Acupuncture has only been used for 60 years in Korean medicine. However, since the treatment was commercialized, many studies have confirmed its efficacy. Although herbal acupuncture developed from acupuncture, its mechanism of action differs somewhat. Herbal acupuncture smooths the flow of blood, which is referred to as “energy” in oriental medicine. Furthermore, the medicine contains concentrated herbal ingredients that work simultaneously, thus surpassing the efficacy of acupuncture itself.
Until recently, there was no proper English word for herbal acupuncture. However, the term “pharmacopuncture” was registered in the 2017 medical academic information classification system (MeSH), which is used by the US National Library of Medicine (NLM) to link academic information in the healthcare field. Additionally, the term “pharmacopuncture” has been added to the new index of PubMed, which is the world’s largest medical journal database.
Pharmacopuncture has strong anti-inflammatory and pain-relieving effects because it directly treats the acupuncture point. In one survey of patients who had visited oriental medicine hospitals, 48% of responders preferred pharmacopuncture to other oriental medicine treatments, because it caused a rapid decrease in pain [
The venom of the European honey bee
Previous studies have demonstrated that the purification of bee venom is a challenging task, as it requires a series of separation and purification steps [
Crude bee venom was purchased from various manufacturers based on quality test results. The medicines were then compared with crude bee venom and with each other. Ultimately, four manufacturers were chosen: Chung-Jin Biotech, Bi-sen, and two local producers from Bong-hwa and Kyung Buk, South Korea.
High-performance liquid chromatography (HPLC) was performed with a C18-5E YMC packed column (5
Melittin, the main active ingredient of bee venom, is a protein that is reduced or destroyed by heat, acids, bases, and so on. In the present study, ethanol was used as a solvent because it does not affect the melittin content during purification and analysis of raw bee venom. More specifically, the stability of melittin in 50% aqueous ethanol solution was investigated, and ethanol was used as a developing solvent in this experiment, because it did not change the melittin content in aqueous solution. In addition, the apamin content decreased in 50% aqueous ethanol solution.
Crude bee venom was isolated and purified in a g/mL dilution. This 10% diluted sample was subjected to a stepped-gradient open column (ODS-A, 120 Å, and 150 meshes) that was eluted using 0%–80% ethanol, affording 13 fractions.
Each of the separated materials obtained through the open column, as well as their purity, were determined using HPLC. The separated components and their degree of purification were then compared with standard reagents. Apamin, PLA2, and melittin standard reagents were prepared at concentrations of 0.1 mg/mL, and their contents were confirmed. HPLC was carried out using a reversed-phase YMC C18 (5
After removal of the allergen from raw bee venom and filtering of the purified melittin using membrane filters (pore size: 0.45–0.2
Changes in the melittin’s composition were observed by applying the above manufacturing process and the quality control method to the prototype product using the raw materials for distribution. To confirm the stability of the BVP using various additives, we used the pH compensator that was used for preparation.
To find high quality raw material, the melittin content of different products was determined using HPLC analysis. Of the four crude bee venoms used, we found that the Bi-sen product contained the highest amount of melittin (35.75%; Table
Comparing raw bee venom by production area.
Division | Apamin (%) | PLA2 (%) | Melittin (%) |
---|---|---|---|
Chung-Jin | 37.240 | 12.631 | 32.245 |
Bi-sen | 44.019 | 14.016 | 35.751 |
Local 1 | 37.959 | 10.771 | 34.432 |
Local 2 | 13.772 | 1.935 | 1.292 |
Changes in melittin content were measured using ethanol, which does not affect melittin content during the analysis and purification of bee venom raw materials. HPLC confirmed that, in a 50% ethanol aqueous solution, the melittin content was stable, but the apamin content was significantly decreased (Figure
Crude bee venom in distilled water
Crude bee venom in 50% ethanol layer
To isolate and purify the active component of crude bee venom (10 g/mL), the raw venom was partitioned into 13 fractions (Table
Separate substances (detected compound) according to their solvent formulations.
Fr. | Solvent gradient | Amount | Detected compound |
---|---|---|---|
(H2O : ethanol) | (mL) | ||
1 | 0 | 50 (each) | ND |
2 | 0 | ND | |
3 | 0 | ND | |
4 | 0 | ND | |
5 | 10% | ND | |
6 | 20% | Apamin | |
7 | 30% | Apamin, PLA2 | |
8 | 40% | Apamin, PLA2 | |
9 | 50% | PLA2 | |
10 | 60% | PLA2, melittin | |
|
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11 | 70% | 100 | Melittin |
|
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12 | 70% | 50 | Melittin |
|
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13 | 80% | 100 | Melittin |
ND: not detected.
The composition of each fraction obtained through open column chromatography was determined by HPLC analysis, using apamin, PLA2, and melittin as standard compounds (Figure
Component content eluted by fractions.
Change in component contents | ||||||
---|---|---|---|---|---|---|
Fraction number | 6 | 7 | 8 | 9 | 10 | 11 |
Apamin (%) | 42.32 | 2.04 | 2.19 | 0.35 | 0.4 | 0.62 |
PLA2 (%) | 0.59 | 20.17 | 24.64 | 6.16 | 1.59 | 0.16 |
PLA2 (%) | 1.66 | 77.79 | 67.93 | 88.15 | 8.75 | 0.33 |
Melittin (%) | - | - | 5.25 | 5.35 | 89.26 | 98.89 |
Purification process of bee venom pharmacopuncture (BVP) from raw material. The product was packaged at a good manufacturing practice (GMP) facility.
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.
Fractions 1–5 (10% ethanol)
Fraction 6 (20% ethanol)
Fraction 7 (30% ethanol)
Fraction 8 (40% ethanol)
Fraction 9 (50% ethanol)
Fraction 10 (60% ethanol)
Fraction 11 (70% ethanol)
Fraction 13 (80% ethanol)
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.
Melittin standard
Isolated melittin from bee venom purity
The purified bee venom was concentrated and lyophilized (concentrated under reduced pressure) to produce a powder. The melittin content in the purified bee venom was determined using HPLC. The venom was then diluted to a concentration of 0.1 mg/mL, which is used in BVP, using water that had been injected through a 0.2
To ensure that the BVP was safe, we compared the efficacy and safety of original bee venom with those of purified bee venom that had been filtered for PLA2 and histamine, as reported previously [
Bee venom for BVP is produced using a medicine preparation process that ensures safety and lack of heavy metals. Thus, the evaluation items are the purity test and the heavy metal test. The purity test confirmed that the herbicide had dissolved and that there were no heavy metals (lead, cadmium, arsenic, and mercury), insoluble particulate matter, insoluble water, sterility, or endotoxins. Thus, based on these standards, the purified bee venom appeared to be appropriate (Table
Safety evaluation of bee venom pharmacopuncture.
Bee venom pharmacopuncture | |
---|---|
Purity test | |
Dissolution state | ND |
Lead | 0 ppm |
Cadmium | 0.0 ppm |
Arsenic | 0 ppm |
Mercury | 0.0 ppm |
Insoluble particulate matter | ND |
Soluble particulate matter | ND |
Sterility test |
ND |
Endotoxin test |
ND |
Changes in melittin composition and purity were observed by applying the above manufacturing process and the quality control method to the prototype product using the raw materials for distribution. The pH compensator was used to confirm the stability of BVP produced using various additives (Figure
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.
As oriental medicine develops, social interest and research into its effects are growing. In addition, unlike injections, pharmacopuncture uses acupuncture points to reduce pain and quickly identify its cause. However, because pharmacopuncture has not been standardized, regulated, and industrialized, it is not clear whether the procedure is safe and stable. The most important issue for pharmacopuncture is safety. Therefore, if pharmacopuncture is to be a pharmaceutical industry, safe medicine should be manufactured and standardized. Currently, China is actively producing and supplying medicinal herbs. To publicize this oriental medicine, China is also actively investing in the medicine business. Chinese pharmacopuncture often uses two or more kinds of medicines from a single medicinal herb or material; these are administered to patients in various formulations.
Therefore, to ensure the safety and the stability of this treatment, it is urgent that researchers standardize pharmacopuncture. In this way, the therapy could be popularized through the pharmaceutical industry.
To our knowledge, the present study was the first that aimed to standardize and improve the raw materials, preparation, and efficacy of BVP, which is a highly effective oriental medicinal treatment. Crude bee venom (Bi-sen) was isolated and purified in a 1 g/mL dilution. In total, 13 fractions were isolated from the crude bee venom. Pure melittin was obtained in the 70%–80% ethanol layer. In comparison with the melittin standard, its purity was 99.4%, and melittin content of our purified bee venom was 99% higher than the commercial standard. Our total melittin yield was 63% and its purity was 92%–99% after separation and purification. The content of melittin in our purified bee venom was determined by HPLC; the melittin was then diluted to 0.1 mg/mL in preparation for BVP. All these procedures were performed at an aseptic GMP facility.
This experiment aimed to separate melittin from crude bee venom to produce safe, effective, and high-concentration standardized medicines for pharmacopuncture. We standardized the manufacturing process to provide safe and stable BVP by increasing the concentrations of the effective components and eliminating allergens. Thus, this study will be seminal in the industrialization and regulation of BVP.
The authors have no conflicts of interest to declare regarding the publication of this paper.
This work was supported by the Standardization Project of Korean Medicine Acupuncture, which is funded by the Korean Ministry of Health and Welfare (3243-302).