Standardization of the Manufacturing Process of Bee Venom Pharmacopuncture Containing Melittin as the Active Ingredient

Abstract

Background: Pharmacopuncture is a unique treatment in oriental medicine that combines chemical stimulation with conventional acupuncture. However, there are no standardized methods for preparing the herbal medicines used in pharmacopuncture, and it is not clear whether the active ingredients are safe and stable. Several studies have investigated nonstandardized preparation processes, but few investigations have addressed safety and preparation methods. Pharmacopuncture may provide an alternative treatment for incurable diseases. However, it must be as valid and safe as standardized medicine. In this way, the present project may contribute to the industrialization of medicine in Korea. It may also expand health insurance coverage by promoting evidence-based medical insurance benefits. Thus, the present study attempted to standardize and improve the raw materials, preparation, and efficacy of bee venom pharmacopuncture (BVP), which is a highly effective technique in oriental medicine.

Method: To purify the crude bee venom, the extract was subjected to a stepped-gradient open column (ODS-A; 120 Å, 150 mesh). Using this method, the yield of melittin was significantly increased and the allergen proteins were effectively removed. The melittin content of the purified bee venom was determined using HPLC, and the product was then diluted to 0.1 mg/mL using injection water in preparation for BVP.

Results: In the present study, we standardized the purification process to provide safe and stable BVP by increasing the main effective components and eliminating allergens. This study will be seminal in the industrialization and regulation of BVP.

Conclusion: We developed an effective strategy for melittin purification and allergen removal from bee venom to create safe BVP.

Figure 1

Solvent stability: Ethanol was considered a good solvent for reducing the side effects of BVP therapeutics. Detection wavelength: UV 220 nm column (YMC C18; 5 μm, 4.6 × 150 mm), flow rate: 0.4 mL/min, sample injection amount: 30 μL, mobile phase conditions: 0.1% trifluoroacetic acid in H₂O, and 0.1% trifluoroacetic acid in acetonitrile (gradient).

Figure 2

Purification process of bee venom pharmacopuncture (BVP) from raw material. The product was packaged at a good manufacturing practice (GMP) facility.

Figure 3

Separation of sequential compounds according to solvent polarity (apamin, PLA2, and melittin). In total, 13 fractions were isolated from the crude bee venom. Pure melittin was obtained in the 70%–80% ethanol layer.

Figure 4

Determination of the purity of the separated melittin (a) and comparison with standard commercial melittin (b). In the comparison with the melittin standard, the purity was 99.4%, and the melittin content of purified bee venom was 99% higher than the commercial standard.

Figure 5

Evaluation of the stability of the bee venom with different additives. The changes of melittin components were examined for 6 months; it was found that melittin was highly stable in pH- and salinity-free pharmacopuncture.