Microbiology and Biotechnology Letters (한국미생물·생명공학회지)

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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The Beneficial Effects of Extract of Pinus densiflora Needles on Skin Health

솔잎추출물의 피부건강 개선효과

  • Choi, Jieun (Department of Life Science & BK21-Plus Research Team for Bioactive Control Technology, Chosun University) ;
  • Kim, Woong (Department of Life Science & BK21-Plus Research Team for Bioactive Control Technology, Chosun University) ;
  • Park, Jaeyoung (Department of Life Science & BK21-Plus Research Team for Bioactive Control Technology, Chosun University) ;
  • Cheong, Hyeonsook (Department of Life Science & BK21-Plus Research Team for Bioactive Control Technology, Chosun University)
  • 최지은 (조선대학교 자연과학대학 생명과학과) ;
  • 김웅 (조선대학교 자연과학대학 생명과학과) ;
  • 박재영 (조선대학교 자연과학대학 생명과학과) ;
  • 정현숙 (조선대학교 자연과학대학 생명과학과)
  • Received : 2016.03.24
  • Accepted : 2016.04.11
  • Published : 2016.06.28
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Abstract

Pinus densiflora Sieb. et Zucc. (P. densiflora) contains several phenolic compounds that exhibit biological activities, such as antimicrobial, antioxidant, and antihypertensive effects. However, the anti-inflammatory effect of P. densiflora on skin has rarely been reported. Malassezia furfur (M. furfur) is a commensal microbe that induces skin inflammation and is associated with several chronic disorders, such as dandruff, seborrheic dermatitis, papillomatosis, and sepsis. The aim of our study was to identify the anti-inflammatory effects of P. densiflora needle extracts on skin health subjected to M. furfur-induced inflammation. The methanolic extract of the pine needles was partitioned into n-hexane, EtOAc, n-BuOH, and water layers. We measured the anti-inflammatory effects (in macrophages) as well as the antioxidant, antifungal, and tyrosinase inhibitory activity of each of these layers. The antioxidant activity of the individual layers was in the order EtOAc layer > n-BuOH layer > water layer. Only the n-BuOH, EtOAc, and n-hexane layers showed antifungal activity. Additionally, all the layers possessed tyrosinase inhibition activity similar to that of ascorbic acid, which is used as a commercial control. The EtOAc layer was not cytotoxic toward the RAW 264.7 cell line. Interleukin 1 beta and tumor necrosis factor (TNF)-α expression levels in M. furfur-stimulated RAW 264.7 cells treated with the EtOAc layer were decreased markedly compared to those in cells treated with the other layers. Taken together, we believe that the needle extracts of P. densiflora have potential application as alternative anti-inflammatory agents or cosmetic material for skin health improvement.

솔잎은 동의보감과 향약생약대사전에 간, 위, 심, 피부 등 질환 치료에 효과가 있다고 하여 예로부터 솔잎을 노화방지와 피부질환 치료에 활용하여 왔다. Malassezia furfur (M. furfur)는 친유성 곰팡이로 인간 피부에 존재하는 흔한 균이다. 이 균은 대게 피부의 각질층에서 주로 발견되며, 어우러기, 지루성 피부염, 비듬, 모낭염, 건성 등의 각종 피부질환의 주요요인이다. 본 연구는 사람의 대부분의 염증실험에서 사용되는 Raw 264.7 세포를 이용하여 솔잎의 항염증 활성을 확인하였다. 솔잎을 에탄올, 물, 메탄올로 추출하여 항산화 효과를 확인한 결과, 메탄올 추출물에서 항산화능이 높게 나타났으며, 이에 따라 헥산, 에틸아세테이트, 부탄올, 물 층으로 나누었다. 이 4층을 이용하여 노화의 원인이 되는 자유라디칼의 소거능을 확인한 결과, 부탄올과 에틸아세테이트 층에서 강한 항산화 활성이 나타났으며, M. furfur에 대한 항균활성을 확인한 결과 부탄올, 에틸아세테이트층과 헥산층에서 항균활성이 나타났다. 또한 티로시나아제 저해에 따른 미백활성은 4층 모두 대조군인 비타민C와 유사하게 나타났다. 세포실험을 위해 먼저 대식세포인 Raw 264.7에 솔잎 분획물을 처리하여 세포독성을 확인 한 결과 Hexane층에서 농도별로 높은 독성이 나타났으며 나머지 층에서는 독성이 나타나지 않음을 확인하였다. 이후 Malassezia furfur로 염증반응이 유도된 Raw 264.7에 에틸아세테이트, 부탄올, 물층을 처리하여 염증에 관련된 유전자들의 발현량을 Real-Time PCR을 통하여 확인하였다. 이때 사용된 유전자는 IL-1β, TNF-α로 에틸아세테이트층이 염증과 깊은 연관이 있는 IL-1β와 TNF-α의 발현량을 낮추는 것으로 확인되었다. 모든 실험결과들을 종합해 보았을 때, 솔잎은 천연자원으로써 피부 건강과 관련된 건강식품, 건강보조제, 화장품 등 폭 넓은 범위로 사용될 수 있을 것으로 사료된다.

Keywords

Introduction

Natural products isolated from plants are used as important resources for medical treatment or health improvement [37]. The natural habit at of Pinus densiflora Sieb. et Zucc. (red pine) are Korea, Japan, China, and southeastern Russia, various parts of red pine are used as supplementary health food to improve health [39]. Also, the leaves of red pine are consumed as traditional medicine for liver diseases, skin diseases, and hypertension [19, 22]. Volatile chemicals of red pine possess antioxidant activity and growth inhibition activities against human intestinal bacteria [13, 29].

The skin is the primary barrier between the body and the external environment and defenses against external factors such as microbial and chemical agents [18]. Oxidative stress disrupts the protective function of skin and causes roughness and wrinkling [6]. The use of antioxidants is an effective treatment to prevent symptoms associated with the aging of skin [24].

Common skin diseases associated with inflammation such as pityriasis versicolor are caused by a fungus of the genus Malassezia [12, 25]. Among the Malassezia species, Malassezia furfur is associated with pityriasis versicolor [15], folliculitis [1], seborrheic dermatitis [2], atopic dermatitis [38], and psoriasis [31]. At high concentrations, this yeast can diminish the normal protective function of skin and affect the body’s ability to regulate inflammation [33]. As such, the goals of treatment are to regulate M. furfur’s growth and associated inflammation, and to prevent the outbreak of secondary infections [9, 11].

In the inflammatory response, macrophages recognize the infection and the secreted several pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin(IL)-1β, IL-4, IL-6, and IL-8 induces inflammation response [27]. Among them, TNF-α and IL-1β are important factor to chronic inflammatory disorders [3]. Kesavan [17] investigated the influence of the Malassezia on the production of IL-1, IL-6, and TNF-α by peripheral blood mononuclear cells (PBMC). Of these, TNF-α plays a particularly important role in skin diseases related to inflammation [21, 36]. Inflammatory responses to TNF-α are controlled through expression of IL-1, as well as via more pro-inflammatory cytokines [34]. The natural product research has developed a variety of therapeutic agents that are proven to be therapeutically effective against a wide range of diseases [23]. Current interest has led to the search for novel natural products with anti-inflammatory activity [40].

In this study, layers with potential anti-inflammatory action were isolated from the methanolic extract of P. densiflora needles. Subsequently, we confirmed their various functional activities relating to skin health: antifungal activity against M. furfur (causative factor of seborrheic dermatitis), antioxidant activity directed against aging [24], and tyrosinase inhibition activity associated with the whitening of skin color [35]. Cytotoxicity and anti-inflammatory activity were confirmed from the measurement of IL-1β and TNF-α expression levels in M. furfur-activated macrophages.

 

Materials and Methods

Preparation of needle extracts from Pinus densiflora

Fresh needles of red pine were picked in Gokseong province, Jeollanam-Do, South Korea. Harvested needles of red pine were washed clean with tap water (cleaned with 5% charcoal) and dehydrated using a spindrier. The dehydrated pine needles (100 kg) were dried in the shade. The dried pine needles (49.97 kg) were treated with 500 L of 80% methanol (MeOH) at 69℃ for 3 h. The resultantmethanolic extracts were concentrated to 20 L. This crude extract was partitioned successively to yield layers of n-hexane (3 × 10 L), EtOAc (3 × 10 L), n-BuOH (3 × 10 L) and water [28].

2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay

The DPPH assay was performed according to a method described by Nenadis and Tsimidou [26] with some modifications. Briefly, a methanolic DPPH solution (0.04 mM; 180 μl) was added to 20 µl of different concentrations of extracts and fractions. The mixture was shaken vigorously and a decrease in absorbance was measured at 517 nm after 30 min of incubation in the dark. The blank solution contained the same amount of DPPH reagent and 20 µl of methanol and each test was performed in triplicate. The percentage of DPPH inhibition was calculated as follows:

DPPH = (1 − As/Ac) × 100

Where Ac and As are the absorbance of the control and test samples, respectively. Ascorbic acid was used as the reference.

M. furfur and Raw 264.7 culture conditions

M. furfur strain KCTC7546 was used in all experiments. It was cultured either on modified YPD plates or liquid medium supplemented with Tween 80 (1% yeast extract, 1% peptone, 2% glucose, 1% Tween 80) for 1 day at 30℃ under aerobic conditions. The conditions were based on published information [7] regarding nutrients and trace components that promote the growth of M. furfur.

Mouse lymphoid microvascular epithelium immortalized cell line, Raw 264.7, was obtained from the ATCC (TIB-71). Raw 264.7 was grown in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS), 2 mM glutamine, and 100 μg/ml penicillinstreptomycin at 5% CO2 and 37℃ humidified atmosphere.

Antifungal activity against M. furfur

Once grown, M. furfur in liquid medium was harvested from a 1 ml culture and suspended in 100 µl PBS. The suspended solutions were plated onto modified YPD agar medium using a sterile spreader. Plant extracts were dissolved in dimethyl sulfoxide (DMSO) to a concentration of 100 mg/ml. Needle extract of P. densiflora (50 µl) and control respectively were impregnated into the sterile paper discs (8 mm diameter) and incubated at 30℃ for 1 day. M. furfur was visualized and images of the plates were captured. The presence of a clear zone around the discs indicated the sensitivity of M. furfur to the P. densiflora samples. The total diameter of each clear zone was measured.

Tyrosinase inhibition analysis

The activity of tyrosinase was determined by following the L-DOPA assay protocol [15]. The analysis used 1 L of the reaction mixture, which was composed of 1 mM sodium phosphate buffer (pH 6.8, 1 M Na2HPO4, 1 M NaH2PO4), 10 mM of L-DOPA and 200 unit/ml tyrosinase. Ascorbic acid was used as the positive control.

Cytotoxicity analysis

3-[4,5-Dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide (MTT) assay was performed using the method previously described by Boncler et al. [4]. Raw 264.7 cells were seeded at 1 × 105 cells per well in a 96-well plate and these cells were grown in a humidified atmosphere of 5% CO2 at 37℃. The cells were then exposed to varying concentrations of the pine needle extract (0 to 200 μg/ml) for 24 h. After that, 100 µl of fresh medium was added to the cells. MTT solution (5 mg/ml of PBS) was added and the plate was placed in an optimal atmosphere at 37℃ to allow the metabolically active cells to reduce MTT to blue formazan crystals. After 4 h, MTT-formazan crystals were dissolved in 50% ethanol and 50% DMSO and the absorbance was measured at 570 nm on a multifunctional plate reader (Eon, Bio-tek, USA). Comparisons were made with untreated cells.

Determination of IL-1β and TNF-α mRNA expression

Raw 264.7 cells (1 × 105 cells/well) were seeded in a 6-well culture plate containing DMEM. The cells were pre-treated with various concentrations of pine needle extracts (0 to 100 μg/ml) for 2 h and then incubated in the absence or presence of M. furfur for 6 h. After incubation, they were collected by centrifugation and total RNA was isolated from pine needle extract-treated cells using Hybrid-R (GeneAll, Korea) according to the manufacturer's protocol. To synthesize cDNA, 2 μg of total RNA was primed with oligo dT and made to react with Hyperscript mix (GeneAll, Korea). To measure the mRNA level of inflammatory cytokines including IL-1β and TNF-α, we designed primers for the target genes (Table 2). cDNA was amplified and the PCR products were visualized using fluorescent dye on a UV transilluminator. mRNA expression of target genes was analyzed by real-time PCR using SYBR Green (Takara, Japan).

 

Results

Antioxidant activity

The methanolic extract of P. densiflora needles showed stronger antioxidant activity than the water or ethanol extracts. The results of dose-dependent DPPH scavenging activity are presented in Fig. 1. After partitioning the pine needle methanolic extract into n-hexane, EtOAC, n-BuOH, and H2O layers, the antioxidant activity of the individual layers was measured and was found to be in the order EtOAc layer > n-BuOH layer > water layer (Fig. 2).

Fig. 1.DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging activity of P. densiflora extracts. PNW; Pine needle water extraction, PNM; Pine needle methanol extraction, PNE; Pine needle ethanol extraction. Ascorbic acid was used as an antioxidant and an experimental control.

Fig. 2.DPPH radical scavenging activity of various layers from P. densiflora needle methanolic extract. PME; Ethyl acetate layer of Pine needle methanolic extract, PMB; n-utyl alcohol layer of Pine needle methanolic extract, PMH; n-hexane layer of pine needle methanolic extract, PNW; Water layer of pine needle methanolic extract.

Comparison of M. furfur growth in various culture media

For M. furfur culture, variable compositions of liquid and solid medium were tested to achieve optimum conditions. The growth of M. furfur in liquid culture was monitored by measuring OD600. After about 24 h, the organism was incubated on agar plates for another 24 h. The media compositions have been described in Table 1. Medium B and E did not have yeast extract or glucose. M. furfur did not grow well in these media. In liquid Medium A and C containing olive oil, M. furfur grew well, but those cultures were extremely oily to spread over the agar plates. Among the five media formulations, Medium D, composed of yeast extract, peptone, and glucose but without olive oil, provided the best conditions for M. furfur growth in both agar and liquid culture.

Table 1.aM. furfur was cultured in liquid medium for 24 h at 30℃ and the growth of liquid cultured M. furfur were monitored by OD600.bD [5] possessed the optimal conditions for liquid and solid medium to culture M. furfur.

Table 2.Sequence of pro-inflammatory gene primer designs.

Antifungal activity of pine needle extracts against M. furfur

To determine fungicidal activities of pine needle extracts, the extracts were applied at 100 mg/ml onto paper discs, which were then placed on M. furfur culture plates. Fig. 3 shows the inhibitory effect of pine needle extract on M. furfur growth. The presence of a clear zone around the disc (8 mm diameter) indicates growth inhibition. The total diameter of the zone of inhibition was measured for each disc and the results are given in Table 3. DMSO was used as an experimental control and to dissolve pine needle extract. The antifungal activity of its individual layers were observed to increase in the order n-hexane layer > EtOAc layer > n-BuOH layer > water layer (Fig. 3, Table 3).

Fig. 3.Antifungal activity of Pinus densiflora against M. furfur. Antifungal activity of P. densiflora needle methanolic extract layers. PME; Ethyl acetate layer of pine needle methanolic extract, PMB, n-butyl alcohol layer of pine needle methanolic extract, PMH; n-hexane layer of pine needle methanolic extract, PNW; Water layer of pine needle methanolic extract. DMSO was used as solvent of P. densiflora.

Table 3.Diameter of inhibition zones caused by P. densiflora needle methanolic extract layers (100 mg/ml) in M. furfur culture plates.

Tyrosinase inhibition activity of pine needle extract

The tyrosinase inhibition activity of the pine needle extract is shown in Fig. 4. All the partitioned layers of the methanolic extract revealed significant tyrosinase inhibitory activity, comparable to that exhibited by ascorbic acid. The layers revealed dose-dependent activity, and the water layer showed the strongest tyrosinase inhibition (Fig. 4).

Fig. 4.Tyrosinase inhibition activity of P. densiflora needle methanolic extract layers. PME; Ethyl acetate layer of Pine needle methanolic extract, PMB; n-butyl alcohol layer of pine needle methanolic extract, PMH; n-hexane layer of pine needle methanolic extract, PNW; Water layer of pine needle methanolic extract. All layers showed dose-dependent tyrosinase inhibition activity similar to that exhibited by ascorbic acid.

Cytotoxicity of pine needle extract in Raw 264.7 cell line

The cytotoxic effects of pine needle extract at molecular and cellular levels were investigated in Raw 264.7 cultured cells via the MTT assay. The results indicated that at concentrations of 25 to 100 μg/ml, the n-hexane layer of the methanolic extract of pine needles displayed cytotoxicity while the EtOAc, n-BuOH, and water layers were not cytotoxic (Fig. 5).

Fig. 5.Viability of RAW 264.7 cells upon exposure to P. densiflora needle methanolic extract layers. All layers were applied to the cells for 24 h and cell viability was assessed by MTT assay as described in the text. n-hexane layer and 200 μg/ml of EtOAc layer were cytotoxic and were excluded from the following experiments.

Anti-inflammatory activity of pine needle extract

IL-1β and TNF-α are pro-inflammatory cytokines that are released from macrophages upon exposure to M. furfur or other inflammatory conditions. Macrophages were infected by M. furfur (1:30) for 20 hours in the presence or absence of methanolic extract of pine needles, including the EtOAc, n-BuOH and water layers which had no cytotoxicity (100 μg/ml pretreatment for 2 h). Total RNA was then extracted. The effect of the three layers on IL-1β and TNF-α mRNA expression levels in M. furfur-induced macrophages was measured by real-time PCR and RT-PCR using mouse IL-1β- and TNF-α-specific sense and antisense primers, as shown in Table 2. The relative intensity of each mRNA quantified was normalized against the mRNA expression of human β-actin. The expression levels of IL-1β and TNF-α increased in M. furfur-treated macrophages, which concurs with previously published data [16]. However, as shown Fig. 6A, when macrophages were treated with M. furfur plus EtOAc layer, the EtOAc layer significantly downregulated the M. furfur-induced IL-1β mRNA expression in macrophages. Accordingly, the results of RT-PCR also showed that pre-treatment with pine needle extract strongly inhibited M. furfur-induced production of IL-1β in macrophages (Fig. 6B). Moreover, we determined that the EtOAc layer significantly inhibited M. furfur-induced TNF-α mRNA expression level in macrophages (Fig. 7A). Correspondingly, as shown in Fig. 7B, RT-PCR findings indicate that the M. furfur-induced increase in expression levels of TNF-α is attenuated by EtOAc layer of pine needle extract among the other layers. Then, Macrophages were infected by M. furfur (1:30) for 20 hours in the presence or absence of methanolic extract of pine needles, including the EtOAc layer (0−100 μg/ml pretreatment for 2 h). Total RNA was then extracted. The effect of the EtOAc layer on IL-1β and TNF-α mRNA expression levels in M. furfur-induced macrophages was measured by real-time PCR and RT-PCR. As shown in Fig. 8A, when macrophages were treated with M. furfur plus EtOAc layer, the EtOAc layer significantly downregulated the M. furfur-induced IL-1β mRNA expression in macrophages. Accordingly, the results of RT-PCR also showed that pre-treatment with pine needle extract inhibited M. furfur-induced production of IL-1β in macrophages (Fig. 8B). Moreover, we determined that the EtOAc layer slightly inhibited M. furfur-induced TNF-α mRNA expression level in macrophages (Fig. 9A). Correspondingly, as shown in Fig. 9B, RT-PCR findings indicate that the M. furfur-induced increase in expression levels of TNF-α is slightly attenuated by EtOAc layer of pine needle extract in a dose-dependent manner.

Fig. 6.Inhibition of M. furfur-induced IL-1β expression by layers from P. densiflora needle methanolic extract. (A) The mRNA expression level of inflammatory genes was determined by real-time PCR. Treatment of EtOAc layer of P. densiflora strongly reduced mRNA expression level of IL-1β, (B) Treatment of EtOAc layer of P. densiflora strongly reduced mRNA expression level of IL-1β.

Fig. 7.Inhibition of M. furfur-induced TNF-α expression by layers from P. densiflora needle methanolic extract. (A) Treatment of EtOAc layer of P. densiflora strongly decreased mRNA expression level of TNF-α, (B) Treatment of EtOAc layer of P. densiflora strongly decreased mRNA expression level of TNF-α.

Fig. 8.Inhibition of M. furfur-induced IL-1β expression by EtOAc layer from P. densiflora needle methanolic extract. (A) Treatment of EtOAc layer of P. densiflora dose-dependently reduced mRNA expression level of IL-1β, (B) Treatment of EtOAc layer of P. densiflora dose-dependently reduced mRNA expression level of IL-1β.

Fig. 9.Inhibition of M. furfur-induced TNF-α expression by EtOAc layer from P. densiflora needle methanolic extract. (A) Treatment of EtOAc layer of P. densiflora slightly decreased mRNA expression level of TNF-α, (B) Treatment of EtOAc layer of P. densiflora slightly decreased mRNA expression level of TNF-α.

 

Discussion

The purpose of this study was to evaluate the effects of P. densiflora on skin health and its anti-inflammatory activity. A wide range of diseases are caused by oxidative stress. Accelerated cell oxidation even leads to wrinkled skin [8]. In recent years, there has been increasing interest in examining natural antioxidants that can protect skin against oxidative stress. In this study, we confirm the antioxidant activity of P. densiflora needles, specifically themethanolic extract. We partitioned four layers from the extract and showed that the BuOH and EtOAc layerspossess the strongest antioxidant activity. This was in contrast to the findings that the highest scavenging effects are exhibited by the water extract from P. densiflora needlesin a study by Park et al. [29]. The discrepancy may be attributed to the differences in the extraction method.

The skin is colonized by over 500 commensal microbial species estimated to form over 95% [32]. Malassezia, particularly M. furfur, a saprophyte occurring widely on human skin, are generally regarded as the causative agents of a number of common dermatological disorders relate to inflammation [20]. In this study, antifungal activity against M. furfur was investigated via the clear zone test using paper discs and n-BuOH, EtOAc, and n-hexane layers of P. densifloraneedle methanolic extract were shown to possess fungicidal activity.

Tyrosinase is the enzyme responsible for transfer of the substrate tyrosine into melanin by melanocytes [10]. Inhibition of tyrosinase can lead to reduce or no melanin synthesis and whitening of skin. In our study, all layers of the methanolic extract were found to exhibit tyrosinase inhibition activity.

IL-1β and TNF-α are multi-functional cytokines with widely overlapping activities. These inflammatory cytokines have a central role in the pathology of chronic inflammatory diseases [30] through their regulation of the immune response to inflammatory stimuli. The macrophage is one of the major cell types in inflammatory response and influences many chronic inflammatory diseases. IL-1β and TNF-α expression levels in M. furfur-treated macrophages, which concurs with previously published data [16]. Thus, it is of therapeutic interest to develop an efficient strategy using P. densiflora extract to down-regulate the expression of M. furfur-induced pro-inflammatory cytokines. The mRNA expression levels of IL-1β and TNF-α in M. furfur-stimulated RAW 264.7 cells decreased with increasing concentrations of EtOAc layerin the concentration range of 0–100 µg/ml. We clearly demonstrated significant suppression of IL-1β levels and a slight attenuation of TNF-α levels by the EtOAc layer in M. furfur-induced RAW264.7 murine macrophages.

In conclusion, the EtOAc and n-BuOH layer of P. densiflora had antioxidant activity and the various layers of the methanolic extract were found to have antifungal activity against the skin pathogen M. furfur. Additionally, all layers of the extract had tyrosinase inhibition activity. We have also shown that the EtOAc layer partitioned from the methanolic extract of P. densiflora pine needles has dose-dependent anti-inflammatory activity in M. furfur-stimulated Raw 264.7 cells through the down-regulation of IL-1β expression levels. Thus, it can be inferred that P. densiflora needles and their components have the potential to be used as alternative anti-inflammatory agents and cosmetic materials for skin health improvement.

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