Protective Effects of Triphala on Dermal Fibroblasts and Human Keratinocytes

<div><p>Human skin is body’s vital organ constantly exposed to abiotic oxidative stress. This can have deleterious effects on skin such as darkening, skin damage, and aging. Plant-derived products having skin-protective effects are well-known traditionally. Triphala, a formulation of three fruit products, is one of the most important rasayana drugs used in Ayurveda. Several skin care products based on Triphala are available that claim its protective effects on facial skin. However, the skin protective effects of Triphala extract (TE) and its mechanistic action on skin cells have not been elucidated <i>in vitro</i>. Gallic acid, ellagic acid, and chebulinic acid were deduced by LC-MS as the major constituents of TE. The identified key compounds were docked with skin-related proteins to predict their binding affinity. The IC₅₀ values for TE on human dermal fibroblasts (HDF) and human keratinocytes (HaCaT) were 204.90 ± 7.6 and 239.13 ± 4.3 μg/mL respectively. The antioxidant capacity of TE was 481.33 ± 1.5 mM Trolox equivalents in HaCaT cells. Triphala extract inhibited hydrogen peroxide (H₂O₂) induced RBC haemolysis (IC₅₀ 64.95 μg/mL), nitric oxide production by 48.62 ± 2.2%, and showed high reducing power activity. TE also rescued HDF from H₂O₂-induced damage; inhibited H₂O₂ induced cellular senescence and protected HDF from DNA damage. TE increased collagen-I, involucrin and filaggrin synthesis by 70.72 ± 2.3%, 67.61 ± 2.1% and 51.91 ± 3.5% in HDF or HaCaT cells respectively. TE also exhibited anti-tyrosinase and melanin inhibition properties in a dose-dependent manner. TE increased the mRNA expression of collagen-I, elastin, superoxide dismutase (SOD-2), aquaporin-3 (AQP-3), filaggrin, involucrin, transglutaminase in HDF or HaCaT cells, and decreased the mRNA levels of tyrosinase in B16F10 cells. Thus, Triphala exhibits protective benefits on skin cells <i>in vitro</i> and can be used as a potential ingredient in skin care formulations.</p></div

Radical scavenging effect of TE on HDF cells against H2O2-induced cell damage.

<p>(<b>A</b>) HDF cells were pre-treated with various concentrations of TE for 1 h and exposed to 10 μM H2O2, and further incubated for 24 h at 37°C. (<b>B</b>) HDFs were co-treated with various concentrations of TE and 10 μM H2O2, incubated for 24 h at 37°C. Cell viability was determined by MTT assay and the results expressed as mean ± SEM (n = 3). <b>(C) Protective effect of TE on DNA damage in HDF cells by release of OH radicals from Fenton reaction.</b> The DNA were isolated from HDF cells and treated with two concentrations of TE (50 and 100μg/mL), FeSO₄ (2mM) and H₂O₂ (1 mM), incubated for 1 h at 37°C. DNA bands were resolved in 1% agarose gel stained with ethidium bromide. Densitometry analysis showing the protective effect of TE on H₂O₂-induced DNA damage. Values shown depict arbitrary units. Data is expressed as mean ± SEM (n = 3).</p

Effect of TE on expression of selected genes involved in biological functions related to aging.

<p>HDF and/or HaCaT cells were treated with different doses of TE for 24 h at 37°C. RNA was then isolated and qPCR carried out using oligo-dT primers for first strand synthesis followed by gene specific primers as described in the section 2.14. The gene expression was quantified and normalized to GAPDH and the data represented as mean ± SD (n = 2). The image shown is a representative from among two replicates. * indicate P < 0.05 and ** indicate P < 0.01. (<b>A</b>) Collagen-I gene expression (<b>B</b>) Elastin gene expression (<b>C</b>) SOD-2 gene expression (<b>D</b>) AQP-3 gene expression <b>(E)</b> Filaggrin gene expression <b>(F)</b> Transglutaminase (TGM1) gene expression <b>(G)</b> Tyrosinase gene expression <b>(H)</b> GAPDH gene expression (<b>I</b>) Involucrin gene expression.</p

(A) Effect of TE on H₂O₂-induced RBC hemolysis. RBC cells were treated with different concentrations of TE and ascorbic acid (AA) (25–100 μg/mL) and the cells were induced with 100 μM H₂O₂ for cell lysis. Data was expressed as percentage of control. Results are shown as mean ± SEM of three experiments. (B) Reducing power activity of TE at different concentrations (0–400 μg/ml).

<p>Absorbance was read at 700 nm and the data are represented as ascorbic acid equivalents. Results expressed as mean ± SEM (n = 3).</p

LC-MS/MS characteristics of Triphala and its constituents.

<p>The Triphala extracts (TE) were subjected to LC-MS analysis. Sample was injected into the ES ionization source using a syringe pump at a flow rate of 30μl/min. LC-MS/MS conditions are described in the text. The mass spectrum was acquired in the negative mode with a voltage of -4500 V and declustering potential -20 V. All the batch acquisition and data processing were controlled by Analyst 1.5 version software.</p

Anti-tyrosinase and melanin inhibitory properties of TE.

<p>The B16F10 cells were cultured in the 40mm petri plates (1×10⁵ cells/well) using the DMEM medium supplemented with 10% FBS for 24 h. <b>(A) Tyrosinase inhibition assay:</b> The cells were treated with different concentrations of TE and then incubated for 24 h with or without 100 nM α-MSH. The cells were lysed, centrifuged, and supernatant collected was monitored at 492 nm. Percentage of tyrosinase inhibition was calculated and expressed as mean ± SEM (n = 3) (<i>p</i> ≤ 0.01, p ≤ 0.05). <b>(B) Melanin inhibition/spitting assay:</b> The cells were treated with different concentrations of TE and 100 μM concentration of forskolin for the stimulation of melanin for 72 h and detached by 0.05% trypsin-EDTA. The cell pellets obtained were dissolved in 1 N NaOH for 1 h at 65°C and melanin content was measured at 405 nm. For % tyrosine inhibition and melanin content analysis, kojic acid (100 μM) was used as a positive control. Mean ± SEM (n = 3) (<i>p</i> ≤ 0.01, p ≤ 0.05). <b>(C) Melanin inhibition assay in Human melanoma (A-375) cells.</b> The experimental conditions remain same as above.</p

(A) Effect of TE on collagen-I production in HDF cells. The HDF cells were treated with two concentrations of TE (50 and 100μg/ml) and incubated for 96 h at 37°C. Collagen-I in cell supernatants were estimated by ELISA and results expressed as percentage of control. (B) Effect of TE on involucrin production in HaCaT cells. HaCaT were treated with two concentrations of TE (50 and 100 μg/ml) and incubated for 96 h at 37°C. Involucrin content in cell supernatants was estimated by ELISA and results expressed as percentage of control. (C) Effect of TE on filaggrin production in HaCaT cells.

<p>HaCaT were treated with two concentrations of TE (50 and 100 μg/ml) and incubated for 96 h at 37°C. Filaggrin content in cell supernatants was estimated by ELISA and results expressed as percentage of control. Mean ± SEM (n = 2); (<i>p</i> ≤ 0.01, p ≤ 0.05).</p

Effect of TE on NO production in LPS-stimulated RAW264.7 cells.

<p>Cells were treated with TE (50 and 100 μg/mL) and dexamethasone (Dexa) (100 μM), with/without LPS (1μg/mL) and incubated for 24 h at 37°C. After incubation, the cell supernatant was used to determine NO level by Griess reagent method. The values are expressed as mean ± SEM of three experiments (<i>p</i> ≤ 0.01, p ≤ 0.05).</p