Synergistic Effect of Madecassoside and Rosmarinic Acid Against Ultraviolet B-Induced Photoaging in Human Skin Fibroblasts

Abstract

Madecassoside (MD) and rosmarinic acid (RA) are well-known compounds with wound healing and antiaging effects. We demonstrated the synergistic protective activity of the MD–RA combination in Hs68 cells against ultraviolet B (UVB)-induced photoaging. The cell viabilities of MD, RA, and MD–RA combinations at various ratios (9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, and 1:9, v/v) were measured to compare their protective effects against UVB radiation. The synergistic interaction between MD and RA was confirmed using a combination index. The strongest effect of the MD–RA combination was observed at a ratio of 3:7. The combination of MD–RA 3:7 exerted a synergistic effect against UVB-induced changes in cell viability, as well as superoxide dismutase activity, reactive oxygen species, glutathione, catalase activity, and malondialdehyde levels. Moreover, the inhibitory effect of the MD–RA combination (3:7) on matrix metalloproteinases and total collagen production was higher than that of MD or RA alone. These results demonstrated that the MD–RA combination (3:7) generated a strong synergistic effect against UVB-induced photoaging in Hs68 cells. Overall, our results provide scientific evidence to support the development of a new combination therapy for skin protection against UVB-induced photoaging through the synergistic interaction between MD and RA. These natural compounds are promising options for antiaging and skin protection in the cosmetic and pharmaceutical industries.

INTRODUCTION

The skin is the largest organ in the human body, acting as protective barrier against harmful pathogens, microorganisms, toxins, and ultraviolet (UV) light.¹ Skin aging is induced by intrinsic and extrinsic factors. Intrinsic aging refers to natural skin aging, whereas extrinsic aging is stimulated by various environmental conditions.² The most harmful of these factors is UV exposure, which can induce wrinkles, premature aging, and skin cancer.³ UV light consists of three different spectra: ultraviolet A (UVA) (320–400 nm), ultraviolet B (UVB) (280–320 nm), and ultraviolet C (UVC) (100–280 nm).¹

UVB radiation reacts directly with moisture in the skin to promote generation of reactive oxygen species (ROS).⁴ Excess ROS activates the phosphorylation of mitogen-activated protein kinase (MAPK) resulting in the production of matrix metalloproteinases (MMPs) through an increase in activator protein-1 (AP-1).⁵ Furthermore, AP-1 suppresses expression of the type 1 collagen gene.⁶ ROS also impairs the antioxidant defense system and promotes oxidative stress, which accelerates aging.⁷ Inhibiting ROS production using various functional compounds in plants could be an efficient way to prevent or delay the progression of skin aging.⁸

Natural compounds have emerged as promising candidates for prevention of photoaging due to their antioxidant and anti-inflammatory properties. Centella asiatica (L) Urban is a herbal remedy used in traditional Chinese medicine to address various diseases.⁹ C. asiatica contains several bioactive components such as triterpenes, and phenolic compounds.¹⁰ In particular, triterpenes exhibit antioxidant, collagen enhancement, and UV protection activities.¹¹

Based on a previous study, madecassoside (MD) is the most abundant triterpene in C. asiatica and believed to be its major bioactive compound.¹² The Korean mint, Agastache rugosa, is a perennial herb found in many East Asian nations.¹³ Rosmarinic acid (RA), found in A. rugosa, has been shown to exert therapeutic potential and exhibit various health benefits such as anti-inflammation, antioxidant, and antibacterial properties.^14,15 RA reduces skin damage and accelerates wound healing.¹⁶

In recent years, synergistic studies in phytomedicine have allowed for the characterization of key novel activities. One of the main objectives of synergistic research is to find a scientific rationale for the therapeutic superiority of multiple plant extracts over a single compound.¹⁷ Various diseases can be treated or prevented more effectively with well-chosen combinations of bioactive compounds than with single compounds. Although the exact mechanisms remain unclear, several studies have investigated the synergistic effects of phytochemicals.^18
–20 This study aimed to compare the protective activity of MD, RA, and MD–RA combinations on UVB-induced skin damage and to elucidate the synergistic effect and application of these natural compounds as cosmeceuticals for the treatment and prevention of skin aging.

MATERIALS AND METHODS

Our study did not include any animal experiments or clinical trials.

Materials

MD, RA, thiobarbituric acid (TBA), glutathione (GSH), glutathione reductase, 5-sulfosalicylic acid dihydrate, 2′,7′dichlorofluorescein diacetate (DCFH-DA), dimethyl sulfoxide, 5,5′-dithiobis-2-nitrobenzoic acid (DTNB), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma Chemical Co. (St. Louis, MO, USA).

Sample preparation

Combinations of MD and RA were prepared at different volume ratios (10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, and 0:10, v/v). They were then diluted in medium and subjected to serial dilutions.

Cell culture and UVB irradiation

Hs68 cells were seeded in 96-well plates at a density of 5.0 × 10⁴ cells/mL. Hs68 cells were pretreated with or without the samples in a serum-free medium for 24 h. The cells were washed with phosphate-buffered saline and irradiated with UVB (30 mJ/cm²) using a UVB lamp (Sankyo Denki Lamps, GL20SE, Marine, Japan). After irradiation, the cells were treated with or without samples in serum-free medium for an additional 24 h. Cell viability and protective effects were determined by using MTT reagent (500 μg/mL).

Measurement of ROS, GSH, malondialdehyde, superoxide dismutase, and catalase

The levels of ROS were measured by DCFH-DA assay using a fluorescence spectrophotometer (Perkin-Elmer, Norwalk, CT, USA), following the established method.²¹ GSH levels were determined using DTNB-GSSG reductase recycling. Lipid peroxidation levels were determined using the TBARS assay. The superoxide dismutase (SOD) activity was measured by the reduction of 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide mediated by xanthine/xanthine oxidase. For catalase (CAT) activity, the concentration of H₂O₂ remaining was determined in the sample after CAT action.

Measurement of MMP-1, MMP-3, and total collagen levels

The levels of MMPs were assessed through the utilization of ELISA kits provided by Merck & Co., Inc., (Whitehouse Station, NJ, USA). Collagen level was measured using a Sircol Soluble Collagen Assay Kit from Biocolor (Carrickfergus, Northern Ireland).

Statistical analysis

The results are expressed as means ± standard errors. Statistical analysis of the results was performed using a one-way analysis of variance, followed by Duncan's test using SAS version 9.4 (SAS Institute, Inc., Cary, NC, USA). The synergistic activity was assessed using the combination index (CI) model described by Chou (2018).²² The interaction of combinations was calculated using “CompuSyn” software (CompuSyn, Inc., Paramus, NJ, USA) and CI was determined by the following isobologram equation: $C I = d_{1} ∕ D_{1} + d_{2} ∕ D_{2} .$

In this equation, D ₁ and D ₂ are the concentrations of the individual constituents required to produce a chosen effect level, whereas d ₁ and d ₂ are the concentrations of the mixture required to produce the same effect. The obtained CI values indicate synergistic (CI <1) effect.

RESULTS AND DISCUSSION

Protective effect of MD and RA against UVB-induced photoaging in Hs68 cells

C. asiatica and A. rugosa are medicinal plants traditionally used for various health benefits.^23,24 The active compounds, MD from C. asiatica and RA from A. rugosa, have been demonstrated to possess various biological effects such as antioxidant, anti-inflammation, antiphotoaging, and wound healing properties.^25
–27 Recently, we reported that a combination of C. asiatica and A. rugosa extracts exhibited synergistic effects on skin protection.²⁸ However, the synergistic effects of these bioactive compounds on photoaging remain unclear.

To understand their synergistic effects on skin protection, we confirmed the protective effects of MD, RA, and MD–RA combinations against UVB-irradiated skin damage in human skin fibroblasts. Cell viability was not significantly altered after 48 h of incubation with MD and RA at concentrations up to 10 μM (Fig. 1). Exposure to UVB decreased cell viability compared with the control (P < .001). However, MD and RA treatment enhanced cell viability by 21.3% and 33.7%, respectively, compared with the UVB exposure group at a concentration of 10 μM (Fig. 1A, B).

FIG. 1.

The protective effects of (A) MD, (B) RA, and (C, D) MD–RA in UVB-induced Hs68 cells and cell cytotoxicity of (E) MD, (F) RA, and (G) MD–RA in normal Hs68 cells. ASA (200 μM) was used as a positive control. Each value is expressed as the mean ± standard error (n = 3). Variables labeled with the same letter indicate that there is no statistically significant difference between their means. Different letters above the bars indicate significant differences based on the Duncan's test (P < .05). ASA, ascorbic acid; MD, madecassoside; RA, rosmarinic acid; MD–RA, combined compounds of MD and RA; UVB, ultraviolet B.

To determine the optimal combination ratio for skin protection, MD (10 μM), RA (10 μM), and MD–RA combinations (9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, and 1:9, v/v) were used to treat UVB-irradiated Hs68 cells, as previously described (Fig. 1C).²⁹ The combination of MD–RA 3:7 showed a greater protective effect against UVB exposure than the other ratios. As shown in Figure 1D, treatment with MD–RA 3:7 enhanced cell viability dose dependently, indicating a potential synergism. Therefore, we used MD–RA 3:7 in subsequent experiments.

Synergistic effect of the MD–RA combinations

A synergistic effect is a phenomenon in which the combined effect of two or more agents is greater than the sum of their individual effects.²² Synergistic combinations are known to enhance efficacy, decrease toxicity, and offer a multitarget mode of action.³⁰ Interactions between specific chemical compounds in natural products are presumed to contribute to their synergistic effects. However, the mechanism by which these compounds interact to produce an overall effect often remains unclear. Although the concentration of active compounds in natural products may be too low to exhibit any clinical activity, it is common practice to assess the single compounds responsible for the observed effect.³¹

Previous studies have explored the potential of natural product combinations to improve inflammation and oxidative stress,^30,32 however, the synergistic activity of the combination of MD and RA on photoaging is unclear. In this study, the type of interaction between MD and RA was analyzed using a CI model and the isobologram method. The positive interactions between MD and RA manifested as an increased protective effect in Hs68 cells compared with MD or RA alone (Fig. 2A, B). As shown in Figure 2C, the MD–RA 3:7 combinations at all concentrations showed CI <1 (0.16–0.50).

FIG. 2.

(A) Dose–effect curves of MD, RA, and MD–RA 3:7 combination. (B) Medium–effect plot of MD, RA, and MD–RA 3:7 combination. (C) CI values were plotted as a function of fraction of cell viability (Fa) by “CompuSyn” software. (D) Isobologram curves of MD, RA, and MD–RA 3:7 combination. Each value is expressed as the mean ± standard deviation (n = 3). CI, combination index.

For 90% cell viability, the combination of MD and RA was needed in the amount of 2.39 and 5.58 μM, respectively, which was lower than the concentration for single treatment of MD (298.26 μM) or RA (25.50 μM) (Fig. 2D). Hence, our results indicate that synergy occurred during treatment with MD–RA 3:7, and the application of this mixture for skin protection may be more beneficial than the use of a single compound.

Effect of the MD–RA combinations on ROS, GSH, malondialdehyde, SOD, and CAT levels

Exposure to UVB radiation can indirectly damage biomolecules by promoting ROS generation, which can oxidize lipids, proteins, and DNA.³³ Oxidized products, such as lipid hydroperoxides, have been implicated in the onset of skin-related diseases.³⁴ Certain major antioxidant enzymes including SOD and CAT have been reported to be depleted in UVB-induced skin damage. To evaluate the potential protective effects of the combination of MD–RA 3:7 against UVB-induced oxidative stress in Hs68 cells, we compared the effects of MD, RA, and MD–RA 3:7 treatment on ROS generation and GSH and malondialdehyde (MDA) levels.

As shown in Figure 3, Hs68 cells exposed to UVB radiation exhibited a significant increase in ROS production (P < .01), GSH depletion (P < .05), and MDA levels (P < .001) compared with control cells. However, treatment with the MD–RA combination markedly reduced ROS production, GSH depletion, and MDA levels induced by oxidative stress compared with single treatment with MD or RA. Our findings suggest that the combination of MD–RA was more effective in preventing increases in ROS production, GSH depletion, and MDA levels than treatment with either compound alone.

FIG. 3.

Effects of MD, RA, and MD–RA 3:7 combination on (A) time course of ROS generation, (B) ROS production at 80 min, (C) GSH, and (D) MDA levels, (E) SOD activity, and (F) CAT activity in UVB-induced Hs 68 cells. Data are presented as the mean ± standard error (n = 3). Variables labeled with the same letter indicate that there is no statistically significant difference between their means. Different letters above the bars indicate significant differences based on the Duncan's test (P < .05). CAT, catalase; GSH, glutathione; MDA, malondialdehyde; ROS, reactive oxygen species; SOD, superoxide dismutase.

Furthermore, we measured antioxidant activities, such as SOD and CAT, in MD, RA, and the MD–RA 3:7 combination. Exposure to UVB markedly reduced SOD and CAT activities; however, treatment with MD–RA significantly restored SOD and CAT activities compared with single treatments with MD or RA (P < .05). The beneficial effects of various medicinal plants have been attributed to phenolic compounds. High proportions of MD in C. asiatica have been shown to be responsible for restoring the antioxidant capacity and inhibiting ROS production.³⁵

A previous study demonstrated that RA could suppress UV-induced skin damage through its antioxidant activity.³⁶ Moreover, RA exerts considerable ROS-scavenging activity by activation of the Nrf2-antioxidant system.³⁷ Luo et al. reported that MD substantially diminished MDA levels and increased antioxidant activity in rats.³⁸ Joardar et al. reported that RA reduces lipid peroxidation levels in the cell membrane and increases GSH and GSH peroxidase levels as antioxidant defenses.³⁹ Overall, MD–RA 3:7 ameliorated UVB-induced oxidative stress by regulating intracellular ROS, GSH, MDA, SOD, and CAT levels, which affected the maintenance of the normal redox status of Hs68 cells.

Effect of the MD–RA combinations on MMPs and total collagen production

UVB irradiation induces collagen breakdown in the dermis by stimulating collagenase activity, resulting in increased wrinkle formation.⁴⁰ MMP-1 is a collagenase that plays a critical role in photoaging.⁴¹ MMP3 activates MMP1, which, in turn, degrades the main components of the extracellular matrix, including collagen.⁴² Therefore, the inhibition of MMPs may be effective in protecting against UVB-induced skin wrinkling and sagging. UVB irradiation markedly increased MMPs production compared with that in control cells (Fig. 4A, B).

FIG. 4.

Effect of MD, RA, and MD–RA 3:7 combination on (A) MMP-1, (B) MMP-3, (C) total collagen productions in UVB-induced Hs68 cells. Data are presented as the mean ± standard error (n = 3). Variables labeled with the same letter indicate that there is no statistically significant difference between their means. Different letters above the bars indicate significant differences based on the Duncan's test (P < .05). MMP, matrix metalloproteinase.

However, the MD and RA treatments significantly decreased MMP production. Treatment with MD–RA 3:7 synergistically decreased the UVB-induced increase in MMP levels by UVB irradiation in Hs68 cells compared with treatment with MD or RA alone. We also assessed the effect of treatment with MD, RA, or MD–RA 3:7 on total collagen production and found that MD–RA 3:7 significantly increased total collagen compared with single treatments with MD or RA (P < .05) (Fig. 4C). Wu et al. reported that MD and asiaticoside markedly enhanced the mRNA levels of collagen types I and III through activation of the TGF-β/Smad pathway.⁴³

RA from Thymus vulgaris inhibits UVB-induced skin injury by regulating the antioxidant defense systems and suppressing the MAPK/AP-1 pathway.⁴⁴ Sutkowska et al. showed that RA exerts beneficial effects on MMPs and type I collagen.⁴⁵ It is widely known that various combinations of bioactive compounds can exert synergistic effects.⁴⁶ These synergistic effects may be attributed to their combined effects on different targets and signaling pathways, leading to enhanced biological activity. Based on previous studies and our results, the MD–RA 3:7 combination was effective in improving skin wrinkles by activating the collagen synthesis pathway through MD and inhibiting the collagen degradation pathway through RA. However, additional research is needed to verify the underlying mechanisms.

SUMMARY

This study showed that the combination of MD and RA had a synergistic effect on restoring cell viability in UVB-induced Hs68 fibroblasts. The synergistic effect of MD–RA 3:7 contributed to increased activity to decrease ROS production, GSH depletion, and lipid oxidation. Moreover, the combination treatment significantly increased enzyme activities of SOD and CAT, reduced levels of MMPs, and increased collagen compared with the single compound treatment.

Our results suggest that the combination of MD and RA in a specific composition can act as a more powerful agent for protecting the skin against UVB-induced oxidative stress and skin aging and may lead to the development of new therapies for preventing skin photoaging. The synergistic effects of MD and RA could offer valuable insights for devising innovative therapeutic strategies to prevent and treat a wide range of diseases.

Footnotes

AUTHOR'S CONTRIBUTIONS

Preparing first draft of the article and data collection were contributed by M.K.; methodology was done by H.H. and S.H.; supervision and review of the study were by J.L.; writing, review, editing, and supervision were carried out by H.L.

AUTHOR DISCLOSURE STATEMENT

No competing financial interests exist.

FUNDING INFORMATION

This study was self-funded.

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