In this study, we explored the effects of unripe fruit extracts of Mangifera indica L. on the anti-aging activity in skin cells. Mangifera indica L. is a popular economical and medicinal plant with numerous health-beneficial properties. The aqueous extracts of unripe fruit of Mangifera indica L. were obtained and subjected to HPLC and NMR analyses for the identification of bioactive compounds. The anti-glycation effect of Mango unripe fruit extracts was monitored by in vitro model system of AGEs (Advanced glycation end products) formation. Mango unripe fruit extracts significantly inhibited the AGEs formation in a dose-dependent manner. Meanwhile, Mango unripe fruit extracts possessed a comparable efficiency to commercialized Kojic acids in the inhibition of melanogenesis in B16-F10 melanoma cells. The UVA-induced cell damages can be prevented and repaired by Mango unripe fruit extracts in skin fibroblast CCD-966SK. Compared to the untreated control, Mango unripe fruit extracts significantly increased the cell viability while being applied before (36%) or after (43%) UVA irradiation. These results verified the potential application of Mango unripe fruit extracts in the skin protection and recovery from UVA irradiation, as well as the suppression of AGEs formation and melanogenesis.
Mangifera indica L. (Mango) is a fruit crop with high economic values, cultivated throughout the tropical and subtropical areas. Mango is notable for its excellent quality of taste and nutrition, and particularly for a wide range of health protective properties. Mango fruit is rich in antioxidants, such as polyphenols, carotenoids and ascorbic acid, which have highlighted its applications in pharmaceutical, nutraceutical and cosmetic industries [
The major polyphenolic compounds found in Mango fruit include quercetin, kaempferol, tannin, Chlorogenic acid, gallic acid, vanillic acid and mangiferin. Among these polyphenolics, mangiferin is a major bioactive constituent of M. indica. Mangiferin is a xanthone glycoside possessing a wide range of pharmacological properties such as antioxidant, antiaging, anticancer, antimicrobial, antidiabetic and immunomodulatory [
Skin aging is mainly due to intrinsic and extrinsic aging. Intrinsic aging of the skin is an inevitable and natural consequence. The two greatest exogenous factors of skin aging are smoking and exposure to UV light [
UV irradiation results in photo-aged skin and accumulation of Advanced glycation end products (AGEs). AGEs are generated via the nonenzymatic glycation or oxidation of proteins, lipids, and nucleic acids. UV-induced intracellular accumulation of AGEs generates reactive oxygen species (ROS) which triggers inflammation and causes dermal protein damages [
In order to unravel the role of fruit extracts of M. indica in the anti-aging of skin, we chose the unripe fruit as materials to avoid excess solubilization of pectic polymers during fruit ripening. We identified the bioactive components from whole fruit extracts by HPLC and NMR analyses. Our investigation focused on the elucidation of antiglycation, anti-melanogenesis and anti-UV irradiation potency of Mango unripe fruit extract. With these preliminary data, we provided useful information and further insights on the application of Mango fruit extracts in skin care.
CCD-966SK (CRL-1881) and B16-F10 (CRL-6475) cell lines were derived from the American Type Culture Collection (Manassas, VA, USA). NMR: 1D and 2D NMR experiments were performed on Varian 400 MHz 400 FT-NMR spectrometer (Varian, Inc. Palo Alto, CA); Chemical shifts are reported in δ (ppm), and TMS was used as an internal standard. All chemicals used in HPLC and NMR were of analytical grade commercial preparations purchased from Merck (Merck Ltd. Taipei, Taiwan). All cell culture media and reagents including MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] were purchased from Sigma-Aldrich (MO, USA) or Gibco (Thermo Fisher Scientific, Inc., CA, USA) and were of reagent grade or cell-culture grade.
The unripe fruit of Mangifera indica L. of size ranging from 3 to 7 cm in length was purchased from the local farmers in Pingtung, Taiwan. The whole fruit was homogenized and extracted in distilled water containing 0.5% citric acid at 85˚C. The fruit extract composed of fruit homogenates and distilled water in a ratio of 1:3 (v/v). The supernatant of fruit extract was collected by centrifugation and filtered to obtain the filtrate. The total volume of filtrates was evaporated by vacuumed centrifugation at 50˚C to the scale of Brix14 (One-degree Brix is 1 g of the dissolved solid content in 100 g of solution, for approximate calculation). The concentrates were directly proceeded to antiglycation assay, or proceeded to freeze drying to obtain extract powders that can be quantified and applied in content analysis and cell culture assays.
The HPLC system consisted of a Hitachi L-2310 series pump (Hitachi Ltd., Tokyo, Japan) equipped with a Hitachi L-2420 UV-VIS detector. Absorbance was recorded at 254 nm. LC analyses were performed on a Mightysil RP-18 GP 250 C18 reversed-phase column (10 mm × 250 mm, 5 µm, Kanto Chemical Co, Inc., Tokyo, Japan) and the column temperature was set at 25˚C. The mobile phase consisted of MilliQ water containing 0.1% formic acid (solvent A) and methanol containing 0.1% formic acid (solvent B). The flowing conditions of mobile phase were 1.0 ml/min flow rate and 20 µl injection volume. The column was equilibrated (A:B; v/v) in 85:15 (5 min), and elution was carried out with the following steps; 85:15 (5 min), a linear gradient increasing from 15% B to 100% (20 min), and 100% B (5 min). Fractions of major peaks were collected for preliminary bioactivity evaluations. Compounds with bioactivities were further identified by NMR spectroscopic analysis under the conditions as described in Section 2.1, and by comparing with the reported data in the literature to resolve the planar structures.
The antiglycation activity was evaluated following the method proposed by Yamaguchi et al. [
B16-F10 mouse melanoma cells were cultured in Dulbecco’s modified Eagle’s medium containing 1% Penicillin-streptomycin and 10% Fetal bovine serum. The B16-F10 cells were seeded at a density of 1.5 × 105 cells per well in 6-well culture plates, and incubated at 37˚C under a 5% CO2 atmosphere for 24 h. Renew the medium with fresh 3 ml of DMEM medium containing 0.25 mg/ml Kojic acid, or 0.25 mg/ml Mango unripe fruit extract, or blank solvent as control. Cells were harvested after incubation at 37˚C for 48 h. Rinse the collected cells with 1 × PBS twice and then resuspend in 200 μl of 1 × PBS. The cell lysates were obtained by freeze-thaw in liquid nitrogen for 10 min and room temperature for 30 minutes. Collect the pellets of cell lysates by centrifugation at 12,000 × g (Thermo ScientificTM HeraeusTM FrescoTM 17 Microcentrifuge, Langenselbold, Germany) for 30 minutes. Suspend the pellets with 120 μl of 1 N NaOH in 60˚C dry bath for 1 hour. Take 100 μl of samples to 96-well plate for spectrophotometric measurement at 405 nm. The amounts of melanin were calculated as: Relative melanin formation (%) = (ODsample/ODcontrol) × 100%.
Human skin fibroblast CCD-966SK (5 × 103/well) were seeded in 96-well plates and incubated at 37˚C for 24 hours. For UVA protection, the medium was replaced with fresh medium alone or containing 0.5 mg/ml Mango unripe fruit extract, and then incubate cells at 37˚C for 24 hours. Irradiate the sample plate with UVA by using ultraviolet radiation chamber under 12 J/cm2 for 1 hour. This condition would cause LD50 (Lethal Dose, 50%), which presents the dose of ionizing radiation leading half percentages of cell death. Set up sample plates without UVA irradiation as control. For UVA resistance, cells were irradiated before treatment of 0.5 mg/ml Mango unripe fruit extract. Cell viability was assessed through MTT assay. Briefly, 15 µl of MTT (4 mg/ml) was added and the cells were incubated for an additional 4 h. Remove medium and add 50 µl/well of DMSO to resolve formazan crystal. Place the plate on shaker and incubate for 10 min and measure the absorbance at 570 nm. Cells viability in response to treatment was calculated as: Cell viability (%) = (ODsample/ODcontrol) × 100%.
All values are expressed as mean ± SD. The statistical significance of the differences between two sample populations was determined by unpaired two-tailed Student’s t-test. Statistical significance was considered at P value < 0.05.
We identified three polyphenols from the aqueous extracts of Mango unripe fruit by HPLC and NMR analyses: Gallic acids, Mangiferin and penta-O-galloyl-β-d-glucose (PGG) (
However, whole Mango extracts are more potent than pure isolated mangiferin, which may be owing to the synergism between mangiferin and other Mango polyphenols [
High levels of AGEs contribute to impaired dermal regeneration, reduced collagen solubility and wrinkles of aging skin [
Although Mango fruit contains high amounts of polyphenols, whether the whole fruit extracts exhibit the potential of anti-melanogenic activity remain uncertain [
formation rate was 82% relative to the untreated control, which was comparable to that of Kojic acid (78%) (
Mango is a potential source of polyphenolic compounds that are responsible for high antioxidant activities that protect the body against oxidative damages [
In conclusion, we evaluated the antiaging activity of Mango unripe fruit extracts on skin cells. We demonstrated that Mango unripe fruit extracts protected dermal fibroblast from UVA irradiation and repair the damages after UVA exposure,
which might exert on the basis of antiglycation and anti-melanogenic activities of the extracts. Our preliminary studies demonstrated a novel potentiality of Mango unripe fruit extracts for promoting skin-whitening, fibroblast regeneration and anti-aging of skin.
This research was entirely funded by TCI Co., Ltd.
The authors declare no conflicts of interest regarding the publication of this paper.
Yu, C.-H., Lin, Y.-T., Su, H.-L., Kan, K.-W., Liu, F.-C., Lin, S.-Y., Chung, Y.-M., Hsu, H.-F. and Lin, Y.-H. (2019) Unripe Fruit Extracts of Mangifera indica L. Protect against AGEs Formation, Melanogenesis and UVA-Induced Cell Damage. Food and Nutrition Sciences, 10, 188-197. https://doi.org/10.4236/fns.2019.102014