Compounds isolated from Eriobotrya deflexa leaves protect against ultraviolet radiation B-induced photoaging in human fibroblasts

Highlights

• Using bio-guided method, 4 active compounds were isolated from Eriobotrya deflexa.

• Activity of these compounds was evaluated using UV-B irradiated WS-1 cells.

• Hyperin reduced MMP-1, intracellular ROS and increased procollagen type-I, TIMP-1.

• LC-MS/MS was used for the quantitative analysis of compounds in active fraction.

Abstract

Ultraviolet (UV) irradiation leads to skin photoaging because of the upregulation of matrix metalloproteinase (MMP)-1 and downregulation of type I collagen and tissue inhibitor of metalloproteinase (TIMP)-1. Eriobotrya deflexa (Hemsl.) Nakai (Rosaceae) is a flowering plant endemic to Taiwan, and its leaves have been used as an expectorant and in antitussive folk remedy. Our previous studies have demonstrated that an E. deflexa leaf extract functions as a free radical scavenger. The current evaluated the antiphotoaging effect of partitioned fractions and specific compounds from the leaves of E. deflexa by using bioguided isolation, compound identification, and biological activity testing with UVB-irradiated human fibroblasts (WS-1 cells). E. deflexa leaves were extracted with 95% ethanol and then partitioned using a sequential treatment of n-hexane, ethyl acetate, and n-butanol (n-BuOH). The bioactive n-BuOH fraction was used for isolation and purification through chromatography. The compounds were identified by analyzing their physical and spectroscopic properties. We identified eight compounds from this fraction; of these compounds, 3-O-α-l-rhamnopyranosyl-(1‴ → 6″)-β-d-galactopyranoside (1), hyperin (2), afzelin (5), and cryptochlorogenic acid methyl ester (7) were isolated from E. deflexa for the first time, and they exhibited MMP-1 inhibition activity. The IC₅₀ values were 96.5, 89.5, 93.4, and 92.8 μM for 1, 2, 5, and 7, respectively. These compounds also enhanced the expression of procollagen type I, and TIMP-1 and hyperin (2) were found to be most effective with IC₅₀ values of 56.7 and 70.3 μM, respectively. Hyperin (2) could reduce intracellular reactive oxygen species production in UVB-irradiated WS-1 cells, with the corresponding IC₅₀ value being 80.7 μM. Liquid chromatography triple-quadrupole mass spectrometry was used for the quantitative and chemical fingerprint analysis of active compounds. Quercetin 3-O-α-l-rhamnopyranosyl-(1‴ → 6″)-β-d-galactopyranoside (1), hyperin (2), afzelin (5), and cryptochlorogenic acid methyl ester (7) constituted 24.2 ± 3.9, 5.5 ± 1.0, 3.4 ± 0.3, and 67.1 ± 8.1 mg/g of dry weight in the active n-BuOH fraction, respectively. Our results demonstrate that the extract and the isolated active compounds from E. deflexa leaves possess the potential for protection against skin photoaging.

Introduction

Skin aging has numerous causes including heritable factors, exposure to ultraviolet (UV) irradiation, hormonal imbalance, and disruption of certain metabolic pathways [1]. These factors can alter the skin structure, function, and appearance and cause cumulative alterations such as wrinkles, solar elastosis, and age spots. Photoaging, defined as the accelerated aging of the skin due to chronic sun exposure, is characterized by changes in the dermal connective tissue [2]. UV irradiation can cause skin damage by altering genome integrity. More specifically, UVB radiation (315–280 nm) can penetrate the epidermal and dermal layers and cause sunburn and genotoxicity due to the induction of phototoxic reactions [3]. The UVB radiation in sunlight is mostly responsible for radiation-induced oxidative stress, reactive oxygen species (ROS) formation, DNA damage, premature skin aging, immunosuppression, and many skin diseases such as nonmelanoma and melanoma skin cancers [4].

Histopathological studies on photoaging skin have documented increased expression and activity of matrix metalloproteinases (MMPs or matrixins), which lead to malfunction of the extracellular matrix (ECM), a complex network of fibrillar proteins and proteoglycans in the dermis [5]. Excessive MMP activity in the skin leads to wrinkles, loss of elasticity, and dilation of small surface-layer capillaries [5]. The various proteins in the MMP family belong to one of four functional groups, according to substrate specificity: collagenases, gelatinases, stromelysins, and membrane-type MMPs. Overexpression of MMP-1 (a collagenase) may lead to ECM degradation because it initiates the cleavage of type I collagen, a major structural protein of the ECM, which is secreted by fibroblasts [6]. Tissue inhibitors of metalloproteinases (TIMPs) are endogenous protein inhibitors that regulate the activities of MMPs in vivo. TIMP-1 upregulation can reduce collagenolysis and elastolysis that occur during skin photoaging. In addition, TIMP-1 regulates proliferation, apoptosis, differentiation, migration, and angiogenesis though different signaling pathways in skin dermal fibroblasts [7].

The Eriobotrya genus of flowering plants, commonly called loquat, is widely used as a folk remedy in China, Japan, and Taiwan to treat asthma, chronic bronchitis, and gastroenteric disorders. Eriobotrya japonica is also known for its edible fruit. Studies have reported that extracts of these plants have antioxidant, anti-inflammatory [8], anticancer [9], and antiosteoclastogenic [10] effects. Other studies have identified the main bioactive components as triterpenoids such as corosolic acid, ursolic acid, and oleanolic acid [11]; flavonoids such as hesperetin [12], kaempferol 3-O-β-glucoside, naringenin, quercetin, and quercetin 3-O-α-rhamnoside [11]; and phenolics such as gallic acid [12].

E. deflexa (Hemsl.) Nakai (Rosaceae), commonly known as Taiwan loquat, is an evergreen ornamental tree. The leaves of E. deflexa have been used as an expectorant and in antitussive folk remedy in Taiwan [13], [14]. A recent study used this species to examine the molecular mechanism of flowering; however, knowledge on the biochemical activities of extracts is limited [15]. A previous study reported the presence of several monoterpene glycosides and triterpene acids in a methanolic extract of E. deflexa leaves [14]. More recently, we demonstrated that an E. deflexa leaf extract functions as a free radical scavenger and also substantially inhibits MMP activity in cultured human skin cells after UVB irradiation [5].

In the present study, we first isolated and identified the chemical compounds of E. deflexa leaves through bioguided chromatography. Subsequently, we examined the photoprotective effects of these compounds on human dermal fibroblasts (WS-1 cells) that were subjected to UVB irradiation to induce photoaging and MMP-1 secretion.