The possible pre -and post -UVA radiation protective effect of amaranth oil on human skin fibroblast cells
Katarzyna Wolosik1, Ilona Zareba2, Arkadiusz Surazynski2, Agnieszka Markowska3
1 Laboratory of Cosmetology, The Faculty of Pharmacy with the Division of Medical Analytics, Medical University of Bialystok, Białystok, Poland
2 Laboratory of Medicinal Chemistry, The Faculty of Pharmacy with the Division of Medical Analytics, Medical University of Bialystok, Białystok, Poland
3 Laboratory of Organic Chemistry, The Faculty of Pharmacy with the Division of Medical Analytics, Medical University of Bialystok, Białystok, Poland
|Date of Submission||08-Dec-2015|
|Date of Acceptance||09-Feb-2016|
|Date of Web Publication||11-Jul-2017|
Laboratory of Cosmetology, The Faculty of Pharmacy, Medical University of Bialystok, ul. Akademicka 3, 15-267 Bialystok
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: The health effects of Amaranth Oil (AO) are attributed to its specific chemical composition. That makes it an outstanding natural product for the prevention and treatment of ultraviolet (UV) irradiation-related pathologies such as sunburn, photoaging, photoimmunosuppression, and photocarcinogenesis. Most of the studies are taken on animal model, and there is a lack of research on the endogenous effect of AO on fibroblast level, where UVA takes it harmful place. Objective: The aim of this study was evaluation if AO can protect or abolish UVA exposure effect on human skin fibroblast. Materials and Methods: The 0.1% AO, 0.25% AO, and 0.5% AO concentration and irradiation for 15 min under UVA-emitting lamp were studied in various condition. In all experiments, the mean values for six assays ± standard deviations were calculated. Results: Pretreatment with various concentrations of AO was tested. The highest concentration of AO where cell survival was observed was 0.5%. Cytotoxicity assays provided evidence for pre- and post-UVA protective effect of 0.1% AO among three tested concentrations. The results also provide evidence that UVA has inhibitory effect on collagen biosynthesis in confluent skin fibroblast, but presence of 0.1% AO abolishes pre- and post-UVA effect comparing to other used AO concentration. The assessment results on DNA biosynthesis show the significant abolished post-UVA effect when 0.1% and 0.5% of AO were added. Conclusion: AO gives pre- and post-UVA protection in low concentration. This provides the evidence for using it not as a main protective factor against UV but as one of the combined components in cosmetic formulation.
Abbreviations used: AO: Amaranth Oil.
Keywords: Amaranth Oil, collagen, fibroblast, in vitro-ultraviolet A protective effect
|How to cite this article:|
Wolosik K, Zareba I, Surazynski A, Markowska A. The possible pre -and post -UVA radiation protective effect of amaranth oil on human skin fibroblast cells. Phcog Mag 2017;13, Suppl S2:339-43
|How to cite this URL:|
Wolosik K, Zareba I, Surazynski A, Markowska A. The possible pre -and post -UVA radiation protective effect of amaranth oil on human skin fibroblast cells. Phcog Mag [serial online] 2017 [cited 2021 May 8];13, Suppl S2:339-43. Available from: http://www.phcog.com/text.asp?2017/13/50/339/210193
- The recommended Amaranth Oil (AO) concentration in cosmetic formulation is between 0.1 and 5%
- Pretreatment with various concentrations of AO suggests to use the highest 0.5% concentration of AO in human skin fibroblast cultures
- The 0.1% of AO in fibroblast cultures, protects and abolishes effect of ultraviolet A (UVA) exposure
- UVA has inhibitory effect on collagen biosynthesis in skin fibroblast, but presence of 0.1% AO abolishes pre- and post-UVA effect
- The abolished post-UVA effect occurs when 0.1% and 0.5% of AO were added on DNA biosynthesis.
| Introduction|| |
The commercially photoprotective products with appropriate sun protection factor (SPF) values are more efficient in solar erythema and sunburn protection, which are caused by high-energy ultraviolet (UV) photons produced by UVB radiation. However, their efficacy in preventing photoaging and skin cancer depends on their ability to block low-energy UVA radiation. It is also general thinking that commercial sunscreens give good UVA and UVB protection. However, the photostability of the sunscreen in the UVA range is not always adequate. Most sunscreens offer good protection against UVB, whereas the UVA photostability of some products decreases during UV exposure. Ideal sunscreening agents should be safe, chemically inert, nonirritating, nontoxic, photostable, and able to provide complete protection to the skin against damage from solar radiation. Although considered safe, sunscreening agents are not free from adverse effects. Phototoxic and photoallergic reactions accompany in increased use of cosmetic products containing of synthetic UV filters, for example,. contact dermatitis, allergic, irritant, and phototoxic and photoallergic reactions. Since a photoprotective cosmetic formulation is applied on large skin surfaces, they may cause the penetration and bioaccumulation of significant quantities of UV chemical absorbers. These percutaneous absorption processes may cause various adverse health effects such as endocrine disruption activity, decrease in superoxide dismutase activity, and even mutagenic and carcinogenic effects. There have been also reports of increased incidence of melanoma as a result of sunscreen use. The reason for this may be because sunscreens absorb UVB almost completely but transmit large quantities of UVA., Effects of UVA manifest usually after a long duration of exposure, even if doses are low. The UV generation of ROS and reactive nitrogen species can outcome in the structural and functional alteration of cutaneous proteins, for example, collagen, elastin, and glycosaminoglycans, which may contribute to photoaging. It has been postulated that UVA upregulates the formation of matrix metalloproteinases (MMPs), enzymes that degrade the matrix protein's elastin and collagen. Degradation in these proteins can result in marked reduction in skin elasticity and increased wrinkling. UVA radiation also produces nuclear structural to the DNA damage,, impairs the immune system, and leads to cancer. It has been linked to 67% of malignant melanoma.
Numbers of conventional and novel herbal cosmetics are useful to treat UV-damaged skin. The steady increase in the incidence of melanoma, nonmelanoma cutaneous neoplasia and preneoplasic disorders has contributed to the demand for more effective protection from the sun.,,, Although modern sunscreens containing UV filters are highly efficient to protect the skin from the deleterious effects of the sun, herbal sunscreens are rapidly replacing them due to mentioned above associated side effects with UV filters.
Some researchers have found that some plant oils contain natural sunscreens. For example, sesame oil resists 30% of UV rays, whereas coconut, peanut, olive, and cottonseed oils block out about 20%. Plant oils have already received much attention as source of antioxidants, but the unique composition of Amaranth Oil (AO) makes it an outstanding natural product for the prevention and treatment of UV irradiation-related pathologies such as sunburn, photoaging, photoimmunosuppression, and photocarcinogenesis The diverse health effects of AO is attributed to its specific chemical composition, i.e., a high level of linoleic acid (up to 50%), tocopherols/tocotrienols and squalene (up to 8%).
The efficacy of sunscreen products has been recognized as an important public health issue. Adequate methods for assessment of the level of protection should be developed and standardized. While the SPF COLIPA testing method in vivo has been used for years, preference should be given to in vitro testing methods as in vivo methods raise ethical concern. The in vitro screening methods may represent a fast and reasonable tool reducing the number of in vivo experiments and risks related to UV exposure of human subjects.
Due to searching of a new nontoxic, compatible with the skin, natural sun filters and post-UV exposure ingredients with proven endogenous protective properties, we wanted to asses if AO abolishes effect of UVA radiation.
| Materials and Methods|| |
Amaranth Oil characteristics
AO was a gift from Amaranth Bio Company (San Diego, CA, USA). Gifted AO had a clean yellow-goldish color. Its sensory characteristics are produced by cold-pressing using organic amaranth grain. The oil was obtained from Amaranthus cruentus seeds. Relative density at 25°C: 0.91–0.98; refraction index at 25°C: 1.20–1.60; melting point: −27°C; heavy metals: maximum 20 ppm; preservatives: none; saponification value: 165–190; acid number: maximum 30 mg KOH/g; peroxide number: maximum 10 mmol/kg. Fatty acid profile: palmitic 16%–22%, oleic 20%–26%, linoleic 40%–50%, and stearic 2%–5%. Squalene: 4%–8%. Vitamin E: 5–10 mg/100 g. AO can be stored for up to 12 months after production. It is recommend to store it in closed containers, away from light, and at a temperature of 5–10°C.
Cell lines and culture
All studies were performed on human skin fibroblasts which were purchased from the American Type Culture Collection (Rockville, MD, USA). The fibroblast cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% fetal bovine serum, 50 U/ml penicillin, and 50 μg/ml streptomycin at 37°C in a 5% CO2 incubator. Fibroblast cells were incubated for 24 h in DMEM without glucose and glutamine with varying concentrations of AO and irritated with UVA (365 nm). First, 0.1 ml stock solution of AO was prepared in 9.9 ml of ethanol (v/v). Stock solution was diluted in growth medium to final concentration of 0%, 0.1%, 0.25%, 0.5%, and 1%. Human skin fibroblasts were exposed to above prepared concentrations of AO for 24 h without refreshing the medium. The maximal concentrations of AO were determined by a cell viability study. The highest concentration of AO where cell survival was observed was 0.5%.
Toxicity of tested substances was determined by the method of Carmichael et al. Fibroblast cells were maintained as described above in 24-well plates. Cells were incubated for 24 h with AO before and after irritation of UVA, medium was discarded, and the cells were rinsed 3 times with phosphate-buffered saline (PBS). Then, the cells were incubated for 4 h in 1 ml of PBS with 25 μl of MTT (5 mg/ml). Medium was removed from the wells, and the cells were lysed 1 ml of DMSO with 20 μl of Sorensen's buffer (0.1 mol/l glycine with 0.1 mol/l NaCl, pH 10.5). The absorbance was measured at the wavelength of 570 nm. Component-treated cells were calculated as a percent of each control cell lines.
[3 H]-thymidine incorporation/DNA biosynthesis assay
To examine the effect of different concentration of AO before and after irritation with UVA on the fibroblast cells proliferation, the cells were plated in 24-well tissue culture dishes at 1 × 105 cells/well with 1 ml of growth medium. After 48 h, the plates were incubated with 0.5 μCi of [3 H] thymidine for 24 h at 37°C. Cells were rinsed 3 times with PBS, solubilized with 1 ml of 0.1 mol/l sodium hydroxide containing 1% SDS, then scintillation fluid “Ultima Gold XR” was added, and incorporation of the tracer into DNA was measured in scintillation counter.
Incorporation of radioactive precursor into proteins was measured after the labeling of confluent cells cultured in growth medium with 5[3 H]-proline (5 μCi/ml and 28 Ci/mM) for 24 h. Incorporation of tracer into collagen was determined by digesting proteins with purified Clostridium histolyticum collagenase, according to the method of Peterkofsky et al. Results are shown as combined values for cell plus medium fractions.
In all experiments, the mean values for six assays ± standard deviations (SD) were calculated. The results were submitted to the statistical analysis using the Student's t-test accepting P < 0.05 as statistically significant.
| Results|| |
Cytotoxicity assay results/cell viability
The pre-UVA protective effect of different concentration of AO was tested under condition, when fibroblast was incubated for 24 h with 0.1% AO, 0.25% AO, and 0.5% AO, then irradiated for 15 min under UVA-emitting lamp. To assess the post-UVA protective effect of different concentration of AO, fibroblast was irradiated for 15 min under UVA-emitting lamp, then incubated for 24 h with 0.1% AO, 0.25% AO, and 0.5% AO.
Preincubation of the fibroblasts with 0.1% and 0.25% concentrations of AO statistically increased (P < 0.05) cell proliferation by 35% (132%± SD, n = 6) in both groups when compared to control cell line. In contrast to the 0.5% AO concentration which statistically (P < 0.05) inhibited cell formation by 50% (48%± SD, n = 6). This provides evidence for pre-UVA protective effect of 0.1% AO [Figure 1]. The 15 min UVA irritation did not inhibit cell formation when 0.1% and 0.25% AO concentrations were added straight after. Results were significant (P < 0.05) for increased cell formation when 0.1% AO was used, and results were significant (P < 0.05) for inhibition of cell formation when 0.5% AO was used. This provides evidence for post-UVA protective effect of 0.1% AO concentration [Figure 2].
|Figure 1: Cytotoxicity assay results in confluent human skin fibroblasts incubated with different concentrations of Amaranth Oil with pre- and post-irritation of ultraviolet A. Component-treated cells were calculated as a percent of each control cell lines. Error bars represent ± standard deviation, n = 6|
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|Figure 2: Collagen biosynthesis measured as 5[3H]-proline incorporation into proteins susceptible to the action of bacterial collagenase in confluent human skin fibroblasts incubated for 24 h with different concentrations of Amaranth Oil. Error bars represent ± standard deviation, n = 6|
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Collagen biosynthesis results
The pre- and post-UVA effect of 0.1%, 0.25%, and 0.5% of AO on collagen synthesis in human skin fibroblast was measured. To assess the activity of AO on collagen synthesis in human skin fibroblasts, cells were incubated for 24 h in 0.1%, 025%, and 0.5% AO for 24 h and irradiated with UVA for 15 min after incubation, as well as irradiated with UVA for 15 min before 24 h incubation in AO.
In control cultures, collagen biosynthesis was intensive. Exposure of the fibroblasts to different concentrations of the AO led to the inhibition of the newly synthesized collagen by 7.64% (98.59% ± SD; n = 6), 83.91% (22.32 ± SD; n = 6), and 59.12% (47.11% ± SD; n = 6), respectively, when compared with the control value (106.23% ±10.93, n = 6). It has been shown that AO significantly (P < 0.05) affects collagen synthesis in 0.25 and 0.5% concentration [Figure 2].
The results show that UVA significantly affects collagen synthesis in both cases of pre- and post-presence of AO. The results provide evidence that UVA has an inhibitory effect on collagen biosynthesis in confluent skin fibroblast, but presence of 0.1% AO abolishes pre- and post-UVA effect comparing to other used AO concentration with control group. In 0.1% AO + UVA group, the collagen biosynthesis decreased only by 18% (82%± SD, n = 6) and by 28% (72%± SD, n = 6) in UVA + 0.1% AO group comparing to control group (c). In contrast, 0.25% AO collagen biosynthesis decreased by 79% (21%± SD, n = 6) in 0.25% AO + UVA group and by 67% (33%± SD, n = 6) UVA + 0.25% AO group. The results were significant [Figure 3].
|Figure 3: Collagen biosynthesis measured as 5[3H]-proline incorporation into proteins susceptible to the action of bacterial collagenase in confluent human skin fibroblasts incubated for 24 h with different concentrations of Amaranth Oil with pre- and post-ultraviolet A irritation. Error bars represent ± standard deviation, n = 6|
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[3H]-thymidine incorporation/DNA biosynthesis assay
To assess the activity of AO on DNA biosynthesis in human skin fibroblasts, cells were incubated for 24 h in 0.1%, 025% and 0.5% AO for 24 h, and irradiated with UVA for 15 min after incubation, as well as irradiated with UVA for 15 min before 24 h incubation in AO.
In control cultures, DNA biosynthesis was intensive. Exposure of the fibroblasts to different concentrations of the AO led to the inhibition of the synthesized DNA by 22% (78 ± SD; n = 6), 18% (82%± SD; n = 6), and increased DNA biosynthesis by 5% (105%± SD; n = 6), respectively, when compared with the control value [Figure 4].
|Figure 4: DNA biosynthesis measured as [3H]-thymidine incorporation into DNA in human skin fibroblasts (control) incubated with different concentrations of Amaranth Oil without the presence of ultraviolet A. Error bars represent ± standard deviation, n = 6|
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The assessment results of pre- and post-UVA effect of different concentration of AO on DNA biosynthesis in human skin fibroblast show the significant abolished post-UVA effect when 0.1% and 0.5% of AO were added [Figure 5].
|Figure 5: DNA biosynthesis measured as [3H]-thymidine incorporation into DNA in human skin fibroblasts (control) incubated with different concentrations of Amaranth Oil in pre- and post-presence of ultraviolet A. Error bars represent ± standard deviation, n = 6|
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| Discussion|| |
Many liquid oils, extracted from fruit and vegetable seeds, are light, low in viscosity, and less occlusive than hydrocarbon oils. Their penetrating and carrying properties, as well as their natural content of tocopherols, carotenoids, and essential fatty acids, make them highly valuable. Several natural-based sunscreen lotions, including the oils of almond, avocado, coconut, cottonseed, olive, peanut, sesame, and soybean, have been reported to have UV filters.
Amaranth grains contain only 1%–7.7% lipids, but these lipids are extremely valuable because of the presence of ingredients such as squalene, unsaturated fatty acids, Vitamin E as tocopherols, tocotrienols, and phytosterols, which are not seen together in other common oils (e.g.: Olive oil).
While much research on plant oils is focused on the effects of UVB radiation, there are not much studies on their effect under UVA condition. The aim of our study was to compare pre- and post-UVA properties of different concentrations of AO on human skin fibroblasts. The cytotoxicity, collagen, and DNA biosynthesis under different condition were studied. Our research confirms both protective and abolishing effect of AO when UVA exposure occurs.
The recommended AO concentration in cosmetic formulation is between 0.1 and 5%. Our pretreatment with various concentrations of AO suggested to use the highest 0.5% concentration of AO in human skin fibroblast cultures. The addition of 0.1% of AO suggests its protective and abolishing effect when UVA exposure occurs.
The early sign of skin aging is due to wrinkle formation. The collagen is the main component of the skin dermis, and its reduction result in aging of the skin. The collagen is the chief structural unit of the extracellular matrix (ECM). The type I collagen helps in the maintenance of the skin dermis structure., Aging and irradiation accelerate the degradation of the ECM, resulting in a decrease in dermal collagen and an increase in the level of the matrix MMP-1, which cleaves interstitial collagen leads the skin appear to be aged.,,, The presented research results show that exposure of human skin fibroblast to UVA exposure is less harmful on collagen formation when 0.1% AO concentration is used.
The assessment results of pre- and post-UVA effect of different concentration of AO on DNA biosynthesis in human skin fibroblast show the significant abolished post-UVA effect when 0.1% and 0.5% of AO were added. The sunscreen products available in the market contain UV absorbers that have been controlled at protecting against UV-induced sunburn and DNA damage. Since the biological endpoint for the determination of the SPF is the UV erythema. The SPF label is the indicator only for a protection against erythemally-effective solar UV, largely confined to the UVB, and partially short-wavelength UVA radiation. There is a continuous need of quantitative determination of different parameters, such as SPF, protection against UV radiations, to support the efficacy and safety of the products.,
There are many factors affecting the UV protective properties such as the use of different solvents, in which the sunscreens are dissolved; the combination and concentration of the sunscreens; the type of emulsion; the effects and interactions of vehicle components such as esters, emollient and emulsifiers used in the formulation; the interaction of the vehicle with the skin; the addition of other active ingredients; and the pH system and the emulsion rheological properties, among other factors, which can increase or decrease UV absorption of each sunscreen. Natural oil and AO represent good quality ingredient in the above-mentioned specification.,,,
| Conclusion|| |
Our study shows that AO gives acceptable pre- and post-UVA protection in low concentration. This provides the evidence for using AO not as a main protective factor against UV but as one of the combined components in cosmetic formulation. It was shown that using only one natural component is not enough for skin protection. Combination of several different natural substances is a right solution. The SPF value for the AO should be a measure to support the efficacy and safety of the cosmetic products also more detailed studies on collagen 1 and MMP-1 activity should be taken. This can provide an evidence for using AO in antiaging cosmetic creams as a wrinkle reducing and natural SPF component.
This work was supported in part by grants from Medical University of Bialystok (144-31829 F).
Financial support and sponsorship
Authors would like to thank for Amaranth Bio Company (San Diego, CA, USA) for foundation of AO for research.
Conflicts of Interest
There are no conflicts of interest.
| References|| |
Kapoor S, Saraf S. Efficacy study of sunscreens containing various herbs for protecting skin from UVA and UVB sunrays. Pharmacogn Mag 2009;19:238-48.
Latha MS, Martis J, Shobha V, Sham Shinde R, Bangera S, Krishnankutty B, et al.
Sunscreening agents: A review. J Clin Aesthet Dermatol 2013;6:16-26.
Lacatusua I, Niculaea G, Badeaa N, Stana R, Popab O, Opreaa O. Design of soft lipid nanocarriers based on bioactive vegetable oils with multiple health benefits. Chem Eng J 2014;246:311-21.
Faurschou A, Beyer DM, Schmedes A, Bogh MK, Philipsen PA, Wulf HC. The relation between sunscreen layer thickness and Vitamin D production after ultraviolet B exposure: A randomized clinical trial. Br J Dermatol 2012;167:391-5.
Roy A, Sahu RK, Matlam M, Deshmukh VK, Dwivedi J, Jha AK.In vitro
techniques to assess the proficiency of skin care cosmetic formulations. Pharmacogn Rev 2013;7:97-106.
Saewan N, Jimtaisong A. Photoprotection of natural flavonoids. J Appl Pharm Sci 2013;3:129-41.
Farmer KC, Naylor MF. Sun exposure, sunscreens, and skin cancer prevention: A year-round concern. Ann Pharmacother 1996;30:662-73.
Diffey BL, Tanner PR, Matts PJ, Nash JF.In vitro
assessment of the broad-spectrum ultraviolet protection of sunscreen products. J Am Acad Dermatol 2000;43:1024-35.
Couteau C, Faure A, Fortin J, Paparis E, Coiffard LJ. Study of the photostability of 18 sunscreens in creams by measuring the SPF in vitro
. J Pharm Biomed Anal 2007;44:270-3.
Korac RR, Khambholja KM. Potential of herbs in skin protection from ultraviolet radiation. Pharmacogn Rev 2011;5:164-73.
Semen KO, den Hartog GJ, Kaminskyy DV, Sirota TV, Maij NG, Yelisyeyeva OP, et al
. Redox modulation by amaranth oil in human lung fibroblasts. Nat Prod Chem Res 2013;2:122.
Bendová H, Akrman J, Krejcí A, Kubác L, Jírová D, Kejlová K, et al. In vitro
approaches to evaluation of Sun Protection Factor. Toxicol In Vitr
Carmichael J, DeGraff WG, Gazdar AF, Minna JD, Mitchell JB. Evaluation of a tetrazolium-based semiautomated colorimetric assay: Assessment of chemosensitivity testing. Cancer Res 1987;47:936-42.
Peterkofsky B, Chojkier M, Bateman J. Determination of collagen synthesis in tissue and cell culture system. In: Fufthmar M, editor. Immunochemistry of the Extracellular Matrix. Boca Raton: CRC; 1982. p. 19-47.
Kaur CD, Saraf S.In vitro
sun protection factor determination of herbal oils used in cosmetics. Pharmacognosy Res 2010;2:22-5.
Wolosik K, Knas M, Zalewska A, Niczyporuk M, Przystupa AW. The importance and perspective of plant-based squalene in cosmetology. J Cosmet Sci 2013;64:59-66.
Cho HS, Lee MH, Lee JW, No KO, Park SK, Lee HS, et al.
Anti-wrinkling effects of the mixture of Vitamin C, Vitamin E, pycnogenol and evening primrose oil, and molecular mechanisms on hairless mouse skin caused by chronic ultraviolet B irradiation. Photodermatol Photoimmunol Photomed 2007;23:155-62.
Jung E, Lee J, Baek J, Jung K, Lee J, Huh S, et al.
Effect of Camellia japonica
oil on human type I procollagen production and skin barrier function. J Ethnopharmacol 2007;112:127-31.
Langton AK, Sherratt MJ, Griffiths CE, Watson RE. A new wrinkle on old skin: The role of elastic fibres in skin ageing. Int J Cosmet Sci 2010;32:330-9.
Uitto J. The role of elastin and collagen in cutaneous aging: Intrinsic aging versus photoexposure. J Drugs Dermatol 2008;7 2 Suppl: s12-6.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]