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Antiaging effect of Curcuma longa L. essential oil on ultraviolet-irradiated skin
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Antiaging effect of Curcuma longa L. essential oil on ultraviolet-irradiated skin Yating Zheng , Chunxing Pan , Zejun Zhang , Wenqian Luo , Xiaoxin Liang , Yaohui Shi , Linjie Liang , Xi Zheng , Lanyue Zhang , Zhiyun Du PII: DOI: Reference:
S0026-265X(19)33360-0 https://doi.org/10.1016/j.microc.2020.104608 MICROC 104608
To appear in:
Microchemical Journal
Received date: Accepted date:
29 November 2019 5 January 2020
Please cite this article as: Yating Zheng , Chunxing Pan , Zejun Zhang , Wenqian Luo , Xiaoxin Liang , Yaohui Shi , Linjie Liang , Xi Zheng , Lanyue Zhang , Zhiyun Du , Antiaging effect of Curcuma longa L. essential oil on ultraviolet-irradiated skin, Microchemical Journal (2020), doi: https://doi.org/10.1016/j.microc.2020.104608
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2020 Published by Elsevier B.V.
HIGHLIGHTS
The anti-aging properties of Essential oil derived from the rhizome of Curcumin longa (CL-EO) were studied in this research article, and the main components CL-EO were analyzed.
The chemical analysis of CL-EO led to the identification of 56 components with the major compounds of ar-turmerone (36.04 %), curlone (8.78 %), β-turmerone (7.05 %), 8,9-dehydro-9-formyl-cycloisolongifolene (5.69 %), β-sesquiphellandrene (5.39 %), germacrone (4.51 %), ar-curcumene (2.19 %), α-himachalene (2.14 %), and ledane (2.13 %) using GC-MS analysis.
The anti-aging activity of CL-EO was determined by performing ultraviolet B (UVB) -induced skin aging assays. Hematoxylin and eosin staining showed that CL-EO reduced the thickness of the epidermis. Immunohistochemistry showed that CL-EO inhibited the expression of interleukin-1β and tumor necrosis factor-α.
Conclusions: CL-EO can reduce cutaneous photoaging in a UVB-irradiated nude mouse model.
1
Antiaging effect of Curcuma longa L. essential oil on ultraviolet-irradiated skin Yating Zhenga, Chunxing Pana, Zejun Zhanga, Wenqian Luoa, Xiaoxin Lianga, Yaohui Shia, Linjie Lianga, Xi Zhengb, Lanyue Zhanga*, Zhiyun Dua* a
Institute of Natural Medicine & Green Chemistry, School of Biomedicine,
Guangdong University of Technology, Guangzhou 510006, China b
Susan Lehman Cullman Laboratory for Cancer Research, Department of Chemical
Biology, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, 164 Frelinghuysen Road, Piscataway, NJ 08854, USA *
Corresponding author
Lanyue Zhang, Institute of Natural Medicine & Green Chemistry, School of Biomedical and Pharmaceutical Science, Guangdong University of Technology, Guangzhou 510006, China
E-mail address: [email protected] Telephone：13556166784 Zhiyun Du, Institute of Natural Medicine & Green Chemistry, School of Biomedicine, Guangdong University of Technology, Guangzhou 510006, China
E-mail address: [email protected] Telephone：13560456449 2
Funding
This research was funded by the National Natural Science Foundation of China (Grant No., 21272043) and Guangdong Science and Technology Department (Grant No.2015KQNCX024)
Abstract
Objective: Essential oil derived from the rhizome of Curcumin longa (CL-EO) has marked anti-inflammatory, antioxidant, antimicrobial, anticancer, and antiviral activities. However, its effect on photodamaged skin has not yet been evaluated. In this study, the anti-skin photoaging activity of CL-EO was determined by performing an ultraviolet B (UVB)-induced skin aging assay.
Methods: CL-EO was extracted by hydrodistillation and characterized using gas chromatography–mass spectrometry. The anti-skin aging effect was determined by topically applying 150 μl of CL-EO diluted to 1, 5, and 10 % with ethanol to the dorsal area of UVB-irradiated nude mice every day except on Sunday for 8 weeks. Histological and immunohistochemical analyses were performed.
Results: In total, 56 compounds, which accounted for 94.36 % of the contents of CL-EO, were detected. The major compounds in CL-EO were ar-turmerone (36.04 %), curlone (8.78 %), β-turmerone (7.05 %), 8,9-dehydro-9-formyl-cycloisolongifolene (5.69 %), β-sesquiphellandrene (5.39 %), germacrone (4.51 %), ar-curcumene (2.19 %), α-himachalene (2.14 %), and ledane 3
(2.13 %). Hematoxylin and eosin staining of tissue sections revealed that CL-EO decreased epidermal skin thickness. Immunohistochemistry showed that CL-EO inhibited interleukin-1β and tumor necrosis factor-α expression.
Conclusions: Thus, CL-EO can reduce cutaneous photoaging in a UVB-irradiated nude mouse model. Therefore, CL-EO could be used in the formulation of skin care and functional cosmetic products.
Keywords: Curcuma longa L., essential oil, nude mice, ultraviolet B, skin damage
Introduction
The rhizome of Curcuma longa L. (Zingiberaceae family), called Jianghuang or Huangjiang in China, contains curcuminoids, which are extensively used in various industries (1, 2). C. longa has been used for centuries as an edible dye and in traditional medicines to treat numerous diseases (3). As a popular Chinese herb, C. longa rhizome is often used to treat gastric ulcers, parasitic infections, skin disorders, sprains, joint inflammation, and cold and flu symptoms(4, 5). In general, C. longa shows anti-inflammatory, antioxidant, antimicrobial, anticancer, and antiviral effects (6).
The essential oil of C. longa (CL-EO) shows all the aforementioned biological activities (7, 8). CL-EO was characterized using gas chromatography–mass spectrometry (GC-MS) and was found to be composed of many compounds(9). The major components in CL-EO were ar-tumerone (35.17 %), tumerone (11.93 %), β-sesquiphellandrene (11.5 %), and curcumene (7.29 %) (10,11,12). CL-EOmight 4
effectively repair skin aging caused by UV irradiation (13). However, its effect on UV radiation-induced skin damage has not yet been evaluated. Therefore, this study aimed to determine the protective effect of CL-EO against UV radiation-induced skin damage in mice. In this study, we used nude mice as an animal model to investigate the effect of CL-EO in repairing UV irradiation-induced damage in vivo.
Materials and methods
Plant materials and chemicals Fresh rhizomes of C. longa were collected from the internship farm of Zhongkai University of Agriculture and Engineering and manually cleaned with water to remove adherent soil and extraneous matter. Next, the rhizomes were sliced, air-dried at room temperature (<25°C), and ground into powder. The powder (50 g) was subjected to hydrodistillation for 3.5 h by using a Clevenger-type apparatus. The distilled CL-EO was then dried over anhydrous MgSO4 and preserved in dark tubes at 4°C until use. The CL-EO yield was determined using the following formula: CL-EO yield (%) = obtained CL-EO (g)/dried rhizome sample (g) × 100 (14). All chemicals used in this study were of analytical grade and purchased from Aladdin (Shanghai, China).
Extraction of CL-EO and GC-MS analysis A GC/MS analysis of CL-EO was performed using DSQ-II Ultra GC–MS (Thermo, USA) with helium as the carrier gas at a flow rate of 1.0 ml/min. The
5
GC-MS system was fitted with a DB-5MS capillary column with 0.25 μm film thickness (Agilent, USA). The sample injection volume was 1 μl, and the split ratio was 100:1. The temperature program was set at 280°C with increments of 5°C/min for a total duration of 5 min (15). The following conditions were maintained: electron energy, 70 eV; ion source temperature, 230°C, and mode, electron-impact mode (16). The CL-EO components were identified by comparing their retention indices and mass spectra with data reported in general-purpose terpene ThermoQuest and NIST libraries and literature.
Anti-skin photoaging activity In total, 30 female nude mice (aged 8 weeks) were purchased from Sun Yat-sen University Laboratory Animal Center. The mice were treated according to the ethical guidelines of the animal center. The mice were randomly divided into five groups of three mice each. The 30 mice were randomly divided into the following five groups of six mice each: (a) 1 % CL-EO (CL-L), (b) 5 % CL-EO (CL-M), (c) 10 % CL-EO (CL-H), (d) Model control group (MC), and (e) vehicle-only (SC). Before exposure to UVB radiation, three mice were housed per cage, maintained on a standard laboratory diet, and provided water ad libitum. The animal cages were placed in a room with a controlled temperature of 23°C and humidity of ∼60 % under a 12-h light/dark cycle for at least 7 days before the experiments(17). The mice were treated according to the ethical guidelines of the Sun Yat-sen University Laboratory Animal Center, and all procedures were performed in accordance with the relevant laws and institutional 6
guidelines. All experimental protocols were approved by the Committee for Animal Care and Use at Guangzhou University of Technology with reference to the European Community guidelines and regulations of the National Institutes of Health, USA (approval number SCXK (Guangzhou)-2008–0020). All animals received humane care in accordance with the Guide for the Care and Use of Laboratory Animals, published by the US National Institutes of Health.
The anti-skin photoaging activity of CL-EO was evaluated by performing a UVB-induced skin aging assay. A UVP CL-1000 ultraviolet crosslinker was used for UV-B irradiation. UVB light with a peak wavelength of 312 nm was used as the UVB source. The spectral irradiance of the minimal erythemal dose (MED) was 175 mJ/cm2 for UVB. The mice were exposed to UV light three times per week (Monday, Wednesday, and Friday) for a total of 8 weeks. The radiation dose was increased weekly from 1 MED to 3 MED and maintained at 3 MED until the end of the experiment. CL-EO (150 μl) diluted at the three concentrations with ethanol was topically applied to the dorsal area of the mice every day except on Sunday for 8 weeks (18). On the day of UV irradiation, the mice were treated with CL-EO after UV exposure.
Evaluation of skin thickness and wrinkle formation At weeks 0, 2, 6, and 8 of the study period, the mice were fixed in a homemade apparatus, and the dorsal skin was lifted up by pinching gently to measure the skin thickness by using a Vernier caliper. Three measurements were performed for each 7
mouse. In addition, mice in the UVB-treated and untreated groups were photographed for the analysis of dorsal skin wrinkles.
Histology and immunohistochemistry The mice were killed by cervical dislocation under anesthesia at the end of the experiment. Dorsal skin tissues were fixed in 4 % formaldehyde, embedded in paraffin, and stained with hematoxylin-eosin (H&E) for analysis (19). The sections were visualized using a standard light microscope. For immunohistochemistry analysis, a 10 mM sodium citrate buffer with 0.05 % Tween-20 (pH 6.0) was used to treat the dorsal skin tissue sectionsat 100°C for 20 min. The sections were then incubated with antibodies against tumor necrosis factor (TNF)-α (dilution 1:200) and interleukin (IL)-1β (dilution 1:200) at 4°C overnight. The sections were then treated with a biotin-conjugated horseradish peroxidase antibody (dilution 1:200) at 25°C for 1 h. Following incubation with the secondary antibody, the sections were developed with 3,3ʹ-diaminobenzidine solution (20). The number of positive cells was calculated using image analysis software after observation under a fluorescence microscope (21).
Statistical analysis
All statistical analyses were performed using the unpaired t-test and analysis of variance (ANOVA); significance was set at P< 0.05.
Results and discussion 8
GC-MS analysis
The CL-EO yield was 4.56 % (Table 1). The relative level of each component of CL-EO was determined by comparing its area with the total areas from the detected peaks. The major components of CL-EO were ar-turmerone (36.04 %), curlone (8.78 %), β-turmerone (7.05 %), 8,9-dehydro-9-formyl-cycloisolongifolene (5.69 %), β-sesquiphellandrene (5.39 %), germacrone (4.51 %), ar-curcumene (2.19 %), α-himachalene (2.14 %), ledane (2.13 %), diepicedrene-1-oxide (2.02 %), p-cymene (1.91 %), cubenol (1.49 %), p-cymene (1.46 %), (E)-nuciferol (1.30 %), santalol (1.19 %), cis-α-santalol (1.19 %), caryophylleneoxide (1.09 %), and curdione (1.09 %) (Figure 1).
The main biological activity of any essential oil is determined by its major components. For example, the essential oil of fresh ginger (Zingiber officinale) contains mainly hydrocarbon sesquiterpenes, such as a-zingiberene (28.9 %), β-sesquiphellandrene (13.1 %), Z-γ-bisabolene (12.5 %), ar-curcumene (11.3 %), and D-germacrene 23)
(2.7 %) and has strong antifungal and antioxidant effects (22,
.Similarly, ar-turmerone, curlone, β-turmerone,
8,9-dehydro-9-formyl-cycloisolongifolene, β-sesquiphellandrene, and germacrone play a major role in the anti-skin photoaging activity of CL-EO.
CL-EO inhibits UVB irradiation-induced skin thickening and wrinkle formation in nude mice 9
Exposure to UV radiation induces oxidative and inflammatory stress, causing non-melanoma skin cancers (24, 25). We evaluated the antiphotoaging activity of CL-EO in nude mice after exposing them to UVB radiation for 8 weeks. The UVB-irradiated MC group and SC group showed symptoms such as rough skin, erythema, and edema. In contrast, the CL-EO-treated mice showed less severe symptoms, with the skin condition improving visibly in a dose-dependent manner (Figure 2(A)). Repetitive UV irradiation was associated with a gradual increase in skin thickening in the MC group and SC group. CL-EO treatment suppressed skin hyperplasia in a dose-dependent manner (Figure 2(B)). Furthermore, topical application of CL-EO at any concentration did not cause any notable phenotypic or behavioral adverse effects.
CL-EO treatment suppresses epidermal hyperplasia
The antiphotoaging effect of CL-EO was evaluated by staining the dorsal skin sections with H&E(26). After UV irradiation for 8 weeks, the MC group and SC group showed prominent histopathological features of photodamaged skin. The epidermis showed abnormal hyperplasia with a thickened stratum corneum(Figure 3(d)(e)). This finding was consistent with that reported previously that significant epidermal hyperplasia is induced during photoaging due to UVB irradiation (27). UVB irradiation induces epidermal cell growth (28). However, in the mice treated with different concentrations of topical CL-EO, epidermal hyperplasia significantly decreased compared to that in the MC and SC group. CL-EO significantly suppressed epidermal 10
thickening. These findings suggest that CL-EO suppresses epidermal hyperplasia and indicate its protective effect against skin photoaging in nude mice.
CL-EO inhibits the production of proinflammatory cytokines
The anti-UVB-induced inflammatory action of CL-EO was further evaluated by performing immunohistochemical and histological analyses by using skin biopsy tissues and assessing the production of proinflammatory cytokines, namely, IL-1β and TNF-α (29). After UVB irradiation for 8 weeks, IL-1β and TNF-α levels increased in the tissues of the mice of the SC and MC group (Figure 4A(d)(e) and 4B(d)(e)). However, in the mice treated with increasing concentrations of CL-EO, the levels of IL-1β and TNF-α in the dorsal skin tissues significantly decreased compared with those in the tissues of the mice of the SC and MC group. This result is consistent with previous findings (26, 27). These results suggest that topical treatment with CL-EO can suppress UVB-induced inflammatory reactions in the skin.
Conclusion
This study revealed the pharmacological potential of C. longa, which is extensively used in traditional Chinese medicine. The GC-MS analysis showed that CL-EO is composed of approximately 56 compounds, and its major components are ar-turmerone, curlone, β-turmerone, 8,9-dehydro-9-formyl-cycloisolongifolene, β-sesquiphellandrene, germacrone, ar-curcumene, α-himachalene, ledane, and 11
diepicedrene-1-oxide. As secondary metabolites, essential oils are influenced by many factors such as genetic factors, environmental conditions, geographic variations, and physiological factors (30, 31). CL-EO has antioxidant, antimicrobial, anticancer, and anti-inflammatory effects (14). In this study, we showed that CL-EO could reduce cutaneous photoaging in a UVB-irradiated nude mouse model. We believe that ar-turmerone, curlone, and β-turmerone were the major contributors to the anti-skin aging effect of CL-EO. Our findings suggest that CL-EO derived from C. longa has potential for application in the formulation of skin care and functional cosmetic products.
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Figure 1. The chromatograms of the compounds of CL-EO analyzed by GC-MS.
17
B
Figure 2. Inhibition of UVB-induced cutaneous photoaging by CL-EO in nude mice. The dorsal skin surface of the nude mice was exposed to UVB radiation three times a week for 8 weeks, and various concentrations of topical CL-EO were applied every day except on Sunday. Each treated group consisted of six mice. (A) Representative photographs showing the appearance of the dorsal skin area at the end of the 8-week experimental period. Increase in skin thickness (B) owing to repetitive UVB irradiation and its suppression by CL-EO in a dose-dependent manner. The increase in skin thickness was significantly different between the Model control group and the groups treated with 5% and 10% CL-EO (P < 0.01). UV: ultraviolet; CL-EO: essential oil of Curcuma longa
18
Figure 3. CL-EO suppresses the UVB-induced increase in epidermal thickness. (A) Representative photographs of hematoxylin and eosin-stained dorsal skin sections obtained at the end of the experiment from the following groups: (a) 1% CL-EO (CL-L), (b) 5% CL-EO (CL-M), (c) 10% CL-EO (CL-H), (d) Model control group (MC), and (e) vehicle-only group (SC). Magnification: 200-fold; scale bar: 200 μm. UV: ultraviolet; CL-EO: essential oil of Curcuma longa
19
Figure 4. CL-EO inhibits the UVB-induced production of inflammatory cytokines. The levels of interleukin-1β (A) and tumor necrosis factor-α (B) in the dorsal skin tissues obtained at the end of the experiment from the following groups were determined using immunohistochemical staining: (a) 1% CL-EO (CL-L), (b) 5% CL-EO (CL-M), (c) 10% CL-EO (CL-H), (d)Model control group (MC), and (e) vehicle-only group (SC). (C) Data represent the mean ± SEM values (n = 6 per group). Magnification: 200-fold; scale bar: 200 μm. *: significantly different from the vehicle-only group (P < 0.05). UV: ultraviolet; CL-EO: essential oil of Curcuma longa
Table 1 Retention indexes (RI) and relative peak areas (%) of compounds in essential oils derived from Curcuma longa rhizomes obtained from different habitats.
20
21
22
23
24
Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
25
Antiaging effect of Curcuma longa L. essential oil on ultraviolet-irradiated skin Yating Zheng , Chunxing Pan , Zejun Zhang , Wenqian Luo , Xiaoxin Liang , Yaohui Shi , Linjie Liang , Xi Zheng , Lanyue Zhang , Zhiyun Du PII: DOI: Reference:
S0026-265X(19)33360-0 https://doi.org/10.1016/j.microc.2020.104608 MICROC 104608
To appear in:
Microchemical Journal
Received date: Accepted date:
29 November 2019 5 January 2020
Please cite this article as: Yating Zheng , Chunxing Pan , Zejun Zhang , Wenqian Luo , Xiaoxin Liang , Yaohui Shi , Linjie Liang , Xi Zheng , Lanyue Zhang , Zhiyun Du , Antiaging effect of Curcuma longa L. essential oil on ultraviolet-irradiated skin, Microchemical Journal (2020), doi: https://doi.org/10.1016/j.microc.2020.104608
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2020 Published by Elsevier B.V.
HIGHLIGHTS
The anti-aging properties of Essential oil derived from the rhizome of Curcumin longa (CL-EO) were studied in this research article, and the main components CL-EO were analyzed.
The chemical analysis of CL-EO led to the identification of 56 components with the major compounds of ar-turmerone (36.04 %), curlone (8.78 %), β-turmerone (7.05 %), 8,9-dehydro-9-formyl-cycloisolongifolene (5.69 %), β-sesquiphellandrene (5.39 %), germacrone (4.51 %), ar-curcumene (2.19 %), α-himachalene (2.14 %), and ledane (2.13 %) using GC-MS analysis.
The anti-aging activity of CL-EO was determined by performing ultraviolet B (UVB) -induced skin aging assays. Hematoxylin and eosin staining showed that CL-EO reduced the thickness of the epidermis. Immunohistochemistry showed that CL-EO inhibited the expression of interleukin-1β and tumor necrosis factor-α.
Conclusions: CL-EO can reduce cutaneous photoaging in a UVB-irradiated nude mouse model.
1
Antiaging effect of Curcuma longa L. essential oil on ultraviolet-irradiated skin Yating Zhenga, Chunxing Pana, Zejun Zhanga, Wenqian Luoa, Xiaoxin Lianga, Yaohui Shia, Linjie Lianga, Xi Zhengb, Lanyue Zhanga*, Zhiyun Dua* a
Institute of Natural Medicine & Green Chemistry, School of Biomedicine,
Guangdong University of Technology, Guangzhou 510006, China b
Susan Lehman Cullman Laboratory for Cancer Research, Department of Chemical
Biology, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, 164 Frelinghuysen Road, Piscataway, NJ 08854, USA *
Corresponding author
Lanyue Zhang, Institute of Natural Medicine & Green Chemistry, School of Biomedical and Pharmaceutical Science, Guangdong University of Technology, Guangzhou 510006, China
E-mail address: [email protected] Telephone：13556166784 Zhiyun Du, Institute of Natural Medicine & Green Chemistry, School of Biomedicine, Guangdong University of Technology, Guangzhou 510006, China
E-mail address: [email protected] Telephone：13560456449 2
Funding
This research was funded by the National Natural Science Foundation of China (Grant No., 21272043) and Guangdong Science and Technology Department (Grant No.2015KQNCX024)
Abstract
Objective: Essential oil derived from the rhizome of Curcumin longa (CL-EO) has marked anti-inflammatory, antioxidant, antimicrobial, anticancer, and antiviral activities. However, its effect on photodamaged skin has not yet been evaluated. In this study, the anti-skin photoaging activity of CL-EO was determined by performing an ultraviolet B (UVB)-induced skin aging assay.
Methods: CL-EO was extracted by hydrodistillation and characterized using gas chromatography–mass spectrometry. The anti-skin aging effect was determined by topically applying 150 μl of CL-EO diluted to 1, 5, and 10 % with ethanol to the dorsal area of UVB-irradiated nude mice every day except on Sunday for 8 weeks. Histological and immunohistochemical analyses were performed.
Results: In total, 56 compounds, which accounted for 94.36 % of the contents of CL-EO, were detected. The major compounds in CL-EO were ar-turmerone (36.04 %), curlone (8.78 %), β-turmerone (7.05 %), 8,9-dehydro-9-formyl-cycloisolongifolene (5.69 %), β-sesquiphellandrene (5.39 %), germacrone (4.51 %), ar-curcumene (2.19 %), α-himachalene (2.14 %), and ledane 3
(2.13 %). Hematoxylin and eosin staining of tissue sections revealed that CL-EO decreased epidermal skin thickness. Immunohistochemistry showed that CL-EO inhibited interleukin-1β and tumor necrosis factor-α expression.
Conclusions: Thus, CL-EO can reduce cutaneous photoaging in a UVB-irradiated nude mouse model. Therefore, CL-EO could be used in the formulation of skin care and functional cosmetic products.
Keywords: Curcuma longa L., essential oil, nude mice, ultraviolet B, skin damage
Introduction
The rhizome of Curcuma longa L. (Zingiberaceae family), called Jianghuang or Huangjiang in China, contains curcuminoids, which are extensively used in various industries (1, 2). C. longa has been used for centuries as an edible dye and in traditional medicines to treat numerous diseases (3). As a popular Chinese herb, C. longa rhizome is often used to treat gastric ulcers, parasitic infections, skin disorders, sprains, joint inflammation, and cold and flu symptoms(4, 5). In general, C. longa shows anti-inflammatory, antioxidant, antimicrobial, anticancer, and antiviral effects (6).
The essential oil of C. longa (CL-EO) shows all the aforementioned biological activities (7, 8). CL-EO was characterized using gas chromatography–mass spectrometry (GC-MS) and was found to be composed of many compounds(9). The major components in CL-EO were ar-tumerone (35.17 %), tumerone (11.93 %), β-sesquiphellandrene (11.5 %), and curcumene (7.29 %) (10,11,12). CL-EOmight 4
effectively repair skin aging caused by UV irradiation (13). However, its effect on UV radiation-induced skin damage has not yet been evaluated. Therefore, this study aimed to determine the protective effect of CL-EO against UV radiation-induced skin damage in mice. In this study, we used nude mice as an animal model to investigate the effect of CL-EO in repairing UV irradiation-induced damage in vivo.
Materials and methods
Plant materials and chemicals Fresh rhizomes of C. longa were collected from the internship farm of Zhongkai University of Agriculture and Engineering and manually cleaned with water to remove adherent soil and extraneous matter. Next, the rhizomes were sliced, air-dried at room temperature (<25°C), and ground into powder. The powder (50 g) was subjected to hydrodistillation for 3.5 h by using a Clevenger-type apparatus. The distilled CL-EO was then dried over anhydrous MgSO4 and preserved in dark tubes at 4°C until use. The CL-EO yield was determined using the following formula: CL-EO yield (%) = obtained CL-EO (g)/dried rhizome sample (g) × 100 (14). All chemicals used in this study were of analytical grade and purchased from Aladdin (Shanghai, China).
Extraction of CL-EO and GC-MS analysis A GC/MS analysis of CL-EO was performed using DSQ-II Ultra GC–MS (Thermo, USA) with helium as the carrier gas at a flow rate of 1.0 ml/min. The
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GC-MS system was fitted with a DB-5MS capillary column with 0.25 μm film thickness (Agilent, USA). The sample injection volume was 1 μl, and the split ratio was 100:1. The temperature program was set at 280°C with increments of 5°C/min for a total duration of 5 min (15). The following conditions were maintained: electron energy, 70 eV; ion source temperature, 230°C, and mode, electron-impact mode (16). The CL-EO components were identified by comparing their retention indices and mass spectra with data reported in general-purpose terpene ThermoQuest and NIST libraries and literature.
Anti-skin photoaging activity In total, 30 female nude mice (aged 8 weeks) were purchased from Sun Yat-sen University Laboratory Animal Center. The mice were treated according to the ethical guidelines of the animal center. The mice were randomly divided into five groups of three mice each. The 30 mice were randomly divided into the following five groups of six mice each: (a) 1 % CL-EO (CL-L), (b) 5 % CL-EO (CL-M), (c) 10 % CL-EO (CL-H), (d) Model control group (MC), and (e) vehicle-only (SC). Before exposure to UVB radiation, three mice were housed per cage, maintained on a standard laboratory diet, and provided water ad libitum. The animal cages were placed in a room with a controlled temperature of 23°C and humidity of ∼60 % under a 12-h light/dark cycle for at least 7 days before the experiments(17). The mice were treated according to the ethical guidelines of the Sun Yat-sen University Laboratory Animal Center, and all procedures were performed in accordance with the relevant laws and institutional 6
guidelines. All experimental protocols were approved by the Committee for Animal Care and Use at Guangzhou University of Technology with reference to the European Community guidelines and regulations of the National Institutes of Health, USA (approval number SCXK (Guangzhou)-2008–0020). All animals received humane care in accordance with the Guide for the Care and Use of Laboratory Animals, published by the US National Institutes of Health.
The anti-skin photoaging activity of CL-EO was evaluated by performing a UVB-induced skin aging assay. A UVP CL-1000 ultraviolet crosslinker was used for UV-B irradiation. UVB light with a peak wavelength of 312 nm was used as the UVB source. The spectral irradiance of the minimal erythemal dose (MED) was 175 mJ/cm2 for UVB. The mice were exposed to UV light three times per week (Monday, Wednesday, and Friday) for a total of 8 weeks. The radiation dose was increased weekly from 1 MED to 3 MED and maintained at 3 MED until the end of the experiment. CL-EO (150 μl) diluted at the three concentrations with ethanol was topically applied to the dorsal area of the mice every day except on Sunday for 8 weeks (18). On the day of UV irradiation, the mice were treated with CL-EO after UV exposure.
Evaluation of skin thickness and wrinkle formation At weeks 0, 2, 6, and 8 of the study period, the mice were fixed in a homemade apparatus, and the dorsal skin was lifted up by pinching gently to measure the skin thickness by using a Vernier caliper. Three measurements were performed for each 7
mouse. In addition, mice in the UVB-treated and untreated groups were photographed for the analysis of dorsal skin wrinkles.
Histology and immunohistochemistry The mice were killed by cervical dislocation under anesthesia at the end of the experiment. Dorsal skin tissues were fixed in 4 % formaldehyde, embedded in paraffin, and stained with hematoxylin-eosin (H&E) for analysis (19). The sections were visualized using a standard light microscope. For immunohistochemistry analysis, a 10 mM sodium citrate buffer with 0.05 % Tween-20 (pH 6.0) was used to treat the dorsal skin tissue sectionsat 100°C for 20 min. The sections were then incubated with antibodies against tumor necrosis factor (TNF)-α (dilution 1:200) and interleukin (IL)-1β (dilution 1:200) at 4°C overnight. The sections were then treated with a biotin-conjugated horseradish peroxidase antibody (dilution 1:200) at 25°C for 1 h. Following incubation with the secondary antibody, the sections were developed with 3,3ʹ-diaminobenzidine solution (20). The number of positive cells was calculated using image analysis software after observation under a fluorescence microscope (21).
Statistical analysis
All statistical analyses were performed using the unpaired t-test and analysis of variance (ANOVA); significance was set at P< 0.05.
Results and discussion 8
GC-MS analysis
The CL-EO yield was 4.56 % (Table 1). The relative level of each component of CL-EO was determined by comparing its area with the total areas from the detected peaks. The major components of CL-EO were ar-turmerone (36.04 %), curlone (8.78 %), β-turmerone (7.05 %), 8,9-dehydro-9-formyl-cycloisolongifolene (5.69 %), β-sesquiphellandrene (5.39 %), germacrone (4.51 %), ar-curcumene (2.19 %), α-himachalene (2.14 %), ledane (2.13 %), diepicedrene-1-oxide (2.02 %), p-cymene (1.91 %), cubenol (1.49 %), p-cymene (1.46 %), (E)-nuciferol (1.30 %), santalol (1.19 %), cis-α-santalol (1.19 %), caryophylleneoxide (1.09 %), and curdione (1.09 %) (Figure 1).
The main biological activity of any essential oil is determined by its major components. For example, the essential oil of fresh ginger (Zingiber officinale) contains mainly hydrocarbon sesquiterpenes, such as a-zingiberene (28.9 %), β-sesquiphellandrene (13.1 %), Z-γ-bisabolene (12.5 %), ar-curcumene (11.3 %), and D-germacrene 23)
(2.7 %) and has strong antifungal and antioxidant effects (22,
.Similarly, ar-turmerone, curlone, β-turmerone,
8,9-dehydro-9-formyl-cycloisolongifolene, β-sesquiphellandrene, and germacrone play a major role in the anti-skin photoaging activity of CL-EO.
CL-EO inhibits UVB irradiation-induced skin thickening and wrinkle formation in nude mice 9
Exposure to UV radiation induces oxidative and inflammatory stress, causing non-melanoma skin cancers (24, 25). We evaluated the antiphotoaging activity of CL-EO in nude mice after exposing them to UVB radiation for 8 weeks. The UVB-irradiated MC group and SC group showed symptoms such as rough skin, erythema, and edema. In contrast, the CL-EO-treated mice showed less severe symptoms, with the skin condition improving visibly in a dose-dependent manner (Figure 2(A)). Repetitive UV irradiation was associated with a gradual increase in skin thickening in the MC group and SC group. CL-EO treatment suppressed skin hyperplasia in a dose-dependent manner (Figure 2(B)). Furthermore, topical application of CL-EO at any concentration did not cause any notable phenotypic or behavioral adverse effects.
CL-EO treatment suppresses epidermal hyperplasia
The antiphotoaging effect of CL-EO was evaluated by staining the dorsal skin sections with H&E(26). After UV irradiation for 8 weeks, the MC group and SC group showed prominent histopathological features of photodamaged skin. The epidermis showed abnormal hyperplasia with a thickened stratum corneum(Figure 3(d)(e)). This finding was consistent with that reported previously that significant epidermal hyperplasia is induced during photoaging due to UVB irradiation (27). UVB irradiation induces epidermal cell growth (28). However, in the mice treated with different concentrations of topical CL-EO, epidermal hyperplasia significantly decreased compared to that in the MC and SC group. CL-EO significantly suppressed epidermal 10
thickening. These findings suggest that CL-EO suppresses epidermal hyperplasia and indicate its protective effect against skin photoaging in nude mice.
CL-EO inhibits the production of proinflammatory cytokines
The anti-UVB-induced inflammatory action of CL-EO was further evaluated by performing immunohistochemical and histological analyses by using skin biopsy tissues and assessing the production of proinflammatory cytokines, namely, IL-1β and TNF-α (29). After UVB irradiation for 8 weeks, IL-1β and TNF-α levels increased in the tissues of the mice of the SC and MC group (Figure 4A(d)(e) and 4B(d)(e)). However, in the mice treated with increasing concentrations of CL-EO, the levels of IL-1β and TNF-α in the dorsal skin tissues significantly decreased compared with those in the tissues of the mice of the SC and MC group. This result is consistent with previous findings (26, 27). These results suggest that topical treatment with CL-EO can suppress UVB-induced inflammatory reactions in the skin.
Conclusion
This study revealed the pharmacological potential of C. longa, which is extensively used in traditional Chinese medicine. The GC-MS analysis showed that CL-EO is composed of approximately 56 compounds, and its major components are ar-turmerone, curlone, β-turmerone, 8,9-dehydro-9-formyl-cycloisolongifolene, β-sesquiphellandrene, germacrone, ar-curcumene, α-himachalene, ledane, and 11
diepicedrene-1-oxide. As secondary metabolites, essential oils are influenced by many factors such as genetic factors, environmental conditions, geographic variations, and physiological factors (30, 31). CL-EO has antioxidant, antimicrobial, anticancer, and anti-inflammatory effects (14). In this study, we showed that CL-EO could reduce cutaneous photoaging in a UVB-irradiated nude mouse model. We believe that ar-turmerone, curlone, and β-turmerone were the major contributors to the anti-skin aging effect of CL-EO. Our findings suggest that CL-EO derived from C. longa has potential for application in the formulation of skin care and functional cosmetic products.
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Figure 1. The chromatograms of the compounds of CL-EO analyzed by GC-MS.
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B
Figure 2. Inhibition of UVB-induced cutaneous photoaging by CL-EO in nude mice. The dorsal skin surface of the nude mice was exposed to UVB radiation three times a week for 8 weeks, and various concentrations of topical CL-EO were applied every day except on Sunday. Each treated group consisted of six mice. (A) Representative photographs showing the appearance of the dorsal skin area at the end of the 8-week experimental period. Increase in skin thickness (B) owing to repetitive UVB irradiation and its suppression by CL-EO in a dose-dependent manner. The increase in skin thickness was significantly different between the Model control group and the groups treated with 5% and 10% CL-EO (P < 0.01). UV: ultraviolet; CL-EO: essential oil of Curcuma longa
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Figure 3. CL-EO suppresses the UVB-induced increase in epidermal thickness. (A) Representative photographs of hematoxylin and eosin-stained dorsal skin sections obtained at the end of the experiment from the following groups: (a) 1% CL-EO (CL-L), (b) 5% CL-EO (CL-M), (c) 10% CL-EO (CL-H), (d) Model control group (MC), and (e) vehicle-only group (SC). Magnification: 200-fold; scale bar: 200 μm. UV: ultraviolet; CL-EO: essential oil of Curcuma longa
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Figure 4. CL-EO inhibits the UVB-induced production of inflammatory cytokines. The levels of interleukin-1β (A) and tumor necrosis factor-α (B) in the dorsal skin tissues obtained at the end of the experiment from the following groups were determined using immunohistochemical staining: (a) 1% CL-EO (CL-L), (b) 5% CL-EO (CL-M), (c) 10% CL-EO (CL-H), (d)Model control group (MC), and (e) vehicle-only group (SC). (C) Data represent the mean ± SEM values (n = 6 per group). Magnification: 200-fold; scale bar: 200 μm. *: significantly different from the vehicle-only group (P < 0.05). UV: ultraviolet; CL-EO: essential oil of Curcuma longa
Table 1 Retention indexes (RI) and relative peak areas (%) of compounds in essential oils derived from Curcuma longa rhizomes obtained from different habitats.
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Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
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