Development of ELISA Method for the Determination of Compound K

Compound K 측정을 위한 ELISA법 개발

  • Received : 2015.11.02
  • Accepted : 2015.12.02
  • Published : 2015.12.31

Abstract

In order to quantify compound K(CK), anticancer component of Panax ginseng C. A. Meyer, high titer rabbit polyclonal antibodies (pAbs) were raised against a conjugate of CK and bovine serum albumin coupled by a periodate oxidation method. Coating antigen (CK-OVA) was also prepared by the same method with OVA. As a result of optimization of antiserum dilution (2,000 fold), coating antigen ($25{\mu}g/ml$) and other condition (incubation time, temperature and washing method), ELISA method for the determination of CK was established. The measuring range extended from 0.5 ng/ml to 25 ng/ml of CK. The antibodies exhibited minor or even no cross reactivities with protopanaxatriol (1.56%) and other tested ginsenosides, $GRb_1$ (0.11%), $GRg_1$ (0.07%) except protopanaxadiol (87.2%) from the structural similarity. And the antibody showed good correlation (r=0.987) between the assay values obtained by this ELISA method and HPLC. Therefore, the ELISA method could be very useful tools for the determination of CK in biological fluids because of their high sensitivity and specificity.

Keywords

References

  1. Yosioka, I., Sugawara, T., Imai, K. and Kitagawa, I. (1972) Soil bacterial hydrolysis leading to genuine aglycone. V. On ginsenosides $Rb_1$, $Rb_2$ and Rc of the ginseng root saponins. Chem. Pharm. Bull. 20: 2418-2421. https://doi.org/10.1248/cpb.20.2418
  2. Zhou, W., Feng, M. Q., Li, J. Y. and Zhou, P. (2006) Crystal structure and bioactivity of ginsenoside compound K. J. Asian Nat. Prod. Res. 8: 519-527. https://doi.org/10.1080/10286020500208600
  3. Zhang, Z., Du, G. J., Wang, C. Z., Wen, X. D., Calway, T. and Li, Z. (2013) Compound K, a ginsenoside metabolite, inhibits colon cancer growth via multiple pathways including p53-p21 interactions. Int. J. Mol. Sci. 14: 2980-2995. https://doi.org/10.3390/ijms14022980
  4. Joh, E. H., Lee, I. A., Jung, I. H. and Kim, D. H. (2011) Ginsenoside Rb1 and its metabolite compound K inhibit IRAK-1 activation-the key step of inflammation. Biochem. Pharmacol. 82: 278-286. https://doi.org/10.1016/j.bcp.2011.05.003
  5. Chen, J., Wu, H., Wang, Q., Chang, Y., Liu, K, Song, S., Yuan, P., Fu, J., Sun, W., Huang, Q., Liu, L., Wu, Y., Zhang, Y., Zhou, A. and Wei, W. (2014) Ginsenoside metabolite compound K alleviates adjuvant-induced arthritis by suppressing T cell activation. Inflammation 37: 1608-1615. https://doi.org/10.1007/s10753-014-9887-0
  6. Kim, S., Kang, B. Y., Cho, S. Y., Sung, D. S., Chang, H. K., Yeom, M. H. et al. (2004) Compound K induces expression of hyaluronan synthase 2 gene in transformed human keratinocytes and increases hyaluronan in hairless mouse skin. Biochem. Piophys. Res. Commun. 316: 348-355. https://doi.org/10.1016/j.bbrc.2004.02.046
  7. Shin, Y. W. and Kim, D. H. (2005) Ginsenoside Rb1 and compound K in scratching behavior mouse models. J. Pharmacol. Sci. 99: 83-88. https://doi.org/10.1254/jphs.FP0050260
  8. Kim, K., Park, M., Lee, Y. M., Rhyu, M. R. and Kim, K. Y. (2014) Ginsenoside metabolite compound K stimulates glucagon-like peptide-1 secretion in HCI-H716 cells via bile acide receptor acivation. Arch. Pharm. Res. 37: 1193-1200. https://doi.org/10.1007/s12272-014-0362-0
  9. Lee, H. U., Bae, E. A., Han, M. J., Kim, N. J. and Kim, D. H. (2005) Hepatoprotective effect of ginsenoside Rb1 and compound K on tert-butyl hydroperoxide-induced liver injury. Liver Int. 25: 1069-1073. https://doi.org/10.1111/j.1478-3231.2005.01068.x
  10. Yang, X.-D., Yang, Y.-Y., Ouyang, D.-S. and Yang, G.-P. (2015) A review of biotransformation and pharmacology of ginsenoside compound K. Fitoterapia 100: 208-220. https://doi.org/10.1016/j.fitote.2014.11.019
  11. Zhou, W., Luo Z. and Zhou, P. (2005) Determination of ginsenoside compound- K by reversed- phase high performance liquid chromatography. Chinese journal of chromatography. 23: 270-272.
  12. Jeoung, M. K., Chung, Y. B., Lee, P. S., Kim, C. S., Kim, N. H., Son, D. J., Hong, J. T. and Moon, D.-C. (2005) Determination of a ginseng saponin metabolite, IH901, in rat plasma by liquid chromatography- tandem mass spectrometry. J. Liquid Chrom. Related Tech. 28: 3205-3216. https://doi.org/10.1080/10826070500330885
  13. Lee, P. S., Song, T. W., Sung, J.-H., Moon D.-C., Song, S. and Chung, Y. B. (2006) Pharmacokinetic characteristics and hepatic distribution of IH-901, a novel intetinal metabolite of ginseng saponin, in rats. Plant Med. 72: 204-210. https://doi.org/10.1055/s-2005-916201
  14. Paek, B., Moon, Y., Kim, J., Jia, H. Y., Kim, S. A. Sohn, D. H., Kim, J. B. and Lee, H. S. (2006) Pharmacokinetics of a ginseng saponin metabolite compound K in rats. Biopharm. Drug Dispos. 27: 39-45. https://doi.org/10.1002/bdd.481
  15. Akao, T., Kanaoka, M. and Kobashi, K. (1998) Appearance of compound K, a major metabolite of ginsenoside Rb1 by intestinal bacteria, in rat plasma after oral administrationmeasurement of compound K by enzyme immunoassay. Biol. Pharm. Bull. 21: 245-249. https://doi.org/10.1248/bpb.21.245
  16. Poulev, A., Deus-Neumann, B. and Zenk, M. H. (1993) Immunoassays for the quantitative determination of colchicine. Planta Med. 60: 77-83.