Scutellariae radix ( S. radix) is one of the most important crude drugs used in Kampo medicines (KMs). A part of its pharmaceutical properties is due to flavone glycoside, baicalin (BI). A technique named eastern blotting was developed for the specific and easy identification of BI in the extracts of crude drugs and KMs using anti-BI monoclonal antibody (MAb). BI separated by silica gel thin-layer chromatography (TLC) transferred to a polyethersulfone (PES) membrane was treated with a NaIO 4 solution and reacted with bovine serum albumin (BSA) preparing BI-BSA conjugate on the PES membrane. Anti-BI MAb was bound and then antibody labeled with peroxidase directed against anti-BI MAb. Finally, a substrate was added and then BI was detected. As little as 1 mg of BI was still detected on the PES membrane under immunostaining method. Various samples of S. radix and KMs which contain S. radix were qualitatively analyzed, and BI was visually detected by eastern blotting technique. Furthermore, this method was applied for the immunohistochemical study to investigate the distribution of BI in the fresh root of Scutellaria baicalensis using immunoblotting by transferred from fresh root to the PES membrane.
References
[1]
Kawashima, K.; Nomura, A.; Makino, T.; Saito, K.; Kano, Y. Pharmacological properties of traditional medicine (XXIX): Effect of Hange-shashin-to and the combinations of its herbal constituents on rat experimental colitis. Biol. Pharm. Bull. 2006, 29, 1599–1603.
[2]
Koda, A.; Watanabe, S.; Yanagihara, Y.; Nagai, H.; Sakamoto, K. A comparative study of the anti-allergic effects of disodium baicalein 6-phosphate (BPS) and disodium cromoglycate (DSCG). Jpn. J. Pharmacol. 1977, 27, 31–38, doi:10.1254/jjp.27.31.
Li, B.Q.; Fu, T.; Yan, Y.D.; Baylor, N.W.; Ruscetti, F.W.; Kung, H.F. Inhibition of HIV infection by baicalin-a flavonoid compound purified from Chinese herbal medicine. Cell Mol. Biol. Res. 1993, 39, 119–124.
[5]
Wu, J.A.; Attele, A.S.; Zhang, L.; Yuan, C.S. Anti-HIV activity of medicinal herbs: usage and potential development. Am. J. Chin. Med. 2001, 29, 69–81, doi:10.1142/S0192415X01000083.
[6]
Konoshima, T.; Kokumai, M.; Kozuka, M.; Iinuma, M.; Mizuno, M.; Tanaka, T.; Tokuda, H.; Nishino, H.; Iwashima, A. Studies on inhibitors of skin tumor promotion. XI. Inhibitory effects of flavonoids from Scutellaria baicalensis on Epstein-Barr virus activation and their anti-tumor-promoting activities. Chem. Pharm. Bull. 1992, 40, 531–533, doi:10.1248/cpb.40.531.
[7]
Shieh, D.E.; Liu, L.T.; Lin, C.C. Antioxidant and free radical scavenging effects of baicalein, baicalin and wogonin. Anticancer Res. 2000, 20, 2861–2865.
[8]
Morimoto, S.; Tateishi, N.; Matsuda, T.; Tanaka, H.; Taura, F.; Furuya, N.; Matsuyama, N.; Shoyama, Y. Novel hydrogen peroxide metabolism in suspension cells of Scutellaria baicalensis Georgi. J. Biol. Chem. 1998, 273, 12606–12611.
[9]
Morimoto, S.; Tateishi, N.; Inuyama, M.; Taura, F.; Tanaka, H.; Shoyama, Y. Identification and molecular characterization of novel peroxidase with structural protein-like properties. J. Biol. Chem. 1999, 274, 26192–26198.
[10]
Lin, M.C.; Tsai, M.; Wen, K.C. Supercritical fluid extraction of flavonoids from Scutellariae radix. J. Chromatogr. A 1999, 830, 387–395, doi:10.1016/S0021-9673(98)00906-6.
[11]
Zhang, L.; Xing, D.; Ding, Y.; Wang, R.; Wang, X.; Du, L. A chromatographic method for baicalin quantification in rat thalamus. Biomed. Chromatogr. 2005, 19, 494–497, doi:10.1002/bmc.467.
[12]
Kotani, A.; Kojima, S.; Hakamata, H.; Kusu, F. HPLC with electrochemical detection to examine the pharmacokinetics of baicalin and baicalein in rat plasma after oral administration of a Kampo medicine. Anal. Biochem. 2006, 350, 99–104.
[13]
Zhang, H.; Tian, K.; Tang, J.; Qi, S.; Chen, H.; Chen, X.; Hu, Z. Analysis of baicalein, baicalin and wogonin in Scutellariae radix and its preparation by microemulsion electrokinetic chromatography with 1-butyl-3-methylimizolium tetrafluoborate ionic liquid as additive. J. Chromatogr. A 2006, 1129, 304–307, doi:10.1016/j.chroma.2006.08.016.
[14]
Weiler, E.W.; Kruger, H.; Zenk, M.H. Radioimmunoassay for the determination of the steroidal alkaloid solasodine and related compounds in living plants and herbarium specimens. Planta Med. 1980, 39, 112–124, doi:10.1055/s-2008-1074912.
[15]
Kido, K.; Morinaga, O.; Shoyama, Y.; Tanaka, H. Quick analysis of baicalin in Scutellariae radix by enzyme-linked immunosorbent assay using a monoclonal antibody. Talanta 2008, 77, 346–350, doi:10.1016/j.talanta.2008.06.034.
[16]
Paudel, M.K.; Putalun, W.; Sritularak, B.; Morinaga, O.; Shoyama, Y.; Tanaka, H.; Morimoto, S. Development of a combined technique using a rapid one-step immunochromatographic assay and indirect competitive ELISA for the rapid detection of baicalin. Anal. Chim. Acta. 2011, 701, 189–193, doi:10.1016/j.aca.2011.05.054.
[17]
Tanaka, H.; Putalun, W.; Tsuzaki, C.; Shoyama, Y. A simple determination of steroidal alkaloid glycosides by thin-layer chromatography immunostaining using monoclonal antibody against solamargine. FEBS Lett. 1997, 404, 279–282, doi:10.1016/S0014-5793(97)00106-3.
[18]
Taki, T.; Kasama, T.; Handa, S.; Ishikawa, D. A simple and quantitative purification of glycosphingolipids and phospholipids by thin-layer chromatography blotting. Anal. Biochem. 1994, 223, 232–238.
[19]
Shan, S.; Tanaka, H.; Shoyama, Y. Enzyme-linked immunosorbent assay for glycyrrhizin using anti-glycyrrhizin monoclonal antibody and an eastern blotting technique for glucuronides of glycyrrhetic acid. Anal. Chem. 2001, 73, 5784–5790, doi:10.1021/ac0106997.
