Standardization of the Manufacturing Process of Bee Venom …downloads.hindawi.com/journals/ecam/2018/2353280.pdf · 2019. 7. 30. · Standardization of the Manufacturing Process

Research ArticleStandardization of the Manufacturing Process of Bee VenomPharmacopuncture Containing Melittin as the Active Ingredient

Yoonmi Lee , Sung-Geun Kim, In-Su Kim, and Hwa-Dong Lee

Traditional Korean Medicine Technology Division, R&D Development, Korean Medicine Preparation Team, National DevelopmentInstitute of Korean Medicine (NIKOM), 94 Hwarang-ro, Gyeongsan-si, Gyeongsangbuk-do, Republic of Korea

Correspondence should be addressed to Hwa-Dong Lee; [email protected]

Received 25 August 2017; Revised 6 December 2017; Accepted 24 December 2017; Published 25 February 2018

Academic Editor: Woojin Kim

Copyright © 2018 Yoonmi Lee et al. This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Background. Pharmacopuncture is a unique treatment in oriental medicine that combines chemical stimulation with conventionalacupuncture. However, there are no standardized methods for preparing the herbal medicines used in pharmacopuncture, and it isnot clear whether the active ingredients are safe and stable. Several studies have investigated nonstandardized preparation processes,but few investigations have addressed safety and preparationmethods. Pharmacopuncturemay provide an alternative treatment forincurable diseases. However, it must be as valid and safe as standardized medicine. In this way, the present project may contributeto the industrialization of medicine in Korea. It may also expand health insurance coverage by promoting evidence-based medicalinsurance benefits. Thus, the present study attempted to standardize and improve the raw materials, preparation, and efficacy ofbee venom pharmacopuncture (BVP), which is a highly effective technique in oriental medicine.Method. To purify the crude beevenom, the extract was subjected to a stepped-gradient open column (ODS-A; 120 A, 150mesh). Using this method, the yield ofmelittin was significantly increased and the allergen proteins were effectively removed. The melittin content of the purified beevenom was determined using HPLC, and the product was then diluted to 0.1mg/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 themain effectivecomponents and eliminating allergens. This study will be seminal in the industrialization and regulation of BVP. Conclusion. Wedeveloped an effective strategy for melittin purification and allergen removal from bee venom to create safe BVP.

1. Introduction

Acupuncture has only been used for 60 years in Koreanmedicine. However, since the treatment was commercialized,many studies have confirmed its efficacy. Although herbalacupuncture developed from acupuncture, its mechanism ofaction differs somewhat. Herbal acupuncture smooths theflow of blood, which is referred to as “energy” in orientalmedicine. Furthermore, the medicine contains concentratedherbal ingredients that work simultaneously, thus surpassingthe efficacy of acupuncture itself.

Until recently, there was no proper English word forherbal acupuncture. However, the term “pharmacopuncture”was registered in the 2017medical academic information clas-sification system (MeSH), which is used by the US NationalLibrary of Medicine (NLM) to link academic information in

the healthcare field. Additionally, the term “pharmacopunc-ture” has been added to the new index of PubMed, which isthe world’s largest medical journal database.

Pharmacopuncture has strong anti-inflammatory andpain-relieving effects because it directly treats the acupunc-ture point. In one survey of patients who had visited orientalmedicine hospitals, 48% of responders preferred pharma-copuncture to other oriental medicine treatments, because itcaused a rapid decrease in pain [1, 2]. Moreover, the safetyinvestigation suggested that acupuncture/pharmacopunctureled to a lower range, frequency, and severity of significantadverse events [3].

The venom of the European honey bee (Apis mellifera)comprises a mixture of proteins, peptides, and other smallmolecules. In bee venom pharmacopuncture (BVP), whichhas pain-relieving and anti-inflammatory effects, the venom

HindawiEvidence-Based Complementary and Alternative MedicineVolume 2018, Article ID 2353280, 7 pageshttps://doi.org/10.1155/2018/2353280

2 Evidence-Based Complementary and Alternative Medicine

is injected at appropriate doses onto acupuncture pointsthat are selected through syndrome differentiation [4]. BVPhas significant therapeutic effects on degenerative knee andrheumatoid arthritis [5–9]. The main active component ofbee venom pharmacopuncture (BVP) is melittin: a peptidewith antimicrobial, antitumor, and anti-inflammatory effects.In oriental medicine, honey bee venom products containingabout 50% melittin are widely used for BVP. However, theseproducts also contain the proteins phospholipase A2 (PLA2)and apamin, which are major allergens as they are capable ofinducing the IgE response in susceptible individuals, accord-ing to the International Union of Immunological Societies(IUIS) [10–12]. Thus, to protect patients against side effectsof BVP, both of these allergens must be effectively removed.

Previous studies have demonstrated that the purificationof bee venom is a challenging task, as it requires a series ofseparation and purification steps [13–15]. Thus, researchershave not yet established the appropriate separation conditionsfor completely removing the allergen proteins while stillobtaining a high yield of melittin. In the present study,we developed an effective strategy for melittin purificationfrom bee venom. Using this method, the yield of melittinsignificantly increased, and the allergen proteins (apaminand PLA2) were effectively removed. The current study mayhelp researchers to develop high quality BVP medicines.It may also expand the coverage of medical insurance byproviding a basis for quality control, standardization, andgood manufacturing practice (GMP) of BVP drugs.

2. Materials and Methods

2.1. Bee Venom. Crude bee venom was purchased fromvarious manufacturers based on quality test results. Themedicines were then compared with crude bee venom andwith each other. Ultimately, four manufacturers were chosen:Chung-Jin Biotech, Bi-sen, and two local producers fromBong-hwa and Kyung Buk, South Korea.

2.2. General. High-performance liquid chromatography(HPLC) was performed with a C18-5E YMC packed column(5 𝜇m, 4.6 × 150mm) using a Waters Alliance UV detector.Solvents for extraction, partition, thin-layer chromatography(TLC), and HPLC were distilled from HPLC grade solvents.The TLC plates used were Silica gel 60 F254 (Art. 1.05554,Merck) and RP-18 F254s (Art. 1.05560, Merck).

2.3. Isolation and Purification

2.3.1. Solvent Stability Test. Melittin, the main active ingredi-ent of bee venom, is a protein that is reduced or destroyedby heat, acids, bases, and so on. In the present study, ethanolwas used as a solvent because it does not affect the melittincontent during purification and analysis of raw bee venom.More specifically, the stability of melittin in 50% aqueousethanol solution was investigated, and ethanol was usedas a developing solvent in this experiment, because it didnot change the melittin content in aqueous solution. Inaddition, the apamin content decreased in 50% aqueousethanol solution.

Table 1: Comparing raw bee venom by production area.

Division Apamin (%) PLA2 (%) Melittin (%)Chung-Jin 37.240 12.631 32.245Bi-sen 44.019 14.016 35.751Local 1 37.959 10.771 34.432Local 2 13.772 1.935 1.292Content Standards. Standard product apamin, PLA2, and melittin (0.1mgcontent).

2.3.2. Isolation Scheme. Crude bee venom was isolated andpurified in a g/mL dilution. This 10% diluted sample wassubjected to a stepped-gradient open column (ODS-A, 120 A,and 150meshes) that was eluted using 0%–80% ethanol,affording 13 fractions.

2.3.3. Isolation and Purity Verification. Each of the separatedmaterials obtained through the open column, as well as theirpurity, were determined using HPLC. The separated compo-nents and their degree of purification were then comparedwith standard reagents. Apamin, PLA2, andmelittin standardreagents were prepared at concentrations of 0.1mg/mL, andtheir contents were confirmed. HPLC was carried out usinga reversed-phase YMC C18 (5 𝜇m, 4.6 × 150mm) that waseluted using a 10%–90% methanol-gradient menu system.

2.4. BVP Manufacturing. After removal of the allergen fromraw bee venom and filtering of the purified melittin usingmembrane filters (pore size: 0.45–0.2 𝜇m), the melittin wassubdivided into 2.25mL vials.

2.5. BVP Quality Management

2.5.1. Safety and Stability Evaluation. Changes in the melit-tin’s composition were observed by applying the above man-ufacturing process and the quality control method to theprototype 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.

3. Results

3.1. Bee Venom. To find high quality raw material, themelittin content of different products was determined usingHPLC analysis. Of the four crude bee venoms used, we foundthat the Bi-sen product contained the highest amount ofmelittin (35.75%; Table 1).

3.2. Isolation and Purification

3.2.1. Solvent Stability. Changes in melittin content weremeasured using ethanol, which does not affect melittincontent during the analysis and purification of bee venom rawmaterials. HPLC confirmed that, in a 50% ethanol aqueoussolution, the melittin content was stable, but the apamincontent was significantly decreased (Figure 1). These resultssuggest that ethanol is a good solvent for reducing the sideeffects of BVP.

Evidence-Based Complementary and Alternative Medicine 3

0

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(a) Crude bee venom in distilled water

0

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(b) Crude bee venom in 50% ethanol layer

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; 5𝜇m, 4.6 × 150mm), flow rate: 0.4mL/min, sample injection amount: 30 𝜇L, mobile phase conditions: 0.1%trifluoroacetic acid in H2O, and 0.1% trifluoroacetic acid in acetonitrile (gradient).

Table 2: Separate substances (detected compound) according totheir solvent formulations.

Fr. Solvent gradient Amount Detected compound(H2O : ethanol) (mL)

1 0

50 (each)

ND2 0 ND3 0 ND4 0 ND5 10% ND6 20% Apamin7 30% Apamin, PLA28 40% Apamin, PLA29 50% PLA210 60% PLA2, melittin11 70% 100 Melittin12 70% 50 Melittin13 80% 100 MelittinND: not detected.

3.2.2. Isolation of the Compounds. To isolate and purify theactive component of crude bee venom (10 g/mL), the rawvenom was partitioned into 13 fractions (Table 2). Accordingto the corresponding HPLC profiles, Fractions 1–5 (∼10%ethanol layer) contained null compounds. In Fraction 6,apamin appeared for the first time, and Fraction 7 containedboth apamin and PLA2. Melittin was eluted in Fraction 10;however, it was mixed with PLA2. Puremelittin was obtainedin the 70%–80% ethanol layer.

3.2.3. Purity Verification. The composition of each fractionobtained through open column chromatography was deter-mined by HPLC analysis, using apamin, PLA2, and melittinas standard compounds (Figure 2). Using the standardcomponents, apamin was detected at 12 minutes, PLA2 intwo peaks at 18 and 19 minutes, and melittin at 27 minutes.Melittin was detected from Fraction 11 (70% ethanol layer)and the peak area (%) was found to be about 98% (Figure 3,

Table 3). The standard purity of the melittin was 99.4%,and melittin content of the purified bee venom was 99%higher than the commercial standard (Figure 4). The totalmelittin yield was 63%, and its purity was about 92%–99%after separation and purification.

3.3. BVP Manufacturing. The purified bee venom was con-centrated and lyophilized (concentrated under reduced pres-sure) to produce a powder. The melittin content in thepurified bee venomwas determined using HPLC.The venomwas then diluted to a concentration of 0.1mg/mL, which isused in BVP, using water that had been injected through a0.2 𝜇mmembrane. Vials were filled with 2.25mL of this drugsolution. All these procedures were performed at an asepticGMP facility (Figure 2).

3.4. BVP Quality Management

3.4.1. Safety Evaluation. To ensure that the BVP was safe, wecompared the efficacy and safety of original bee venom withthose of purified bee venom that had been filtered for PLA2and histamine, as reported previously [10].

Bee venom for BVP is produced using a medicinepreparation process that ensures safety and lack of heavymetals. Thus, the evaluation items are the purity test andthe heavy metal test. The purity test confirmed that theherbicide had dissolved and that there were no heavy metals(lead, cadmium, arsenic, and mercury), insoluble particulatematter, insoluble water, sterility, or endotoxins. Thus, basedon these standards, the purified bee venom appeared to beappropriate (Table 4).

3.4.2. Stability Evaluation. Changes in melittin compositionand purity were observed by applying the above manu-facturing process and the quality control method to theprototype product using the raw materials for distribution.The pH compensator was used to confirm the stability ofBVP produced using various additives (Figure 5). Changesin the melittin content were examined for 6 months, and it

4 Evidence-Based Complementary and Alternative Medicine

Table 3: Component content eluted by fractions.

Change in component contentsFraction number 6 7 8 9 10 11Apamin (%) 42.32 2.04 2.19 0.35 0.4 0.62PLA2 (%) 0.59 20.17 24.64 6.16 1.59 0.16PLA2 (%) 1.66 77.79 67.93 88.15 8.75 0.33Melittin (%) - - 5.25 5.35 89.26 98.89

70%

Pure melittin

1~4 5 6 7~8 9 10

0% 10% 20% 30~40% 50% 60%

11~13

Apamin ApaminPLA2 PLA2 PLA2

Melittin Melittin

Frac.

EtOH%

Detectedcompd

ODS open column

Purity test

BioactivityCytotoxicity test

SafetyStability test

90%

Raw bee venom powder (10 g)

H2O : EtOH

Dilution0.2 mg/mL

>90% melittin

Ultrafiltration2 times(0.4–0.2 uM)

Packing2.2 mL

Figure 2: Purification process of bee venompharmacopuncture (BVP) from rawmaterial.The product was packaged at a goodmanufacturingpractice (GMP) facility.

was found that melittin was highly stable in both pH-free andsalinity-free pharmacopuncture.

4. Discussion

As oriental medicine develops, social interest and researchinto its effects are growing. In addition, unlike injections,pharmacopuncture uses acupuncture points to reduce pain

and quickly identify its cause. However, because pharma-copuncture has not been standardized, regulated, and indus-trialized, it is not clear whether the procedure is safe andstable. The most important issue for pharmacopuncture issafety. Therefore, if pharmacopuncture is to be a pharma-ceutical industry, safe medicine should be manufacturedand standardized. Currently, China is actively producingand supplying medicinal herbs. To publicize this oriental

Evidence-Based Complementary and Alternative Medicine 5

−50

050

100150200250

(mAU

)

10 20 30 40 500(min)

(a) Fractions 1–5 (10% ethanol)

0250500750

1000125015001750

(mAU

)

10 20 30 40 500(min)

(b) Fraction 6 (20% ethanol)

0250500750

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0 20 30 40 5010(min)

(c) Fraction 7 (30% ethanol)

−50

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(mAU

)10 20 30 40 500

(min)

(d) Fraction 8 (40% ethanol)

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(e) Fraction 9 (50% ethanol)

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(f) Fraction 10 (60% ethanol)

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(g) Fraction 11 (70% ethanol)

0200400600800

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(h) Fraction 13 (80% ethanol)

Figure 3: Separation of sequential compounds according to solvent polarity (apamin, PLA2, and melittin). In total, 13 fractions were isolatedfrom the crude bee venom. Pure melittin was obtained in the 70%–80% ethanol layer.

medicine, China is also actively investing in the medicinebusiness. Chinese pharmacopuncture often uses two or morekinds 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 thistreatment, it is urgent that researchers standardize pharma-copuncture. In this way, the therapy could be popularizedthrough the pharmaceutical industry.

6 Evidence-Based Complementary and Alternative Medicine

20 400(min)

0

500

1000

(mAU

)

240 260 280 300 320 340 360 380220(nm)

Calculated

2827(min)

(a) Melittin standard

0

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400

600

(mAU

)

20 400(min)

Calculated

2826(min)

240 260 280 300 320 340 360 380220(nm)

(b) Isolated melittin from bee venom purity

Figure 4: Determination of the purity of the separatedmelittin (a) and comparison with standard commercial melittin (b). In the comparisonwith themelittin standard, the puritywas 99.4%, and themelittin content of purified bee venomwas 99%higher than the commercial standard.

To our knowledge, the present study was the first thataimed to standardize and improve the raw materials, prepa-ration, and efficacy of BVP, which is a highly effective orientalmedicinal treatment. Crude bee venom (Bi-sen) was isolatedand purified in a 1 g/mL dilution. In total, 13 fractionswere isolated from the crude bee venom. Pure melittin wasobtained in the 70%–80% ethanol layer. In comparison withthe melittin standard, its purity was 99.4%, and melittincontent of our purified bee venom was 99% higher than thecommercial standard. Our total melittin yield was 63% andits purity was 92%–99% after separation and purification.Thecontent ofmelittin in our purified bee venomwas determined

by HPLC; the melittin was then diluted to 0.1mg/mL inpreparation for BVP. All these procedures were performed atan aseptic GMP facility.

5. Conclusions

This experiment aimed to separate melittin from crude beevenom to produce safe, effective, and high-concentrationstandardized medicines for pharmacopuncture. We stan-dardized the manufacturing process to provide safe andstable BVP by increasing the concentrations of the effectivecomponents and eliminating allergens. Thus, this study willbe seminal in the industrialization and regulation of BVP.

Evidence-Based Complementary and Alternative Medicine 7

Table 4: Safety evaluation of bee venom pharmacopuncture.

Bee venom pharmacopuncturePurity test

Dissolution state NDLead 0 ppmCadmium 0.0 ppmArsenic 0 ppmMercury 0.0 ppm

Insoluble particulate matter NDSoluble particulate matter NDSterility test1 NDEndotoxin test2 ND1Sterility test: direct method using liquid thioglycolic acid medium andsoybean casein digestion medium. 2Endotoxin test: using Pierce� LALChromogenic Endotoxin Quantitation Kit (determination coefficient [𝑅2] ≥0.9932).

0 days30 days60 days

Melittin amount (g/mL)

0102030405060

+

Ｊ（

+．

aCl+

．aC

l

+

ＪH

Crud

e pow

der

Crud

e pow

der

Crud

e pow

der

Crud

e pow

der

Figure 5: Evaluation of the stability of the bee venom with differentadditives. The changes of melittin components were examined for6 months; it was found that melittin was highly stable in pH- andsalinity-free pharmacopuncture.

Conflicts of Interest

The authors have no conflicts of interest to declare regardingthe publication of this paper.

Acknowledgments

This work was supported by the Standardization Projectof Korean Medicine Acupuncture, which is funded by theKorean Ministry of Health and Welfare (3243-302).

References

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[3] M.-R. Kim, J.-S. Shin, J. Lee et al., “Safety of acupunctureand pharmacopuncture in 80,523 musculoskeletal disorderpatients: A retrospective reviewof internal safety inspection andelectronicmedical records,”Medicine (United States), vol. 95, no.18, p. e3635, 2016.

[4] Y.-J. Ahn, J.-S. Shin, J. Lee et al., “Safety of essential bee venompharmacopuncture as assessed in a randomized controlleddouble-blind trial,” Journal of Ethnopharmacology, vol. 194, pp.774–780, 2016.

[5] H. K. Ko, “Experimental Studies on the Effect of Bee VenomTheraphy on the Analgesic, Antipyretic and Anti-inflammatoryAction,” Korean journal of oriental medicine, vol. no. 1, pp. 283–292, 1992.

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[7] J.-A. Lim, K. Sung-Nam, and L. Sung-Young, “The clinical studyon bee venom acupuncture treatment on osteoarthritis of kneejoint,” Journal of Pharmacopuncture, vol. 8, no. 2, pp. 29–37,2005.

[8] W. Wu-Hao, A. Kyu-Beom, L. Jin-Kang, and J. Hyoung-Seok,“Clinical investigation compared with the effects of the bee-venom acupuncture on knee joint with osteoarthritis,” Journalof Pharmacopuncture, vol. 3, pp. 101–103, 2001.

[9] K. M. Lee, K. S. Lee, S. C. Yem et al., “A Clinical study ofBee-venomacupuncture treatment on protrusion disc Patients,”Journal of Korean Acupuncture Moxibustion Society, vol. 5, pp.13–25, 2004.

[10] M.Moreno andE.Giralt, “Three valuable peptides frombee andwasp venoms for therapeutic and biotechnological use:Melittin,apamin and mastoparan,” Toxins, vol. 7, no. 4, article no. A020,pp. 1126–1150, 2015.

[11] E. Spoerri, J. Jentsch, and P. Glees, “Apamin from bee venom.Effects of the neurotoxin on subcellular particles of neuralcultures,” FEBS Letters, vol. 53, no. 2, pp. 143–147, 1975.

[12] C. M. Freeman, C. R. A. Catlow, A. M. Hemmings, and R. C.Hider, “The conformation of apamin,” FEBS Letters, vol. 197, no.1-2, pp. 289–296, 1986.

[13] Y. Maulet, U. Brodbeck, and B. W. Fulpius, “Purification frombee venom of melittin devoid of phospholipase A2 contamina-tion,” Analytical Biochemistry, vol. 127, no. 1, pp. 61–67, 1982.

[14] W. Zhu, B. Wang, and X. Zhu, “Isolation and purification ofBV I -2H from bee venom and analysis of its biological action,”Chinese Science Bulletin, vol. 47, no. 11, pp. 910–914, 2002.

[15] D.-O. Moon, S.-Y. Park, K.-J. Lee et al., “Bee venom and melit-tin reduce proinflammatory mediators in lipopolysaccharide-stimulated BV2 microglia,” International Immunopharmacol-ogy, vol. 7, no. 8, pp. 1092–1101, 2007.

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