Journal of Ethnopharmacology 154 (2014) 408–418

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Prevention of UV radiation-induced cutaneous photoaging in mice by topical administration of patchouli oil Rong-Feng Lin 1, Xue-Xuan Feng 1, Chu-Wen Li 1, Xiao-Jun Zhang, Xiu-Ting Yu, Jiu-Yao Zhou, Xie Zhang, You-Liang Xie, Zi-Ren Su n, Janis Ya-Xian Zhan n School of Chinese Materia Medica, Guangzhou University of Chinese Medicine, Guangzhou, People's Republic of China

art ic l e i nf o

a b s t r a c t

Article history: Received 31 January 2014 Received in revised form 20 March 2014 Accepted 9 April 2014 Available online 18 April 2014

Ethnopharmacological relevance: Pogostemon cablin has been widely used in traditional Chinese medicine for the treatment of many diseases, including skin disorders. In the skin beauty and care prescriptions, Pogostemon cablin is one of the top ten frequently used traditional Chinese medicines. Aim of the study: The present study was aimed to investigate the protective effects of the essential oil of Pogostemon cablin (patchouli oil, PO) against UV-induced skin photoaging in mice. Materials and methods: To ensure the quality of PO, the chemical compositions of PO were identified, and the content of its chemical marker patchouli alcohol was determined, which was around 28.2% (g/g) in PO. During the experiment period, the dorsal depilated skin of mice was treated with PO for two hours prior to UV irradiation. Then the protective effects of PO on UV-induced skin photoaging were determined by macroscopic and histological evaluations, skin elastic test, collagen content determination and biochemical assays of malondiaidehyde (MDA) content, activities of anti-oxidative indicators including superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT). Results: Compared to UV exposure groups, present results showed that topical administration of PO, especially at dose of 6 mg/mouse and 9 mg/mouse, significantly inhibited the increase in skin wrinkle formation, alleviated the reduction in skin elasticity and increased the collagen content by about 21.9% and 26.3%, respectively. We also found that application of 6–9 mg/mouse PO could not only decrease the epidermal thickness by about 32.6%, but also prevent the UV-induced disruption of collagen fibers and elastic fibers. Furthermore, the content of MDA was decreased by almost 26.5% and activities of SOD, GSH-Px and CAT were significantly up-regulated after the treatment of PO. Conclusion: Results of present study revealed that PO was capable of maintaining skin structural integrity caused by UV irradiation and it was useful in preventing photoaging. These protective effects of PO were possibly due to its anti-oxidative property. Therefore, we suggested that PO should be viewed as a potential therapeutic agent for preventing photoaging. & 2014 Elsevier Ireland Ltd. All rights reserved.

Keywords: Skin photoaging Ultraviolet rays Oxidative damage Patchouli oil Antioxidant

1. Introduction Photoaging is a process of premature aging of the skin attributed to continuous and long-term exposure to solar ultraviolet (UV) radiation including UVA (400–320 nm) and UVB (320– 280 nm) (Fisher et al., 2002), which is clinically characterized by sunburn, coarse wrinkling, loss of elasticity and actinic keratosis.

n Correspondence to: School of Chinese Materia Medica, Guangzhou University of Chinese Medicine, 232 Wai Huan Dong Road, Guangzhou Higher Education Mega Center, Guangzhou 510006, People's Republic of China. Tel.: þ 86 20 3935 8517; fax: þ86 20 3935 6390. E-mail addresses: [email protected] (Z.-R. Su), [email protected] (J.-X. Zhan). 1 Rong-Feng Lin, Xue-Xuan Feng and Chu-Wen Li contributed equally to this work.

http://dx.doi.org/10.1016/j.jep.2014.04.020 0378-8741/& 2014 Elsevier Ireland Ltd. All rights reserved.

In particular, chronic exposure to UV radiation results in prominent histological changes in the extracellular matrix (ECM) of the connective tissue, reflecting a clinically photoaged skin with disintegration of elastic fibers and excessive deposition of abnormal collagen (Yaar and Gilchrest, 2007). The unifying pathogenic agent for these changes is UV-generated high level of reactive oxygen species (ROS) (Yaar and Gilchrest, 2007). The dramatically increased ROS will not only induce lipid peroxidation in fibroblasts cells and trigger a cascade of signal transduction pathways (Pillai et al., 2005; Ngo et al., 2011), but also deplete skin's antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT) and glutathion peroxidase (GSH-Px) (Pillai et al., 2005). If these enzymes are overwhelmed, there will be extra free radicals that can stimulate generation of matrix metalloproteinases, suppress collagen gene expression, and consequently lead abnormal matrix degradation, which eventually result in sagging and wrinkle formation

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(Pillai et al., 2005; Kim et al., 2009). Hence, according to the understanding that enzymatic antioxidant defense systems may regulate ROS and scavenge free radicals, treatment with antioxidants is believed to be an effective approach to prevent skin from photo damage. Herba Pogostemonis is the dried aerial of Pogostemon cablin (Blanco) Benth. (Labiatae), which is native to tropical regions of Asia and is now extensively cultivated in China, Indonesia, India and Malaysia (Liu et al., 2009). It is commonly used as traditional Chinese medicine for centuries not only to cure exogenous fever, hypertension, diabetes and diarrhea, but also to cure facial diseases according to the ancient Chinese medical books such as yv yao yuan fang (YuuKo, 2009; Li et al., 2011, 2012; Xian et al., 2011). Importantly, in the skin beauty and care prescriptions, Pogostemon cablin is one of the top ten frequently used traditional Chinese medicines (YuuKo, 2009). The dry leaves of Pogostemon cablin on hydrodistillation give an essential oil known as patchouli oil (PO). To date, PO becomes one of the most important ingredients of cosmetic products due to its herbaceous notes and fixative properties. Pharmacological studies have showed that PO contains various bioactive components to exhibit anti-allergic and anti-acne activities, antibacterial activity on skin, as well as antioxidative and anti-inflammatory effects (DePo Yang, 1998, 2000; Liu et al., 2009; Kim et al., 2010; Xian et al., 2011; Li et al., 2012). Wei and Shibamoto have reported that PO inhibited the oxidation of hexanal to hexanoic acid and scavenged DPPH free radicals (Wei and Shibamoto, 2007). Kim and Cho et al. have also revealed that PO could effectively protect human neuroglioma cell line A172 against both the necrotic and apoptotic cell death induced by ROS as a powerful ROS scavenger (Kim et al., 2010). Chemically, PO mainly possesses terpenoids. Among them, sesquiterpenes compounds can block the signaling pathways involved in oxidative stress so that protect cell components from the harmful actions of free radicals (Murakami, 2009; Ghantous et al., 2010; Manoj et al., 2012). However, to the best of our knowledges, no previous studies have proved the anti-oxidant properties of PO against UV-caused photoaging. The present study aimed to investigate whether topically applied with PO could alleviate UV-induced phothoaging by macroscopic, histological and collagen content evaluations, and to determine whether PO could attenuate oxidative stress by testing MDA content and activities of various oxidative indicators such as SOD, CAT and GSH-Px.

2. Materials and methods 2.1. Materials and chemicals Patchouli oil (PO), was purchased from Nanhai Zhongnan Co., Ltd. (Lot 121101, Foshan, China). PO was dissolved in ethanolpropylene glycol (2:8, v/v) to yield three different concentrations: 25 mg/ml, 50 mg/ml and 100 mg/ml. Commercial kits used for determination of SOD, GSH-Px, CAT, and malondialdehyde (MDA), as well as the mouse hydroxyproline (Hyp) assay kit were purchased from Jiancheng institution of Biotechnology (Nanjing, China). All other chemicals and reagents used in this study were analytical grade. 2.2. Qualitative and quantitative analyses of PO For the quality control of PO, the content of a chemical markerpatchouli alcohol was determined using Varian-3900 gas chromatography–flame ionization (GC–FID) instrument (Varian Corporation, Palo Alto, CA, USA). The analytical conditions described in Pharmacopoeia of the People's Republic of China were applied: samples were

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diluted with n-hexane (10 mg/ml) prior to injection. Helium was used as carrier gas at a constant flow rate of 1 ml/min with split ratio 10:1. DB-5 ms, 30 m  0.32 mm ID  0.25 μm capillary column (Agilent Corporation, Santa Clara, CA, USA) was used. Oven temperature program was initiated at 180 1C, held for 10 min then raised at 5 1C/ min to 230 1C, held for 3 min with injector temperature 280 1C and detector temperature 280 1C. Octadecane solution (1.5 mg/ml) was used as the internal standard (China Pharmacopoeia Committee, 2010). Further, the chemical compositions of PO were analyzed by a gas chromatography with mass spectrometry detector (GC–MS). It was performed on Agilent 6890-5975 model gas chromatograph– mass spectrometer (Agilent Corporation, Santa Clara, CA, USA) and fitted with a non-polar column of HP-5MS (30 m  0.25 mm ID  0.25 μm). The essential oil samples were diluted with nhexane at a ratio of 1:6 prior to injection. The carrier gas was helium at a constant flow rate of 1 ml/min with split ratio 60:1; and the injector temperature was 280 1C. The temperature was programmed from 140 1C to 160 1C at a rate of 1 1C/min, and then raised to 230 1C at 5 1C/min. Thereafter, the conditions were held for 20 min. The mass spectrometer measurement was scanned from 50 to 400 m/z. The ionization source temperature was 280 1C. The injection volume was 1 μl. Individual compounds were identified through comparison of substance mass spectrum with the NIST database.

2.3. Grouping of animals Sixty three female Kunming mice (20–22 g) were obtained from the animal center of Guangzhou University of Chinese Medicine (Guangzhou, China). All animals received humane care in accordance with the Guide for the Care and Use of Laboratory Animals, published by the US National Institution of Health. All experimental protocols were approved by the Committee for Animal Care and Use at Guangzhou University of Chinese Medicine with reference to the European Community guidelines and the regulations of the National Institute of Health of USA (Approval number SCXK (Guangzhou)-2008–0020). The mice were maintained under a natural light/dark cycle and housed in a room with controlled temperature (23 72 1C) and humidity (50% 710%). The mice were fed with food and water ad libitum and were acclimatized for one week before the experiment. At the beginning of the experiment, mice were randomly divided into seven groups with nine mice each (Table 1). Dorsal skins were shaved with lady shavers (Philipss) for 2.5  3 cm2 after the mice were anesthetized by ether inhalation. Thereafter, the shaving was performed as required.

Table1 Treament schedule of the study. Group

Shave UV radiation Vehicle PO (mg/mouse) 120 μl/mouse 3 6 9

Naive Control (NC) Sham Control (SC) Model Control (MC) Vehicle Control (VC) PO Low Dose (PO-L) PO Middle Dose (PO-M) PO High Dose (PO-H)

 þ þ þ þ þ þ

 : without treatment. þ : with treatment.

  þ þ þ þ þ

   þ þ þ þ

    þ  

     þ 

      þ

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2.4. UV-irradiation and PO treatment In order to establish the photoaging model, simulated solar irradiation was provided by an array of seven UVB lamps, with an emission spectrum between 285 and 350 nm (peak at 310– 315 nm), surrounding with three UVA lamps (Waldmann UV800, Germany) emitting exclusively UVA in the range of 320–400 nm (peak at 365 nm). The ratio of UVA: UVB was measured by a Waldmann UV meter (Waldmann Lichttechnik GmbH, Germany) and was about 9.3:0.7. Mice in the flat and round cages were irradiated under the UV lamp keeping the distance at 30 cm. The frequency of exposure was set at four times a week (except Monday, Wednesday and Friday) for ten weeks. The integrated UV irradiance was measured by a UV meter, and the minimal erythemal dose (MED) was 70 mJ/cm2 in our research. The intensities of UV were increased by 1 MED per week until the fourth week, and then 4 MED kept constant for the remaining period of exposure. The total radiation dose was about 9.52 J/cm2 (Kim et al., 2009; Ngo et al., 2011). In two hours before UV irradiation, the shaved dorsal skins of mice were applied topically with PO (3, 6 and 9 mg/mouse) or vehicle (120 μl/mouse) every day for 10 weeks. Treatment schedule was shown in Table 1.

2.5. Macroscopic evaluation of dorsal skins The UV-exposed dorsal skin of each mouse which was under anesthesia was photographed once a week for 10 weeks. The macroscopic visual scores were accessed according to the grading scale showed in Table 2 by an observer who was blind to the grouping (Kim et al., 2009; Agrawal and Kaur, 2010). 2.6. Skin recovery ability test The Skin recovery ability test (pinch test) was performed weekly using a modified protocol based on the method described by Tsukahara in order to investigate the elasticity of the dorsal skin (Tsukahara et al., 2005; Agrawal and Kaur, 2010). Briefly, the midline of the dorsal skin of the anesthetized mouse was picked up with fingers to a degree that its feet just lightly touched the table (see Fig. 4A-Method). Pinch was subsequently released and the skin recovery time was calculated immediately.

2.7. Histopathology studies The skin samples were excised from the shaved dorsal skin of the mice immediately after they were sacrificed at the end of 10th week. Then samples were fixed with 10% formalin neutral buffered solution for at least 24 h. The representative samples were embedded in paraffin, cut into 5 μm sections and stained with

Table 2 Grading scale for evaluation of photoaging. Grade

Evaluation criteria

0

Smoothness without any wrinkles; fine striations running the length of the body fine striations A few shallow wrinkles; disappearance of all fine striations Shallow wrinkles across the dorsal skin Deep and coarse wrinkles with laxity Increased deep wrinkles Surface accompanied with severe wrinkles; development of lesions

1 2 3 4 5 6

Hematoxylin-Eosin (H&E) staining (Levy and Barlow, 1989; Uhm et al., 2010) for routine histology study. Moreover, the skin specimens were stained using the elastic Gomori's aldehyde fuchsin technique to evaluate the elastic fibers as previously described (Proctor and Horobin, 1983). To quantify epidermal hyperplasia following UV exposure, the thickness of the epidermis was measured at 10 randomly selected locations per slide using an optical microscope (Leica DMLB) with 200  magnification. Each specimen was photographed under a Leica DC 300 camera. Histological alterations were evaluated and quantified through the image analysis program Image J 1.36 (Wayne Rasband, National Institutes of Health, Bethesda, MD) (Manoj et al., 2012). 2.8. Total collagen content determination To study the alterations of collagen content, 100 mg of the shaved dorsal skin was used for the total hydroxyproline (Hyp) assay, and Hyp assay was performed according to the manufacturer's instructions (Jiancheng Inst. of Biotechnology, Nanjing, China). Hyp usually can be converted to the equivalent of collagen through multiplication by the factor 7.46, considering Hyp is the almost exclusive amino acid of collagen and accounts for 13.4 7 0.24% of mammalian collagen as previously described (Neuman and Logan, 1950; Kong et al., 2013).

2.9. Biochemical indexes' assays The shaved dorsal skin (0.4 g) was cut into pieces and homogenized with Ultra Turrax (T18 Basic, IKA) in 9 volumes of cold normal saline at 4 1C to get the 10% skin tissue homogenate. Before centrifugation, 0.15 ml of the skin tissue homogenate was taken out for the MDA assay and the rest was centrifuged at 3000g for 20 min at 4 1C. The total supernatant was used for SOD, GSH-Px and CAT assays. All of the biochemical assays mentioned above were carried out following the manufacturer's protocols of the corresponding diagnostic kits (Nanjing Jiancheng Bioengineering Inst., Nanjing, China).

2.10. Statistical analysis Experimental values were analyzed by one-way ANVOA. All data were expressed as mean 7 standard deviation (SD). A value of po 0.05 was considered to be statistically significant. All analyses were performed using Statistical Analysis Software (SPSS 17.0).

3. Results 3.1. Chemical compositions of patchouli oil To ensure the quality of PO, the content of patchouli alcohol, the major chemical marker of PO, was determined by GC–FID. As illustrated in Fig. 1, the content of patchouli alcohol was 28.2% (g/g), which met the requirements of Pharmacopoeia of the People's Republic of China (26%). Moreover, results of the phytochemical analysis were presented in Table 3 and Fig. 2. A total of 18 constituents were identified in the essential oil constituting 88.3% of the total oil composition. The oil composition mainly consisted of terpenoids, of which the monoterpenes and sesquiterpenes accounted for more than 50% (peak area percentage). Apart from patchouli alcohol (29.3%), the major components were β-gurjunene (17.4%) and β-guaiene (12.7%), and the other minor components

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Fig. 1. (A) GC-FID chromatogram of patchouli alcohol in PO and internal standard (octadecane). (B) GC-FID chromatogram of patchouli alcohol standard substance and internal standard (octadecane).

were β-patchoulene (5.8%), α-patchoulene (5.6%) and Δ-patchoulene (4.3%). 3.2. PO reduced the skin lesions induced by UV radiation The representative photographs of the UV-induced macroscopic skin lesions were showed in Fig. 3A. The mice in the SC group only shaved but not irradiated showed healthy skin with no wrinkles, which demonstrated that the shaving did not cause macroscopic skin lesions. Eight out of nine (88.9%) mice in MC group began to appear wrinkles, erythemas and leathery appearance from the fourth week. Likewise, approximately seven out of nine (77.8%) mice in the VC group exhibited the same situations. However, application with PO at high dose (9 mg/mouse, PO-H group) had a tendency to suppress the UV-induced extensive erythemas and deep wrinkles after six weeks treatment. In addition, the PO-M group (6 mg/ mouse) did not exhibit its inhibited effect on skin wrinkle formation until the eighth week. As shown in Fig. 3A, at the end of this experiment, the mice in the PO-H group showed more smoothness appearance than that in the VC group. And mice in the PO-M group showed no lesions but a few shallow wrinkles judging by Fig. 3A POM. In the PO-L group, six mice (66.67%) showed coarse wrinkles and the other three exhibited slight erythemas. Statistically, Fig. 3B showed that the visual scores of the MC group and the VC group were not different, but both were obviously higher than that in the SC group. However, the scores

Table 3 Chemical compositions of patchouli oil used in present study. Peak no.

Components

Retention time (min)

Relative percentage peaka (% in PO)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

β-Patchoulene β-Elemene cis-Thujopsene Isocaryophyllene β-Gurjunene α-Patchoulene Δ-Patchoulene β-Caryophyllene cis-β-Guaiene γ-Gurjunene β-Guaiene β-Selinene Elemol Globulol trans-Longipinocarveol Caryophyllene oxide Patchouli alcohol Aristolone

19.519 20.074 20.593 21.073 21.909 22.453 22.593 23.412 23.870 24.288 24.584 24.943 26.384 27.224 27.476 29.408 30.709 55.568

5.8 0.7 0.8 2.1 17.4 5.6 4.3 0.4 0.6 2.3 12.7 0.5 0.6 0.4 0.7 0.3 29.3 3.8

a

Relative percentage as internal normalization of total peaks was observed.

of the PO-H group and PO-M group were significantly lower than that of the VC group, indicating that topic application of PO prevented the UV-induced macroscopic skin damages.

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Fig. 2. GC–MS total ion chromatogram (TIC) of the PO. The component labels in the chromatogram correspond to the peak number given in Table 3.

Fig. 3. (A) Physical appearances of UV-irradiated mice with different treatments at the last week. (B) The trend of macroscopic changes reflected in the changes of visual scores was expressed by a histogram. Data represents means 7 SD (n¼9). (#): p o 0.05 compared with the SC group. (n): p o0.05 compared with the VC group.

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Fig. 4. Evaluation of skin elasticity by pinch testing. (A) Photographs of pinch test. Method: the method of pinch test described by Tsukahara. (B) Recovery time was evaluated weekly. Data represents means 7SD (n¼ 9). (#): p o 0.05 compared with the SC group. (n): p o 0.05 compared with the VC group.

3.3. PO recovered the skin elasticity in the pinch test To evaluate the dorsal skin elasticity of the mice with different treatments, pinch test was carried out weekly and the photographs of mouse dorsal skin after being stretched were taken (Fig. 4A). As shown in Fig. 4B, the recovery times of the mice needed in the MC and VC groups were not significantly different, but were significantly longer than that of the SC group. Although the PO-L group did not reduce the recovery time significantly as compared to the VC group, the recovery times of the mice in the PO-M and PO-H groups were significantly shorter than that in the VC group. These results demonstrated that treatment with PO (especially 6–9 mg/mouse) could promote the skin elasticity. 3.4. PO alleviated UV-induced epidermal hyperplasia Epidermal thickness is used as a parameter to directly reflect inhibitory effect of PO on UV-induced epidermal hyperplasia which is thought to cause wrinkles (Pillai et al., 2005). As can be seen in Fig. 5,

epidermal thickness showed no significant difference either between NC and SC groups or between MC and VC groups, but there was an around 3-fold increase of epidermal thickness in the MC group compared with the SC group (po0.05 vs. SC). However, topical application of PO-M and PO-H significantly decreased the thickness to 49.8 μm and 45.5 μm compared with the VC group. Hence, these results together highlighted that PO (6 and 9 mg/mouse) significantly alleviated UV-induced epidermal hyperplasia. 3.5. PO inhibited the abnormal histological alterations Repeated UV exposure to the mice resulted in the skin structure alterations. Based on histological observation by H&E staining of dorsal skins, non-irradiated groups (NC and SC) revealed relatively complete structures (Fig. 6). The epidermis was a stratified squamous epithelium, covered by fine and thin stratum corneum. The dermal–epidermal junction (DEJ) interlocked to form fingerlike projection that supported the adhesion of the dermis to the epidermis (Khavkin and Ellis, 2011; Kong et al.,

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Fig. 5. Application of PO prevented hyperplasia of epidermis. (A) Photographs of epidermal hyperplasia observed by H&E staining (all of the photographs are magnified at 200  ). Epidermal thickness was shown here via the double-headed black arrows. The scale bar represents 50 μm. (B) Histogram accompanied with error bar represents means 7 SD (n ¼6). (#): p o 0.05 compared with the SC group. (n): po 0.05 compared with the VC group.

2013). Moreover, the dermis was tightly connected to the epidermis through a basement membrane and showed organized distribution and uniformed thickness of collagen which stained as pink deposits. Arranged and branched elastic fibers were exhibited clearly in the dermis of the mice in the NC and SC groups (Fig. 7). Deeper dermis contained clusters of sebaceous glands attached to the regular hair follicles. Diffuse inflammatory infiltration could not be observed (Fig. 6NC and SC). After ten weeks of UV-irradiation, the histopathological features of the MC and VC groups were quite similar. Both of them showed prominent epidermal hyperplasia with abnormal keratinization and hyperkeratosis. Beneath the epidermis, a clear flattening of the DEJ paralleling with disappearance of dermal papillae was found in Fig. 6MC (a). Flattening DEJ caused approximately a half decrease in surface contact area causing fragile skin (Khavkin and Ellis, 2011). In the dermis of MC group mice, as well as VC group mice, a large proportion of the collagen fiber bundles showed twisted and disorganized, some of which even showed destruction (Fig. 6MC (b) and VC). Within the papillary dermis, there were masses of tangled and degraded elastic fibers (Fig. 7MC and VC). In addition, as illustrated in Fig. 6MC (b), hemorrhage was observed. This finding was consistent with the previous reports finding that UV-caused lesions could be reflected in the broken micrangium to some extent (Agrawal and Kaur, 2010; Ngo et al., 2011). Such lesions had further invited an inflammatory infiltration with a number of macrophages and leukocytes formed in and underneath the dermis as shown in Fig. 6MC(c).

The skin of the mice treated with 3 mg/mouse PO (PO-L) displayed thickened stratum corneum, epidermis and flattened DEJ. Although they were not as serious as that in the MC and VC groups, the onset of repairs of denatured and fractured collagen and elastic fibers were also found. But diffuse inflammation was ameliorated. However, the application with 6 mg/mouse PO (PO-M) inhibited the abnormal epidermal hyperplasia and showed thinner stratum corneum. In addition, DEJ became wavy as compared to that of the VC group on account of the well-marked appearance of dermal papillae and epidermal rete ridge. On the other hand, collagen and elastic fibers in dermis interwove closely, oriented randomly and embedded with each other. Diffuse inflammation was not present. When applied with 9 mg/mouse PO (PO-H), epidermal thickness was decreased by about 34.9%, compared with that in the VC group. The epidermis was covered by a slightly thickened stratum corneum and the structure of the DEJ returned to near-normal level (Fig. 6). In addition, the upper portion of the dermis showed an order arrangement of hair follicles, the content of collagen was markedly increased and collagen bundles were thickened. Moreover, the accumulation of elongated and threadlike elastic fibers could be visually observed (Fig. 7PO-H) and inflammatory infiltration did not exhibit in the Fig. 6PO-H. 3.6. PO elevated collagen content in photoaged mice Collagen is a major component of animal tissues. The results showed that the collagen content between NC and SC groups did

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Fig. 6. Hematoxylin & Eosin (H&E) staining of mouse skin in: NC (100  ); SC: having the similar features to that in the NC group (200  ); MC (a): showing destroyed structure (200  ); MC (b): tangled and sparse collagen accompanied with hemorrhage (400  ); MC (c): inflammation infiltrate through dermal and subcutaneous tissue (400  ); VC: having the similar characteristic to that in the MC group (200  ); PO-L (200  ); PO-M (200  ); PO-H (200  ). ED, epidermis; DR, dermis; ST, subcutaneous tissue; DEJ, dermal–epidermal junction; SC, stratum corneum; HF, hair follicle; IFI, inflammation infiltration.

not have significant difference. Compared with the SC mice, the MC mice and VC mice showed the same decrease of skin collagen content, which demonstrated that the vehicle solution did not affect the UV-induced decrease of collagen content (Table 4). After treated with 3 mg/mouse PO (PO-L), the collagen content in the UV-irradiated mice skin increased slightly. However, PO treatment at the doses of 6 and 9 mg/mouse significantly enhanced the collagen content by 21.9% and 26.3% respectively as compared to the VC treatment. These results indicated that PO (especially 6 and 9 mg/mouse) alleviated the UV-induced collagen damage. 3.7. PO inhibited UV-induced oxidative stress Activities of antioxidant enzymes are generally considered as parameters to evaluate the antioxidant levels of organism. As illustrated in Table 4, there were no statistically significant differences in SOD, CAT and GSH-Px activities either between NC and SC groups or between MC and VC groups, indicating that depilation and solvent did no bias to these indicators in our study. In comparison with the SC group, activities of SOD, CAT and GSHPx in the MC group were significantly decreased (po 0.05). However, applying with PO-M and PO-H (6 and 9 mg/mouse PO) increased SOD and GSH-Px activities obviously (po 0.05, vs. VC). Meanwhile, PO-L and PO-H significantly enhanced CAT activity by 29.8% and 90.7%, respectively though PO-M did not show significant enhancement. These results revealed that PO could protect activities of antioxidant enzymes to scavenge free radicals and further inhibit UV-induced oxidative stress.

3.8. PO decreased the skin malonaldehyde (MDA) content MDA is the aldehydic decomposition product of lipid oxidation, the content of which can reflect the lipid peroxide level well. Table 4 showed that the MDA contents were similar either between NC and SC groups, or between MC and VC groups. Moreover, upon irradiation of UV, around 3-fold increase of MDA content in the VC group was observed as compared to that in the SC group. However, the increased MDA content was reduced by about 26.5% by topically administration of PO (all p o0.05, vs. VC), especially in the PO-M and PO-H groups. These results indicated that PO could suppress lipid peroxidation of UV-irradiated mice.

4. Discussion Photoaged skin, attributed to chronic UV irradiation, is manifested as the increase in skin thickness, formation of wrinkles and reduction in skin elasticity, which fundamentally associated with reduction in the content of collagen and elastic fibers. Specifically, UV irradiation degrades the collagen and elastic fibers mainly by generating the ROS (Kim et al., 2011; Fan et al., 2013). Thus, strategies to apply with antioxidants are valuable for preventing skin photoaging. In the present study, the anti-photoaging effects of PO were evaluated by assessing macroscopic grade, doing histopathological study and detecting the collagen content, MDA content and activities of SOD, CAT and GSH-Px, which were

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Fig. 7. Gomori's aldehyde fuchsin staining of mouse skin (original magnification 400  ). NC&SC: elastic fibers manifested as purple fine line samples showing integrated and interlaced situation. MC&VC: showing fractured, separated and degraded elastic fibers. EF, elastic fibers. (For interpretation of references to color in this figure legend, the reader is referred to the web version of this article.) Table 4 Effects of PO on biochemical indexes of photoaged mice skin. Group

SOD Ua/mgprot

GSH-Px Ub/mgprot

CAT Uc/mgprot

MDA Nmold/mgprot

Collagen (μg/mg)e

NC SC MC VC PO-L PO-M PO-H

35.167 6.88 32.22 7 7.20 25.187 5.201;# 23.777 4.491;# 26.84 7 6.18 33.887 7.78n 30.42 7 7.05n

656.677 101.08 681.83 7 97.44 340.177 45.171;# 361.377 82.371;# 345.417 48.11 526.117 75.55n 642.537 96.05n

14.81 7 1.25 13.93 7 1.35 6.02 7 0.901;# 5.617 0.921;# 7.28 7 1.16n 6.25 7 0.62 10.707 0.82n

6.87 7 1.23 8.047 1.36 19.99 7 1.831;# 18.98 7 2.001;# 14.95 7 1.78n 17.81 7 1.68n 9.117 1.55n

27.50 7 3.42 24.877 4.38 16.05 7 3.891;# 18.30 7 4.131;# 20.69 7 4.00 22.317 2.69n 23.117 3.06n

Each value represents the mean 7 SD of 9 mice per group. n

significantly different from vehicle control group (np o 0.05). significantly different from sham group (#p o0.05). a One unit of SOD activity was defined as the amount of the enzyme inhibiting the oxidation by 50%. b One unit of glutathione peroxidase is defined as the amount of the enzyme leading 1 μmol GSH oxidized per min. c One unit of catalase activity was defined as the amount of enzyme that reduces 1 μmol of H2O2 per second under defined conditions. d MDA content was expressed as nmol per mg protein. e Collagen content was expressed as μg per mg skin. #

considered as important indexes for oxidative stress evaluation (Kim et al., 2009; Kong et al., 2013). Our results indicated that PO had photo-protective effects in a mouse model, which effectively accelerated the recovery of the macroscopic and histological skin

lesions, prevented collagen and elastic fibers' degradation and elevated the activities of SOD, CAT and GSH-Px. Chronic UV exposure is known to directly damage the skin surface to cause photoaging, which is characterized by an increase in the

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number of wrinkles and a decrease in the resilience (Agrawal and Kaur, 2010; Sayama et al., 2010). According to the previous reports, macroscopic grading and pinch test have had wide application in the skin surface evaluation (Agrawal and Kaur, 2010; Sayama et al., 2010). Comparison of untreated (SC) group and the vehicle (VC) group in our study, VC group showed an increase in skin thickness, sagging of the skin and an decrease in skin elasticity, which were consistent with the previous reports (Oba and Edwards, 2006). However, in comparison with the VC group, the PO treated groups had an appreciable skin repair such as less wrinkling, suggesting that PO could effectively attenuate photo damage of the skin surface. In addition, many recent studies suggested that the wrinkled appearance and the formation of sagging skin mainly resulted from the reduction of collagen and the loss of integrity of elastic fibers in the skin dermis (Kang et al., 2009; Hughes et al., 2011; Khavkin and Ellis, 2011). Our histological data was consistent with the results from the macroscopic analysis. Based on histological observation, continuous UV irradiation induced fragmented collagen fibers as well as decreased the number of elastic fibers and changed elastic fibers into tangled and degraded formation. Collagen content determination also revealed that UV significantly reduced the collagen content. However, PO treatment apparently prevented UV-induced collagen reduction, rebuilt the integrity of collagen structure and remodeled elastic fibers. These findings were in accordance with the results in pinch test and suggested that PO prevented mice skin from wrinkles and sagging, mainly by repairing collagen and elastic fibers and accelerating the collagen synthesis which was further confirmed by Hyp content determination. Accordingly, decreased the density of collagen observed in H&E staining and reduced collagen content detected by Hyp content determination implied that there might be an increased secretion of MMPs since they are directly responsible for the degradation of extracellular matrix (Son et al., 2012; Haarmann-Stemmann et al., 2013). Moreover, MMPs are fundamentally stimulated by UVinduced ROS (Kim et al., 2005; Bickers and Athar, 2006). In the physiological situation, endogenous antioxidant enzymes (SOD, CAT and GSH-Px) will secret sufficiently to catalyze ROS into O2 and H2O (Prasad et al., 2007; Hou et al., 2009). Our investigation showed that when UV irradiation induced the excessive increase of ROS in mice skin, the defense of these antioxidant enzymes was overwhelmed and their activities were decreased. However, topic application with PO could prevent the decrease in enzymes' activities. These results revealed that the anti-photoaging property of PO was due to its protective effects on antioxidant enzymes, by which the ROS level could be decreased. Such antioxidative efficacy of PO in vivo was supported by some reports that various essential oil of pogostemon possessed antioxidant properties, and sesquiterpenoids such as patchouli alcohol in PO also exhibited free radical scavenging activities (Wei and Shibamoto, 2007; Ghantous et al., 2010; Manoj et al., 2012). Furthermore, it has been known that ROS can aggravate lipid peroxidation in membranes, cellular components (i.e. lipids, proteins and DNA), consequently cause serious skin pathologies (Fisher et al., 2002; Masaki, 2010; Kong et al., 2013). MDA is the lipid peroxidation product and usually quantified to identify lipid peroxidation (Pillai et al., 2005; Fan et al., 2013). It was reported that the continuous exposure of the mice skin to UV radiation obviously increased MDA level (Hou et al., 2009), which was also observed in our study. However, topical PO treatment significantly down regulated the MDA level in mice skin. These results reflected that PO suppressed propagation of lipid peroxidation. In addition, Zhuang et al. had reported that inhibition of lipid peroxidation could protect membranes of fibroblast cells, and consequently suppressed the degradation of collagen (Zhuang et al., 2009; Ngo et al., 2011). This statement seems to be in collaboration with respect to results stated above and further testifies the significant protective effect of PO on skin visual appearance.

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Furthermore, taking the results of GC study into consideration, we hypothesized the anti-photoaging activity of PO in the present study could be due to the bioactivities of its chemical compounds. Among 18 identifiable components in PO, patchouli alcohol, guaiene, isocaryophyllene, caryophyllene and selinene were reported to have antioxidant activities. Manoj and Manohar et al. have reported that essential oil of pogostemon paniculatus had DPPH free-radical scavenging activity and this activity was attributed to patchouli alcohol and guaiene because they were the major components (Manoj et al., 2012). Quassinti and Lupidi et al. have also reported cis-β-guaiene, selinene and caryophyllene were the most representative components of essential oil of Hypericum hircinum L. subsp. majus (Aiton) N. Robson which showed significant effect of anti-oxidation (Quassinti et al., 2013). In addition, previous studies have revealed moderate inhibitory activities of caryophyllene against lipid peroxidation and scavenging activities against hydroxyl radical and superoxide anion (Calleja et al., 2013). Hence, compounds in PO, especially sesquiterpene which possesses potential anti-oxidant and anti-inflammatory effects, are crucial for PO's anti-photoaging effect, and the specific chemical compounds responsible for anti-photoaging effect of PO need to be further identified. In summary, this is the first study to comprehensively demonstrate the protective effects of topical administration of PO on UVinduced photoaged mouse skin. Our work gives evidence that PO can be used as potential functional cosmetic products for skin care.

Acknowledgments This work was supported by grants from the Guangdong International Cooperation Projects in 2012, Guangdong Province, PR China (Project no. 2012B050300002), China Postdoctoral Science Foundation (Grant no. 2014M552188) and the Innovation and Entrepreneurship Training Program of College Students of Guangdong (No. 1057213031).

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