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ISSN : 1598-5504(Print)
ISSN : 2383-8272(Online)
Journal of Agriculture & Life Science Vol.49 No.1 pp.125-135

Anti-wrinkle Effect of PLA2-free Bee Venom against UVB-irradiated Human Skin Cells

Hyunkyoung Lee1, Seong Kyeong Bae1, Min-Jung Pyo1, Yunwi Heo1, Choul Goo Kim2, Changkeun Kang1,3, Euikyung Kim1,4*
1College of Veterinary Medicine, Gyeongsang National University, Jinju, Korea
2Chung Jin Biotech Co., Ltd., Hanyang University Business Center, Anshan-si, Korea
3Inst. of Agric. & Life Sci., Gyeongsang National University, Jinju, Korea
4Research Institutes of Life Science, Gyeongsang National University, Jinju, Korea
Corresponding author : Euikyung Kim, Tel.: +82-55-772-2355, Fax: +82-55-772-2349,
November 24, 2014 December 10, 2014 December 22, 2014


The use of bee venom (Apis mellifera L., BV) occasionally causes side effects such as inflammation and allergic reactions in the recipients. Several case reports also suggested the treatment of BV has some limitations in its clinical uses, due to the occurrence of dermal necrosis and anaphylatic reactions. It is generally understood that bee venom allergy is mainly the result of its allergic component, phospholipase A2 (PLA2). The present study was aimed to generate PLA2-free bee venom (PBV) and evaluate its efficacy as skin care and cosmetic preparation, comparing with original bee venom (BV). Our results showed that both BV and PBV exhibited significant protective effects in UVB-irradiated human keratinocyte (HaCaT) and human dermal fibroblast (HDF) cells and they also induced type I collagen synthesis in UVB-irradiated HDF cells except BV at 3 μg/ml. Furthermore, BV and PBV showed the inhibition of UVB-stimulated matrix metalloproteinase-1 (MMP-1), a major collagen degrading enzyme in skin. However, BV, unlike PBV, exhibited strong cytotoxicities in skin cells (both HaCaT and HDF) at its working concentrations of anti-wrinkle effect. The underlying cell signaling mechanisms of anti-wrinkle effects of BV and PBV were demonstrated by the activation of ERK1/2, and p38. Conclusively, PBV appears to be the bee venom of choice with less cytotoxicity and higher efficacy on UVB-irradiated skin cells in comparison with original bee venom (BV). Therefore, PBV can better be used as a cosmetic ingredient exhibiting excellent anti-wrinkle effect against photoaging than original BV.


    Ontario Ministry of Agriculture, Food and Rural Affairs


    Skin aging is a natural process of chronological changes of our skin. On the other hand, premature aging is an unnatural aging process and mostly caused by a long-term exposure to Ultraviolet (UV) irradiation which is probably due to increased outdoor lifestyle and wide use of tanning devices for cosmetic purposes. It is referred to as photoaging and is characterized by rough wrinkles, epidermal thickness, increased levels of MMPs, inflammation and collagen degradation (Fineschi et al., 2007; Gilchres, 1989). The UV spectrum is generally divided into three types, UVA (320-400 nm), UVB (280-320 nm), and UVC (200-280 nm). UVC is the most powerful energy, but it is almost completely absorbed by the ozone layer. Whereas UVA and UVB reach the Earth’s surface. UVB is a lesser extent energy reaching to the earth’s surface, it is 500-800 times more harmful than UVA (Pillai et al., 2005). Several studies demonstrated that UVB is the most dangerous light causing skin cancer in experimental animals and inducing DNA damage (Yoshino et al., 2002; Tzung and Rünger, 1998). In addition, UVB irradiation is responsible for epidermal thickness and degradation of extracellular matrix (ECM), leading to damage of skin tissue integrity, formation of wrinkle and inflammation (Rittié and Fisher, 2002). Therefore, the protection of skin from UVB irradiation may contribute to prevent the processes of wrinkle formation, photoaging and inflammatory reactions of the skin.

    Matrix metalloproteases (MMPs) are usually secreted from fibroblasts and keratinocytes, the major target cells of UVB irradiation in skin. MMPs degrade ECM that plays a pivotal role in the maintenance of dermal skin layers (Kim et al., 2004). Skin is mainly composed of collagen (70-80% dry weight) and the overproductions of MMPs can induce abnormal degradation of ECM, resulting in loss of elasticity, integrity and wrinkle formation in skin. In addition, continuous exposure of UVB may generate inflammatory mediators, resulting in the activation of MMPs. Accordingly, the inhibition of the MMPs can be one of the best strategies for the protection and prevention of wrinkle formation against UVB irradiation.

    In recent years, several natural reagents have been reported to protect UVB-mediated skin damage and their application on skin care products has been increasing (Bae et al., 2008; Afaq and Mukhtar, 2006). Carnosic acid, a phenolic diterpene, inhibits the UVB-induced MMPs in human skin fibroblasts and keratinocytes through suppression of ROS production and ERK/AP-1 activation (Park et al., 2013). The green tea polyphenols have been also reported to protect UV-induced oxidative damage and modulate MMPs expression at low concentrations without tachyphylaxis in a human study, proposing its therapeutic potential as photoaging inhibitor (Vayalil et al., 2004).

    Bee venom (BV) has caught people’s attention as a cosmetic ingredient due to its protective, antibacterial and anti-inflammatory effects on skin (Han et al., 2013). Previous studies have demonstrated that BV reduces protein levels as well as mRNA expressions of UVB-stimulated MMP-1 and -3 and it also protect damage of UVB-irradiated human dermal fibroblasts (Han et al., 2007). However, sometimes the BVcontaining products have been accused of having adverse effects (erythema and allergy reaction) that have created a profound disturbance in its utilization. For the safe use of BV in skin care products, we prepared PLA2-free bee venom (PBV) by removing PLA2 from natural BV using ultrafiltration and investigated its therapeutic potentials in comparison with natural BV, with the focus on its beneficial (anti-wrinkle) effect and adverse (cytotoxic) effect. These results would have shed light on establishing the developmental strategy of anti-wrinkle skin care products, such as an inhibitor of collagen-degrading enzymes or a stimulator of skin cell proliferation in skin aging.

    II.Materials and methods


    Dimethyl sulphoxide (DMSO), 3-(4,5-dimethyl-2-yl)- 2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Dulbecco’s modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), Bovine serum albumin (BSA), penicillin, streptomycin and trypsin were purchased from Gibco-BRL (Grand Island, NY, USA). Fibroblast Growth Media BulletKit (FGMTM-2 BulletKitTM) was obtained from Lonza (Walkersville, MD, USA). Antibodies for phospho-ERK, phospho-p38 and GAPDH were from Cell Signaling Technology (Beverly, MA, USA). All other reagents used were of the purest grade available.

    2.2.Preparation of PLA2-free bee venom

    BV and PBV prepared from honey bee (Apis mellifera L.) were supplied from Chungjin Biotech (Anshan-si, Korea). Briefly, BV was collected using bee venom collector (Chungjin Biotech, Ashan-si, Korea) in a sterile manner under strict laboratory conditions. BV collector was placed on the hive and bees were given electronic shock to cause them to sting onto a glass plate of BV collector from which dried BV was later scraped off. Collected BV was dissolved in distilled water, and then using 3.0 μm filtration membrane remove big debris like dust and pollen. Subsequently, 0.45 and 0.2 μm membrane filtration were used to eliminate if any tiny debris. The filtered bee venom (BV) was lyophilized and stored at -20°C for later use. PBV in this experiment were prepared according to the flowing procedure. The filtration was conducted using a stirred ultrafiltration (Millipore series 8400, Merck kGaA, Darmastadt, Germany) cell with a 10 kDa molecular weight cut-off membrane (Ultracel PL regenerated cellulose, 76 mm, Millipore Corporation, Bedford, MA, USA). After filtration, to confirm whether PLA2 is sufficiently excluded in PBV, PLA2 assay and SDS-PAGE were performed for PBV and BV and compared with each other. PBV was lyophilized and stored at -20°C for later use.

    2.3.Cell culture

    For this experiment, human keratinocyte (HaCaT) cells were cultured in DMEM supplemented with antibiotics (100 U/ml of penicillin A and 100 U/ml streptomucin) and 10% FBS at 37℃ in a humidified atomosphere with 95% air and 5% CO2. Human dermal fibroblast (HDF) cells were cultured in FGMTM-2 BulletKitTM using same conditions. HaCaT cells were supplied by Dr. T-J Yoon (University of Gyeongsang, Jinju, Korea) and HDF cell was obtained from MTCC (Modern cell & Tissue Technologies, Seoul, Korea).

    2.4.UVB irradiation

    HaCaT and HDF and were seed to 80% confluence in well plates and starved in serum-free media. These cells were pretreated with different concentrations (1.5 and 3.0 μg/ml) of BV or PBV for 2 h. Medium was removed and then, cells were washed with phosphate-buffered saline (PBS) thrice and replaced with fresh PBS. These cells were irradiated with 40 mL/cm2 of UVB light using at 312 nm UVB meter (VL-6.M ; Vilber Lourmat, Marne-la-Vallée Cedex1, France). Cell viability and measurement of collagen synthesis were performed at 24 h and the other assay were collected 6 h after UVB irradiation.

    2.5.Cell viability

    HaCaT and HDF cells were seeded to 1×105 cells/mL in 24 well culture plates in growth medium. After allowing cells to attach, the medium was removed and the cells washed twice with PBS. The cells were then treated with different concentrations (1.5 and 3.0 μg/mL) of BV or PBV and incubated for 24 h in serum free medium. The cell viability was determined using MTT assay. Briefly, MTT dye (5 mg/mL) was added to the cell culture media and they were incubated for additional 3 h. After the medium was removed, DMSO was added to the cells for the solubilization of generated formazan salts. The amount of formazan salt was determined by measuring the optical density (OD) at 540 nm using spectrophotometer microplate (Power WaveTMXS, BioTek Instruments, Inc., Winooski, VT, USA). Relative cell viability of treatment was calculated as a percentage of vehicle-treated control ([OD of treated cells/OD of control] × 100).

    To compare the protective effects of BV and PBV against UVB-induced damage, HaCaT and HDF cell viability exposed with a various levels of UVB or BV or PBV preparations for 24 h. Subsequently, HaCaT and HDF cells were pretreated with BV or PBV for 2 h, following irradiated with 40 mJ/cm2 of UVB as described above and cultured in fresh serum-free DMEM. MTT assay was performed at 24 h post-irradiation.

    2.6.Measurement of collagen synthesis

    The quantity of Type I collagen in HDF cells were measured using ELISA kit (Takara Bio Inc., Japan). The level of Type I collagen synthesis can be determined by detecting procollagen Type I carboxyterminal peptide (PIP) using polyclonal antibodies, rather than the direct measurement of collagen. HDF cells were plated in complete growth media and incubated for 24 h. HDF cells were pre-treated with BV or PBV for 2 h and then exposed to UVB. After incubation of 24 h in serum-free DMEM, the culture supernatants were collected and measure with an immunoassay kit following the manufacturer’s instruction. The absorbance measurement was detected with a spectrophotometer microplate at 450 nm.

    2.7.Western blot analysis

    After HaCaT and HDF cells were seeded on well plates, the cells were pre-incubated with different concentrations of BV or PBV for 2 h, and then exposed to UVB. The cells were allowed with additional incubation for 6 h after irradiated with UVB and, then the treated cells were collected by scraping with 200 μL of RIPA buffer containing protease inhibitor cocktail. Cell lysates were separated 12% SDS-polyacrylamide gel, transferred to PVDF membranes (Bio-Rad, C.A. USA) and subsequently subjected to immunoblot analysis using specific primary antibodies for overnight at 4℃. After washing, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody (Cell Signaling Technology, Beverly, MA) for 1 h at room temperature. The blots were visualized by using an enhanced chemiluminescence method (ECL, Amersham Biosciences, Buckinghamshire, UK) and analyzed using ChemiDoc XRS (Bio-Rad, C.A. USA). Densitometry analysis was performed with a Hewlett-Packard scanner and NIH Image software (Image J).

    2.8.Statistical analysis

    The results are expressed as a mean ± standard deviation (S.D.). A paired Student’s t-test was used to assess the significance of differences between two mean values. p<0.05 was considered to be statistically significant.


    3.1.Comparison of protective effects between BV and PBV on UV-irradiated cells

    To examine if BV and PBV protect skin cells against UVB-irradiated cell damage, the MTT assays were conducted in HaCaT and HDF cells. The UVB irradiation at 40 mJ/cm2 reduced cell viability approximately 40% in HaCaT and 60% in HDF cells comparing their respective controls (without UVB). For both BV and PBV, the cell viabilities were not affected by up to 1 μg/ml of the treatments, whereas a significant loss of cell viability was observed at 3 μg/ml concentrations for in BV but not in PBV (data not shown). The cells (HaCaT cell and HDF cell) were pretreated with BV or PBV for 2 h, then exposed to UVB irradiation as previously prescribed at above. The results showed that PBV perfectly protected the cells against UVB-mediated cell damage based on cell viability. Unlike PBV, however, the pretreatment of BV (3 μg/ml) resulted in even a higher cytotoxicity than UVB irradation alone (Fig. 1). From this experiment, PBV appears to have superior protective effects but much lower cytotoxic adverse effects than BV in UVB-associated cell damages.

    3.2.Effects of BV and PBV on UVB-induced type I collagen production and protein expression

    To evaluate the effects of BV and PBV on type I procollagen production in UVB-irradiated HDF cells, the cells were exposed with UVB 40 mJ/cm2 and the quantities of collagen synthesis were measured using a type-I procollagen assay kit. Both BV and PBV significantly restored procollagen synthesis in UVB-irradiated HDF cells except for BV 3 μg/ml (Fig. 2A). In consistent with the result of procollagen synthesis, the protein levels of the collagen were enhanced in UVB-irradiated HDF cells treated with BV or PBV in a concentration-dependent manner (Fig. 2B & 2C).

    3.3.Effects of BV and PBV on MMPs expressions on UV-irradiated cells

    It has been recently reported that UVB induces skin aging process through MMPs induction. We investigated whether the increased level of collagen in treatment of BV or PBV is associated with the modulation of MMPs expression of the skin cells. UVB caused inductions of MMP-1 and -13 with respect to control HaCaT cells, in which BV and PBV treatments significantly attenuated the MMPs expressions (Fig. 3A & 3C). In HaCaT cells, BV and PBV exerted the greatest inhibitory effects on UVB-induced MMP-13 expression than MMP-1. The inhibitory effects of BV and PBV on MMPs expressions were also determined in HDF cells. Unlike HaCaT cells, there were other MMPs expressions (MMP-1, -2 and -3) in HDF cells (Fig. 3B & 3D). Especially, MMP-1 is the most important degradation enzymes of collagen and initiates degradation of type I collagen at first. UVBirradiated HDF cells produced the highest expression level of MMP-1 than the other MMPs and PBV exhibited the strongest suppressive effect aganist UVB-induced MMP-1 expression (approximately up to 80%) at 1.5 μg/ml without cytotoxicity. However, the expressions of MMP-3 were only slightly affected by the treatments of BV and PBV.

    3.4.Effect of BV and PBV on UVB-induced ERK and p38 activation

    Several investigators have reported that the major mechanism of UVB-induced MMPs expressions is associated with the activation of mitogen-activated protein kinases (MAPKs) signaling (Rittié and Fisher, 2002; Moon et al., 2008; Scharffetter-Kochanek et al., 2000). The effects of BV and PBV on UVB-induced activation of MAPKs signalings were examined for their molecular mechanisms of cellular protection in UVB-irradiated HaCaT and HDF cells. Both BV and PBV inhibited the UVB-stimulated phosphorylations of ERK1/2 and p38, but not JNK1/2 in both HaCaT and HDF cells (Fig. 4A & 4B). Both BV and PBV demonstrated a greater suppression of the phosphorylation of p38 than that of ERK1/2 in skin cells.


    BV has been used for the treatments of chronic inflammation (rheumatoid arthritis) and relief of pain in Oriental medicine for thousands of years (Han et al., 2007; Park et al., 2004). BV contains at least 18 pharmacologically active components, including melittin, apamin, adolapin, phospholipase A2, histamine and epinephrine (Ali, 2012). Previous studies have demonstrated that BV possesses various pharmacological effects, including anti-inflammatory and rapid cicatrizing effect of wound in rats (Han et al., 2011). In addition, BV reduces protein level as well as mRNA expression of UVB-induced MMP-1 and -3 and protects human dermal fibroblast against UVB-irradiated damages (Han et al., 2007). Therefore, many people have tried to make cosmetics and skin care products using BV. However, it is important to guarantee its safety since cosmetics composed of BV occasionally can induce unwanted side effect following skin contact. PLA2 has been considered as a major allergen of BV, which promotes inflammation and stimulates cross-linking of immunoglobin E on mast cells and basophils (Dudler et al., 1995). In this study, we therefore try to develop BV without such side effect and prepare PLA2-free bee venom (PBV) using ultrafiltration. Then the therapeutic profiles of PBV have been explored for its biological activity and molecular mechanism of actions in comparison with BV.

    Recently, many researchers have been extensively studied on the developments of protective compounds derived from natural resources against UVB-induced skin damages. Although UVB reaching the Earth’s surface is only 8%, it is well known to be absorbed mainly by epidermis and can cause predominant photoaging. UVB irradiation induces the damage of skin cells and disturbs ECM preservation, and accelerates skin aging through increased production of MMPs (Scharffetter-Kochanek et al., 2000). There are three predominant groups of MMPs: collagenases, gelatinase, and stromelysins. The collagenase (MMP-1, -8, -13, and -18) cleave interstitial (structural) collagens, with MMP-1 as the predominant one. Gelatinases, MMP-2 and -9, degrade denatured structural collagens and further degrade collagen fragments generated by collagenase. The stromelysins (MMP-3, -10, -11 and -19) degrade basement membrane collagens as well as proteglycans and matrix glycoproteins, with MMP-3 as the activator of proMMP-1. UVB-induced MMPs activities and expressions cause reduction of collagen production and induction of its degradation, which led to loss of elasticity of skin structure and formation of wrinkles (Makrantonaki & Zouboulis, 2007). Hence, we investigated effects of BV and PBV on the natural target cells of UVB in epidermal and dermal potions, such as keratinocyte (HaCaT) and fibroblast (HDF), then focused on whether BV and PBV regulate the expressions of MMP-1, -2, -3 and -13 in UVBirradiated skin cells. HaCaT cells showed the increases in the expressions of MMP-1 and -13 (Fig. 3A), while HDF cells revealed the excessive expressions of MMP-1, -2 and -3 on UVB irradiation (Fig. 3B). In HaCaT cells, both BV and PBV showed a strong inhibition for the expression of MMP-1. Furthermore, BV and PBV in HDF cells have consistent effects with MMP-1 modulation in HaCaT cells. Although both BV and PBV seem to be excellent inhibitors of MMP-1, BV at high concentration (3 μg/mL) showed a strong cytotoxicity. In cell viability data, PBV (up to 3 μg/mL) showed a clear protection against UVB with non-cytotoxic effects, whereas BV 3 μg/mL exhibited cytotoxicity in both HDF and HaCaT cells. PBV treatment in HDF cells induced down-regulation of MMP-1 more than 75%, whereas only 50% in HaCaT cells. MMP-1 is a representative collagenase responsible for the degradation of dermal collagen type I, II and III. Several studies have reported that MMP-1 expression is increased intrinsically with age or extrinsically by UV irradiation. Down-regulation of MMP-1 expression could be a useful indicator in determining potent anti-wrinkle ingredients (Makrantonaki & Zouboulis, 2007). Interestingly, PBV decreased MMP-1 expression and other MMPs associated with destruction of collagen in skin. Furthermore, PBV increased level of type I collagen synthesis in HDF cells. Hence, PBV appears to be a suitable reagent which protects human keratinocytes and fibroblasts and prevents skin wrinkle formation.

    Next, we attempted to characterize the molecular mechanism underlying the inhibitory effect of BV and PBV on MMPs modulations. Major signaling pathways known to mediate UVB-induced biological response involve the mitogen-activated protein kinases (MAPKs). MAPKs are consist of three types, such as ERKs, p38 and JNK (Bode & Dong, 2003) and regulate a wide range of intracellular signaling molecules that are involved in various biological processes, such as, inflammation, cell proliferation, differentiation and apoptosis. In our data, UVB alone appeared to activate ERK1/2 and p38 but not JNK in HaCaT and HDF cells. UVB-induced activation of ERK1/2 and p38 were down regulated by BV or PBV treatments in HaCaT and HDF cells. In comparison with level of ERK1/2 and p38, both BV and PBV affected the regulations of MMPs through phosphorylations of p38 than ERK1/2 (Fig. 4C & 4D).

    In conclusion, both BV and PBV restore cell damage and collagen production and inhibit MMP-1 and -13 of HaCaT cells and MMP-1, -2 and -3 of HDF cells under UVB exposure. However, BV shows adverse cytotoxic effect, whereas PBV may have some advantages to prevent skin wrinkle formation without cytotoxicity. Therefore, application with PBV in cosmetic products appears to be promising strategies in preventing skin wrinkling and protecting exposure to UVB. However, further studies are necessary to understand more detailed molecular mechanism of anti-wrinkle effect of PBV in vitro and evaluate its effect in suitable in vivo models.


    This research was supported by ‘Agricultural Biotechnology Development Program’, Ministry of Agriculture, Food and Rural Affairs.



    Comparison of protective effects of BV and PBV on (A) HaCaT and (B) HDF cells against UVB-irradiation. The cells were pretreated with BV or PBV for 2 h before UVB irradiation. After 24 h, cell viability was measured using MTT assay and expressed as a percentage of control. The data shown are the means ± SD of three experiments. *p<0.05, #p<0.05 as compared with control group and UVB irradiated group, respectively


    Effect of BV and PBV on the type I collagen synthesis and expression. (A) HDF cells were pretreated with BV or PBV for 2 h and then exposed to UVB (40 mJ/cm2). After 24 h of UVB exposure, release of type I collagen into the media was evaluated by ELISA, and (B) the expression of type I collagen protein was detected by Western blotting. (C) The protein expression of type I collagen was quantified by ImageJ. GAPDH protein levels were used as control. The data shown are the means ± SD of three experiments. *p<0.05, #p<0.05 as compared with control group and UVB irradiated group, respectively


    Modulation of MMPs expression in UVB-irradiated cells by BV and PBV. (A) HaCaT and (B) HDF cells were pretreated with the indicated concentrations of BV or PBV for 2 h, and then irradiated with UVB (40 mJ/cm2). The cell viabilities were determined after 24 h of the treatment. Protein extracts were prepared and subjected to Western blot assay using the primary antibodies for MMP-1, -2, -3 and -13, then they were quantified by Image J (C and D). GAPDH protein levels were used as control. p<0.05, #p<0.05 as compared with control group and UVB irradiated group, respectively


    Modulation of MAPKs signaling pathway by BV and PBV. (A) HaCaT and (B) HDF cells were pretreated with the indicated concentrations of BV or PBV for 2 h, and then further irradiated with UVB for 6 h. Protein extracts were prepared and subjected to Western blot assay using the primary antibodies for phospho-ERK and phospho-p38, then they were quantified by Image J (C and D). GAPDH protein levels were used as control. The data shown are the means ± SD of three experiments. p<0.05, #p<0.05 as compared with control group and UVB irradiated group, respectively



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