In vitro antimicrobial and anti-inflammatory effects of herbs against Propionibacterium acnes

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In vitro antimicrobial and anti-inﬂammatory effects of herbs against

Propionibacterium acnes

Tsung-Hsien Tsai

a

_{, Tzung-Hsun Tsai}

b

_{, Wen-Huey Wu}

c

_{, Jonathon Te-Peng Tseng}

a

_{, Po-Jung Tsai}

_c,*

a

Department of Dermatology, Taipei Medical University–Wanfang Hospital, Taipei, Taiwan, ROC b

Department of Dentistry, Keelung Chang-Gung Memorial Hospital, Keelung, Taipei, Taiwan, ROC c

Department of Human Development and Family Studies, National Taiwan Normal University, Taipei 10610, Taiwan, ROC

a r t i c l e

i n f o

Article history: Received 8 March 2009

Received in revised form 23 June 2009 Accepted 31 July 2009 Keywords: Herbs Propionibacterium acnes Cytokines Anti-inﬂammation

a b s t r a c t

Propionibacterium acnes play an important role in the pathogenesis of acne by inducing certain inflamma-tory mediators and comedogenesis. The objective of this study was to evaluate the antimicrobial and anti-inflammatory effects of herbal extracts against P. acnes. Among the ten tested herbs, methanolic extracts of rose (Rosa damascene), duzhong (Eucommia ulmoides Oliv.), and yerba mate (Ilex paraguarien-sis) were found to inhibit the growth of P. acnes with respective minimum inhibitory concentrations of 2, 0.5, and 1 mg/ml. In addition, duzhong and yerba mate extracts reduced the secretion of pro-inflamma-tory cytokines such as tumour necrosis factor-a, interleukin (IL)-8, and IL-1b by human monocytic THP-1 cells pretreated with heat-killed P. acnes at a concentration of 0.1 mg/ml. Our results suggested that duz-hong and yerba mate extracts possess both antimicrobial and anti-inflammatory effects against P. acnes and can possibly be used as therapeutic agents for acne.

1. Introduction

Acne vulgaris is a common skin disease involving pilosebaceous follicles. The pathogenesis of acne vulgaris is multifactorial, includ-ing increased sebum production, comedogenesis,

Propionibacte-rium acnes proliferation, and inﬂammation (Leyden, 2003).

P. acnes play an important role not only in the process of

inflamma-tion but also in the formainflamma-tion of comedones. P. acnes contribute to the inflammatory nature of acne by inducing monocytes to secrete pro-inflammatory cytokines including interleukin (IL)-1b, IL-8, and tumour necrosis factor (TNF)-

a

(Kim, 2005). The major classes of therapeutic agents are topical and systemic retinoids, antimicro-bial agents, and systemic hormonal drugs. Bacterial resistance is an ongoing problem in the treatment of acne vulgaris. Recently, new retinoids with additional anti-inﬂammatory action are being co-administered with antibiotics to reduce the risk of bacterial resistance (Leyden, 2003). Therefore, an agent which can inhibit

P. acnes growth and suppress the inﬂammatory response will

pro-vide promising beneﬁts to patients with acne vulgaris.

Herbs have been used for many purposes, including medication, nutrition, ﬂavouring, beverages, and fragrance. Much of the early interest in functional foods and nutraceuticals was based on the

medicinal uses of herbs. Herbal tea products commonly consumed in Taiwanese daily life are commonly applied in folk medicine or traditional Chinese medicine, like honeysuckle (Lonicera japonica), juhua (Chrysanthemum morifolium), duzhong (Eucommia ulmoides Oliv.), and jiaogulan (Gynostemma pentaphyllum). Honeysuckle and juhua are two very popular herbal teas during the summer-time. Honeysuckle possesses a wide range of antibacterial proper-ties including action against Staphylococcus aureus, streptococci,

Bacillus dysenterii, and Salmonella typhi. In addition, honeysuckle

shows hepatoprotective effects against CCl4-induced hepatic injury

(Huang, 1998). Juhua is widely used as a remedy for the common

cold, headaches, and hypertension (Huang, 1998). The duzhong ex-tract has antihypertensive, antioxidative, and antigastric ulcer ef-fects, and promotes collagen synthesis (Takeshi, Sanse, &

Yoshihisa, 2001). Jiaogulan has been found to have many

pharma-cological effects, such as hypoglycemic (Norberg et al., 2004), hyp-olipidemic (Cour, Molgaard, & Yi, 1995), and antiallergic (Huang

et al., 2008) functions.

Scented teas are popular not only in Western countries but also in Taiwan, like jasmine (Jasminum sambac), lavender (Lavandula

angustifolia, formerly Lavandula ofﬁcinalis), osmanthus (Osmanthus fragrans Lour), and rose (Rosa damascene). They are usually used

alone or mixed with other herbal teas to provide fragrance and a pleasant taste. In addition to the role of fragrance or pleasant smells, they are thought to be functional for human health. For example, lavender is approved for balneotherapy for circulatory disorders. Lavender oil inhibits mast cell degranulation and has

*Corresponding author. Address: Department of Human Development and Family Studies, National Taiwan Normal University, 162 Hoping E. Rd., Sec. 1, Taipei 10610, Taiwan, ROC. Tel.: +886 2 23636425x55; fax: +886 2 23639635.

E-mail address:[email protected](P.-J. Tsai).

Contents lists available atScienceDirect

Food Chemistry

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antimicrobial and antiphlogistic effects (Thornfeldt, 2006). Fur-thermore, lemongrass (Cymbopogon citrates; Cheel, Theoduloz,

Rodrigues, & Schmeda-Hirschmann, 2005) and yerba mate (Ilex

paraguariensis;Gugliucci, 1996) exhibit antioxidant activities and are becoming more popular in Taiwan.

Our previous study showed that herbal extracts from jasmine, jiaogulan, lemongrass, honeysuckle, duzhong, and yerba mate pos-sess antimicrobial activity against cariogenic Streptococcus

sangui-nis (Tsai, Tsai, Chien, Lee, & Tsai, 2008). However, until now, their antimicrobial activities against P. acnes have not been reported. Moreover, aqueous extracts of lavender, sweet osmanthus, lemon-grass, rose, and juhua showed anti-inﬂammatory effects which were attributed to the moderate inhibitory activity of nitric oxide production in LPS-stimulated Raw 264.7 macrophages (Tsai, Tsai,

Yu, & Ho, 2007). Since the P. acnes-mediated inﬂammatory

re-sponse and comedogenesis are known to be involved in the path-ogenesis of acne vulgaris, this spurred our interest in examining possible anti-inflammatory effects of herbal extracts on the inflam-matory reaction specifically incited by P. acnes. In the present study, the ability of ten herbal extracts to inhibit the growth of P.

acnes was examined, and the anti-inﬂammatory effects of these

herbal extracts were further evaluated. 2. Materials and methods

2.1. Materials

The strain of P. acnes (BCRC10723, isolated from facial acne) was obtained from the Bioresource Collection and Research Center (Hsinchu, Taiwan). P. acnes were cultured in brain heart infusion (BHI) broth (Difco, Detroit, MI, USA) with 1% glucose. The bacteria were cultured in an anaerobic atmosphere using BBL GasPak sys-tems (Becton Dickinson Microbiology Syssys-tems, Cockeysville, MD, USA).

The human monocytic THP-1 cell line (BCRC 60430) was ob-tained from the Bioresource Collection and Research Center (Hsin-chu, Taiwan). Cells were maintained in RPMI 1640 (Gibco, Carlsbad, CA, USA) supplemented with 10% heated-inactivated fe-tal bovine serum (FBS, Gibco), penicillin (100 U/ml), and strepto-mycin (100

l

g/ml). The human ﬁbroblast Hs68 cell line (BCRC 60038) was obtained from the Bioresource Collection and Research Center. The immortalised human HaCaT keratinocyte cell line was developed byBoukamp et al. (1988). Cells were maintained in Dul-becco’s modiﬁed Eagle’s medium (DMEM, Gibco) supplemented with 10% heated-inactivated FBS, penicillin (100 U/ml), and strep-tomycin (100

l

g/ml). These cells were incubated at 37 °C in a humidiﬁed atmosphere with 5% CO2.

The assay kits for TNF

a

, IL-1b, and monocyte chemoattractant protein (MCP)-1 were purchased from eBioscience (San Diego, CA, USA). The IL-8 assay kit was purchased from R&D (Minneapolis, MN, USA). All chemicals were of analytical-grade purity.

2.2. Preparation of extracts

Ten dried herbs including juhua, honeysuckle, jasmine, laven-der, rose, osmanthus, duzhong, jiaogulan, lemongrass, and yerba mate were purchased from a local supermarket in Taipei, Taiwan. The dried herbs were ground up and then extracted with methanol according to our previous study (Tsai et al., 2008). Brieﬂy, 10 g of each dried herb was extracted with 50 ml of methanol at room temperature for 3 h. After extraction, the mixture was ﬁltered, and the residue was re-extracted with 50 ml of fresh methanol overnight. The combined methanolic solution was centrifuged at 12,000g for 10 min and evaporated on a rotary evaporator. The methanolic extract was reconstituted in dimethyl sulfoxide

(DMSO) to a concentration of 400 mg/ml for the subsequent experiments.

2.3. Determination of antimicrobial activity

The herbal extracts were tested against P. acnes by determining the minimum inhibitory concentration (MIC) values obtained by a microdilution broth method as previously described (Tsai et al., 2008). Brieﬂy, P. acnes was incubated in BHI broth with 1% glucose for 72 h under anaerobic conditions and adjusted to yield approx-imately 1 108_{colony-forming units (CFU)/ml. In sterile 96-well}

microtiter plates, 100

l

l of a plant extract was diluted with broth and added to wells containing 100

l

l of the bacterial suspension in broth. Twofold serial dilutions were made in broth over a range to give concentrations of 0.06–8 mg/ml of the methanolic extracts. To adjust the interference by plant pigments, a parallel series of mixtures with un-inoculated broth was prepared. Triplicate sam-ples were performed for each test concentration. After incubation for 72 h at 37 °C under an anaerobic condition, microbial growth was determined by absorbance at 600 nm using a microplate read-er (Biotek Instruments, Winooski, VT, USA). The MIC was deﬁned as the lowest concentration of a test compound which inhibited the growth of P. acnes. The experiments were performed in triplicate.

2.4. Preparation of heat-killed P. acnes

P. acnes were cultured in BHI broth with 1% glucose for 72 h at

37 °C under an anaerobic condition. The log-phase bacterial culture was harvested, washed three times with PBS, and incubated at 80 °C for 30 min to kill the bacteria. The heat-killed P. acnes were stored at 4 °C until use.

2.5. Measurement of cytokine production in human monocytic cells

Human monocytic THP-1 cells were seeded at 1 106_cells/ml

in 24-well plates with serum-free medium, and were stimulated with heat-killed P. acnes (wet weight 100

l

g/ml) alone or in com-bination with different concentrations (0.01, 0.05, and 0.1 mg/ml) of herbal extracts for an 18-h incubation. Cell-free supernatants were collected, and concentrations of MCP-1, TNF

a

, 1b, and IL-8 were analysed with respective enzyme immunoassay kits.

2.6. In vitro cytotoxicity assay

HaCaT and HS68 cells were cultured in DMEM containing 10% FBS and penicillin–streptomycin at 37 °C in a humidiﬁed 5% CO2

atmosphere. HaCaT (1 105cells/ml) and HS68 (1 104cells/ml) were seeded on 96-well plates, and extract treatment began 24 h after seeding. Cell viability was evaluated by conventional 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) reduction assays. After a 24-h incubation with various con-centrations of herbal extracts, 0.1 ml of MTT (5

l

g/ml) was added to each well. After 2 h of incubation, the supernatant was removed, and the precipitate was dissolved in 100 ml of acidic isopropanol. The optical density (OD) of the resulting solution was measured spectrophotometrically at 540 nm. The concentration which reduced the cell viability by 50% (TC50) for each extract was

calculated from ﬁtted dose–response curves. The experiments were performed in triplicate.

2.7. Statistical analysis

All data are presented as the mean ± standard deviation (SD). Statistical analyses were performed using the SPSS 13.0 statistical package (Chicago, IL, USA). The Mann–Whitney U-test was used to

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compare differences between the DMSO vehicle and herbal treat-ments. A p-value of <0.05 was considered statistically signiﬁcant. 3. Results and discussion

3.1. Antimicrobial activity of herbal extracts against P. acnes

We tested the antimicrobial activities of ten herbal extracts against P. acnes (Table 1). Extracts of rose, duzhong, and yerba mate exhibited notable antimicrobial activity against P. acnes. The duzhong extract showed the greatest antimicrobial activity against P. acnes with an MIC of 0.5 mg/ml. The yerba mate extract showed moderate antibacterial activity against P. acnes at an MIC of 1 mg/ml. The rose extract was less effective against P. acnes with an MIC of 2 mg/ml. In vitro antimicrobial activities of essential oils, medicinal plants, and chemicals against P. acnes have been re-ported (Chomnawang, Surassmo, Nukoolkarn, & Gritsanapan, 2005; Docherty, McEwen, Sweet, Bailey, & Booth, 2007; Kim,

Kim, Lee, & Hyun, 2008; Kim et al., 2008). However, a limited

num-ber of studies have been performed to assess the anti-P. acnes activities of herbal tea extracts. The essential oil of rosemary

(Ros-marinus ofﬁcinalis L.) had antibacterial activity against P. acnes with

an MIC value of 0.56 mg/ml (Fu et al., 2007). Guava (Psidium

guaj-ava) and walnut (Juglans regia) leaf extracts also showed anti-P. acnes activity (Qadan et al., 2005). Anti-P. acnes activities of ex-tracts from rose, duzhong, and yerba mate were demonstrated herein. However, the beneﬁcial effect of consuming these herbal extracts in the treatment of P. acnes infection remains to be evalu-ated in further studies.

3.2. Anti-inﬂammatory activities of the herbal extracts

We describe here for the first time the antimicrobial activities of extracts of rose, duzhong, and yerba mate against P. acnes. To fur-ther ascertain whefur-ther these selected extracts possess biological properties against inflammatory acne, subsequent experiments were conducted to determine their inhibitory effects on the pro-inflammatory mediator secretion in co-culture of THP-1 cells with heat-killed P. acnes.

P. acnes contribute to the inﬂammatory nature of acne by

induc-ing monocytes to secrete pro-inﬂammatory cytokines includinduc-ing TNF

a

, IL-1b, and IL-8 (Vowels, Yang, & Leyden, 1995). MCP-1 is associated with modulating monocyte migration in response to inﬂammation (Graves & Jiang, 1995). Therefore, to investigate the anti-inﬂammatory potential of these herbal extracts, we performed an ELISA for TNF

a

, IL-1b, IL-8, and MCP-1 in supernatants of heat-killed P. acnes-stimulated THP-1 monocytes. To account for any reduction in pro-inﬂammatory cytokines resulting from cytotoxic effects of the extracts, the cytotoxicity induced by these extracts was determined by MTT assays in THP-1 cells. Methanolic extracts

of rose, duzhong, and yerba mate had low cytotoxic effects at a concentration of 0.1 mg/ml (data not shown). In addition, follow-ing an 18-h incubation period, methanolic extracts from rose, duz-hong, and yerba mate (up to 0.2 mg/ml) did not increase the secretion of either TNF-

a

, IL-8, IL-1b, or MCP-1 by THP-1 cells in the absence of heat-killed P. acnes (data not shown).

As shown inFigs. 1 and 2, THP-1 cells treated with heat-killed P.

acnes showed increases in TNF

a

, IL-1b, IL-8, and MCP-1 secretion. These results confirmed that P. acnes can stimulate pro-inflamma-tory mediators and also that it plays an important role in the path-ogenesis of inflammatory acne. All three tested extracts suppressed the secretion of TNF

a

, one of the most important pro-inﬂammatory cytokines, in dose-dependent manners (Fig. 1A). Furthermore, duz-hong and yerba mate extracts also inhibited IL-1b (Fig. 1B) and IL-8

(Fig. 2A) secretion. The duzhong extract also decreased MCP-1

pro-duction, while neither rose nor yerba mate had an effect on MCP-1 release (Fig. 2B). It is worth noting that rose extract inhibited TNF

a

(Fig. 1A), but increased IL-1b (Fig. 1B) and IL-8 (Fig. 2A), secretion.

In acne, the host response to P. acnes can result in the production of pro-inﬂammatory cytokines and contribute to the clinical manifes-tations of the disease. TNF

a

is a pleiotropic cytokine produced by activated macrophages. IL-8 along with other P. acnes-induced che-motactic factors may play important roles in attracting neutrophils to the pilosebaceous unit (Kim et al., 2002). Due to the functional redundancy and pleiotropic effects of inflammatory mediators, it is difficult to identify a single molecule as the best candidate for anti-inflammatory therapeutics. It is a better strategy to inactivate a range of inflammatory mediators not just a single cytokine

Table 1

Minimal inhibition concentrations (MICs) of the methanolic extracts of herbs against Propionibacterium acnes.

Common name Botanical name Part examined MIC (mg/ml) Honeysuckle Lonicera japonica Flowers >4

Jasmine Jasminum sambac Flowers >4

Juhua Chrysanthemum

morifolium

Flowers >4 Lavender Lavandula ofﬁcinalis Flowers >4 Osmanthus Osmanthus fragrans Lour Flowers >4

Rose Rosa damascene Flowers 2

Duzhong Eucommia ulmoides Oliv. Leaves 0.5 Jiaogulan Gynostemma pentaphyllum Leaves >4 Lemongrass Cymbopogon citrates Leaves >4 Yerba mate Ilex paraguariensis Leaves 1

Concentration of TNF α (pg/mL) 0 100 200 300 400 500 * * * * * * * * Concentration of IL-1 β (pg/mL) 0 200 400 600 (-) P. acnes (+) P. acnes 0.01 mg/mL 0.05 mg/mL 0.1 mg/mL P. acnes Duzhong Rose Mate * * * * * * *

(A)

(B)

* DMSO control

Fig. 1. Effects of herbal extracts on the production of pro-inﬂammatory cytokines such as TNFa(A) and IL-1b (B) in heat-killed Propionibacterium acnes-treated THP-1 cells. Data are expressed as the mean ± SD.*p < 0.05 compared to the DMSO vehicle; ( ) control, no treatment of heat-killed P. acnes.

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through blocking activation of common transcription factors such as NF-

j

B involved in their induction. Thus our data suggest that duzhong and yerba mate exhibited anti-inﬂammatory properties and may be regarded as health-beneﬁting substances.

Duzhong leaves contain many phytochemicals, such as poly-phenolics, ﬂavonoids, and triterpines (Kawasaki, Uezono, &

Nakaz-awa, 2000; Tsai et al., 2008). Aucubin isolated from duzhong

showed a protective effect in preventing ultraviolet B (UVB)-induced oxidative stress (Ho et al., 2005a) and had an inhibitory effect on matrix metalloproteinase (MMP)-1 production in UVB-irradiated human ﬁbroblasts (Ho et al., 2005b). The methanol extract of duzhong leaves also stimulated collagen synthesis in an aged-rat model (Li et al., 1998). Natural plant extracts are often added to skin care and cosmetic products. Therefore, duzhong extract or its component might be considered potential agents to use in anti-acne, anti-ageing, and skin care formulae.

Yerba mate tea reduced lipid peroxidation and TNF

a

in mice ex-posed to cigarette smoke and is considered a potential anti-inflam-matory and nutritional resource against cigarette smoke-induced inflammation (Lanzetti et al., 2008). Yerba mate tea-derived com-pounds inhibit proteasome activity, suggesting that it may be used in psoriasis and inflammatory disorders (Arbiser et al., 2005). Although an anti-inflammatory property of yerba mate against P.

acnes was identiﬁed, its dermatologic beneﬁts still need to be

investigated.

Even though we identiﬁed antibacterial and anti-inﬂammatory effects of duzhong and yerba mate extracts against P. acnes, we did not determine their mechanisms of action. P. acnes lipase is recog-nised as one of the major factors in the pathogenesis of acne, being responsible for the hydrolysis of sebum and release of

inflamma-tory compounds (Higaki, 2003). In addition, P. acnes has three separate clusters of genes that encode enzymes involved in extra-cellular polysaccharide biosynthesis, suggesting that it can form an extracellular bioflim matrix (Brüggemann et al., 2004). New data are needed to clarify the antimicrobial and anti-inflammatory properties found in this study by analysing the effects of these se-lected herbal extracts on bacterial morphology, bacterial mem-brane integrity, biofilm formation, and lipase activity of P. acnes.

3.3. In vitro cytotoxicity of herbal extracts using human skin cells

Due to the possibility of introducing herbal ingredients as top-ical agents for acne, the cytotoxicity of these selected extracts was examined in human skin keratinocytes and ﬁbroblasts using

in vitro MTT assays (Fig. 3). The respective TC50values of rose,

duz-hong, and mate against HaCaT keratinocytes were 2.54, 1.74, and 0.82 mg/ml. The respective TC50values of rose, duzhong, and yerba

mate against HS68 fibroblasts were >2.5, 2.49, and 0.80 mg/ml. Rose extracts showed the relatively lowest cytotoxic effects on both skin cells compared to the other tested compounds. Cell via-bility was significantly reduced after exposure of both cell lines to the yerba mate extract at a concentration of 1 mg/ml. Although the two cell types responded similarly, HaCaT keratinocytes were more sensitive than HS68 fibroblasts to these compounds. Toxicity data reported by this study can potentially be used to assess the human topical risk exposure to these selected herbal extracts. Further studies are needed to clarify the risk of these materials as well as the suitability of their applications for human use.

Concentraion of IL-8 (ng/mL) 0 5 10 15 20 25 30 (-) P. acnes (+) P. acnes 0.01 mg /mL 0.05 mg /mL 0.1 mg /mL * * * * * Concentrations of MCP-1 (ng/mL) 0 5 10 15 20 25 * Duzhong

DMSO Rose Mate

* P. acnes *

(A)

(B)

control

Fig. 2. Effects of herbal extracts on the production of pro-inﬂammatory cytokines such as IL-8 (A) and MCP-1 (B) in heat-killed Propionibacterium acnes-treated THP-1 cells. Data are expressed as the mean ± SD.*

p < 0.05 compared to the DMSO vehicle; ( ) control, no treatment of heat-killed P. acnes.

Cell viability (% of control)

0 20 40 60 80 100 120 Rose Duzhong Mate

(A) HaCaT cells

Concentration (mg/mL)

0.0 0.5 1.0 1.5 2.0 2.5

Cell viability (% of control)

0 20 40 60 80 100

(B) HS 68 cells

Fig. 3. In vitro cytotoxicity of herbal extracts against human HaCaT keratinocytes (A) and HS68 ﬁbroblasts (B). HaCaT or HS68 cells were cultured for 24 h in medium with the indicated contractions of herbal extracts. The cellular cytotoxicity was determined according to an MTT assay, and results are expressed as the mean ± SD from three independent experiments.

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4. Conclusions

The above results suggest that duzhong and yerba mate may be useful in the treatment of acne vulgaris. Although the antibacterial and anti-inflammatory effects of duzhong and yerba mate extracts against P. acnes were demonstrated, their mechanism remains un-known. Further study is needed to clarify the active constituents and their possible inhibitory mechanisms against pro-inflamma-tory cytokines. Cosmeceuticals and nutraceuticals are growing areas of interest and controversy. Developing new active botanical extracts and compounds to provide dietary supplements and cos-metics as anti-acne agents is still a field with great potential. Acknowledgements

This work was supported by research grants from the National Taiwan Normal University (Contract No. 96C04) and the National Science Council, Taipei, Taiwan (NSC97-2320-B-003-005-MY3). References

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