1. INTRODUCTION
Astaxanthin (AST) (3,3-dihydroxy-β,β-carotene-4,4-dione), a red xanthophyll carotenoid, has been acknowledged as one of the most effective antioxidants. AST consists of the two β-ionone-type rings at both ends and one polyene chain in the structure; hence AST easily attaches to the cell or organelle membrane (Fig. 1) [1,2]. Hence, AST can protect cells by neutralizing or quenching free radicals produced inside or outside the cells [3,4]. Haematococcus pluviaslis, unicellular microalgae, is the best natural, commercial source of AST [3]. AST is commonly absorbed through the oral route to ameliorate Parkinson’s disease, stroke, high cholesterol, liver diseases, aging [1,3,5]. In the cosmetic field, AST was used as an anti-aging agent to protect skin from adverse effects of ultraviolet (UV) irradiation and reactive oxygen species (ROS) [6,7]. UV irradiation and ROS can break down DNA in skin cells, the extracellular matrix (ECM), such as collagen, elastin, and up-regulate enzymes that degrade skin ECM, such as matrix metalloproteinase-1 (MMP-1), neutral endopeptidase (NEP). These modifications lead to cell death, senescence, and aging skin [8–11]. AST was proved to prevent DNA damage, reduce the expression, and activity of MMP-1 and NEP in UV-induced fibroblast in vitro [12,13]. However, the protective ability of AST against ROS on fibroblast is limited. Currently, AST was applied frequently as an oral nutritional supplement; yet studies in topical application of AST in the cosmetic field have been neglected [6,7]. In this study, we aim to extract AST from a new H. pluviaslis strain isolated in Vietnam and determine its protective effects against ROS (H2O2) on fibroblasts (in vitro) and against UV irradiation on mouse skin (in vivo).
 | Figure 1: Structure of astxanthin [3]. [Click here to view] |
2. MATERIALS AND METHODS
2.1. Astaxanthin Extraction
The green algae Haematococcus pluvialis was cultivated in 750-ml sterilized glass columns containing 450 ml of modified BG-11 medium. The flasks were illuminated at 40 μmol.m−2.s−1 of white light (Philips, TLD 18W) with a dark: light cycle of 12 ours:12 hours, 22°C° and continuously aerated by the bubbling air. Cell number was counted by a hemacytometer cell counting chamber. For astaxanthin (AST) induction, logarithmic cells were incubated in BG-11 with nitrogen deficiency at high light intensity (170 μmol.m−2.s−1) with a light: dark cycle of 24:0 for 2–3 weeks [14]. Astaxanthin was extracted according to Sarada [15] with some modifications. Freeze-dried algae were treated by HCl 2 M for 2 minutes, ground, and extracted in acetone. AST concentration was evaluated by High-performance liquid chromatography (HPLC) [16].
2.2. ABTS Assay
The ABTS radical solution was prepared by oxidation of ABTS (7 mM) by K2S2O8 (2.45 mM). This solution was incubated for 14–16 hours in dark and then diluted in ethanol to obtain a working solution that has an absorbance of 0.7 ± 0.02 at the wavelength of 734 nm. Next, 80 μl of AST solution in different concentrations was mixed with ABTS working solutions and the OD values at 734 nm were record after 60 minutes. Trolox was used as a positive control. The inhibition percentage was calculated as the following formulation: Percentage (%) = [1−(Afinal/Ainitial)] × 100%, where Ainitial and Afinal are the absorbances at 734 nm at the beginning and final step [17,18].
2.3. Tyrosine Inhibition Test
Astaxanthin in different concentrations was added to wells of 96-well plates (50 µl/well), followed by adding 50 µl tyrosinase (25 U/well). The mixture was stored at room temperature for 15 minutes, and then supplemented with L-DOPA (100 µl) and left at room temperature. After 15 minutes, OD values at 492 nm were recorded. The percentage of inhibition was evaluated as the following formula: %inhibition = [(ODblank-A−ODblank-B) − (ODsample-A − ODsample-B)]/(ODblank-A − ODblank-B) × 100 where ODblank-A, ODblank-B is the absorbance of blank after and before test, ODsample-A, ODsample-B is the absorbance of the sample after and before test [17].
2.4. In Vitro Cytotoxicity Assay
3T3 cells were cultured in cell culture medium containing 90% DMEM/F12, 10% fetal bovine serum (FBS), 1% antibiotics in 37°C°, 5% CO2. The cells were seeded into a 96-wells plate at a density of 4 × 103 cells/well. After 24 hours, the cells were treated with AST in the range of 1–10 µg/ml (5 wells/group), the final DMSO concentration, which was used to dissolve AST, was 0.5%. Cell culture medium containing 0.5% DMSO was used as a control. After 1 day, cytotoxicity was evaluated by WST-1 assay according to the manufacturer’s instruction [19]. OD values were recorded at wavelength of 440 nm.
2.5. In Vitro Evaluation of the Protective Ability of AST against H2O2-Induced Oxidative Stress
3T3 cells were pre-treated with various AST concentrations (1, 2.5, 5, or 10 μg/ml) for 12 hours before the H2O2 treatment. H2O2-induced oxidative stress in fibroblast was performed according to Yokozawa et al. [20] with some modifications. First, 3T3 cells were seeded on a 96-wells plate at a density of 10 × 103 cells/well. One day later, cells were pre-treated with AST 1, 2.5, 5, and 10 μg/ml or commercial AST (trAST) (10 μg/ml) for 12 hours, cell culture medium containing 0.5% DMSO was used as a H2O2 control. Next, all cells (AST, trAST, and H2O2 control groups) were challenged by 200 µM H2O2 dissolved in DMEM/F12 5% FBS for 90 minutes. Then, the medium was replaced. Viable cells were examined by Hemocytometer and seeded on a 4-well plate with a concentration of 3 × 104 viable cells/well. Four days later, WST-1 assay, DAPI/ phalloidin staining, and senescence-associated b-galactosidase (SA-gal) staining were performed to test cell viability, cell nucleus area, and SA-gal expression. Cell culture medium containing 0.5% DMSO (non-treated H2O2) was used as a blank control group (Table 1).
2.6. In Vivo Evaluation of the Protective Ability of AST gainst UV-Induced Skin Lesion
AST was dissolved in commercial Sacha Inchi oil to achieve the concentrations of 20 or 200 μg/ml. Mus musculus var. Albino mice (age of 5–6 weeks, 30–35 g) were purchased from Pasteur Institute of Hochiminh city 1 week before the experiments. The mice were housed at 25°C° with a dark: light cycle of 12 ours:12 hours. The study was approved by the Animal Care and Use Committee (ACUCUS) of University of Science, Vietnam National University of Hochiminh city (approval number 1170B/KHTN-ACUCUS). The experiment was divided into five groups that belong to Table 1. UV radiation was performed using a UV lamp (Exo Terra, UVB 150, 25W), which gives the full solar spectrum with high UVB output. The dorsal skin of the mice was shaved and irradiated by UV light. UVB intensity was 100 mJ/cm2 per time for weeks 1–2 (three3 times/week), 200 mJ/cm2 per time for weeks 3–4 (three3 times/week), and 300 mJ/cm2 per time for weeks 5–6 (three3 times/week) [21]. After 6 weeks, wrinkle formation was evaluated based on the grading scale of Rumjhum Agrawal, and skin was stained by Trichrome staining [22]. In AST groups, dorsal skin was topically treated daily with AST (20 or 200 μg/ml) 8 hours before UV irradiation as the above method.
 | Table 1: Groups in experiment of AST ability against H2O2 treatment. [Click here to view] |
2.7. Statistical Analysis
Statistical analysis was performed using one-way analysis of variance with SigmaPlot software where p < 0.05 was considered to be statistically significant.
3. RESULTS
3.1. Astaxanthin Extraction
Under low light intensity condition (40 μmol.m–2.s–1), H. pluvialis cells predominantly existed in green motile zoospore stage witch 20–30 μm in diameter and 2 flagella. The cell concentration rapidly reached a peak of 9.5 × 105 cells/ml after 14–18 days of culture with inoculation of 1 × 105 cells/ml. Under unfavorable conditions: high light intensity (170 μmol.m−2.s−1) and nitrogen deficiency, the algal cells rapidly changed to the encystment stage. They begin to lose flagella, expand cell volume, and thicken the cell wall (palmella stage). After 7–10 days of induction, AST occurred in the center of the cell and rapidly occupied the entire cytoplasm. Most of the cells developed into aplanospores after 28–35 days of induction. HPLC results showed that AST concentration is 2.9% ± 0.5% of dry weight and free AST accounted for 4.4% ± 0.7% total AST (Fig. 2).
3.2. ABTS Assay
ABTS assay was used for determining the activity of antioxidants which reduce radical ABTS and decolorize its color. After 60 minutes of incubation, ABTS working solution changed to colorless (incubated with Trolox) or light orange (incubated with AST). The inhibition percentage of Trolox and AST was shown in Figure 3. IC50 value of Trolox was 604.8 ± 9.5 μM. The results showed that AST had ABTS scavenging activity in the range of 12.5–160 μg/ml with an IC50 value of 148.02 μg/ml and TEAC value was 0.126 mmol Trolox/g extract (Fig. 3).
3.3. Tyrosinase Inhibition Test
Tyrosinase catalyzes tyrosine to dopaquinone which polymerized to dark pigment having the maximum absorbance at a wavelength of 492 nm [23]. The results showed that the AST could inhibit tyrosinase activity with IC50 of 120.4 µg/ml extraction and the IC50 value of kojic acid (the control) was 25.6 µg/ml (180.9 µM). The results showed AST could inhibit tyrosinase and less potent than kojic acid.
 | Figure 2: Astaxanthin from H. pluvialis. (A) Motile macrozooids (×200), (B) Pamella (×100), (C) Aplanospores (×100) and (D) HPLC of AST extraction. [Click here to view] |
 | Figure 3: The results of ABTS assay. (A) Trolox and (B) AST. [Click here to view] |
 | Table 2: Groups in experiment of AST ability against UV irradiation. [Click here to view] |
3.4. Cytotoxicity Test In Vitro
3T3 cells were exposed to the medium supplemented with AST (0, 1, 5, or 10 µg/ml) (n = 5). OD values of WST-1 assay were shown in Table 2. OD values of all groups were not statistically different (p < 0.05) (Table 2). Moreover, after 1 day, morphological alterations and cell detachment were not observed, 3T3 cells retained fibroblast-like shape with elongate morphology (Fig. 4). It was concluded that AST in the range of 1–10 μg/ml was not cytotoxic to fibroblast cells.
3.5. In Vitro Evaluation of Protection Activity of AST
The experiment was performed in Table 1. In the control group, soon after the stress, the mortality rate increased significantly from 2.5% ± 0.4% (blank group) to 25.4% ± 3.5% of cells (p < 0.05) (Fig. 5A). Next 4 days, the OD values did not increase, it was comparable to day 1 (p > 0.05). The morphological alterations were observed on day 4th: the enlargement of cell nuclear size (430.6 ± 81.8 μm2) and cell size (data not shown) (Fig. 6B). Moreover, the percentage of SA-gal-expressed cells (a biomarker of cell senescence) in this group was 61.9 ± 7.5 which significantly higher than the blank and AST groups (p < 0.05) (Fig. 7). These results showed that H2O2 caused cell death accompanied by cell senescence, including the inhibition of cell proliferation, the increase of nuclear area, cell size, and SA b-Gal expression.
 | Figure 4: 3T3 cells after 1 day treatment with AST. (A) Control (×100), (B–D) AST 10, 25, and 50 µg/ml (×100). [Click here to view] |
 | Figure 5: Graphs show protective ability of AST on fibroblast. (A) cell mortality, (B) WST-1 assay, and (C) SA-b Gal expression. (a,b,c,d: significant difference). [Click here to view] |
 | Figure 6: The cells were stained with DAPI/phalloidin blue. (A) Normal cells, (B) H2O2 treated cells, (C–F) AST 1, 2.5, 5, and 10 μg/ml, and (G) 10 μg/ml (×100). [Click here to view] |
 | Table 3: OD value of WST-1 assay in cytotoxicity test. [Click here to view] |
In the AST group, the cells were sequentially treated with AST (1, 2.5, 5, and 10 µg/ml) and H2O2. The death rate in all AST groups was lower than the control group (p < 0.05). The WST-1 assay showed that OD values in group AST 1, 2.5 rose slightly on day 4th compared to day 1st (p > 0.05) while OD values in group AST 5, 10 on day 4th was higher than day 1st (p < 0.05). The results of DAPI/phalloidin staining showed cells in all AST groups retained their initial shape: spindle-like morphology with a smaller nuclear area than control (p < 0.05). The ratio of SA-gal-positive cells was lower than the control group (p < 0.05) (Figs. 5C and 7). Based on these results, we concluded that AST prevented the adverse effects of H2O2-induced oxidative stress in a dose-dependent manner. Between the various concentrations, AST 5–10 gives the best effect in protecting the fibroblast cells in this experiment.
 | Figure 7: SA-b Gal expression of the cells. (A) Normal cells, (B) H2O2-treated cells, (C–F) AST 1, 2.5, 5, and 10 μg/ml, and (G) 10 μg/ml (×100). [Click here to view] |
3.6. In Vivo Evaluation of the Protective Ability of AST against UV-Induced Skin Lesion
To determine the capacity to protect skin from UV damage, AST is topically applied on dorsal skin before UV irradiation. The visible appearance of the dorsal skin changed after 6 weeks of UV irradiation. The skin of all mice lost its color, became dry and rough. Moreover, some deep wrinkles perpendicular to the spine had appeared since week 4 (Fig. 8 and Table 4). In AST groups, skin remained smooth and reddish color, fine striation appeared (Fig. 8 and Table 4) (score was 4.3 ± 0.5). There is no significant difference between the wrinkle score of mice in AST 20 and 200 μg/ml (2.3 ± 0.5, 2.5 ± 0.5). Trichrome staining was carried out to verify the histological structure of the skin. After 6 weeks, there is a considerable increase in epithelium thickness in the UV group compared to the control group (42.2 ± 8.9 μm, 20.5 ± 4.5 μm). In AST groups (20 or 200 μg/ml), the thickness of these groups was smaller than the UV group and higher than the control group (28.9 ± 3.1 μm, 32.2 ± 4.9 μm) (p < 0.05) (Fig. 9 and Table 4). There is no significant difference between the epithelial layer of mice in AST 20 and 200 μg/ml (p > 0.05). These results showed that AST ameliorated UV-induced lesions on mouse skin.
 | Figure 8: Skin surface at week 6th post-treatment. (A) Control, (B), UV group, (C) oil, (D and E) AST 20, 200 μg/ml. [Click here to view] |
 | Table 4: Results of groups in skin protection from UV damage. [Click here to view] |
4. DISCUSSION AND CONCLUSION
AST is a powerful antioxidant and adheres easily to the membranes of cells, mitochondrial, nucleus [2,4]. Therefore, AST could prevent the harmful effects of free radicals produced in the intracellular and extracellular environment. Haematococcus pluvialis is one of the best natural sources of AST that contains the maximum AST concentration of 5% dry weight [6,24]. In Vietnam, Dang Thi Diem Hong is the first to isolate and culture H. pluvialis. This strain, obtained from the North of Vietnam, can reach the concentration of 0.95 × 106 cells/ml in a modified RM medium after 15 days of inoculation [25]. In this study, we aimed to investigate its ability to protect skin against the harmful effects of H2O2 and UV irradiation. The results showed that AST occupied 2.9% dry weight and existed in two different forms: free (4.4%) and esterified, in which ester form is the predominant part (data not shown). The ABTS assay shows that AST is a potent antioxidant and its TEAC value was 0.126 mmol Trolox/g extract closed to Zuluaga’s et al. [18]. Moreover, AST could reduce tyrosinase activity, which less effective than kojic acid. This result is similar to Chintong et al. [19]. Therefore, AST could be used as a skin-lightening agent.
UV irradiation and ROS are the most harmful factors to the skin. UV, composed of predominant UVA and UVB, can break down DNA double helix and create abnormal covalent bonds or produce ROS [26,27]. ROS is generated in normal metabolic processes or cellular reactions. Its level depends on age and is accelerated by UV induction [28,29]. Many kinds of research prove the protective effect of AST on fibroblast against UV irradiation on fibroblasts in vitro [12,13,30]. However, little research checks on its ability to prevent oxidative stress into fibroblast. H2O2, a powerful oxidizing agent, is usually used in senescent experiments [20,31]. The results showed that H2O2 triggered cell death (approximate 25% of cells), inhibited proliferation, and induced cell senescence. Moreover, over half of the cells expressed SA b-gal, a gold marker for cell senescence [31,32]. These results showed that H2O2 induced oxidative stress damage on fibroblasts [31,33]. In experiment groups, cells were pre-treated with AST (1–10 μg/ml) before H2O2 treatment. The results showed that AST reduced cell death rate, maintained cell proliferation, and suppressed SA b-gal expression. It was concluded that AST (1–10 μg/ml) could defend fibroblast against H2O2-induced oxidative stress, in which the 5 μg/ml was the best effective concentration.
 | Figure 9: Trichrome staining of mouse skin at week 6th post-treatment. (A) Control, (B) UV group, (C) Saccha Inchi oil, and (D and E) AST 20, 200 μg/ml (×200). [Click here to view] |
Skin lesions on Mus musculus var. Albino was caused by UV irradiation, according to Kim et al. [21] with some modifications. We used the Extro UVB 150 lamp, which gives a high output UVB similar to the solar spectrum in deserts. The amount of UV irradiation was calculated based on the UVB intensity of this lamp measured by a UVB meter. After 6 weeks of treatment, some skin aging signs appeared, and the skin surface changed to be rough, sagging, and had many deep and shallow wrinkles. In this study, we want to examine AST as a topical skincare product that can be used one time a day (before sleeping). Hence, AST (20 or 200 μg/ml) was applied on the dorsal skin 8 hours before UV irradiation. The results showed the mouse skin retained initial characteristics, including smooth, reddish, and had many shallow wrinkles. So, AST (20 or 200 μg/ml) ameliorated UV-induced skin lesions. We concluded that AST 20 μg/ml was the best effective concentration in this experiment.
5. CONCLUSION
AST was successfully extracted from H. pluvialis isolated in Vietnam. AST is a potent antioxidant that can prevent dangerous impacts from H2O2 and UV irradiation on the skin.
6. FINANCIAL SUPPORT
The study was supported by The Youth Incubator for Science and Technology Program, managed by Youth Development Science and Technology Center - Ho Chi Minh Communist Youth Union and Department of Science and Technology of Ho Chi Minh City, the contract number is “32/2019/ HÄ-KHCN-VƯ”.
AUTHOR CONTRIBUTIONS
All authors made substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data; took part in drafting the article or revising it critically for important intellectual content; agreed to submit to the current journal; gave final approval of the version to be published; and agree to be accountable for all aspects of the work. All the authors are eligible to be an author as per the international committee of medical journal editors (ICMJE) requirements/guidelines.
CONFLICTS OF INTEREST
The authors report no financial or any other conflicts of interest in this work.
ETHICAL APPROVALS
This study does not involve experiments on animals or human subjects.
PUBLISHER’S NOTE
This journal remains neutral with regard to jurisdictional claims in published institutional affiliation.
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