Bee Venom Cosmetic Helps Treat Acne

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Research Article

Effects of cosmetics containing purified honeybee
(Apis mellifera L.) venom on acne vulgaris
Sang Mi Han1, Kwang Gill Lee1, Sok Cheon Pak2

1. Department of Agricultural Biology, National Academy of Agricultural Science, Suwon 441-100, Korea
2. School of Biomedical Sciences, Charles Sturt University, Bathurst, New South Wales 2795, Australia

OBJECTIVE: Acne vulgaris is a chronic dermatologic problem with multiple factors involved in
its pathogenesis. Alternative solutions to acne treatment were instigated by antibiotic resistance
despite of its extensive use. Purified bee venom (PBV) has been proposed as a promising candidate
for that purpose. The present study was designed to confirm the antibacterial effect of PBV and
access the efficacy of cosmetics containing PBV in subjects with acne vulgaris.
METHODS: The skin bacterium Propionibacterium acnes was incubated with PBV at various
concentrations and bacterial growth was evaluated using the colony forming unit (CFU)
assay. The mechanism of PBV employed in killing P. acnes was examined by scanning
electron microscopy (SEM) and transmission electron microscopy (TEM). In addition, a total
of 12 subjects were randomized in a double-blind, controlled trial to receive either cosmetics
containing PBV or cosmetics without PBV for two weeks. Evaluations included lesion counts
and skin microorganism.
RESULTS: PBV exhibited antimicrobial activity in a concentration-dependent manner, reducing
the number of P. acnes CFU by approximately 6 logs at a concentration of 0.5 mg. When PBV
concentration was higher than 1.0 mg, no P. acnes colonies were spotted on an agar. TEM
and SEM of untreated P. acnes illustrated the normal pleomorphic structure, whereas the PBVtreated bacterium lost the integrity of surface architecture. Significant difference (P=0.027) in
the grading levels based on numbers of lesion counts for inflammatory and noninflammatory
was observed in favour of the PBV group compared with the control group. In terms of average
decrement of skin microorganism, subjects receiving cosmetics containing PBV experienced
a significant 57.5% decrease of adenosine triphosphate levels, whereas participants receiving
cosmetics without PBV experienced a nonsignificant decrease of 4.7%.
CONCLUSION: These results show that the in vitro actions of antimicrobial activity of PBV were
translated in vivo. Cosmetics containing PBV provided a certain degree of efficacy in terms of
lesion counts and skin microorganism concentration compared with cosmetics without PBV in
subjects with acne vulgaris. PBV may be a good candidate compound for developing therapeutic
drug for the treatment of acne vulgaris.
KEYWORDS: bee venoms; acne vulgaris; Propionibacterium acnes; skin; cosmetics
DOI: 10.3736/jintegrmed2013043
Han SM, Lee KG, Pak SC. Effects of cosmetics containing purified honeybee (Apis mellifera L.) venom on
acne vulgaris. J Integr Med. 2013; 11(5): 320-326.
Received November 26, 2012; accepted March 6, 2013.
Open-access article copyright © 2013 Sang Mi Han et al.
Correspondence: Sang Mi Han, PhD; Tel: +82-31-290-8510; E-mail: sangmih@korea.kr

1 Introduction
Bee venom from the honeybee (Apis mellifera L.)
contains a variety of different peptides including melittin,
September 2013, Vol.11, No.5

apamin, adolapin, and mast cell-degranulating peptide[1,2].
Bee venom has been used as a complementary medicine to
treat conditions such as rheumatoid arthritis and cancerous
tumors[1,3,4]. Recently, bee venom also has been used as
a cosmetic ingredient in antiaging, anti-inflammatory,

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Journal of Integrative Medicine


and antibacterial products. Purified bee venom (PBV)
is generally obtained by collecting a large amount of
bee venom from honeybees by electric stunning using
a bee venom collector (a process that does not harm the
honeybees), removing impurities from the collected bee
venom, and lyophilizing purified venom. We previously
reported the skin-photoprotective effect of PBV that
resulted from the reduction in the protein levels of matrix
metalloproteinases, which are the major contributors to
photoaging processes[5].
Acne vulgaris is one of the most common dermatologic
problems, affecting mainly adolescents and often
continuing into adulthood with women being affected
at higher rates than men [6]. Although multiple factors
are involved in acne pathogenesis, the underlining
mechanism for its onset and subsequent development are
not fully elucidated yet. However increasing amounts of
data seem to confirm the involvement of abnormalities
in sebum production, follicular keratinization, bacterial
proliferation and inflammation[7]. According to the current
concept of acne pathogenesis, sebaceous hyperplasia and
follicular hyperkeratinization cause a change of follicular
milieu with consecutive proliferation of bacteria, mainly
Propionibacterium acnes [8] . This leads to increased
production of pro-inflammatory cytokines which stimulate
comedogenesis at the level of follicular keratinocytes. Further
inflammatory responses via the activation of cell-mediated
immune processes lead to the increasing severity of acne.
There are currently a number of proven effective treatments
for acne vulgaris with various degrees of success. Early
and effective therapy based on the treatment of pathogenic
causes described above should be implemented because
scarring caused by severe inflammatory acne is an
extreme concern to the patients. As long as clinical
symptoms can be alleviated and scarring can be prevented
by intervention, it needs to be promoted. This could be
apparent from the use of bee venom since it has recently
been reported to possess antibacterial effect against acneinducing bacteria[9]. The aim of the present study was to
confirm the antibacterial effect of bee venom, and evaluate
the efficacy of cosmetics containing PBV in the treatment
of acne vulgaris.
2 Materials and methods
2.1 Purified honeybee venom collection
All experimental procedures used in the present study
were approved by the Animal Care and Use Committee
at the National Academy of Agricultural Science (NAAS)
and conform to the US National Institutes of Health
Guidelines for the care and use of laboratory animals.
Experimental colonies of natural honeybees (Apis
mellifera L.) used in this study were maintained at NAAS,

Suwon, Korea. Bee venom was collected with a bee
venom collector (Chungjin Biotech, Korea) in a sterile
manner under strict laboratory conditions. In brief, the bee
venom collector was placed on the hive, and the bees were
given enough electric shock to cause them to sting a glass
plate from which dried bee venom was later scraped off.
The collected venom was diluted in cold sterile water and
then centrifuged at 10 000×g for 5 min at 4 ℃ to discard
residues from the supernatant. PBV was lyophilized by
freeze dryer and refrigerated at 4 ℃ for later use.
2.2 Colony forming unit assay
The colony forming unit (CFU) assay was performed
as described by Ernst et al[10]. P. acnes (ATCC 6919) was
obtained from the Korean Culture Center of Microorganisms,
Seoul, Korea. P. acnes was cultured at 37 ℃ on reinforced
clostridial medium (BD, MD, USA) under anaerobic
conditions for 2 d and collected in the mid-log phase. The
bacterium was washed three times with the assay buffer,
which consisted of 10 mmol/L sodium phosphate, supplemented with 0.03% trypticase soy broth (TSB, BD, MD,
USA) and counted by applying a conversion factor of
7.5 × 107 bacteria per milliliter = 1 optical density unit at
600 nm. Various concentrations of PBV were incubated
with 3.75 × 105 bacteria in a final volume of 30 mL at
37 ℃ for 4 h. After incubation, 10-fold dilutions were
prepared and plated on solid media composed of Brucella
broth (BD, MD, USA) with 5% sheep red blood cells. The
plates were incubated for 2 d at 37 ℃ under anaerobic
conditions, then individual colonies were counted, and the
number of CFU per tube was calculated.
2.3 Electron microscopy
P. acnes at a concentration of 2.08 × 107/mL was suspended
in 10 mmol/L phosphate buffer sodium (PBS, pH 7.2)
supplemented with 0.03% TSB and incubated with 100 ng
PBV. Samples were fixed for 30 min with 2% glutaraldehyde
in PBS at room temperature and then washed and suspended
in PBS. Samples for scanning electron microscopy (SEM)
were filtered through a micropore filter. The filters coated
with P. acnes were dehydrated in a graded ethanol series
(50%, 75%, 95% and 100% with 15 min each), followed
by similar exposure to a hexamethyldisilazane reagent
(50%, 75%, and 95% with 30 min each). After this process,
the samples were incubated in 100% hexamethyldisilazane
reagent overnight. SEM samples were coated with gold,
viewed and imaged with a Hitachi scanning electron
microscope (S-2460N, Hitachi, Japan). Transmission electron
microscopy (TEM) samples were dehydrated in graded
ethanol as above, embedded in Epon and sectioned. TEM
samples were stained with uranyl acetate, viewed and
imaged using a Carl Zeiss transmission electron microscope
(LEO912AB, CarlZeiss, Germany).
2.4 Clinical study design and treatment
Korean female and male subjects aged from 12 to 35 years

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participated in a doubled-blind, placebo-controlled,
split face study with left-right randomization that was
carried out June 2012. Twelve subjects would complete
the study for the home-use test procedure to ensure. To
be enrolled, all subjects must have had mild to moderate
acne, as graded by the Korean Acne Grading System
(KAGS), grade of 1 to 3. The severity of acne was assessed
using KAGS, proposed by the consensus conference in
2004[11]. The severity score (grades 1 to 6) was as follows:
grade 1, papules ≤ 10; grade 2, papules 11-30; grade 3,
papules ≥ 31, nodules ≤ 10; grade 4, nodules 11-20,
± mild ongoing scars; grade 5, nodules 21-30, ± moderate
ongoing scars; grade 6, nodules ≥ 30, ± severe ongoing
scars and ± sinus tract. To be enrolled, all subjects had
to have signed a certification that they had no allergy or
sensitivity to bees or bee sting. All subjects were required
to read, understand and sign a written informed consent
and complete a brief demographic medical history form.
Subjects under the age of 18 had to have a parent or a
legal guardian who signed the informed consent form. This
randomized, double-blind controlled study was performed to
evaluate the efficacy and safety of cosmetics containing
PBV in the treatment of acne vulgaris as defined by the
KAGS.
Participants with dermatological conditions which could
interfere with treatment or evaluation were excluded.
The study protocol was approved by institutional review
boards. All volunteers provided written informed consent
prior to entering the study. This study was conducted in
accordance with Good Clinical Practice. Subjects were
randomized in a 1:1 ratio to receive either cosmetics
containing PBV or cosmetics without PBV twice daily in
the morning and evening for two weeks. Cosmetics were
applied topically on the whole face with an amount of
4 mL per day. The concentration of PBV is 0.06 mg/mL in
PBV-containing cosmetics (Table 1).
2.5 Assessment
Efficacy was assessed at baseline and at the end of twoweek treatment. Efficacy variables were the number of
inflammatory and noninflammatory lesions. The definition
of lesion was adapted from KAGS. In addition, any
remaining P. acnes deposited on the skin were measured
using adenosine triphosphate (ATP) swab test. All bacteria
release ATP when the cell wall ruptures, and consecutive
reaction with luciferase from Lumitester PD-10 (KIKOMAN,
Japan) results in the production of light. The light emitted
is measured and expressed in relative light units. This ATP
analysis is therefore an indirect means of measuring the
biomass of P. acnes on the skin. ATP levels were the average
of three measurements.
2.6 Statistical analysis
The data are expressed as the mean ± standard error
of mean. Statistical differences among groups were

Table 1 Formulation of cosmetics
Composition (% in weight)
Ingredient

Control

Purified bee venomcontaining cosmetics

90.365
6.00
3.00

90.359
6.00
3.00

Polyethyleneglycol-60
hydrogenated castor oil

0.30

0.30

Methylparaben
Sodium citrate
Fragrance

0.10
0.08
0.055

0.10
0.08
0.055

Disodium ethylenediaminetertraacetic acid

0.05

0.05

Allantoin
Citric acid
Bee venom

0.03
0.02
-

0.03
0.02
0.006

Water
Butylene glycol
Alcohol

calculated by analysis of variance by SPSS (SPSS 18.0
version, Chicago, IL, USA). The significant differences
with a value less than 0.05 were determined by independent
t-test.
3 Results
3.1 Antimicrobial effects of PBV
3.1.1 Bacterial growth
To confirm whether the antimicrobial PBV kills P.
acnes, the activity of PBV at various concentrations
against bacterial growth was tested. Bacterial growth
was evaluated using the CFU assay. PBV exhibited
antimicrobial activity in a concentration-dependent
manner, reducing the number of P. acnes CFU by
approximately 6 logs at a concentration of 0.5 mg (Figure
1A). When PBV concentration was higher than 1.0 mg,
no P. acnes colonies were spotted on an agar during the
incubation for 4 h at 37 ℃ (Figure 1B).
3.1.2 Observation by electron microscopy
PBV exerts its antimicrobial activity against Gramnegative and Gram-positive organisms[5]. To determine
what mechanism is employed in the killing of P. acnes
by PBV, we examined bacteria that had been treated with
PBV using both SEM and TEM. Both SEM and TEM
of untreated P. acnes illustrate the bacterium’s normal
pleomorphic structure. By SEM, the surface of the
untreated bacteria appeared smooth and rounded, whereas
the PBV-treated bacteria demonstrated a recessed and
withered surface (Figure 2A). Also, differences between
untreated and PBV-treated bacteria were appreciated
by TEM, which demonstrated many “ghost” cells after
12 h of PBV treatment and few surviving bacteria with

322



in subjects receiving cosmetics without PBV (P = 0.363).
Treatment with cosmetics containing PBV caused a
significant success rate (P = 0.001) during the two-week
period.

Figure 1 PBV has antimicrobial activity against P. acnes

A: Various concentrations of PBV were incubated with P. acnes for 4 h at
37 °C and tested for antimicrobial activity using the CFU assay. B: The
antimicrobial activity of PBV in a qualitative CFU assay which involved
plating PBV with P. acnes on solid media and counting individual colonies to determine the CFU. Experiments in A and B are representative of
three experiments.
PBV: purified bee venom; CFU: colony forming unit.

darker and more condensed cytoplasms compared to
untreated bacteria (Figure 2B). As can be seen in Figure 2B,
the untreated bacteria had fimbriae while PBV-treated
bacteria had surface with an absence of fimbriae. TEM
revealed the untreated P. ances had a cell wall with well
demarcated outer and inner dark, lipophilic layers and
a lighter, hydrophilic peptidoglycan layer. In contrast,
after incubation with PBV, P. acnes lost the integrity of
this surface architecture as the lighter, more hydrophilic,
peptidoglycan layer as well as the darker more lipophilic
layers of the cell appear disturbed, losing their crisp, welldefined structures (Figure 2B). In addition, we observed
a wider, likely edematous, space inside of the cell wall,
further suggesting its disruption as well as peripheral
clumping of nuclear material within the cell. In whole,
these images reveal that PBV perturbs the surface
integrity of P. acnes in a manner that may make it porous,
suggesting that this is the likely mechanism by which
PBV kill this organism.
3.2 Observed efficacy
A total of 12 subjects, between 12 and 35 years of age
and in general good health were to be empanelled (Figure
3). The grading levels based on numbers by lesion counts
for inflammatory and noninflammatory at baseline and
after two weeks of treatment are shown in Table 2. Statistically
significant difference from the treatment was not shown

Figure 2 PBV caused architectural changes of cell wall of
P. acnes demonstrated by electron micrographs

A: Scanning electron microscopy analysis demonstrated the effect of
PBV on the surface of P. acnes as compared to the surfaces of untreated
control bacteria (× 20 000). B: Transmission electron microscopy
revealed that PBV treatment disrupted the sharply layered surface
architecture of P. acnes, making the cell wall less distinct and more
permeable to fluid influx (× 1 000, pictures in the square are × 3 150).
PBV: purified bee venom.

Figure 3 Flow diagram of the number of patients included in
this trial

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In terms of average decrement of skin microorganism
from baseline (Table 3), subjects receiving cosmetics
containing PBV experienced a significant 57.5% decrease
of ATP level during two-week study period (P=0.007 from

17.825 ± 3.911 to 7.566 ± 6.082), whereas participants
receiving cosmetics without PBV experienced nonsignificant
decrease of 4.7% from 17.539 ± 3.202 to 16.708 ± 2.268
(P=0.448).

Table 2 The change of KAGS score based on numbers of inflammatory and noninflammatory lesions
KAGS score

Control group (n=6)

PBV group (n=6)

Week 2

Week 0

Week 2

0

0

Week 0

0

0

Comparison of group (P value)*

3

1

2

2

2

2

2

2

3

2

1

3

2

1

2

0

4

0

0

0

0

5

0

0

0

0

6

0

0

0

0

P value

**

*

0.363

0.027

0.001

**

Analyzed by independent t-test; analyzed by paired t-test. KAGS: Korean Acne Grading System; PBV: purified bee venom.

Table 3 The average decrement of skin microorganism level
Class

Control group (n=6)

PBV group (n=6)

Week 0

17.539 ± 3.202

17.825 ± 3.911

Week 2

16.708 ± 2.268

7.566 ± 6.082

P value**

0.448

Comparison of group (P value)*
0.012

0.007
*

Data are expressed as relative light units. Analyzed by independent t-test; **analyzed by paired t-test. PBV: purified bee venom.

Comparison between two groups by independent t-test
confirmed the effect of PBV on P. acnes counts (P=0.012).
Figure 4 illustrates the effect of cosmetics containing PBV
on facial lesions after 2 weeks of treatment.

Figure 4 Photographs of subjects at baseline and week 2 of
treatment for (A) control group and (B) purified bee venom group

4 Discussion
As therapeutic agents for acne vulgaris, antibiotics
are normally administered to inhibit inflammation or
kill bacteria. According to the systemic review, topical
antibiotics such as clindamycin and erythromycin are an
effective treatment for predominance of inflammatory

lesions in mild to severe acne [11]. Oral antibiotics are
recommended for moderate acne patients whose acne
severity has not been adequately reduced by topical
antibiotics. Oral erythromycin demonstrated a similar
efficacy to oral tetracycline in total lesion count reduction.
However, the development of antibiotic resistance is
of concern and limiting use of antibiotics seems to be a
better eco-responsible acne management[12]. To overcome
the problem of antibiotic resistance, natural products with
antimicrobial properties have been extensively studied
for a possible application in acne treatment. PBV has
been proposed as a promising candidate for an alternative
treatment for antibiotic therapy of acne vulgaris[9].
PBV possesses a variety of different peptides with melittin
as the main component[13]. Repetitive chemical therapy
with PBV was able to produce a robust analgesic effect
on chronic neuropathic pain[14]. PBV has been reported
to have anticancer activities. Cancerous tumor growth
was hampered by PBV through induction of apoptosis in
lung cancer cells[15], breast cancer cells[16], hepatocellular
carcinoma cells [17] and prostate cancer cells [18] . We
previously found that PBV possessed antifibrogenic
properties that were mediated by suppression of proinflammatory cytokines and fibrogenic gene expression[19].

324



We also demonstrated that PBV[20] and apamin[21] prevent
the combined lipopolysaccharide and atherogenic dietinduced atherosclerosis via down-regulation of endothelial
adhesion molecules. Recently PBV has also been experimented for antiaging and antibacterial functions making
it ideal for use in skin care and cosmetic preparation. We
reported skin-photoprotective action of PBV through
reduction of protein levels of matrix metalloproteinases
which are main contributors to photoaging processes[22].
Antimicrobial activity of PBV against skin bacteria in
human monocytic cells was also found in our another
study[23]. Furthermore, PBV augmented wound healing
with concomitant inhibition of cytokines associated with
fibrosis, which resulted in decreased wound size and
increasing of epithelial proliferation in a mouse fullthickness excision wound model[24].
In the assessment and evaluation of the safety of
substance, determination of irritant effects on skin and
eyes is an important initial step[24]. PBV was well tolerated
and exhibited no dermal and ocular irritation potential
in rabbits[25], rats and guinea pigs[26]. This randomized,
double-blind controlled study was designed to determine
the clinical benefit of a cosmetics containing PBV twice
daily in the treatment of subjects with acne vulgaris.
Results of this study showed that the addition of PBV to
cosmetics improved the outcome of acne vulgaris in terms
of reduction of both inflammatory and noninflammatory
lesions and decrement of skin microorganism. Seborrhea
in patients with acne leads to a proliferation of bacteria
particularly P. acnes by providing an ideal anaerobic
environment for their growth[8]. Subsequently lymphocytes
are attached and inflammatory cytokines are expressed
to cause comedogenesis. We reported that PBV reduced
the P. acnes-induced secretion of interleukin-8 and tumor
necrosis factor-α[9]. It implies that this anti-inflammatory
activity of PBV is the key mechanism for its antibacterial
property. Our current study confirms the antimicrobial
activity of PBV against P. acnes with cell lysis, indicating
the cell membrane appears to be the primary site of PBV
action.
Our findings suggest that cosmetics containing PBV
provided a certain degree of efficacy in terms of lesion
counts and skin microorganism concentration compared
with cosmetics without PBV in subjects with acne vulgaris.
Long-term treatment with cosmetics containing PBV
could be safe because the irritation potential of PBV is
negligible. In this study, the in vitro actions of antimicrobial
activity of PBV were translated in vivo. Cosmetics containing
PBV provided a certain degree of efficacy in terms of lesion
counts and skin microorganism concentration compared
with cosmetics without PBV in subjects with acne vulgaris.
PBV may be a good candidate compound for developing
therapeutic drug for the treatment of acne vulgaris.

5 Acknowledgements
This work was supported by a grant from BioGreen21
Program, Rural Development Administration (Code #:
PJ009519), Republic of Korea.
6 Competing interests
The authors declare that they have no competing interests.
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jcim@163.com, we will send you a complimentary print issue upon receipt.
Editors-in-Chief: Wei-kang Zhao & Lixing Lao. ISSN 2095-4964. Published by Science Press, China.

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Bee Venom Cosmetic Helps Treat Acne

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