Qualitative and quantitative evaluation of melittin in honeybee venom and drug products containing honeybee venom

Open access


A reverse-phase high-performance liquid chromatographic (RP -HPLC ) method was developed and validated for the analysis of honeybee venom samples and drug products containing honeybee venom. The validation parameters were linearity, sensitivity, precision, and recovery. Melittin is the main component of honeybee venom was extracted with pure water, and then evaluated by RP -HPLC with a photodiode array (PDA ) detector. Separation of the samples was achieved on a Europa Protein C18 column with linear gradient elution of acetonitrile and 0.4% phosphoric acid at 25°C. There was a flow rate of 1 mL/min. Detection was set at 220 nm. Limits of detection (LOD ) and quantification (LO Q) for melittin were 1.1 and 3.2 μg/mL, respectively. The amount of melittin in honeybee venom samples ranged from 21.9 to 66.4 %.

Gauldie J., Hanson J. M., Shipolini R. A., Vernon C. A. (1978) The Structures of some peptides from bee venom. European Journal of Biochemistry 83: 405-410. DOI: 10.1111/j.1432-1033.1978.tb12106.x Gauldie J., Hanson J. M., Rumjanek F. D., Shipolini R.

A., Vernon C. A. (1976) The peptide components of bee venom. European Journal of Biochemistry 61: 369-376. DOI: 10.1111/j.1432-1033.1976. tb10030.x Habermann E. (1972) Bee and wasp venoms. Science 177: 314-322. DOI: 10.1126/science. 177.4046.314

Kim H. W., Kwon Y. B., Ham T. W., Roh D. H., Yoon S. Y., Lee H. J., Han H. J., Yang I. S., Beitz A. J., Lee J. H. (2003) Acupoint stimulation using bee venom attenuates formalin induced pain behavior and spinal cord fos expression in rats. Journal of Veterinary Medical Science 65: 349-355. DOI: 10.1292/jvms.65.349

Kokot Z. J., Matysiak J. (2009) Simultaneous determination of major constituents of honeybee venom by LC-DAD. Chromatographia 69: 1401-1405. DOI: 10.1365/s10337-009-1052-9

Kwon Y. B., Lee H. J., Han H. J., Mar W. C., Kang S. K., Yoon O. B., Beitz A. J., Lee J. H. (2002) The water-soluble fraction of bee venom produces antinociceptive and anti-inflammatory effects on rheumatoid arthritis in rats. Life Sciences 71: 191-204. DOI: 10.1016/S0024-3205(02)01617-X Kwon Y. B., Ham T. W., Kim H. W., Roh D. H., Yoon S. Y., Han H. J, Yang I. S., Kim K. W., Beitz A. J., Lee J. H. (2005) Water soluble fraction (<10 kDa) from bee venom reduces visceral pain behavior through spinal alpha 2-adrenergic activity in mice. Pharmacology Biochemistry and Behavior 80: 181-187. DOI: 10.1016/j.pbb.2004.10.017

Lee M. S., Pittler M. H., Shin B. C., Kong J. C., Ernst E. (2008) Bee venom acupuncture for musculoskeletal pain: A review. The Journal of Pain 9: 289-297. DOI: 10.1016/j.jpain.2007.11.012

Matysiak J., Schmelzer C. E. H., Neubert R. H. H., Kokot Z. J. (2011) Characterization of honeybee venom by MALDI-TOF and nanoESI-QqTOF mass spectrometry. Journal of Pharmaceutical and Biomedical Analysis 54: 273-278. DOI: 10.1016/j. jpba.2010.08.020

Pacáková V., Stulik K. (2000) Validation of a method for determination of phospholipase A2 and melittin in bee venom preparations by capillary electrophoresis. Journal of AOAC International 83: 549-554.

Pacáková V., Štulík K., Hau P. T., Jelínek I., Vinš I., Sýkora D. (1995) Comparison of high-performance liquid chromatography and capillary electrophoresis for the determination of some bee venom compounds. Journal of Chromatography A 700: 187-193. DOI: 10.1016/0021-9673(94)01170-j Park H. J., Lee S. H., Son D. J., Oh K. W., Kim K. H., Song H. S., Kim G. J., Oh G. T., Yoon D. Y., Hong J. T. (2004) Antiarthritic effect of bee venom: inhibition of inflammation mediator generation by suppression of NF-κB through interaction with the p50 subunit. Arthritis and Rheumatism 50: 3504-3515. DOI: 10.1002/art.20626

Putz T., Ramoner R., Gander H., Rahm A., Bartsch G., Thurnher M. (2006) Antitumor action and immune activation through cooperation of bee venom secretory phospholipase A2 and phosphatidylinositol-( 3,4)-bisphosphate. Cancer Immunology, Immunotherapy 55: 1374-1383. DOI: 10.1007/ s00262-006-0143-9

Rader K., Wildfeuer A., Wintersberger F., Bossinger P., Mucke H. W. (1987) Characterization of bee venom and its main components by high performance liquid chromatography. Journal of Chromatography 408: 341-348. DOI: 10.1016/s0021-9673(01)81820-3

Russell P. J., Hewish D., Carter T., Sterling-Levis K., Ow K., Hattarki M., Doughty L., Guthrie R., Shapira D., Molloy P. L., Werkmeister J. A., Kortt A. A. (2004) Cytotoxic properties of immunoconjugates containing melittin-like peptide 101 against prostate cancer: in vitro and in vivo studies. Cancer Immunology,Immunotherapy 53: 411-421. DOI: 10.1007/ s00262-003-0457-9

Rybak M., Skubida P. (2007) Application of coupled electrical and sound stimulation for honeybee venom collection. Journal of Apicultural Science 51: 63-66

Rybak-Chmielewska H., Szczęsna T. (2004) HPLC study of chemical composition of honeybee (Apis mellifera L.) venom. Journal of Apicultural Science 48: 103-109.

Rybak M., Muszyńska J., Skubida P. (1995) A technology for bee venom collection. Pszczelnicze Zeszyty Naukowe 39(2): 223-231.

Son D. J., Lee J. W., Lee Y. H., Song H. S., Lee C. K., Hong J. T. (2007) Therapeutic application of anti-arthritis, pain-releasing, and anti-cancer effects of bee venom and its constituent compounds. Clinical Pharmacology and Therapeutics 115: 246-270. DOI: 10.1016/j.pharmthera.2007.04.004

Szokan G., Harvath J., Almas M., Saftics G., Palocz A. (1994) Liquid chromatographic analysis and separation of polypeptide components from honeybee venoms. Journal of Liquid Chromatography and Related Technologies 17: 3333-3349. DOI: 10.1080/10826079408013516

Zhou J., Zhao J., Zhang S., Shen J., Qi Y., Xue X., Li Y., Wub L., Zhang J., Chen F., Chen L. (2010) Quantification of melittin and apamin in bee venom lyophilized powder from Apis mellifera by liquid chromatography- diode array detector-tandem mass spectrometry. Analytical Biochemistry 404: 171-178. DOI: 10.1016/j.ab.2010.05.014

Ziyavitdinov Z. F., Inogamov U. K., Sagdiev N. Z. (1995) Development of a method for the complex isolation of physiologically active components from bee venom. Chemistry of Natural Compounds 31: 726-730. DOI: 10.1007/bf01386189

Journal of Apicultural Science

The Journal of Research Institute of Horticulture and Apicultural Research Association

Journal Information

IMPACT FACTOR 2017: 0.75
5-year IMPACT FACTOR: 1.007

CiteScore 2017: 0.92

SCImago Journal Rank (SJR) 2017: 0.345
Source Normalized Impact per Paper (SNIP) 2017: 0.461

Cited By


All Time Past Year Past 30 Days
Abstract Views 0 0 0
Full Text Views 186 186 26
PDF Downloads 100 100 16