Accepted Manuscript

Low-dose blue light irradiation enhances the antimicrobial

activities of curcumin against Propionibacterium acnes

Ming-Yeh Yang, Kai-Chih Chang, Liang-Yü Chen, Anren Hu

PII: S1011-1344(18)30156-8
DOI: doi:10.1016/j.jphotobiol.2018.09.021
Reference: JPB 11362
To appear in: Journal of Photochemistry & Photobiology, B: Biology
Received date: 9 February 2018
Revised date: 9 September 2018
Accepted date: 24 September 2018

Please cite this article as: Ming-Yeh Yang, Kai-Chih Chang, Liang-Yü Chen, Anren Hu ,
Low-dose blue light irradiation enhances the antimicrobial activities of curcumin against
Propionibacterium acnes. Jpb (2018), doi:10.1016/j.jphotobiol.2018.09.021

This is a PDF file of an unedited manuscript that has been accepted for publication. As
a service to our customers we are providing this early version of the manuscript. The
manuscript will undergo copyediting, typesetting, and review of the resulting proof before
it is published in its final form. Please note that during the production process errors may
be discovered which could affect the content, and all legal disclaimers that apply to the
journal pertain.
 ACCEPTED MANUSCRIPT

Low-Dose Blue Light Irradiation Enhances the Antimicrobial

Activities of Curcumin against Propionibacterium acnes

Ming-Yeh Yang1, Kai-Chih Chang2,3, Liang-Yü Chen 4,*

PT
loknath@mail.mcu.edu.tw, Anren Hu2,** anren@mail.tcu.edu.tw

RI
SC
1
Institute of Medical Sciences, Tzu-Chi University, Hualien 970, Taiwan

2
Department of Laboratory Medicine and Biotechnology, Tzu-Chi
NU
University, Hualien 970, Taiwan
MA

3
Department of Laboratory Medicine, Buddhist Tzu-Chi General Hospital,
D

Hualien, Taiwan
TE

4
Department of Biotechnology, Ming-Chuan University, Taoyuan City
EP

333, Taiwan

*
C

Correspondence to, L. Y. Chen No.5, Deming Rd., Guishan Dist.,

AC

Taoyuan City 333, Taiwan (R.O.C.)

**
Correspondence to A. Hu, No.701, Sec. 3, Zhongyang Rd., Hualien City,

Hualien County 970, Taiwan (R.O.C.)

Abstract
 ACCEPTED MANUSCRIPT

Propionibacterium acnes (P. acnes) is an opportunistic infection in

human skin that causes acne vulgaris. Antibiotic agents provide the

effective eradication of microbes until the development of drug-resistant

microbes. Photodynamic inactivation (PDI) is a non-antibiotic therapy for

PT
microbial eradication. In this study, the visible blue light (BL, λ max = 462

RI
nm) was used to enhance the antimicrobial activities of curcumin, a

SC
natural phenolic compound. Individual exposure to curcumin or BL

irradiation does not generate cytotoxicity on P. acnes. The viability of P.

NU
acnes was decreased significantly in 0.09 J/cm 2 BL with 1.52 μM of
MA

curcumin. Furthermore, the low-dose blue light irradiation triggers a

D

series of cytotoxic actions of curcumin on P. acnes. The lethal factors of

TE

photolytic curcumin were investigated based on the morphology of P.

EP

acnes by SEM and fluorescent images. The membrane disruption of

C

microbes was observed on the PDI against P. acnes. Chromatography and

AC

mass spectrometry techniques were also used to identify the photolytic

metabolites. Curcumin could be photolysed into vanillin through BL

irradiation, which presents a strong linear relationship in quantitation.

Because the safety of blue light in mammalian cell has been proven, the
 ACCEPTED MANUSCRIPT

photolytic curcumin treatment could support non-antibiotic therapy to

eradicate P. acnes on clinical dermatology.

Keywords: Photodynamic inactivation; Acne vulgaris; Synergistic effect;

PT
Photolysis; Vanillin

RI
SC
NU
MA
D
TE
C EP
AC
 ACCEPTED MANUSCRIPT

1. Introduction

Propionibacterium acnes (P. acnes) is an opportunistic pathogen on

the human skin surface. It can infect pilosebaceous units of skin and

induce pro-inflammatory factors, and may result in skin disorders, such as

PT
folliculitis and acne vulgaris [1]. P. acnes is also considered to be the

RI
relative pathogen of some surgical complications, arthritis, and

SC
sarcoidosis [2-4].

Antibiotic therapy has been used to treat microbial infections for

NU
several decades. However, the first drug-resistant P. acnes was reported in
MA

1979 in the US. P. acnes could resist tetracycline in 2004 in Sweden [5].
D

In 2009, P. acnes could resist clindamycin, co-trimoxazole, and

TE

erythromycin in European patients [6]. In 2014, P. acnes was found to

EP

have high resistance to azithromycin and doxycycline in Indian patients

C

[7].
AC

Many non-antibiotic medicines, such as isotretinoin and benzoyl

peroxide, were developed as alternative therapies for the inhibition of P.

acnes [7-9]. Despite their efficacy, these small molecule drugs may have

unpredictable side effects, causing the skin irritation and even

reproductive toxicity [10, 11]. Photodynamic inactivation (PDI) is a

 ACCEPTED MANUSCRIPT

minimally invasive therapy for bacterial infections, and is a potential

solution to the problem of bacterial resistance [12, 13]. The use of

anti-inflammatory natural products and photodynamic therapy has

attracted widespread interest.

PT
Curcumin is a yellow pigment extracted from the rhizome of

RI
Curcuma longa, which is a traditional herb medicine in Asia. The

SC
phyto-phenol also has been widely used in clinical treatments, due to its

anti-inflammatory, anti-oxidation, anti-proliferative, and anti-bacterial

NU
bioactivity [14]. Curcumin has antimicrobial activity against various
MA

microbes, including gram positive bacteria, gram negative bacteria

D

[15-18], and fungi [19]. In particular, curcumin can inhibit the biofilm
TE

formation of Enterococcus faecalis on the tooth substrates [20], and

EP

enhance the antibiotic susceptibility to pathogens of urinary tract

C

infection [21].
AC

As shown in Fig. 1, curcumin has different degradation and

metabolic pathways in vitro [22] and in vivo [23, 24]. Curcumin

conjugates with glucuronic acid and transforms to curcumin glucuronide

in plasma after oral administration [25]. The structure of curcumin could

also transform to bicyclopentadione through autoxidation reaction [26].

 ACCEPTED MANUSCRIPT

Due to the complicated and reactive matrix, these products are hard to be

characterized in steady states. Although, curcumin and curcuminoids are

photosensitive and chemically unstable [27]. The mechanism by which

visible light irradiation combined with curcumin affects microbial and

PT
cell growth is still not understood.

RI
Curcumin could interact with lipid monolayer and accumulate in the

SC
hydrophobic region of acyl chain [28]. Hence, curcumin also affects the

function of membrane proteins [29] and the gene expression associated

NU
with cell membrane integrity [30]. Curcumin could decrease the bacterial
MA

pathogenicity through involving the cell membrane protein activity

D

[31-34]. These results indicate that curcumin could affect the microbial
TE

metabolism and survival.

EP

The cytotoxicity of reactive oxygen species (ROS) was considered to

C

be the primary antimicrobial mechanism in PDI. In a previous study,

AC

riboflavin photolysis generated ROS and damages DNA [35]. However,

lipophilic curcumin is different from hydrophilic photosensitizers, such as

riboflavin derivatives. Because of these multiple phototoxic mechanism

of microbes, and the photolysis pathway of curcumin cannot be verified,

the dosage of curcumin for PDI therapy must be carefully evaluated. In

 ACCEPTED MANUSCRIPT

this study, we have investigated the influence of visible BL irradiation on

curcumin photolysis and inactivation of P. acnes.

2. Materials and methods

PT
2.1 Chemicals

RI
Curcumin, vanillin, ethidium bromide, fluorescentin isothiocyanate,

SC
glutaraldehyde, cacodylate, tannic acid, osmium tetroxide, bromophenol

blue, sucrose, agarose gel, acetonitrile, dimethyl sulfoxide (DMSO),

NU
AcOH, Na2HPO4, KCl, KH2PO4, and NaCl were purchased from
MA

Sigma-Aldrich (St. Louis, USA). SYTO 9 and PI were purchased from

D

Invitrogen (California, USA). Reinforced clostridium medium (RCM)

TE

and luria-bertani (LB) broth were purchased from BD Biosciences (San

EP

Jose, USA). Uranyl acetate was purchased from Polysciences, Inc.

C

(Warrington, USA).
AC

2.2 Microbes and Growth Conditions

P. acnes BCRC 10723 strain was provided by Prof. K. C. Chang. A

colony of P. acnes was grown on RCM medium at 37℃. This colony of

microbes was inoculated into centrifuge tubes containing 10 ml of

medium. The P. acnes under anaerobic conditions were cultivated on an

 ACCEPTED MANUSCRIPT

agar plate for 72 h. The optical density of microbial growth was measured

in a 10-mm cuvette by spectrophotometer (DU-650, Beckman, USA) at

λ= 600 nm. Thus, P. acnes were applied in the photodynamic experiment

of antimicrobial activity with curcumin.

PT
2.3 Setup of Photo-reaction System

RI
The irradiation unit was arranged in a microbial incubator as a

SC
photo-reaction system as described in previous studies [35, 36]. The

transparent glass tubes were used for photodynamic experiments and

NU
placed in front of light irradiation. The voltage power supply was set as
MA

DC 5V. The light intensity of BL was 3.0 mW/cm 2 and measured by solar
D

power meter (TM-206, Tenmars Electronics, Taiwan). The constant

TE

exposure could be maintained through adjustment of distance between the

EP

glass tubes and the light source. The gas-cooled temperature of the
C

photo-reaction system was maintained at 36 ± 1°C and monitored on an

AC

infrared thermometer (Raytek, USA). The emission spectra (λ max = 462

nm) of LED (VetaLED Company, Taiwan) was from 410 to 510 nm and

measured using a micro-spectrometer (GREEN-Wave UVNb-25,

StellarNet Inc., USA).

2.4 Antimicrobial Effects of Photolytic Curcumin

 ACCEPTED MANUSCRIPT

The microbial viability experiments were evaluated as a cross-factor

design between the curcumin and the BL irradiation. The P. acnes (108

cell/ml for P. acnes) was incubated at 37°C until OD 600 =1.0. The quantity

of P. acnes with 1 ml in antimicrobial experiment was 2×105 cell/ml by

PT
diluted process. The 1 ml of microbial solution was mixed with 1 ml of

RI
curcumin suspension (final concentration of 1.5 to 100 μM) in glass tubes.

SC
Curcumin dissolved in 100% DMSO and the concentration of stock

solution was 200 mM. The microbial broth was used as diluted solution. P.
NU
acnes was treated with 0, 1.56, 12.5, 25, and 100 μM of curcumin. Then,
MA

the treated solutions were irradiated with 3.0 mW/cm 2 of BL for 0, 1/6,
D

1/2, and 1 min (equivalent to radiant exposure of 0, 0.03, 0.09, and 0.18
TE

J/cm 2). After treatment, the microbial solutions were diluted 1000 times
EP

with fresh medium and 100 μl of solution was spread on agar plates and
C

cultured at 37℃ for 72 h. The microbial colony was counted, and the
AC

dilution ratio for quantity of P. acnes was multiplied. This experiment

was performed in triplicate. The relative inhibition ratio was determined

as the following equation:

Inhibition Ratio (%) = [1 - (CFU treatment / CFU control)] × 100%.

2.5 Observations for Microbial Membrane Disruption

 ACCEPTED MANUSCRIPT

The cellular membrane disruption ability of photolytic curcumin was

analyzed through a scanning electron microscope (SEM) and fluorescent

staining. P. acnes was incubated at 37°C for 72 h in RCM medium until

an OD600 of 1.0 was reached. Approximately 108 microbial cells were

PT
treated with 50 μM curcumin and BL irradiation (0 min to 10 min). The P.

RI
acnes culture was incubated at 37°C for 1 h in a dark place as negative

SC
controls. The volume of microbial solution was adjusted to 100 μl. The

sample pretreatments for SEM observation were carried as following.

NU
Microbial pre-fixation was performed using 2.5% (w/v) glutaraldehyde in
MA

0.1 M cacodylate buffer and 1% tannic acid at 4°C for 16 h. Microbial

D

post-fixation was performed using 1% osmium tetroxide in 0.1 M

TE

cacodylate buffer at 4°C for 1 h. The microbial sample was then washed
EP

extensively by phosphate-buffered saline (PBS; 3 mM KCl, 0.15 M NaCl,

C

2 mM KH2PO 4, 8 mM Na2HPO4, pH 7.4) and dehydrated with a graded

AC

ethanol series. After the process of critical-point drying and gold coating,

the sample was observed using SEM (Hitachi H-7500, Tokyo, Japan).

The SYTO 9 and PI as fluorescent probes were used to monitor the

membrane disruption in different treatment conditions. The samples were

pretreatment for fluorescent stain observation as following. The microbial

 ACCEPTED MANUSCRIPT

solutions were centrifuged in 10,000 G at 4 °C for 10 min, and the

suspensions were discarded. The centrifugation was repeated for 3 times.

Each 5 µl of SYTO 9 (3.34 mM) and PI (20 mM) were mixed with 990 µl

DI water as the fluorescent stain solution. The 20 µl of stain solution

PT
resuspension the pellet of centrifuged microbial and react in dark place

RI
for 30 min. Drop 2 µl solution in glass slide that observed by fluorescent

SC
microscope (Nikon E800). The excitation wavelength of SYTO 9 and PI

is 480 nm. The emission wavelengths of SYTO 9 and PI are 500 nm and
NU
635 nm, respectively.
MA

2.6 Liquid Chromatography-Mass Spectrometry (LCMS) Analysis

D

The LCMS (Thermo Scientific, San Jose, USA) was performed on a

TE

triple quadrupole mass spectrometer (TSQ Quantum Access) equipped

EP

with an auto-sampler, a quaternary pump (Accela), electrospray ion

source, and Xcalibur workstation. The column (Accucore C18, 2.6 μm of

C
AC

particle size and 10 mm of length × 2.1 mm of inner diameter, Thermo

Fisher Scientific) was eluted by the mobile phase at 0.3 ml/min. The

mobile phase consisted of 5% acetonitrile in ultrapure water with 0.04%

AcOH (A) and 95% acetonitrile in ultrapure water with 0.04% AcOH (B)

using an isocratic elution mode of 50% (B) in 0 to 5 min. The MS

 ACCEPTED MANUSCRIPT

operation with electrospray ionization in negative mode was set as

follows: injection volume, 10 μl; capillary temperature, 320°C; capillary

voltage, 4 kV; MS scan range, m/z =50–400; the sheath and aux gas (N2)

flow rates were 49 and 35 L/h, respectively. The 50 µM of curcumin

PT
solutions were irradiated by BL of 3.0 mW/cm2 for 0, 1, 2, and 3 h, and

RI
then analyzed by LCMS.

SC
2.7 Photolytic Product of Curcumin was Confirmed by FTMS

The photoproduct was generated from curcumin with BL irradiation

NU
for 3 h and detected using the Fourier transform mass spectrometry
MA

(FTMS, SolariX, Bruker, USA). The resolving power of FTMS is

D

10,000,000. The theoretical molecular mass (MW) was calculated by the

TE

Molecular Weight Calculator (Matthew Monroe) with the most abundant

EP

isotope for each element and the measured bias evaluated by following

the equation: Bias (in ppm) = (Measured MW – Theoretical MW) /

C
AC

(Theoretical MW).

A 10-μM sample of vanillin standard solution was analyzed by

LCMS. The column and chromatographic operations were the same as

that described above. The MS was operated in the positive mode, and the

parameter of MS/MS was set as follows: the precursor ion was set to m/z
 ACCEPTED MANUSCRIPT

153.0; collision energy, 30%; scan time, 0.3 s; Q1 and Q3 peak width,

0.70 Da; product ion scan range from m/z 45.0-160.0; collision gas (N2)

pressure, 1.5 mTorr.

2.8 Statistics

PT
The experiments were performed in triplicate, and the data are

RI
expressed as mean ± standard deviation of three individual experiments.

SC
The data were assessed by analysis of variance (ANOVA) using SPSS

Statistics (IBM, Armonk, NY, USA). A p-value < 0.05 was considered as
NU
significant.
MA

3. Results
D

3.1 Antimicrobial Activity of Curcumin or BL

TE

The toxic effects of curcumin against P. acnes were evaluated

EP

cautiously through the viable count. The viability of P. acnes was

C

measured by colony-forming unit, which is the quantitative evaluation of

AC

microbial survival. The microbes with curcumin treatment were

cultivated in a dark place for 1 min to avoid light irradiation. The toxic

effects of individual variables (curcumin concentrations, and irradiation

dosages) were investigated on P. acnes in this study. According to the

results in Fig. 2(A), the inhibition ratio of P. acnes of curcumin

 ACCEPTED MANUSCRIPT

treatments was below 20% compared to the control. The inhibition ratio

of P. acnes was altered slightly after treatment with curcumin at

micro-levels. Otherwise, the irradiation dosage of visible BL used in this

study was not a major lethal factor.

PT
3.2 Antimicrobial Activity of Curcumin with BL

RI
In Fig. 2(B), the antimicrobial activities of various combinations of

SC
curcumin and BL irradiation were evaluated. The inhibition ratio of P.

acnes was significantly enhanced when curcumin was combined with BL

NU
irradiation. The 50% inhibition ratio of P. acnes was achieved at
MA

approximately 100 μM curcumin under the 0.03 J/cm2 BL irradiation

(equal with the 3.0 mW/cm 2 for 10 s). However, the inhibition ratio of P.
D
TE

acnes could increase to 100% under the BL irradiation of 0.09 (or 0.18)
EP

J/cm 2 with the curcumin concentrations (1.56 ─ 100 μM). Therefore, the
C

inhibition ratio of P. acnes depended on the curcumin concentration under

AC

the low-dose irradiation of BL (0.03 J/cm 2). The BL irradiation acts as a

catalyst or a convertor on the antimicrobial activity of curcumin against P.

acnes.

3.3 Microbial Membrane Disruption under Photodynamic Reaction

The lethal factors of photolytic curcumin were investigated based on

 ACCEPTED MANUSCRIPT

the morphology of P. acnes by SEM. The shape of the microbe displayed

no cellular damage in untreated, individuals of the curcumin and BL

irradiation groups in Fig. 3(a, b, and c). In contrast, exposure of the

microbe to 50 μM of curcumin combined with BL irradiation for 10 min

PT
caused a noteworthy modification of P. acnes cellular morphology such

RI
as the surface blebbing, the surface roughening, and the cellular leakages

SC
as shown in Fig. 3(d), (e), and (f).

Curcumin could interact with the lipid monolayer and accumulates in

NU
the hydrophobic region of the acyl chain [28]. SYTO 9 and PI as
MA

fluorescent probes for nucleic acid staining were used to monitor the
D

microbial membrane perturbation. The SYTO 9 could penetrate healthy

TE

microbes to stain nucleic acid. The PI only could stain nucleic acid while
EP

microbial membrane damage [37]. The individual of 50 µM curcumin

and 0.018 J/cm 2 BL had slight damage to P. acnes as shown in Fig. 3(g)
C
AC

and 3(i). The synergistic effect of photolytic curcumin displayed the

dramatic damage to inactive P. acnes in Fig. 3(h) which showed red

nucleic acid staining in most cells.

The gene expression, associated with the cell membrane integrity of

microbes, was decreased after curcumin treatment [30]. Photolysis of

 ACCEPTED MANUSCRIPT

curcumin could also produce superoxide anion and singlet oxygen in

lipophilic environments [38]. The photodynamic reaction of curcumin

resulted in a survival catastrophe that derived from this aberration of

microbial morphology, disintegration of the cell structure, and microbial

PT
dysfunction. The bacterial outer membrane protein damage is also

RI
observed as one of the PDI effects on Vibrio parahaemolyticus [39].

SC
3.4 Photolytic Products of Curcumin by LCMS Analysis

Curcumin is unstable and undergoes autoxidation in liquid solution at

NU
neutral-basic and alkaline pH [22]. The half-life of curcumin
MA

decomposition in methanol is 92.7 h, which is higher than ethyl acetate

D

(15.1 h), acetonitrile (6.3 h), and chloroform (2.7 h) [40]. Curcumin in
TE

methanol could stabilize in 1450 lux of fluorescent light [41]. The

EP

curcumin aqueous solutions were irradiated by BL (at 3.0 mW/cm 2) for 0,

C

1, 2, and 3 h. The level of photolytic curcumin by BL was evaluated by

AC

LCMS in the negative mode.

The isotopic MW of curcumin (C 21H20O 6) was 368.1. As shown in

Fig. 4(A), the retention time (RT) for curcumin eluted at 0.43 min, m/z =

367.1. It was noted that a photoproduct of curcumin (RT, 0.22 min; m/z =

151.4) could be observed in all cases of BL irradiation in Fig. 4(B) ─

 ACCEPTED MANUSCRIPT

4(D). The abundance of curcumin ion ([M-H]-: 367.1) was decreased

significantly with the increased time of BL irradiation. The signal at m/z =

151.4 was also increased with the irradiation time. The smaller MW of

photoproduct than the curcumin molecule demonstrates a photolysis

PT
when curcumin irradiated with BL. Obviously, the increase of the

RI
photoproduct is correlated to the decrease of curcumin in photolysis. As

SC
quantitative analysis, the specific abundance change between reactant and

product is a strong linear negative correlation displayed in Fig. 4(E).

NU
3.5 Major Photoproduct Identified by FTMS
MA

Curcumin could be degraded by BL irradiation for 3 h to form the

major photoproduct in Fig. 4. The [M-H]- ion of m/z = 151.4 was

D
TE

enhanced as the major photoproduct of curcumin in photolysis. Vanillin

EP

was one of candidates generated from the curcumin decomposition as

C

shown in Fig. 1. Based on the exact isotopic MW (152.04736) and a

AC

similar formula (C8H8O3) of vanillin, which had a minimal bias with the

measured MW of photoproduct (shown in Table 1), the vanillin is the

main suspect for photoproduct of curcumin. Other suspected molecules,

camphor and acenaphthylene have the larger bias in the high-resolution

mass measurements. Finally, standard chemicals of vanillin and curcumin

 ACCEPTED MANUSCRIPT

were used to confirm by chromatography and mass spectrometry. As

shown in Fig. 5, the results of chromatograms and secondary mass

spectrum indicate that vanillin is the major photoproduct generated from

the curcumin photolysis with BL irradiation.

PT
4. Discussion

RI
Curcumin play an antioxidant agent in acidic condition through

SC
hydrogen transfer reaction. Nevertheless, it could be photosensitizer in

higher pH through electron donating ability [42]. The high concentration

NU
and exposure time could enhance the cytotoxicity of curcumin. A total of
MA

105 cells of P. acnes was used to evaluate the cytotoxicity of

photodynamic curcumin treatments. The exposures of 100 μM of

D
TE

curcumin for 1 min could injure 20% of P. acne, but 1.56 μM of curcumin
EP

treatment for 1 min resulted in slight damage as shown in Fig. 2(A).

C

Hence, the individual BL irradiation also had a slight effect on P. acnes

AC

inactivation, as shown in Fig 2(A). The level of microbe infection is

relative to quantity, and a high density of microbes could more easily

resist antimicrobial agents and photodamage.

As shown in Fig. 2(B), the inhibition ratio of P. acnes was enhanced

dramatically with the increased time of BL irradiation. The cytotoxicity

 ACCEPTED MANUSCRIPT

enhancement of curcumin against P. acnes was triggered by BL. High

doses of curcumin with 216 J/cm 2 of BL intra-oral irradiation could

inhibit 25% of microbial survival in orthodontic patients [43]. The dosage

of curcumin should be evaluated cautiously for anti-microbial activity

PT
because high levels of curcumin would chelate the biological iron [44].

RI
PDI could inactive the microbes through DNA damage, membrane

SC
disruption, and protein dysfunction. In addition to DNA oxidative damage

caused by ROS, the curcumin activated by the blue light (BL) could
NU
inhibit the biofilm formation of Candida albicans by the singlet oxygen
MA

generated from the photodecomposition of curcumin [45]. Moreover,

D

curcumin has a potential influence as a metal chelator in the biological

TE

metabolic system and may induce mitotic catastrophe to impede

EP

endothelial cell proliferation [46]. In the first, the individual safe dose of
C

curcumin and BL has been evaluated. Thus, the synergistic effect of

AC

curcumin and BL is significant on antimicrobial activity.

The 108 of P. acnes was chosen to evaluate the damage for membrane

structure and permeation as 50 μM of curcumin with various dosages of

BL in Fig. 3. Otherwise, vanillin is the major photoproduct of curcumin

with BL irradiation, and is primarily a membrane-active compound.

 ACCEPTED MANUSCRIPT

Many studies have also demonstrated that the vanillin also is cytotoxic to

microbe due to the inhibition of respiration and the dissipation of ion

gradients [47-49]. We suggest that the membrane damage of P. acnes is

the main antimicrobial pathway in photodynamic curcumin treatment,

PT
which is consistent with the results of other study [50].

RI
However, the curcumin degradation would generate the complicated

SC
metabolic products, which were unstable by variable conditions,

including pH of solution, temperature, exposed dosage, and incubated

NU
time of photochemical experiments [51]. Through a series of highly
MA

sensitive mass spectrometry techniques, vanillin was identified as the

D

major photolytic product of curcumin after BL irradiation in this study.

TE

A clinical application was reported to treat patient with acne vulgaris

EP

for microbial eradication [52]. Gold et al. had reported a clinical study
C

with self-applied blue light therapy for mild-to-moderate inflammatory

AC

acne that showed a significant improvement of the subjects’ skin

conditions [53]. The photolytic curcumin also had high anti-microbial

ability in P. acnes [54]. The side effect of curcumin as photosensitizer

was low in a non-invasive therapy. Here, we displayed the high efficiency

of antimicrobial ability in low concentration of photolytic curcumin.

 ACCEPTED MANUSCRIPT

5. Conclusions

Our previous study reveals that visible BL is safer than UV for

exposure to mammalian cells [36]. In this study, we found that curcumin

PT
could be photodegraded to generate vanillin under BL irradiation.

RI
Individual exposure to curcumin or BL irradiation does not generate

SC
cytotoxicity on P. acnes. The 1.5 μM of curcumin with 0.09 J/cm 2 of BL

irradiation could eradicate P. acnes significantly. One of the action

NU
mechanisms of P. acnes eradication by photolytic curcumin was observed
MA

as the membrane perturbation in this study. Furthermore, the low-dose

blue light irradiation triggers a series of cytotoxic actions of curcumin on

D
TE

P. acnes. We believe this photodynamic inactivation technique may be a

EP

potential non-antibiotic and safer treatment for skincare applications.

C
AC
 ACCEPTED MANUSCRIPT

Conflict of interest statement

The authors have claimed no conflict of interest in this study.

PT
Acknowledgments

RI
We thank the Electron Microscopy Laboratory of Tzu-Chi University

SC
for technical assistance. This work was supported by grants from the

Ministry of Science and Technology of Taiwan (MOST

NU
105-2113-M-320-001) and TCMMP 105-13, the Buddhist Tzu Chi
MA

Medical Foundation.
D
TE
C EP
AC
 ACCEPTED MANUSCRIPT

References

[1] J. Kim, Review of the innate immune response in acne vulgaris:

activation of Toll-like receptor 2 in acne triggers inflammatory cytokine

responses, Dermatology, 211 (2005) 193-198.

PT
[2] N. Asakawa, K. Uchida, M. Sakakibara, K. Omote, K. Noguchi, Y.

RI
Tokuda, K. Kamiya, K.C. Hatanaka, Y. Matsuno, S. Yamada, K. Asakawa,

SC
Y. Fukasawa, T. Nagai, T. Anzai, Y. Ikeda, H. Ishibashi-Ueda, M. Hirota,

M. Orii, T. Akasaka, K. Uto, Y. Shingu, Y. Matsui, S.I. Morimoto, H.

NU
Tsutsui, Y. Eishi, Immunohistochemical identification of
MA

Propionibacterium acnes in granuloma and inflammatory cells of

D

myocardial tissues obtained from cardiac sarcoidosis patients, PLoS One,

TE

12 (2017) e0179980.
EP

[3] S.R. Nodzo, K.K. Boyle, S. Bhimani, T.R. Duquin, A.O. Miller, G.H.
C

Westrich, Propionibacterium acnes Host Inflammatory Response During

AC

Periprosthetic Infection Is Joint Specific, HSS J, 13 (2017) 159-164.

[4] V. Zeller, A. Ghorbani, C. Strady, P. Leonard, P. Mamoudy, N.

Desplaces, Propionibacterium acnes: an agent of prosthetic joint infection

and colonization, J Infect, 55 (2007) 119-124.

[5] C. Oprica, L. Emtestam, J. Lapins, E. Borglund, F. Nyberg, K.

 ACCEPTED MANUSCRIPT

Stenlund, L. Lundeberg, E. Sillerstrom, C.E. Nord, Antibiotic-resistant

Propionibacterium acnes on the skin of patients with moderate to severe

acne in Stockholm, Anaerobe, 10 (2004) 155-164.

[6] B. Vares, S. Zandi, H. Abdollahi, Determination of microbial agents

PT
of acne vulgaris and Propionibacterium acnes antibiotic resistance in

RI
patients referred to dermatology clinics in Kerman, Iran, Jundishapur

SC
Journal of Microbiology, 4 (2011) 17-22.

[7] K. Sardana, T. Gupta, B. Kumar, H.K. Gautam, V.K. Garg,

NU
Cross-sectional Pilot Study of Antibiotic Resistance in Propionibacterium
MA

Acnes Strains in Indian Acne Patients Using 16S-RNA Polymerase Chain

D

Reaction: A Comparison Among Treatment Modalities Including

TE

Antibiotics, Benzoyl Peroxide, and Isotretinoin, Indian J Dermatol, 61

EP

(2016) 45-52.
C

[8] K.C. Lee, P.A. Lio, Evidence-based recommendations for the

AC

diagnosis and treatment of paediatric acne, Arch Dis Child Educ Pract Ed,

99 (2014) 135-137.

[9] A.E. Ryan-Kewley, D.R. Williams, N. Hepburn, R.A. Dixon,

Non-antibiotic Isotretinoin Treatment Differentially Controls

Propionibacterium acnes on Skin of Acne Patients, Front Microbiol, 8

 ACCEPTED MANUSCRIPT

(2017) 1381.

[10] K.M. Whitney, C.M. Ditre, Management strategies for acne vulgaris,

Clin Cosmet Investig Dermatol, 4 (2011) 41-53.

[11] S. Coberly, E. Lammer, M. Alashari, Retinoic acid embryopathy:

PT
case report and review of literature, Pediatr Pathol Lab Med, 16 (1996)

RI
823-836.

SC
[12] N. Kashef, Y.Y. Huang, M.R. Hamblin, Advances in antimicrobial

photodynamic inactivation at the nanoscale, Nanophotonics-Berlin, 6

NU
(2017) 853-879.
MA

[13] T.A. Skwor, S. Klemm, H.Y. Zhang, B. Schardt, S. Blaszczyk, M.A.

D

Bork, Photodynamic inactivation of methicillin-resistant Staphylococcus

TE

aureus and Escherichia coli: A metalloporphyrin comparison, J Photoch

EP

Photobio B, 165 (2016) 51-57.

C

[14] S. Ghosh, S. Banerjee, P.C. Sil, The beneficial role of curcumin on

AC

inflammation, diabetes and neurodegenerative disease: A recent update,

Food Chem Toxicol, 83 (2015) 111-124.

[15] S.H. Mun, D.K. Joung, Y.S. Kim, O.H. Kang, S.B. Kim, Y.S. Seo,

Y.C. Kim, D.S. Lee, D.W. Shin, K.T. Kweon, D.Y. Kwon, Synergistic

antibacterial effect of curcumin against methicillin-resistant

 ACCEPTED MANUSCRIPT

Staphylococcus aureus, Phytomedicine, 20 (2013) 714-718.

[16] H.H. Tonnesen, H. de Vries, J. Karlsen, G. Beijersbergen van

Henegouwen, Studies on curcumin and curcuminoids. IX: Investigation

of the photobiological activity of curcumin using bacterial indicator

PT
systems, J Pharm Sci, 76 (1987) 371-373.

RI
[17] S. Luer, R. Troller, C. Aebi, Antibacterial and antiinflammatory

SC
kinetics of curcumin as a potential antimucositis agent in cancer patients,

Nutr Cancer, 64 (2012) 975-981.

NU
[18] J. Song, B. Choi, E.J. Jin, Y. Yoon, K.H. Choi, Curcumin suppresses
MA

Streptococcus mutans adherence to human tooth surfaces and

D

extracellular matrix proteins, Eur J Clin Microbiol Infect Dis, 31 (2012)

TE

1347-1352.
EP

[19] K. Neelofar, S. Shreaz, B. Rimple, S. Muralidhar, M. Nikhat, L.A.

C

Khan, Curcumin as a promising anticandidal of clinical interest, Can J

AC

Microbiol, 57 (2011) 204-210.

[20] P. Neelakantan, C. Subbarao, S. Sharma, C.V. Subbarao, F.

Garcia-Godoy, J.L. Gutmann, Effectiveness of curcumin against

Enterococcus faecalis biofilm, Acta Odontol Scand, 71 (2013)

1453-1457.
 ACCEPTED MANUSCRIPT

[21] I.A. Packiavathy, S. Priya, S.K. Pandian, A.V. Ravi, Inhibition of

biofilm development of uropathogens by curcumin - an anti-quorum

sensing agent from Curcuma longa, Food Chem, 148 (2014) 453-460.

[22] Y.J. Wang, M.H. Pan, A.L. Cheng, L.I. Lin, Y.S. Ho, C.Y. Hsieh, J.K.

PT
Lin, Stability of curcumin in buffer solutions and characterization of its

RI
degradation products, J Pharm Biomed Anal, 15 (1997) 1867-1876.

SC
[23] A. Hassaninasab, Y. Hashimoto, K. Tomita-Yokotani, M. Kobayashi,

Discovery of the curcumin metabolic pathway involving a unique enzyme

NU
in an intestinal microorganism, Proc Natl Acad Sci U S A, 108 (2011)
MA

6615-6620.
D

[24] S. Tan, T.W. Rupasinghe, D.L. Tull, B. Boughton, C. Oliver, C.

TE

McSweeny, S.L. Gras, M.A. Augustin, Degradation of curcuminoids by

EP

in vitro pure culture fermentation, J Agric Food Chem, 62 (2014)

C

11005-11015.
AC

[25] M.H. Pan, T.M. Huang, J.K. Lin, Biotransformation of curcumin

through reduction and glucuronidation in mice, Drug Metab Dispos, 27

(1999) 486-494.

[26] O.N. Gordon, P.B. Luis, H.O. Sintim, C. Schneider, Unraveling

curcumin degradation: autoxidation proceeds through spiroepoxide and

 ACCEPTED MANUSCRIPT

vinylether intermediates en route to the main bicyclopentadione, J Biol

Chem, 290 (2015) 4817-4828.

[27] K.I. Priyadarsini, Photophysics, photochemistry and photobiology of

curcumin: Studies from organic solutions, bio-mimetics and living cells,

PT
Journal of Photochemistry and Photobiology C: Photochemistry Reviews,

RI
10 (2009) 81-95.

SC
[28] A. Karewicz, D. Bielska, B. Gzyl-Malcher, M. Kepczynski, R. Lach,

M. Nowakowska, Interaction of curcumin with lipid monolayers and

NU
liposomal bilayers, Colloids Surf B Biointerfaces, 88 (2011) 231-239.
MA

[29] H.I. Ingolfsson, R.E. Koeppe, 2nd, O.S. Andersen, Curcumin is a

D

modulator of bilayer material properties, Biochemistry, 46 (2007)

TE

10384-10391.
EP

[30] A. Kumar, S. Dhamgaye, I.K. Maurya, A. Singh, M. Sharma, R.

C

Prasad, Curcumin targets cell wall integrity via calcineurin-mediated

AC

signaling in Candida albicans, Antimicrobial agents and chemotherapy,

58 (2014) 167-175.

[31] P. Hu, P. Huang, M.W. Chen, Curcumin reduces Streptococcus

mutans biofilm formation by inhibiting sortase A activity, Arch Oral Biol,

58 (2013) 1343-1348.
 ACCEPTED MANUSCRIPT

[32] B.S. Park, J.G. Kim, M.R. Kim, S.E. Lee, G.R. Takeoka, K.B. Oh,

J.H. Kim, Curcuma longa L. constituents inhibit sortase A and

Staphylococcus aureus cell adhesion to fibronectin, J Agric Food Chem,

53 (2005) 9005-9009.

PT
[33] Y. Zong, T.W. Bice, H. Ton-That, O. Schneewind, S.V. Narayana,

RI
Crystal structures of Staphylococcus aureus sortase A and its substrate

SC
complex, J Biol Chem, 279 (2004) 31383-31389.

[34] O. Schneewind, D.M. Missiakas, Protein secretion and surface

NU
display in Gram-positive bacteria, Philos Trans R Soc Lond B Biol Sci,
MA

367 (2012) 1123-1139.

D

[35] J.-Y. Liang, C.-W. Cheng, C.-H. Yu, L.-Y. Chen, Investigations of
TE

blue light-induced reactive oxygen species from flavin mononucleotide

EP

on inactivation of E. coli, Journal of Photochemistry and Photobiology B:

C

Biology, 143 (2015) 82-88.

AC

[36] M.Y. Yang, C.J. Chang, L.Y. Chen, Blue light induced reactive

oxygen species from flavin mononucleotide and flavin adenine

dinucleotide on lethality of HeLa cells, J Photochem Photobiol B, 173

(2017) 325-332.

[37] P. Stiefel, S. Schmidt-Emrich, K. Maniura-Weber, Q. Ren, Critical

 ACCEPTED MANUSCRIPT

aspects of using bacterial cell viability assays with the fluorophores

SYTO9 and propidium iodide, BMC Microbiol, 15 (2015) 36.

[38] B. Yi, H.J. Ka, M.-J. Kim, J. Lee, Effects of curcumin on the

oxidative stability of oils depending on type of matrix, photosensitizers,

PT
and temperature, JAOCS, Journal of the American Oil Chemists' Society,

RI
92 (2015) 685.

SC
[39] J. Wu, H. Mou, C. Xue, A.W. Leung, C. Xu, Q.J. Tang,

Photodynamic effect of curcumin on Vibrio parahaemolyticus,

NU
Photodiagnosis Photodyn Ther, 15 (2016) 34-39.
MA

[40] H.H. Tonnesen, J. Karlsen, Studies on curcumin and curcuminoids.

D

VI. Kinetics of curcumin degradation in aqueous solution, Z Lebensm

TE

Unters Forsch, 180 (1985) 402-404.

EP

[41] L.C. Price, R. Buescher, Decomposition of turmeric curcuminoids as

C

affected by light, solvent and oxygen, Journal of food biochemistry, 20

AC

(1996) 125-133.

[42] S.V. Jovanovic, S. Steenken, C.W. Boone, M.G. Simic, H-atom

transfer is a preferred antioxidant mechanism of curcumin, Journal of the

American Chemical Society, 121 (1999) 9677-9681.

[43] V.H. Panhoca, F.L. Esteban Florez, T.Q. Correa, F.R. Paolillo, C.W.
 ACCEPTED MANUSCRIPT

de Souza, V.S. Bagnato, Oral Decontamination of Orthodontic Patients

Using Photodynamic Therapy Mediated by Blue-Light Irradiation and

Curcumin Associated with Sodium Dodecyl Sulfate, Photomed Laser

Surg, 34 (2016) 411-417.

PT
[44] Y. Jiao, J.t. Wilkinson, X. Di, W. Wang, H. Hatcher, N.D. Kock, R.

RI
D'Agostino, Jr., M.A. Knovich, F.M. Torti, S.V. Torti, Curcumin, a cancer

SC
chemopreventive and chemotherapeutic agent, is a biologically active

iron chelator, Blood, 113 (2009) 462-469.

NU
[45] M.C. Andrade, A.P. Ribeiro, L.N. Dovigo, I.L. Brunetti, E.T.
MA

Giampaolo, V.S. Bagnato, A.C. Pavarina, Effect of different

D

pre-irradiation times on curcumin-mediated photodynamic therapy

TE

against planktonic cultures and biofilms of Candida spp, Arch Oral Biol,
EP

58 (2013) 200-210.
C

[46] S.J. Jackson, L.L. Murphy, R.C. Venema, K.W. Singletary, A.J.
AC

Young, Curcumin binds tubulin, induces mitotic catastrophe, and impedes

normal endothelial cell proliferation, Food Chem Toxicol, 60 (2013)

431-438.

[47] A.E. Patterson, A.J. Flewelling, T.N. Clark, S.J. Geier, C.M. Vogels,

J.D. Masuda, C.A. Gray, S.A. Westcott, Antimicrobial and

 ACCEPTED MANUSCRIPT

antimycobacterial activities of aliphatic amines derived from vanillin,

Can J Chem, 93 (2015) 1305-1311.

[48] H.Y. Chenia, Antimicrobial Activity of Cinnamaldehyde, Vanillin

and Kigelia Africana Fruit Extracts against Fish-Associated

PT
Chryseobacterium and Myroides Spp. Isolates, Afr J Tradit Complem, 12

RI
(2015) 55-67.

SC
[49] R.M. Cava-Roda, A. Taboada-Rodriguez, M.T. Valverde-Franco, F.

Marin-Iniesta, Antimicrobial Activity of Vanillin and Mixtures with

NU
Cinnamon and Clove Essential Oils in Controlling Listeria
MA

monocytogenes and Escherichia coli O157:H7 in Milk, Food Bioprocess

D

Tech, 5 (2012) 2120-2131.

TE

[50] P. Tyagi, M. Singh, H. Kumari, A. Kumari, K. Mukhopadhyay,

EP

Bactericidal activity of curcumin I is associated with damaging of

C

bacterial membrane, PLoS One, 10 (2015) e0121313.

AC

[51] O.N. Gordon, C. Schneider, Vanillin and ferulic acid: not the major

degradation products of curcumin, Trends Mol Med, 18 (2012) 361-363;

author reply 363-364.

[52] C. Horfelt, B. Stenquist, O. Larko, J. Faergemann, A.M. Wennberg,

Photodynamic therapy for acne vulgaris: a pilot study of the

 ACCEPTED MANUSCRIPT

dose-response and mechanism of action, Acta Derm Venereol, 87 (2007)

325-329.

[53] M.H. Gold, A. Andriessen, J. Biron, H. Andriessen, Clinical Efficacy

of Self-applied Blue Light Therapy for Mild-to-Moderate Facial Acne, J

PT
Clin Aesthet Dermatol, 2 (2009) 44-50.

RI
[54] S.R. de Annunzio, L.M. de Freitas, A.L. Blanco, M.M. da Costa, C.C.

SC
Carmona-Vargas, K.T. de Oliveira, C.R. Fontana, Susceptibility of

Enterococcus faecalis and Propionibacterium acnes to antimicrobial

NU
photodynamic therapy, J Photochem Photobiol B, 178 (2018) 545-550.
MA
D
TE
C EP
AC
 ACCEPTED MANUSCRIPT

Fig. 1. Chemical structures of curcumin and metabolic products,

referenced from literatures [22-26].

Fig. 2. Dose-response relationships of curcumin photolysis on inhibition

of P. acnes. (A) P. acnes cells were treated with different dosages

PT
of curcumin or BL irradiation. (B) P. acnes cells were treated with

RI
the different dosages of curcumin and BL irradiation (0.03, 0.09,

SC
and 0.18 J/cm 2). Data are represented as mean ± standard

deviation, where n = 3. Statistical differences (p < 0.05) between

NU
groups are indicated by different letters above the bar.
MA

Fig. 3. Membrane permeation of P. acnes induced by curcumin

D

photolysis and investigated by SEM. P. acnes was grown in SDA

TE

medium and treated under the referred conditions. P. acnes

EP

without treatment as the control (a), were treated by BL irradiation

C

for 1 min without curcumin (b) in a dark place for 10 min (c), by
AC

BL irradiation for 1 min (d), by BL irradiation for 3 min (e), and

by BL irradiation for 10 min (f) with 50 μM curcumin. The red

arrows indicate the fragmented microbial membrane. Fluorescent

images of P. acnes after treating with a 50 µM curcumin (g),

treating with 50 µM curcumin and 0.018 J/cm2 BL (h), and

 ACCEPTED MANUSCRIPT

treating with 0.018 J/cm2 BL (i), were investigated by the

fluorescent staining of SYTO 9 (in green) and PI (in red).

Fig. 4. The LCMS analysis results and total ion chromatogram (TIC) of

curcumin solution with BL irradiation for different time periods.

PT
(A) Curcumin solution in the dark for 3 h; (B) Curcumin solution

RI
with BL irradiation for 1 h; (C) Curcumin solution with BL

SC
irradiation for 2 h; (D) Curcumin solution with BL irradiation for

3 h; (E) Blank with BL irradiation for 3 h. (F) The MS signal

NU
abundances of curcumin (m/z=367.3) and its photoproduct
MA

(m/z=151.4) were counted for each irradiation time.

D

Fig. 5. The extraction ion chromatogram (EIC) of LCMS analysis of

TE

curcumin and its photolytic products. (A) Curcumin standard

EP

solution; (B) The photolytic curcumin solution after BL irradiation

C

for 3 h; (C) Vanillin standard solution. The MS/MS result of (B)

AC

and (C) are shown.

 ACCEPTED MANUSCRIPT

Table 1. The chemical formulas of curcumin and its photolytic products

were validated and analyzed with the molecular mass biases,

derived from the data of FTMS.

Compound [M] Empirical Measured MW Theoretical Biasa

PT
Formula [M-H]- MW [M-H]- (×10-6)

RI
Curcumin C21H20O6 367.11876 367.11815 1.66159

SC
Vanillinb C8H8O3 151.03993c 151.03951 2.78072

Camphorb 151.03993c
NU
C10H16O 151.11228 -

481.799446
MA

Acenaphthyleneb C12H8 151.03993c 151.05477 - 98.24251

D

a
Bias = (Measured MW － Theoretical MW) / (Theoretical MW), in
TE

ppm.
EP

b
The typical compounds are proposed for the empirical formulas of 152
C

Da molecular mass.
AC

c
The molecular mass of major photolytic product measured by FTMS.
 ACCEPTED MANUSCRIPT

Graphical abstract

Highlights

 Curcumin with blue light irradiation can inhibit Propionibacterium acnes

survival.

PT
 Curcumin could photolyse into vanillin through blue light irradiation.

RI
 Membrane disruption dominates the mechanism of photodynamic

SC
inactivation.

 Low doses of photolytic curcumin may act as an antimicrobial agent.

NU
 This photodynamic inactivation was applied in skincare and clinical therapy.
MA
D
TE
C EP
AC
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5