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J Acupunct Res > Volume 34(1); 2017 > Article
Jo and Roh: Major Components of Clinically used Bee Venom Pharmacopuncture※

Abstract

Objectives

The purpose of this study was to analyze the components of the clinically used bee venom (BV) pharmacopuncture.

Methods

Two kinds of bee venom pharmacopuncture (BV-I and II), three kinds of separate purification BV (SPBV) pharmacopuncture (SPBV-I, II, and III), and apitoxin were investigated in this study. We performed a component analysis of melittin, apamin, and phospholipase A2 using high-performance liquid chromatography (HPLC).

Results

  1. BV-I contained approximately 40% more melittin than BV-II did.

  2. In the three separate purification BV pharmacopuncture, SPBV-I, SPBV-II, and SPBV-III, phospholipase A2 content decreased remarkably.

  3. The melittin content in SPBV-I increased by 5% compared to that in BV-I.

  4. The amount of melittin in apitoxin was similar to that in SPBV-I.

Conclusion

The compositions of the BV pharmacopuncture and separate purification BV pharmacopuncture changed depending on the collection method and concentration. Therefore, it is necessary to choose the most suitable BV for each specific medical treatment target. Furthermore, research into the composition of BV may be needed for its safe and effective use.

Abstract

목적

본 연구는 임상에서 사용되는 여러 종류의 봉약침을 분석하여 주요 성분의 비율 차이를 알아보는데 목적이 있다.

방법

두 가지 종류의 봉독 약침(BV-I 과 II), 세 가지 종류의 정제 봉독약침(SPBV I, II 및 III)과 Apitoxin 을 분석 시료로 하고, HPLC를 이용하여 분석하였다. 분석을 위한 표준시료는 Melittin, apamin, and Phospholipase A2를 사용하였다.

결과

1. BV-I은 BV-II 보다 Melittin 함량이 약 40%정도 높게 나타났다.
2. 정제 봉독약침(SPBV I, II 및 III)은 Phospholipase A2 함량이 현저하게 낮게 나타났다.
3. SPBV I의 Melittin함량은 BV-I보다 약 5% 정도 높게 나타났다.
4. Apitoxin 의 Melittin함량은 SPBV I과 유사하였다.

결론

이상의 결과로 봉약침의 주요 성분 비율은 수집방법 또는 농도에 따라 차이가 있을 수 있음을 알 수 있었다. 그러므로 임상에서 치료 목적과 방법에 적합한 봉약침을 선택하여 사용하여야 하며 봉약침의 효과적이고 안전한 사용을 위한 연구가 지속되어야 할 것이다.

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 area1). 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 BV2). 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 countries3).
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 time4).
Previously, Lee5) has studied the main components of bee venom and Kwon6,7) has studied the composition and safe use of sweet bee venom. The study by Kang8) confirmed the stability of Sweet BV. An’s9) 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 A2) in BV and SPBV using HPLC and the results are reported here.

Materials and Methods

1. Materials

BV is produced using electrical and microwave stimulation methods. The BV used for BV pharmacopuncture, mainly used clinically, was used as the analytical sample. Two kinds of BV pharmacopuncture (BV-I and II), three kinds of SPBV pharmacopuncture (SPBV-I, II, and III), and apitoxin were investigated in these experiments. The samples were diluted to the appropriate concentration using distilled water (Table 1).

2. Reagents

Methanol, acetonitrile (both Sigma-Aldrich, USA), and distilled water (Welgene, Korea) were used for the HPLC analyses. Melittin, apamin, and phospholipase A2 (all Sigma-Aldrich, USA) were used as standard products for the HPLC analysis.

3. Methods

An HPLC system (Agilent 1200, USA) was used for the analysis, and it was equipped with a Varian 9300, 510 pump, U6K injector, and ultraviolet (UV) absorbance detector as well as a comulting integrator D520A device. An analytical HPLC column (Shiseido, Japan. 5-μm, C18, 300Å, 3.9 mm × 250 mm) was used. The melittin, apamin, and phospholipase A2 standard solutions were prepared at concentrations of 10, 20, 50, and 100 μg/mL (Table 2).

Results

1. Analysis of main components of BV using HPLC

1) Construction of calibration curve of melittin standard solutions using HPLC

Melittin standard solutions were analyzed at concentrations of 10, 20, 50, and 100 μg/mL. The correlation coefficient of the calibration curve was 0.9984, and the calibration curve had a linear function, y =13960x−21479 (Fig. 1). The retention time of melittin was 32.044 min.

2) Construction of calibration curve of apamin standard solutions using HPLC

The apamin standard solutions were analyzed at concentrations of 10, 20, 50, and 100 μg/mL. The correlation coefficient of the calibration curve was 0.9991, and the calibration curve had a linear function, y =12704x−113929 (Fig. 2). The retention time of apamin was 11.437 min.

3) Construction of calibration curve of phospholipase A2 standard solutions using HPLC

The phospholipase A2 standard solutions were analyzed at concentrations of 10, 20, 50, and 100 μg/mL. The correlation coefficient of the calibration curve was 0.9987, and the calibration curve had a linear function, y =9931x−12956 (Fig. 3). The retention time of phospholipase A2 was 23.702 min.

2. Analysis of main components of BV-I

The analysis results of BV-I are as follows. The retention times of melittin, apamin, and phospholipase A2 were 31.256, 11.240, 23.880 min, respectively. The peak areas of melittin, apamin, and phospholipase A2 were 47.4751, 2.9602, and 26.5647%, respectively (Fig. 4).

3. Analysis of main components of BV-II

The analysis results of BV-II are as follows. The retention times of melittin, apamin, and phospholipase A2 were 32.367, 10.968, 23.779 min, respectively. The peak areas of melittin, apamin, and phospholipase A2 were 5.7384, 3.6380, and 10.6236%, respectively (Fig. 5).

4. Analysis of main components of SPBV-I

The analysis results of SPBV-I are as follows. The retention times of melittin, apamin, and phospholipase A2 were 31.981, 11.141, 23.729 min, respectively. The peak areas of melittin, apamin, and phospholipase A2 were 52.1313, 0.0553, and 1.1427%, respectively. An unknown peak with an area of 38.6727% was observed at 11.763 min (Fig. 6).

5. Analysis of main components of SPBV-II

The analysis results of SPBV-II are as follows. The retention times of melittin, apamin, and phospholipase A2 were 30.721, 11.416, and 23.576 min. The peak areas of melittin, apamin, and phospholipase A2 were 10.2250, 0.0208, and 1.4051%, respectively. An unknown peak with an area of 81.3537% was observed at 11.960 min (Fig. 7).

6. Analysis of main components of SPBV-III

The analysis results of SPBV-III are as follows. The retention times of melittin, apamin, and phospholipase A2 were 32.204, 11.000, and 23.899 min, respectively. The peak areas of melittin, apamin, and phospholipase A2 were 21.8047, 8.6487, and 0.9635%, respectively. An unknown peak with an area of 68.5831% was observed at 10.763 min (Fig. 8).

7. Analysis of main components of apitoxin

The analysis results of apitoxin are as follows. The retention time of melittin or apitoxin was 32.355 min while the peak area of melittin was 51.4417% (Fig. 9).

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 A2, which is the main enzyme in BV, is a polymer with a molecular weight ≥ 10,000. Phospholipase A2 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 A2 induces immunoglobulin E (Ig E) synthesis10). It is also known that people who are allergic to BV have Ig E that responds to phospholipase A211), which destroys cell membranes and causes cell damage.
In a previous study, three major components showed various effects. A study by Ahn study12) investigated the relationship between the efficacy of melittin and lung carcinoma while Kang13) reported the anti-inflammatory efficacy of melittin. The study by Han14) was on the effect of melittin on synovial cells. A study by Kim15) investigated the anticancer effect of apamin while the study by Kwon16) was on the anticancer effect of melittin and apamin. The study by Kim17,18) was on the neuroprotective effect of phospholipase A2. Possible methods for isolating BV components include dialysis, dissolution, filtration, and HPLC3). In particular, HPLC allows the precise analysis of small samples4) and has been used to measure purity after separating pure peptides from BV.
Reversed-phase (RP) HPLC is an important method of evaluating BV tablets19). 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 A2, 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 Kwon5).
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.

Conclusion

We analyzed the components of BV pharmacopuncture, which is widely used in clinical practice, and arrived at the following conclusions.
  1. BV-I contained approximately 40% more melittin than BV-II did.

  2. In the three SPBV pharmacopuncture types (SPBV-I, SPBV-II, and SPBV-III), the phospholipase A2 level decreased remarkably.

  3. The melittin content in SPBV-I increased by 5% compared to that in BV-I.

  4. The amount of melittin in apitoxin was similar to that in SPBV-I.

Therefore, we conclude that BV pharmacopuncture and SPBV pharmacopuncture are suitable for clinical treatment. However, their compositions change depending on the collection method and concentration. Therefore, it is necessary to choose the most suitable BV for each medical treatment target. In addition, further research is needed to evaluate the safety and effectiveness of BV use.

Fig. 1.
Calibration Curve of Melittin (y =13960×−21479, r2=0.9984)
acup-34-1-31f1.tif
Fig. 2.
Calibration Curve of apamin(y =12704×−113929, r2=0.9991)
acup-34-1-31f2.tif
Fig. 3.
Calibration Curve of Phospholipase A2 (y =9931×−12956, r2=0.9987)
acup-34-1-31f3.tif
Fig. 4.
HPLC chromatogram of BV-I
acup-34-1-31f4.tif
Fig. 5.
HPLC chromatogram of BV-II
acup-34-1-31f5.tif
Fig. 6.
HPLC chromatogram of BV-I
acup-34-1-31f6.tif
Fig. 7.
HPLC chromatogram of BV-II
acup-34-1-31f7.tif
Fig. 8.
HPLC chromatogram of BV-I
acup-34-1-31f8.tif
Fig. 9.
HPLC chromatogram of Apitoxin
acup-34-1-31f9.tif
Table 1.
Abbreviation and Conditions about samples
Abbreviation Conditions
BV-I Bee venom pharmacopuncture using electrical stimulation (dilution 1:4000)
BV-II Bee venom pharmacopuncture by microwave stimulation(dilution 1:4000)
SPBV-I Separate purification bee venom pharmacopuncture using electrical stimulation (dilution,1:4000)
SPBV-II Separate purification bee venom pharmacopuncture using microwave stimulation (dilution 1:4000)
SPBV-III Separate purification bee venom pharmacopuncture using microwave stimulation (dilution 1:8000)
Table 2.
Analytical Conditions for measuring of Melittin, apamin, and Phospholipase A2
Item Condition
Column Shiseido, −μ, C18, 300Å, 3.9 mm × 250 mm
Mobile phase A: 0.1% Trifluoroacetic acid (TFA) 2 L B: 0.1% TFA in acetonitrile: water (80:20)
Gradient 5% B-80% B for 40 min
Flow rate 1.5 mL/min
Wave length 220 nm
Sample volume 20 μL

VI. References

1. Yin CS, Koh HG. The first documental record on bee venom therapy in Oriental medicine : 2 prescriptions of bee venom in the ancient Mawangdui books of Oriental medicine. The Acupunct. 1998;15(1):143–7.

2. Kwon GR. The study of the introduction of Bee venom Acupuncture, Biochemistry and Pharmachology have been obtained the following results. The Acupunct. 1994;11(1):159–71.

3. Kim MH. Bee venom therapy and Bee venom acupuncture. First edition. Seoul: Korean Education Planning. 1996:20–72, 133–49, 171–6.

4. Marvin M. HPLC. second edition. New York: Wiley-Interscience. 2007:52–87.

5. Lee JS, Kwon GR, Choi HY. A Study on Major Components of Bee Venom Using HPLC. The Acupunct. 2000;17(4):120–9.

6. Kwon GR, Choi SH, Cha BC. Component Analysis of Sweet BV and Clinical Trial on Antibody Titer and Allergic Reactions. J. of Pharmacopuncture. 2006;9(2):79–86.

7. Kwon GR, Chu CS, Park HS, Kim MK, Cha BC, Lee E. Expermental Studies of quantitative evaluation using HPLC and safety of Sweet Bee Venom. J. of Pharmacopuncture. 2007;10(2):81–6.
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8. Kang KS, Kwon GR. Experimental studies of validation and stability of Sweet Bee Venom using HPLC. J. of Pharmacopuncture. 2009;12(4):33–49.
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9. An KW, Kim NY, Seo KY, Kim CG. Melittin Content Analysis of Korea and Aother Country Bee Venom Using HPLC. Koea J. Apiculture. 2010;25(4):283–90.

10. Korean Pharmacopuncture Institute Pharmacopuncturogy. Second edition. Seoul: Elsevier korea. 2012:151–4.

11. Müller U, Akdis CA, Fricker M, et al. Successful immunotherapy with T-cell epitope peptides of bee venom phospholipase A2 induces specific T-cell anergy in patients allergic to bee venom. J. of Allergy and Clinical Immunology. 1998;101(6):747–54.
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12. Ahn CB, Im CW, Youn HM, Park SJ, Choi YH. Melittin-induced Aapoptosis is Associated with Inhibition of COX-2 and hTERT Expression in Human Lung Carcinoma A549 Cells. The Acupunct. 2003;20(5):93–106.

13. Kang J, Song HS. The Effects of Bee Venom and Melittin on NO, iNOS and MAP Kinase Family in RAW 264.7 Cells. The Acupunct. 2006;21(3):107–19.

14. Han SW, Park KH, Jung TY, Seo JC. The Effect of Bee Venom & Melittin Solution on Cell Death in Synovial Cell Line. The Acupunct. 2002;19(4):74–88.

15. Kim YM, Lee JD, Park DS. The Anti-Cancer Effect of Apamin in Bee-Venom on Melanoma cell line SK-MEL-2 and Inhibitory Effect on the MAP-Kinase Signal Pathway. The Acupunct. 2001;18(4):101–15.

16. Kwon DH, Lee JD, Choi DY. The Study of Aati-cancer Effects of Bee Venom for Aqua-acupuncure. The Acupunct. 2001;18(1):129–45.

17. Kim SM, Jung TY, Leem SC, Seo JC, Han SW. The Effect of Phospholipase A2 Herbal-acupuncture on Neurotransmitters against Meddle Cerebral Artery Occulsion Rats. Korean J. Oriental Physiology & Pathology. 2004;18(5):1322–30.

18. Kim SM, Jung TY, Leem SC, et al. The Protective Effect of Phospholipase Herbal-acupuncture against the Neuronal Damage Induced by Middle Cerebral Artery Occulsion (MCAO) in Rats. Korean J. of Meridian & Acupuncture. 2004;21(3):89–96.

19. Szókán G, Horváth J, Almás M, Saftics G. Liquid Chromatographic Analysis and Separation of Polypeptide Components from Honey Bee Venoms. J. of liquid chromatography. 1994;17(16):3333–49.
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