since the structure of our COX-1 inhibitors are very simple, they may be useful as lead compounds for superior COX-1 inhibitors as analgesic agents without gastric disturbance.
Akihiro Tai, Jun Takebayashi, Ayako Ueno, Eiichi Gohda and Itaru Yamamoto : An isocratic HPLC method for the simultaneous determination of novel stable lipophilic ascorbic acid derivatives and their metabolites, Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences, Vol.840, No.1, 38-43, 2006.
(Summary)
2-O-alpha-D-glucopyranosyl-6-O-hexadecanoyl-L-ascorbic acid (6-sPalm-AA-2G), a novel stable lipophilic ascorbic acid derivative, was hydrolyzed to 2-O-alpha-D-glucopyranosyl-L-ascorbic acid (AA-2G), ascorbyl 6-palmitate (6-sPalm-AA) and ascorbic acid (AA) with alpha-glucosidase and lipase. An HPLC method for the simultaneous determination of AA, AA-2G, 6-sPalm-AA and 6-sPalm-AA-2G was developed using a cyanopropyl column with an isocratic solution of methanol-phosphate buffer (pH 2.1) (65:35, v/v) containing 20mg/l of dithiothreitol at a detection wavelength of 240 nm. The calibration curves were found to be linear in the range of 10-200 microM. Linear regression analysis of the data demonstrated the efficacy of the method in terms of precision and accuracy. This method was satisfactorily applied to the determination of 6-sPalm-AA-2G and its three metabolites in a 6-sPalm-AA-2G solution treated with purified enzymes or a small intestine post-mitochondrial supernatant and to the separation of novel stable lipophilic AA derivatives other than 6-sPalm-AA-2G and their metabolites. AA, AA-2G and other well-known stable AA derivatives, ascorbic acid 2-phosphate and ascorbic acid 2-sulfate, were also separated under the same conditions. The results show that the procedure is rapid and simple and that it can be employed for in vitro metabolic analysis of various AA derivatives.
(Keyword)
Animals / Ascorbic Acid / calibration / Chromatography, High Pressure Liquid / Intestine, Small / Linear Models / Lipids / Male / Rats / Rats, Wistar / Reproducibility of Results / Spectrophotometry, Ultraviolet
Jun Takebayashi, Akihiro Tai, Eiichi Gohda and Itaru Yamamoto : Characterization of the radical-scavenging reaction of 2-O-substituted ascorbic acid derivatives, AA-2G, AA-2P, and AA-2S: a kinetic and stoichiometric study., Biological & Pharmaceutical Bulletin, Vol.29, No.4, 766-771, 2006.
(Summary)
The aim of this study was to characterize the antioxidant activity of three ascorbic acid (AA) derivatives O-substituted at the C-2 position of AA: ascorbic acid 2-glucoside (AA-2G), ascorbic acid 2-phosphate (AA-2P), and ascorbic acid 2-sulfate (AA-2S). The radical-scavenging activities of these AA derivatives and some common low molecular-weight antioxidants such as uric acid or glutathione against 1,1-diphenyl-picrylhydrazyl (DPPH) radical, 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation (ABTS+), or galvinoxyl radical were kinetically and stoichiometrically evaluated under pH-controlled conditions. Those AA derivatives slowly and continuously reacted with DPPH radical and ABTS+, but not with galvinoxyl radical. They effectively reacted with DPPH radical under acidic conditions and with ABTS+ under neutral conditions. In contrast, AA immediately quenched all species of radicals tested at all pH values investigated. The reactivity of Trolox, a water-soluble vitamin E analogue, was comparable to that of AA in terms of kinetics and stoichiometrics. Uric acid and glutathione exhibited long-lasting radical-scavenging activity against these radicals under certain pH conditions. The radical-scavenging profiles of AA derivatives were closer to those of uric acid and glutathione rather than to that of AA. The number of radicals scavenged by one molecule of AA derivatives, uric acid, or glutathione was equal to or greater than that by AA or Trolox under the appropriate conditions. These data suggest the potential usage of AA derivatives as radical scavengers.
Hiroaki Kaji, Akihiro Tai, Kazufumi Matsushita, Hiroshi Kanzaki and Itaru Yamamoto : Activation of murine peritoneal macrophages by water-soluble extracts of Bursaphelenchus xylophilus, a pine wood nematode, Bioscience, Biotechnology, and Biochemistry, Vol.70, No.1, 203-210, 2006.
(Summary)
In our previous study, water-soluble extracts from Bursaphelenchus xylophilus (B. xylophilus), a pine wood nematode, were shown to enhance interleukin (IL)-4 plus lipopolysaccharide-induced polyclonal immunoglobulin (Ig) E production in vitro in mice and to increase serum levels of an antigen-nonspecific IgE in vivo. Here we examined whether the nematode extracts stimulate immunofunctions of murine peritoneal macrophages. In both resident and inflammatory macrophages, Fcgamma receptor-mediated phagocytosis was markedly activated by B. xylophilus extracts, while non-specific phagocytosis was not. The enhancement of specific phagocytosis was accompanied by an increase in the formation of IgG-Fcgamma receptor rosettes. B. xylophilus extracts also stimulated IL-1beta production in both types of macrophages, and enhanced NO production and mRNA expression of inflammatory cytokines in inflammatory macrophages. These results indicate that the extracts of B. xylophilus contain an activating substance(s) for immunofunctions in macrophages, besides an enhancing factor for polyclonal IgE production.
Ming Zhong, Akihiro Tai and Itaru Yamamoto : In vitro augmentation of natural killer activity and interferon-gamma production in murine spleen cells with Agaricus blazei fruiting body fractions, Bioscience, Biotechnology, and Biochemistry, Vol.69, No.12, 2466-2469, 2005.
(Summary)
Aqueous extracts of the Agaricus blazei fruiting body prepared at different temperatures were fractionated by ethanol precipitation with various ethanol concentrations. The original aqueous extracts of A. blazei failed to stimulate natural killer (NK) cell activity in murine spleen cells in vitro, but the strongest effect was observed in a 30% ethanol-soluble-50% ethanol-insoluble fraction prepared from the extract at 40 degrees C (fraction A-50). Fraction A-50 also showed the strongest augmenting effect on interferon (IFN)-gamma production. This augmentation of NK activity and IFN-gamma production by fraction A-50 was completely abrogated by a heat treatment.
Eriko Aoyama, Ritsuko Yoshihara, Akihiro Tai, Itaru Yamamoto and Eiichi Gohda : PKC- and PI3K-dependent but ERK-independent proliferation of murine splenic B cells stimulated by chondroitin sulfate B, Immunology Letters, Vol.99, No.1, 80-84, 2005.
(Summary)
High molecular weight polyanions such as dextran sulfate are known to be weak polyclonal activators of murine B cells, but the molecular mechanism of their mitogenic activitiy is not fully elucidated. Although chondroitin sulfate A (CSA), B (CSB) and C (CSC) are highly charged polyanions, little is known about their effects on the proliferation of B cells. In this study, we demonstrated that CSB stimulated proliferation of murine B cells as markedly as did anti-IgM antibody, more markedly than did dextran sulfate and much more markedly than did CSA, CSC, heparin and hyaluronic acid. CSB caused translocation of protein kinase C (PKC) isoform beta from cytosol to membrane fractions and increased phosphorylation of Akt but not phosphorylation of extracellular signal-regulated kinase (ERK) of B cells. CSB-induced B cell proliferation was almost completely blocked by either the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 or the PKC inhibitor GF109203X but was not significantly inhibited by the ERK kinase inhibitor PD98059. The mitogenic effect of anti-IgM was significantly inhibited by all the three inhibitors, while the mitogenic effect of LPS was inhibited only by LY294002. These findings indicate that CSB stimulated proliferation of murine B cells more markedly than did dextran sulfate and suggest that PKC and PI3K are crucial but that ERK is less important for the mitogenic activity of CSB, the signaling pathways of which may be at least partly distinct from those of anti-IgM and LPS.
(Keyword)
Animals / B-Lymphocytes / Cell Proliferation / Cells, Cultured / Dermatan Sulfate / Female / Glycosaminoglycans / Mice / Mice, Inbred BALB C / Mitogen-Activated Protein Kinases / Phosphatidylinositol 3-Kinases / Phosphorylation / Protein Kinase C / Protein Kinase Inhibitors / signal transduction / Spleen / T lymphocytes
Takahiro Motoki, Yoichiro Takami, Yasuyuki Yagi, Akihiro Tai, Itaru Yamamoto and Eiichi Gohda : Inhibition of hepatocyte growth factor induction in human dermal fibroblasts by tryptanthrin, Biological & Pharmaceutical Bulletin, Vol.28, No.2, 260-266, 2005.
(Summary)
In addition to regulation of normal cell functions, hepatocyte growth factor (HGF) has also been shown to be involved in malignant cell transformation and in growth, invasion and metastasis in cancer cells. Inhibitors of HGF production have a potential for interfering with malignant cell transformation and progression of tumors. We found that tryptanthrin, one of the major compounds extracted from the medicinal plant Polygonum tinctorium, which is known for its antitumor activity, strongly inhibited HGF production stimulated by various HGF inducers in human dermal fibroblasts. HGF production induced by phorbol 12-myristate 13-acetate (PMA) was potently inhibited by tryptanthrin without any appreciable cytotoxic effect. Tryptanthrin also inhibited HGF production induced by epidermal growth factor (EGF) and platelet-derived growth factor. Moreover, proliferation of the fibroblasts induced by the two growth factors was potently suppressed by tryptanthrin to the level of proliferation of unstimulated fibroblasts. However, tryptanthrin did not inhibit HGF production induced by the protein kinase A-activating agents cholera toxin and 8-bromo-cAMP. These effects of tryptanthrin were different from the effects of transforming growth factor beta1 and dexamethasone, both of which inhibit HGF production induced by all the above inducers. Upregulations of HGF gene expression by PMA and EGF were also inhibited by tryptanthrin. Activation of the mitogen-activated protein kinase (MAPK) signaling pathway is crucial for PMA-induced HGF production, but tryptanthrin did not attenuate phosphorylation of MAPK induced by PMA. These results indicate that tryptanthrin potently inhibited induction of HGF production probably through events downstream of MAPK activation.
Akihiro Tai, Satomi Goto, Yutaka Ishiguro, Kazuko Suzuki, Teruhiko Nitoda and Itaru Yamamoto : Permeation and metabolism of a series of novel lipophilic ascorbic acid derivatives, 6-O-acyl-2-O-alpha-D-glucopyranosyl-L-ascorbic acids with a branched-acyl chain, in a human living skin equivalent model, Bioorganic & Medicinal Chemistry Letters, Vol.14, No.3, 623-627, 2004.
(Summary)
A series of novel lipophilic vitamin C derivatives, 6-O-acyl-2-O-alpha-D-glucopyranosyl-L-ascorbic acids possessing a branched-acyl chain of varying length from C(8) to C(16) (6-bAcyl-AA-2G), were evaluated as topical prodrugs of ascorbic acid (AA) with transdermal activity in a human living skin equivalent model. The permeability of 6-bAcyl-AA-2G was compared with those of the derivatives having a straight-acyl chain (6-sAcyl-AA-2G). Out of 10 derivatives of 6-sAcyl-AA-2G and 6-bAcyl-AA-2G, 6-sDode-AA-2G and 6-bDode-AA-2G exhibited most excellent permeability in this model. Measurement of the metabolites permeated from the skin model suggested that 6-bDode-AA-2G was mainly hydrolyzed via 6-O-acyl AA to AA by tissue enzymes, while 6-sDode-AA-2G was hydrolyzed via 2-O-alpha-D-glucopyranosyl-L-ascorbic acid to AA. The former metabolic pathway seems to be advantageous for a readily available source of AA, because 6-O-acyl AA, as well as AA, is able to show vitamin C activity.
Jun Takebayashi, Akihiro Tai and Itaru Yamamoto : pH-dependent long-term radical scavenging activity of AA-2G and 6-Octa-AA-2G against 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation, Biological & Pharmaceutical Bulletin, Vol.26, No.9, 1368-1370, 2003.
(Summary)
The 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation (ABTS (radical +)) decolorization assay was applied to evaluate the stoichiometric radical scavenging activity of ascorbic acid (AA) and two AA derivatives, 2-O-alpha-D-glucopyranosyl-L-ascorbic acid (AA-2G) and 2-O-alpha-D-glucopyranosyl-6-O-octanoyl-L-ascorbic acid (6-Octa-AA-2G). AA rapidly reacted with ABTS (radical +), and the reaction was completed within 10 min. In contrast, AA-2G and 6-Octa-AA-2G continuously reacted with ABTS (radical +), and the reaction was not completed after 2 h. The radical scavenging activity of AA-2G and 6-Octa-AA-2G in aqueous solutions at pH 4.0 and above was higher than that at pH 3.0, whereas AA showed no difference in the pH range 3 to 6. The amounts of ABTS (radical +) scavenged by one molecule of AA, AA-2G and 6-Octa-AA-2G after 2 h of reaction at pH 6.0 were approximately 2.0, 3.4 or 3.9 molecules, respectively. This study demonstrates that the quantity of ABTS (radical +) quenched by AA-2G and 6-Octa-AA-2G is superior to that of AA in a long-term reaction.
Akihiro Tai, Daisuke Kawasaki, Satomi Goto, Eiichi Gohda and Itaru Yamamoto : Vitamin C activity in guinea pigs of 6-O-acyl-2-O-alpha-D-glucopyranosyl-L- ascorbic acids with a branched-acyl chain, Bioscience, Biotechnology, and Biochemistry, Vol.67, No.8, 1675-1682, 2003.
(Summary)
A series of novel acylated ascorbic acid derivatives, 6-O-acyl-2-O-alpha-D-glucopyranosyl-L-ascorbic acids with a branched-acyl chain (6-bAcyl-AA-2G) were recently developed in our laboratory as stable and lipophilic ascorbate derivatives. In this study, the bioavailability of 6-bAcyl-AA-2G was investigated in guinea pigs. Various tissue homogenates from guinea pigs hydrolyzed 6-bAcyl-AA-2G to give ascorbic acid (AA), 2-O-alpha-D-glucopyranosyl-L-ascorbic acid (AA-2G), and 6-O-acyl AA. The releasing pattern of the three hydrolysates suggested that 6-bAcyl-AA-2G was hydrolyzed via 6-O-acyl AA to AA as a main pathway and via AA-2G to AA as a minor pathway. The former pathway seems to be of advantage, because 6-O-acyl AA, as well as AA, can have vitamin C activity. In addition, we found that a derivative with an acyl chain of C(12), 6-bDode-AA-2G, had a pronounced therapeutic effect in scorbutic guinea pigs by its repeated oral administrations. These results indicate that 6-bAcyl-AA-2G is a readily available source of AA in vivo, and may be a promising antioxidant for skin care and treatment of diseases associated with oxidative stress.
Xiaohua Zhou, Akihiro Tai and Itaru Yamamoto : Enhancement of neurite outgrowth in PC12 cells stimulated with cyclic AMP and NGF by 6-acylated ascorbic acid 2-O-alpha-glucosides (6-Acyl-AA-2G), novel lipophilic ascorbate derivatives, Biological & Pharmaceutical Bulletin, Vol.26, No.3, 341-346, 2003.
(Summary)
It has been shown that ascorbate (AsA) and its stable derivative, ascorbic acid 2-O-alpha-glucoside (AA-2G), do not elicit neurite outgrowth in PC12 cells. However, these ascorbates are synergistically enhanced by both dibutyryl cyclic AMP (Bt(2)cAMP)- and nerve growth factor (NGF)-induced neurite outgrowth in this model. In the present study, the effects of a series of novel lipophilic ascorbate derivatives, 6-acylated ascorbic acid 2-O-alpha-glucosides (6-Acyl-AA-2G), on neurite outgrowth induced by Bt(2)cAMP and NGF were examined in PC12 cells. We found that all the tested acylated ascorbate derivatives enhanced neurite formation induced by both agents in a dose-dependent manner. Of the 6-Acyl-AA-2G derivatives, 6-octanoyl ascorbic acid 2-O-alpha-glucoside (6-Octa-AA-2G) enhanced the Bt(2)cAMP-induced phosphorylated MAPK p44 and p42 expression. A alpha-glucosidase inhibitor, castanospermine, completely abrogated the promotion of neurite outgrowth and MAPK expression by 6-Octa-AA-2G. Addition of 6-Octa-AA-2G (0.5 mM) to PC12 cells caused a rapid and significant increase in intracellular AsA content, which reached a maximum and was maintained from 12 to 24 h after the culture. These findings suggest that 6-Acyl-AA-2G is rapidly hydrolyzed to AsA within the cell and enhances neurite differentiation through the interaction with the inducer-activated MAPK pathway.
(Keyword)
Acylation / Animals / Ascorbic Acid / Blotting, Western / Cell Count / Cell Line / Cyclic AMP / Dose-Response Relationship, Drug / Drug Interactions / Enzyme Inhibitors / Extracellular Space / Indolizines / Mitogen-Activated Protein Kinases / Nerve Growth Factor / Neurites / PC12 Cells / Rats
Hiroaki Kaji, Masahiko Kawada, Akihiro Tai, Hiroshi Kanzaki and Itaru Yamamoto : Augmentation by Bursaphelenchus xylophilus, a pine wood nematode, of polyclonal IgE production induced by lipopolysaccharide plus interleukin-4 in murine splenocytes, Journal of Pharmacological Sciences, Vol.91, No.2, 158-162, 2003.
(Summary)
Bursaphelenchus xylophilus (B. xylophilus) is a pine wood nematode that is known to cause pine wilt disease. We report here that B. xylophilus extracts augmented the polyclonal immunoglobulin E (IgE) production induced by lipopolysaccharide (LPS) plus interleukin-4 (IL-4) both in murine splenocytes and purified B cells as determined by ELISA and ELIspot assays, but they did not cause such a promotion in the absence of either LPS or IL-4. We also observed that the antigen-nonspecific IgE levels were increased in sera of mice treated with B. xylophilus extracts, which were comparable to those of Ascaris suum extracts. These findings suggest that administration of B. xylophilus extracts could suppress allergic diseases via a saturation of mast cell Fcepsilon receptors or/and an inhibition of antigen-specific IgE synthesis to the allergen by a polyclonal response.
(Keyword)
Adjuvants, Immunologic / Animals / Ascaris suum / B-Lymphocytes / Dose-Response Relationship, Drug / Enzyme-Linked Immunosorbent Assay / Female / Immunoglobulin E / In Vitro Techniques / Interleukin-4 / Lipopolysaccharides / Mast Cells / Mice / Mice, Inbred BALB C / Nematoda / Receptors, Fc / Spleen / Stimulation, Chemical / Tissue Extracts
(Link to Search Site for Scientific Articles)
● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 12686761
Akihiro Tai, Daisuke Kawasaki, Kenji Sasaki, Eiichi Gohda and Itaru Yamamoto : Synthesis and characterization of 6-O-acyl-2-O-alpha-D-glucopyranosyl-L-ascorbic acids with a branched-acyl chain, Chemical & Pharmaceutical Bulletin, Vol.51, No.2, 175-180, 2003.
(Summary)
We previously reported the chemical synthesis of a series of novel monoacylated vitamin C derivatives, 6-O-acyl-2-O-alpha-D-glucopyranosyl-L-ascorbic acids (6-Acyl-AA-2G) possessing a straight-acyl chain of varying length from C(4) to C(18), as effective skin antioxidants. In this paper, we describe branched type of 6-Acyl-AA-2G derivatives (6-bAcyl-AA-2G) synthesized by use of a 2-branched-chain fatty acid anhydride as an acyl donor. The stability of 6-bAcyl-AA-2G in neutral solution was much higher than that of 6-Acyl-AA-2G, while they were susceptible to enzymatic hydrolysis for exerting vitamin C effect. These branched derivatives as well as 6-Acyl-AA-2G increased the radical scavenging activity against 1, 1-diphenyl-2-picrylhydrazyl and the lipophilicity in octanol/water-partitioning systems with increasing length of their acyl group. In addition, the 6-bAcyl-AA-2G derivative with an acyl chain of C(12), 6-bDode-AA-2G had the excellent solubility to various solvents, suggesting easy handling in cosmetic use. These characteristics of 6-bAcyl-AA-2G may be available for skin care application as an effective antioxidant.
Jun Takebayashi, Akihiro Tai and Itaru Yamamoto : Long-term radical scavenging activity of AA-2G and 6-acyl-AA-2G against 1,1-diphenyl-2-picrylhydrazyl, Biological & Pharmaceutical Bulletin, Vol.25, No.11, 1503-1505, 2002.
(Summary)
Stoichiometric evaluation of the radical scavenging activity of O-substituted derivatives at the C-2 position of ascorbic acid (AA) was conducted. Their reaction with a stable radical, 1,1-diphenyl-2-picrylhydrazyl (DPPH), under an acidic condition was assessed by the colorimetric method. 2-O-alpha-D-Glucopyranosyl-L-ascorbic acid (AA-2G) and a series of 6-O-acyl-2-O-alpha-D-glucopyranosyl-L-ascorbic acids (6-Acyl-AA-2G) had long-term radical scavenging activity against DPPH. The reaction of AA-2G or 6-Acyl-AA-2G with DPPH was very slow when compared with AA. However, one molecule of these derivatives consumed approximately three molecules of DPPH radicals at the end of the experiment (2 h). In contrast, one molecule of AA scavenged two molecules of DPPH radicals, and the reaction ended in the short time (<10 min). The quantity of radicals quenched by AA-2G and 6-Acyl-AA-2G was superior to that of AA in a long-term reaction.
Akihiro Tai, Yoshihito Fujinami, Kyoko Matsumoto, Daisuke Kawasaki and Itaru Yamamoto : Bioavailability of a series of novel acylated ascorbic acid derivatives, 6-O-acyl-2-O-alpha-D-glucopyranosyl-L-ascorbic acids, as an ascorbic acid supplement in rats and guinea pigs, Bioscience, Biotechnology, and Biochemistry, Vol.66, No.8, 1628-1634, 2002.
(Summary)
The bioavailability of a series of novel acylated ascorbic acid derivatives, 6-O-acyl-2-O-alpha-D-glucopyranosyl-L-ascorbic acids (6-Acyl-AA-2G), as an ascorbic acid (AA) supplement was investigated in rats and guinea pigs. Oral administration of 6-Acyl-AA-2G to rats resulted in an increase in the plasma AA level. However, the intact form was not detectable in the plasma by high-performance liquid chromatography, indicating its hydrolysis through the process of absorption. After an intravenous injection to rats of 6-Octa-AA-2G as a representative derivative, the intact form rapidly disappeared from the plasma, being followed by a prolonged and marked elevation of the plasma AA level. Various tissue homogenates from guinea pigs were examined for their releasing activity of AA, 2-O-alpha-D-glucopyranosyl-L-ascorbic acid (AA-2G) and 6-O-acyl-AA from 6-Acyl-AA-2G. High activity was observed in the small intestine. These hydrolytic activities to AA and 6-O-acyl-AA were completely inhibited by castanospermine, an alpha-glucosidase inhibitor, and AA-2G was observed as the only resulting hydrolysate, suggesting the participation of alpha-glucosidase and esterase in the in vivo hydrolysis of 6-Acyl-AA-2G. 6-Octa-AA-2G was found to exhibit an obvious therapeutic effect in scorbutic guinea pigs from its repeated oral administration. These results indicate that 6-Acyl-AA-2G is a readily available source of AA activity in vivo, and may be useful as an effective pharmacological agent and as a promising food additive.
Itaru Yamamoto, Akihiro Tai, Yoshihito Fujinami, Kenji Sasaki and Shino Okazaki : Synthesis and characterization of a series of novel monoacylated ascorbic acid derivatives, 6-O-acyl-2-O-alpha-D-glucopyranosyl-L-ascorbic acids, as skin antioxidants, Journal of Medicinal Chemistry, Vol.45, No.2, 462-468, 2002.
(Summary)
A series of novel monoacylated vitamin C derivatives were chemically synthesized with a stable ascorbate derivative, 2-O-alpha-D-glucopyranosyl-L-ascorbic acid (AA-2G), and acid anhydrides in pyridine. Their solubility in organic phase, thermal stability, radical scavenging activity, and in vitro skin permeability was evaluated. These monoacylated derivatives were identified as 6-O-acyl-2-O-alpha-D-glucopyranosyl-L-ascorbic acids (6-Acyl-AA-2G) by UV spectra, elemental analyses, and nuclear magnetic resonance spectroscopy. The reactions afforded 6-Acyl-AA-2G in high yields (30-60%). 6-Acyl-AA-2G exhibited satisfactory stability in neutral solution comparable to that of a typical stable derivative, AA-2G, and also showed the radical scavenging activity. The lipid solubility of 6-Acyl-AA-2G was increased with increasing length of their acyl group. Increased skin permeability was superior to those of AA-2G and ascorbic acid (AsA). 6-Acyl-AA-2G that is susceptible to enzymatic hydrolysis by tissue esterase and/or alpha-glucosidase produces AA-2G and AsA, which is in the skin tissues. Thus, these findings indicate that the novel vitamin C derivatives presented here, 6-Acyl-AA-2G, may be effective antioxidants in skin care and medicinal use.
Kazuyoshi Kunishiro, Akihiro Tai and Itaru Yamamoto : Effects of rooibos tea extract on antigen-specific antibody production and cytokine generation in vitro and in vivo, Bioscience, Biotechnology, and Biochemistry, Vol.65, No.10, 2137-2145, 2001.
(Summary)
Rooibos tea contains a large amount of flavonoids and acts as a potent antioxidant. In this study, we examined the effects of Rooibos tea extract on antigen-specific antibody production and cytokine generation in vitro and in vivo. The primary in vitro anti-ovalbumin (anti-OVA) or sheep red blood cell (SRBC) antibody production in murine splenocytes was markedly stimulated by the addition of the tea extract at concentrations of 1-100 microg/ml. On the other hand, a nonspecific antibody response elicited with lipopolysaccharide (LPS) in purified splenic B-cells was not modified by the extract. Rooibos tea extract caused an increase in the generation of interleukin 2 (IL-2) both in OVA- and anti-CD3-primed splenocytes at concentrations ranging from 10 microg/ml to 1000 microg/ml. In contrast, this tea extract suppressed the generation of interleukin 4 (IL-4) in OVA-primed splenocytes. Moreover, the reduction of OVA-induced antibody production in serum of the cyclosporin A (CyA) -treated rats can be significantly restored and the IL-2 generation in murine splenocytes was stimulated, following oral administrations of Rooibos tea extract. Thus, our findings suggested that Rooibos tea extract may facilitate the antigen-specific antibody production through selective augmentation of IL-2 generation both in vitro and in vivo. Collectively, Rooibos tea intake may be of value in prophylaxis of the diseases involving a severe defect in Th1 immune response such as cancer, allergy, AIDS, and other infections.
Akihiro Tai, Shino Okazaki, Noriko Tsubosaka and Itaru Yamamoto : Protease-catalyzed monoacylation of 2-O-alpha-D-Glucopyranosyl-L-ascorbic acid in pyridine, Chemical & Pharmaceutical Bulletin, Vol.49, No.8, 1047-1049, 2001.
(Summary)
2-O-alpha-D-Glucopyranosyl-6-O-octanoyl-L-ascorbic acid was enzymatically synthesized from 2-O-alpha-D-glucopyranosyl-L-ascorbic acid (AA-2G) and vinyl octanoate with a protease from Bacillus subtilis in pyridine. Furthermore, with various linear saturated fatty acid vinylesters as acyl donors, AA-2G was also converted to their corresponding 6-O-acyl AA-2G in the same manner. The reactivities of transacylation decreased with increasing length of the acyl groups. Thus, short chain acyl groups were transferred to AA-2G by this protease more efficiently than were long chain acyl groups. This enzymatic method is recommended for the synthesis of 6-Acyl-AA-2G with short or medium length chain acyl groups.
Ming Zhong, Akihiro Tai and Itaru Yamamoto : IgE production is involved in butyrate-enhanced NK cell activity in vivo, Chinese Medical Sciences Journal, Vol.16, No.2, 76-81, 2001.
(Summary)
It has been demonstrated that patients with asthma have a large number of NK cells and show a stronger NK activity. These results indicate that NK cell activity may be related to total IgE level in serum in healthy subjects. Previously, we have found that sodium butyrate (NaBu) markedly enhanced the IL-4-induced IgE production in the LPS-stimulated murine splenocytes in vitro, and inductive rat IgE production in vivo, and enhanced the NK cell activity ex vivo. We hypothesized that the IgE production might be involved in butyrate-enhanced NK cell activity in vivo. Mice were intraperitoneally treated/immunized with NaBu or/and Ascaris suum extract (ASC), and the spleen NK cell activity was evaluated. Furthermore, the effect of serum (NAS) on IL-2- or IFN-gamma-induced spleen NK cell activity was determined. The spleen NK cell activity and IL-2- or IFN-gamma-induced spleen NK cell activity of mice treated/immunized with NaBu or/and ASC were stronger than those of untreated/unimmunized mice. Although IL-4 blocked IL-2 (100 U/ml)- or IFN-gamma (100 U/ml)-induced increase in NK cell activity, these NK cell activities in mice treated/immunized with NaBu/ASC were not inhibited. IgE production showed a tendency to rise in NaBu-treated mice serum, and a synergistic effect was observed with treatment of NaBu and ASC. Moreover, the NAS significantly increased IL-2(25 U/ml)-or IFN-gamma(25 U/ml)-induced NK cell activity, and its effect was inhibited by anti-mouse IgE mAb. These data show that IgE plays an important role in NAS-enhanced IL-2/IFN-gamma-induced NK cell activity, and IL-4 does not inhibit IgE and IL-2/IFN-gamma-induced NK cell activity in mice.
(Keyword)
Animals / Ascaris suum / Butyrates / Female / Immunoglobulin E / Interferon-gamma / Interleukin-2 / Interleukin-4 / Killer Cells, Natural / Mice / Mice, Inbred BALB C / Spleen
(Link to Search Site for Scientific Articles)
● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 12901494
Yoshihito Fujinami, Akihiro Tai and Itaru Yamamoto : Radical scavenging activity against 1,1-diphenyl-2-picrylhydrazyl of ascorbic acid 2-glucoside (AA-2G) and 6-acyl-AA-2G, Chemical & Pharmaceutical Bulletin, Vol.49, No.5, 642-644, 2001.
(Summary)
The radical scavenging activity of the stable derivatives, which are O-substituted at the C-2 position of ascorbic acid (AA), against 1,1-diphenyl-2-picrylhydrazyl (DPPH) was evaluated in buffer under different pH conditions, and compared with those of AA and alpha-tocopherol. AA was shown to have 50% radical scavenging ability (EC50) at a concentration of 2.2 x 10(-5) M against 0.1 mM DPPH in 60% ethanol. Ascorbyl 6-palmitate, a lipophilic AA derivative which has a free endiol group and is therefore unstable, also showed potent radical scavenging activity with EC50 of 2.9 x 10(-5) M. A typical lipophilic antioxidant, alpha-tocopherol gave a similar EC50 value as that of AA. In contrast, ascorbyl 2,6-dipalmitate, AA 2-phosphate and AA 2-sulfate exhibited negligible scavenging activity. On the other hand, 2-O-alpha-D-glucopyranosyl-L-ascorbic acid (AA-2G) and a series of 6-O-acyl-2-O-alpha-D-glucopyranosyl-L-ascorbic acids (6-Acyl-AA-2G) themselves exhibited the radical scavenging activity of EC50: 6.1 x 10(-5) M and 4.4 x 10(-5)-5.9 x 10(-5) M, respectively, although their activities were lower than that of AA. Among 6-Acyl-AA-2G derivatives, the EC50 values tended to decrease with increasing length of their acyl carbon group. Increasing pH of the buffer resulted in decrease in the scavenging activity of all compounds tested as expected. We speculate that the difference in the radical scavenging activity of derivatives O-substituted at the C-2 position of AA may be ascribed to the linkage type of the substituent group to the endiol-lactone resonance system and the degree of dissociation of the C-3 proton.
Akihiro Tai, Kazuyoshi Kawazu and Akio Kobayashi : Species-specificity of an elicitor-active oligosaccharide, LN-3, to leguminous plants, Zeitschrift für Naturforschung. C, A Journal of Biosciences, Vol.51, No.5-6, 371-378, 1996.
Akihiro Tai, Eri Ohsawa, Kazuyoshi Kawazu and Akio Kobayashi : A minimum essential structure of LN-3 elicitor activity in bean cotyledons, Zeitschrift für Naturforschung. C, A Journal of Biosciences, Vol.51, No.1-2, 15-19, 1996.
Akio Kobayashi, Akihiro Tai and Kazuyoshi Kawazu : Structural elucidation of an elicitor-active oligosaccharide, LN-3, prepared from algal laminaran, Journal of Carbohydrate Chemistry, Vol.14, No.6, 819-832, 1995.
Goro Okamoto, Yuichiro Fujii, Ken Hirano, Akihiro Tai and Akio Kobayashi : Pollen tube growth inhibitors from 'Pione' grape pistils, American Journal of Enology and Viticulture, Vol.46, No.1, 17-21, 1995.
100.
Akio Kobayashi, Akihiro Tai, Hiroshi Kanzaki and Kazuyoshi Kawazu : Elicitor-active oligosaccharides from algal laminaran stimulate the production of antifungal compounds in alfalfa, Zeitschrift für Naturforschung. C, A Journal of Biosciences, Vol.48, No.7-8, 575-579, 1993.
(Summary)
Natural polysaccharides were examined for their activity as elicitors of phytoalexins and flavonoids in alfalfa cotyledons. Only two polysaccharides, laminaran and pectic acid, had elicitor activity. Laminaran, which was more active than pectic acid, was hydrolyzed by tunicase and the hydrolysate was subjected to charcoal and gel filtration columns. Introduction of the pyridylamino group into the elicitor-active oligosaccharides was attempted in order to facilitate isolation. The pyridylamino derivatives were found to exhibit higher activity than the original oligosaccharides. Their liquid chromatography mass spectrometry (LC-MS) analysis revealed that the elicitor-active principles form two ion clusters with the same molecular weights, m/z 1070 and 1232. Their high performance liquid chromatography (HPLC) analysis showed three main peaks. The individual peaks (LN-1,2,3) were collected and subjected to the alfalfa cotyledon assay. LN-3 showed the highest activity (minimum effective concentration, 0.8 μg/ml).
Akihiro Tai : Applied research from basic research using ascorbic acid derivatives, Vitamins, Vol.93, No.9, 383-393, Sep. 2019.
(Summary)
L-Ascorbic acid (AsA) is an important antioxidant in food and biological systems, but it is unstable under various oxidative conditions, resulting in its rapid degradation. Therefore, the development of many stable AsA derivatives has been attempted. Among them, ascorbic acid 2-glucoside (AA-2G) has been approved by the Japanese Government as a quasi-drug principal ingredient in skin care and a food additive and it has been used widely in cosmetics and health foods in Japan. AA-2G is a provitamin C agent that exhibits vitamin C activity <i>in vitro</i> and <i>in vivo</i> after its enzymatic hydrolysis to AsA by α-glucosidase. 6-<i>O</i>-Acyl-2-<i>O</i>-α-D-glucopyranosyl-L-ascorbic acids having a straight-acyl chain of varying length from C4 to C18 (6-sAcyl-AA-2G) and a branched-acyl chain of varying length from C6 to C16 (6-bAcyl-AA-2G) are lipophilic stable AsA derivatives for improving the bioavailability of AA-2G. 6-sAcyl-AA-2G and 6-bAcyl-AA-2G exert usually known vitamin C functions efficiently<i> in vitro </i> and <i>in vivo</i>. Recently, AsA derivatives immobilized on a resin to let proteins recognize characteristic chemical structures of AsA have also been synthesized as new tools for identifying target proteins of AsA. In this mini review, the author presents researches using these AsA derivatives in applied fields from basic fields.
Akihiro Tai, Yuji Iwaoka and Hideyuki Ito : シクロデキストリングルカノトランスフェラーゼとアミログルコシダーゼを用いた高効率的かつ位置選択的なエリソルビン酸グルコシドの合成, Vitamins, Vol.87, No.7, 364-366, Jul. 2013.
Akihiro Tai : Antioxidant properties of 2-O-substituted ascorbic acid derivatives, Vitamins, Vol.87, No.2, 70-80, Feb. 2013.
(Summary)
The aim of this study was to characterize the antioxidant properties of three ascorbic acid (AsA) derivatives O-substituted at the C-2 position of AsA: ascorbic acid 2-glucoside (AA-2G), ascorbic acid 2-phosphate (AA-2P), and ascorbic acid 2-sulfate (AA-2S). The radical-scavenging activities of these AsA derivatives and some common low molecular-weight antioxidants such as uric acid and reduced glutathione (GSH) against 1,1-diphenyl-picrylhydrazyl (DPPH) radical, 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation (ABTS^<・+>) or galvinoxyl radical were kinetically and stoichiometrically evaluated under pH-controlled conditions. AA-2G, AA-2P and AA-2S slowly and continuously reacted with DPPH radical and ABTS^<・+>, but not with galvinoxyl radical. They effectively reacted with DPPH radical under acidic conditions and with ABTS^<・+> under neutral conditions. In contrast, AsA immediately quenched all species of radicals tested at all pH values. The reactivity of Trolox, a water-soluble vitamin E analogue, was comparable to that of AsA in terms of kinetics and stoichiometrics. Uric acid and GSH exhibited long-lasting radical-scavenging activity against these radicals under certain pH conditions. The radical-scavenging profiles of AA-2G, AA-2P and AA-2S were closer to those of uric acid and GSH rather than to that of AsA. The number of radicals scavenged by one molecule of the AsA derivatives was equal to or greater than that by AsA or Trolox under the appropriate conditions. Furthermore, inhibitory effects of AA-2G, AA-2P and AA-2S on 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH)-induced oxidative hemolysis of sheep erythrocytes were studied and were compared with those of AsA and other antioxidants. The order of the inhibition efficiency was AA-2S≥Trolox=uric acid≥AA-2P>AA-2G=AsA>GSH. Although the reactivity of the AsA derivatives against AAPH-derived peroxyl radical (ROO^・) was much lower than that of AsA, the derivatives exerted equal or more potent protective effects on AAPH-induced hemolysis. In addition, the AsA derivatives were found to react per se with DPPH radical and ABTS^<・+> and ROO^・, not via AsA as an intermediate. These results suggest that the AsA derivatives per se act as biologically effective antioxidants under moderate oxidative stress and that AA-2G and AA-2P may be able to act under severe oxidative stress after enzymatic conversion to AsA in vivo.
Akihiro Tai : Design and application of novel stable lipophilic ascorbic acid derivatives, Vitamins, Vol.82, No.3, 151-160, Mar. 2008.
(Summary)
L-Ascorbic acid (AsA), vitamin C, possesses many biological activities, but it readily decomposes upon exposure to heat, UV light, metal ions, and oxidants. Therefore, Yamamoto and Muto et al. have developed a stable AsA derivative, 2-O-α-D-glucopyranosyl-L-ascorbic acid (AA-2G). AA-2G has been approved by the Japanese Government as a quasi-drug principal ingredient in skin care and as a food additive and is currently being used as a medical additive in commercial cosmetics. This stable AsA derivative exhibits vitamin C activity in vitro and in vivo after enzymatic hydrolysis to AsA by α-glucosidase. Recently, we have synthesized two types of monoacylated derivatives of AA-2G, 6-O-acyl-2-O-α-D-glucopyranosyl-L-ascorbic acids having a straight-acyl chain (6-sAcyl-AA-2G) and a branched-acyl chain (6-bAcyl-AA-2G), in order to improve the bioavailability of AA-2G. These derivatives showed satisfactory stability in neutral solution comparable to that of AA-2G, and had radical scavenging activity per se. The lipophilicity of 6-sAcyl- and 6-bAcyl-AA-2G was increased with increasing length of their acyl group. 6-sAcyl- and 6-bAcyl-AA-2G with an appropriate length of the acyl chain group exhibited antiscorbutic activity in guinea pigs and skin permeability superior to that of AA-2G in a human living skin equivalent model. At a lower concentration than AA-2G, 6-sAcyl- and 6-bAcyl-AA-2G enhanced both dibutyryl cyclic AMP- and nerve growth factor-induced neurite outgrowth in PC 12 cells, antigen-specific antibody production in murine splenic cells and collagen synthesis in human skin fibroblasts, and inhibited melanogenesis in B16 mouse melanoma cells. It is necessary to metabolize the derivatives by α-glucosidase at least to demonstrate these biological activities. These results indicate that 6-sAcyl- and 6-bAcyl-AA-2G are readily available sources of AsA activity in vitro and in vivo, may be useful as an effective pharmacological agent and as a promising food additive.