Introduction
Bee venom (BV) pharmacopuncture is used to treat diseases and requires the extraction of venom from live bees. BV has been used for 2,500 years, and classic treatment involves injecting the venom of a live bee directly into the affected body area
1). Korean medical doctors mainly use extracted and purified BV for treatment. The BV is injected into the meridian points or disease-related areas according to Korean medical principles. This method combines the effect of the acupuncture with that of the components of the BV
2). BV treatments are currently used in the US, Russia, and Europe to treat patients in the form of formulations such as injections or ointments, mainly for arthritis, atopy, and rheumatic diseases. BV treatment is recognized as an area of alternative medicine in many countries
3).
Chromatography is a method used to separate the components of a mixture using mobile and stationary phases. Among the various types, liquid chromatography is characterized by the use of a liquid as the mobile phase. The mobile phase in which the sample of the test chemical substance is dissolved is pushed by a pump at a constant flow rate under high pressure to facilitate its passage through a fixed phase column containing a filler. Then, the individual chemical substances in the test sample pass through the column at different times according to their affinity for the mobile and fixed phases. Thus, high-performance liquid chromatography (HPLC) is a method for quantifying a particular chemical by measuring the magnitude of its separation over time
4).
Previously, Lee
5) has studied the main components of bee venom and Kwon
6,
7) has studied the composition and safe use of sweet bee venom. The study by Kang
8) confirmed the stability of Sweet BV. An’s
9) study compared the melittin content of BV from domestic sources and foreign countries. All of these studies used HPLC for the analyses. There have been some studies on BV components, but no study has compared BV and separate purification BV (SPBV) pharmacopuncture methods, which are commonly used in clinical practice by Korean medical doctors. Therefore, we analyzed three components (melittin, apamin, and phospholipase A
2) in BV and SPBV using HPLC and the results are reported here.
Discussion
BV is a transparent liquid stored in venom sac. Following exposure to air, the water content evaporates and turns it crystalline. The specific gravity of BV is 1.1313 and, therefore, it is heavier than water while its pH is 5.2–5.5, which is slightly acidic. The effective amount of water removed from the BV is approximately 30%. Each bee has approximately 1 μL of venom, which yields a dry weight of approximately 0.3 mg of crystals. Approximately 5,000–10,000 bees are required to produce 1 g of dried BV. BV is composed of approximately 40 main ingredients including peptides, enzymes, physiologically active amines, carbohydrates, lipids, and amino acids.
The major peptides are melittin, apamin, adolapin, and the mast cell degranulating (MCD) peptide. These peptides have anti-inflammatory, antibacterial, and antipyretic effects. Melittin is the most important component of BV, and it consists of 40–50% of the dry weight of BV. It has cell lysis and hematocytolysis properties, and induces histamine release and increases cortisone levels. Apamin is a representative neurotoxin among the BV components, which promotes nerve conduction and causes hyperactivity. An overdose of apamin causes muscle spasms and respiratory failure. Melittin and apamin stimulate axes including the hypothalamus, pituitary, and adrenal cortex. The action of both substances increases the secretion of adrenocortical hormones.
Phospholipase A
2, which is the main enzyme in BV, is a polymer with a molecular weight ≥ 10,000. Phospholipase A
2 constitutes 10–12% of the weight of dried BV and is a significant antigen of allergic reactions, which is the major cause of the systemic allergic reactions due to BV. Previous studies have shown that phospholipase A
2 induces immunoglobulin E (Ig E) synthesis
10). It is also known that people who are allergic to BV have Ig E that responds to phospholipase A
211), which destroys cell membranes and causes cell damage.
In a previous study, three major components showed various effects. A study by Ahn study
12) investigated the relationship between the efficacy of melittin and lung carcinoma while Kang
13) reported the anti-inflammatory efficacy of melittin. The study by Han
14) was on the effect of melittin on synovial cells. A study by Kim
15) investigated the anticancer effect of apamin while the study by Kwon
16) was on the anticancer effect of melittin and apamin. The study by Kim
17,
18) was on the neuroprotective effect of phospholipase A
2. Possible methods for isolating BV components include dialysis, dissolution, filtration, and HPLC
3). In particular, HPLC allows the precise analysis of small samples
4) and has been used to measure purity after separating pure peptides from BV.
Reversed-phase (RP) HPLC is an important method of evaluating BV tablets
19). Therefore, we analyzed two kinds of BV pharmacopuncture (BVI and II), three kinds of SPBV pharmacopuncture (SPBV-I, II, and III), and apitoxin using HPLC. Most of the previous component analysis studies have been on melittin and apamin. In this study, we analyzed phospholipase A
2, which causes allergic reactions. And this study used two concentrations samples that most commonly used. The analysis results of BV-I revealed that the retention time of melittin was 31.256 min and its composition ratio was 47.4751%. The retention time of apamin was 11.240 min, and its composition ratio was 2.9602%. Melittin was detected at a concentration approximately 16 times higher than that of apamin. The analysis results of BV-II showed that the retention time of melittin was 32.367 min and its composition ratio was 5.7384%. The retention time of apamin was 10.968 min, and its composition ratio was 3.6380%. These values are lower than the average melittin and apamin values for BV. Similar results were also observed in the study of Kwon
5).
We found that BV-I had more melittin than BV-II did at the same concentration. The analysis results of SPBV-I showed the retention time of Melittin was 31.981 min, and its composition ratio was 52.1313%. The retention time of apamin was 11.141 min, and its composition ratio was 0.0553%. Furthermore, the retention time of phospholipase A2 was 23.729 min, and its composition ratio was 1.1427%. The unknown peak observed with a retention time of 11.763 min had a composition ratio of 38.6727%.
The analysis results of SPBV-II showed that the retention time of melittin was 30.721 min and its composition ratio was 10.2250%. The retention time of apamin was 11.416 min, and its composition ratio was 0.0208%. Furthermore, the retention time of phospholipase A2 was 23.576 min, and its composition ratio was 1.4051%. The unknown peak observed with a retention time of 11.960 min had a composition ratio of 81.3537%.
The analysis results of SPBV-III revealed that the retention time of melittin was 32.204 min and its composition ratio was 21.8047%. The retention time of apamin was 11.000 min, and its composition ratio was 8.6487%. Furthermore, the retention time of phospholipase A2 was 23.899 min, and its composition ratio was 0.9635%. The unknown peak observed with a retention time of 10.763 min had a composition ratio of 68.5831%.
In the three SPBV pharmacopuncture analysis, phospholipase A2 decreased remarkably. The analysis results of SPBV-I revealed that the melittin content of SPBV-I increased by 5% compared to that of BV-I. All three SPBV pharmacopuncture types showed an unknown peak. In addition, similar results were obtained in repeated experiments. It would be necessary to conduct further research studies on this. The analysis results of apitoxin showed that the retention time of melittin was 32.355 min and its composition ratio was 51.4417%. The amount of melittin in apitoxin is similar to that in SPBV-I. This study had a limitation in that we did not randomly test BV pharmacopuncture at various concentrations. Therefore, further studies using varying concentrations are needed.