Isolation of maltol derivatives from Stellera chamaejasme and the anti-atopic properties of maltol on skin lesions in DNCB-stimulated mice

The aim of this study was to isolate maltol derivatives from S. chamaejasme and to investigate the anti-atopic dermatitis (anti-AD) effect of maltol in a 2,4-dinitrochlorobenzene (DNCB)-sensitized mouse model of AD. A novel compound, maltol 3-O-(4′-O-cis-p-coumaroyl)-β-d-glucoside (named isosoyamaloside I), and two known maltol derivatives (maltol and soyamaloside I) were isolated from S. chamaejasme using chromatographic methods. Dermal application of maltol to DNCB-sensitized AD mice reduced erythema, pruritus, and lichenification scores. Histopathological examinations revealed significant decline in mast cell infiltration in maltol-treated AD mice. In addition, maltol accelerated skin barrier recovery by reducing TEWL and skin pH and increasing skin hydration. Maltol was also found to suppress atopy-induced IL-4 and IgE elevations in serum, which are known to be essential for the development of atopy. The results of this study show that maltol is a potential therapeutic candidate for the treatment of AD-related skin diseases.


Introduction
Atopic dermatitis (AD) is a relapsing chronic inammatory skin disease that exhibits increasing prevalence in childhood. 1 AD is characterized by episodes of extreme pruritus and features of eczema in localized lesions. 2,3 Atopy-specic helper T cells (T H 2like T cells) play an important role in the initiation of allergic immune response. 4,5 Preferential accumulation of T H 2 cells in acute skin lesions of AD results in signicant increases in the numbers of cells expressing IL-4, IL-5, and IL-13. 3,4 In particular, expression of transgenic IL-4 has been reported to decrease the expressions of multiple genes associated with innate defense, and to trigger an allergic inammation that resembles AD in human skin. 6,7 Topical applications of the immunomodulators tacrolimus and pimecrolimus inhibit the expressions of the inammatory cytokine IL-4, and today the use of nonsteroidal topical agents is viewed to have considerable potential for the treatment of inammatory skin conditions and atopic diseases. 8,9 In this reason, the down-regulation of IL-4 overexpression in AD is considered an important pharmacological target. 10,11 Stellera chamaejasme L. (Thymelaeaceae) is a multi-stemmed perennial weed mainly distributed in northern and southwestern China. 12 The roots of S. chamaejasme, also known as 'Langdu', are used in traditional medicines in many Asian countries, including China, Nepal, India and Japan, as a remedy for stubborn skin ulcers, psoriasis, chronic tracheitis, and tuberculosis. 13,14 Extracts of S. chamaejasme have been reported to exhibit anti-inammatory, analgesic, and wound healing activities. 13,15 and phytochemical investigations of the roots of S. chamaejasme have identied avonoids, diterpenes, and coumarins as main constituents. 16,17 However, no detailed studies have been conducted to determine whether on maltol or on the maltol glycosides, which are found in the aerial parts of S. chamaejasme, have anti-AD properties. During our continuing search for anti-inammatory constituents in plants, we identi-ed maltol and a new compound isosoyamaloside I in an ethanoic extract of the aerial parts of S. chamaejasme and found they acted as strong IL-4 inhibitors. Additional study was undertaken to investigate the anti-atopic effects of maltol, which was the most active IL-4 inhibitor found, in a 2,4-dinitrochlorobenzene (DNCB)-sensitized murine model of AD. Dried aerial parts of S. chamaejasme (6.11 kg) were extracted with 95% EtOH and evaporated under reduced pressure to yield S. chamaejasme EtOH extract (SCAE) (569.8 g). SCAE was suspended in distilled water and sequentially partitioned using n-hexane, ethyl acetate, and n-butanol. The ethyl acetate fraction (20.8 g) was then subjected to silica gel column chromatography using a CH 2 Cl 2 -MeOH gradient (20 : 1 / 100% MeOH) to yield 13 fractions (SCE1$SCE13). Fraction SCE3 (868.4 mg) was suspended in MeOH and ltered to obtain maltol (81.5 mg). Fraction SCE7 (229.1 mg) was suspended in MeOH and ltered to yield soyamaloside I (82.3 mg). The resulting ltrate of SCE7 was subjected to Sephadex LH-20 (MeOH) column chromatography and afforded isosoyamaloside I (33.4 mg).

Cell culture
RBL-2H3 cells (a rat basophilic leukemia cell line) were purchased from the American Type Culture Collection (ATCC, #CRL-2256, Bethesda, MD). Cells were cultured in minimum essential medium (MEM) supplemented with Eagle's salt containing 10% fetal bovine serum (FBS), 2 mM L-glutamine, and antibiotics (100 U mL À1 penicillin and 100 mg mL À1 streptomycin) at 37 C in a humidied 5% CO 2 atmosphere.
2.5. b-Hexosaminidase release activity in RBL-2H3 cells RBL-2H3 cells were incubated in a 24-well plate (1 Â 10 5 cells per well) at 37 C for 2 h, and then incubated with anti-DNP IgE (antidinitrophenyl-immunoglobulin E) overnight. These IgEsensitized cells were then washed with Siraganian buffer, treated with DMSO or maltol derivatives (10 mM) for 1 h, and incubated in DNP-BSA (1 mg mL À1 ) for 30 min to stimulate cell degranulation. To measure b-hexosaminidase activities, culture media were centrifuged and the supernatants obtained were mixed with 1 mM poly-N-acetyl glucosamine (p-NAG) in 0.1 M sodium citrate buffer and incubated for 1 h at 37 C. b-Hexosaminidase release to supernatant were determined by measuring differences in absorbance at 405 nm vs. non-treated controls.

Histological analysis
Histological analysis was performed using formalin-xed paraffin sections (4-6 mm). Briey, dorsal skins of SKH-1 hairless mice were xed in 10% neutral formalin for 24 h, embedded in paraffin, cut into 2-3 mm slices, sectioned using a microtome, mounted on slides, and dried overnight at 37 C. Slices were stained with hematoxylin and eosin to evaluate eosinophil inltration or with toluidine blue to count mast cells number. Histopathological changes were examined under a light microscope (Olympus CX31/ BX51, Olympus Optical Co., Tokyo) and photographed (TE-2000U, Nikon Instruments Inc., Melville, USA).

Transepidermal water loss and skin hydration
Transepidermal water loss (TEWL) was assessed using a Tewameter TM210 unit (Courage and Khazaka, Cologne, Germany) and skin hydration and pH were measured using a SKIN-O-MAT (Cosmomed, Ruhr, Germany). Biophysical skin parameters, that is, TEWL, skin hydration, and skin pH were measured weekly under standard conditions (25 AE 5 C, 55 AE 5% RH).

Determinations of total serum IgE and IL-4 concentrations
Blood was collected from abdominal aortas and centrifuged at 10 000 rpm for 15 min at 4 C. Serum samples prepared from blood obtained on ED21 and stored at À70 C until required for IgE and IL-4 determinations. Total serum IgE and IL-4 levels were quantied using an enzyme-linked immunosorbent assay kit (eBioscience, San Diego).

Statistical analysis
The analysis was conducted using one-way analysis of variance (ANOVA). Results are expressed as means AE SDs, and statistical signicance was accepted for p values of <0.05.

Isolation of maltol derivatives from S. chamaejasme and structural elucidation of isosoyamaloside I
A new maltol glucoside (isosoyamaloside I) and two known maltol derivatives (maltol and soyamaloside I) were isolated from the 95% EtOH extract of the aerial parts of S. chamaejasme (SCAE; Fig. 1a). Maltol and soyamaloside I were identied by comparing 1 H NMR, 13

Maltol inhibited IL-4 expression and mast cell degranulation in RBL-2H3 cells
Maltol signicantly reduced the amounts of IL-4 released by mast cells (Fig. 2a), and inhibited IL-4 release from PIstimulated RBL-2H3 mast cells by 58% and 67% inhibition at concentrations of 10 mM and 30 mM, respectively. Isosoyamaloside I inhibited IL-4 release (by 40%) at 30 mM, but not at 10 mM. b-Hexosaminidase release is a marker of mast cell degranulation and is a good indicator of the extent of allergic reaction. 18 The effects of maltol, soyamaloside I, and isosoyamaloside I on b-hexosaminidase release by RBL-2H3 cells were examined, and both maltol and isosoyamaloside I effectively inhibited b-hexosaminidase release from mast cells (Fig. 2b), though maltol had the greater effect.

Effects of maltol on AD-like skin lesions of DNCBinduced atopic mice
Skin lesions were evaluated using dermatitis scores, as summarized in Fig. 3a. On ED20, AD-like skin lesions, such as, erythema, erosion, and dryness, were observed on the dorsal skins of DNCB-treated mice. In contrast, AD-like symptoms were markedly less severe in the DNCB-maltol group than in the DNCB group (Fig. 3b). Dorsal skin sections collected on day 21 were H&E stained to detect cells inltrating tissues or toluidine blue stained to evaluate mast cell numbers. AD-like symptoms, such as, epithelial and dermal thickening and inammatory cell inltration, were observed in the DNCB group. Numbers of inammatory cells that inltrated corium tissues and epidermal thickening were signicantly lower in the DNCB-maltol group than in the DNCB controls (Fig. 4a), and similar effects were also observed in toluidine blue stained sections of dorsal skin lesions. Mast cell degranulation and inltration were signicantly greater in DNCB controls than in vehicle controls. However, degrees of mast cell inltration were markedly lower in the DNCB-maltol group than in DNCB controls (Fig. 4b). Application of 1% pimecrolimus cream (Elidel) as a positive control improved AD skin symptoms by decreasing dermal thickening (Fig. 4a) and mast cell inltration (Fig. 4b) in DNCB-induced atopic animal model.

Effects of maltol on skin barrier function in atopic mice
TEWL and skin hydration values reect water loss from the body and are commonly as parameters of skin barrier function. TEWL, skin hydration, and skin pH values in stratum corneum on ED0, 7, 14, and 21 are provided in Fig. 5. TEWL and skin pH in the DNCB group were signicantly greater than in vehicle controls on ED6. Skin hydration values were lower in the DNCB group than in the vehicle control group on ED21, but TEWL and skin pH values were signicantly lower in the DNCB-maltol group than in vehicle controls at this time (by 60% and 75%, respectively) ( Fig. 5A and B). Co-treatment with 0.5% maltol was also found to have reduced DNCB-induced skin hydration loss by 29% as compared with DNCB controls (Fig. 5C). On ED21, cotreatment with 1% Elidel, a positive control, reduced the decrease in TEWL observed in the DNCB control by 35% (Fig. 5a) and increased skin hydration by 27% (Fig. 5b).

Effects of maltol on dermal thickness and serum IgE and IL-4 levels in atopic mice
Epidermal thicknesses and mast cell numbers were measured on ED21. As shown in Fig. 6a, dermal skins were signicantly thicker in the DNCB-treated group than in vehicle controls. However, maltol signicantly reduced these DNCB-induced increases in ear thickness. Maltol signicantly inhibited epidermal thickness by 72%. Additionally, mice in the DNCBmaltol group had fewer mast cells in dermis than DNCB controls (Fig. 6b). On ED21, total IgE and IL-4 concentrations were estimated by ELISA. Serum levels of IgE and IL-4 were found to be 8.8-and 3.1-fold higher, respectively, in DNCB controls than in normal controls, but IgE and IL-4 expressions were found to be markedly lower in the DNCB-maltol group than in vehicle controls ( Fig. 6c and d). Topical maltol treatment suppressed IgE elevation to 67% of control (Fig. 6c) and   IL-4 elevation to 87% of control (Fig. 6d). 1% Elidel co-treatment inhibited DNCB-induced IgE increase by 37% (Fig. 6c) and IL-4 increase by 68% (Fig. 6d).

Discussion
Maltol (3-hydroxy-2-methyl-4-pyrone) is produced by many plants and contributes to the food avor. 19,20 Recently, maltol has attracted research interest because of its antioxidant and anti-fatigue effects in medicinal plants, such as, Korean red ginseng. 21 Maltol has been reported to have diverse pharmacological actions, such as, neuroprotective and hepatoprotective effects in animals, 22,23 and maltol and maltol glucoside have been reported to inhibit the overproductions of inammatory cytokines like TNF-a and IL-1b, and thus, to exhibit anti-inammatory activities. 23,24 These research studies shown maltol and maltol derivatives have potential use as therapeutic agents for the treatment of AD. Nevertheless, no reports have been issued on the anti-AD effects of maltol or its derivatives. During our continuing search for phytochemicals with anti-inammatory properties, we found that maltol and a novel compound isosoyamaloside I isolated from the aerial parts of S. chamaejasme attenuated IL-4 expression and b-hexosaminidase release in vitro. We then examined the effects of maltol in vivo because it was observed to strongly inhibit mast cell degranulation (RBL-2H3 cells). Topical application of 0.5% maltol was found to suppress AD-like skin symptoms in DNCBinduced hairless mice by reducing leukocyte and mast cell skin inltration. Interestingly, maltol also signicantly decreased skin TEWL and pH values, increased skin hydration, and reduced DNCB-induced increases in epidermal thickness. It has been well established that AD is a disease characterized by altered skin barrier functions and by immune dysregulation. 25 TEWL and skin hydration values reect loss of water through skin and are commonly used parameters of skin barrier function. 26 Furthermore, it has been reported that epidermal cell hyperplasia and decreased skin barrier function are usually followed by an increase in TEWL in murine models of AD. 27 Our results indicate the anti-AD effect of maltol is closely related to amelioration of skin barrier dysfunction.
Herbal anti-inammatory agents are oen used to prevent and treat skin and allergic diseases. 27 Plant-derived molecules with anti-AD properties are known to exert their effects by disrupting the activities of inammatory cytokines and receptors. 27,28 In the present study, serum IL-4 and IgE levels were elevated in DNCB controls on ED21. However, treatment with maltol from ED7 to ED20 markedly suppressed the serum overexpressions of IL-4 and IgE in atopic mice, which suggests the anti-inammatory effects of maltol were due at least in part to the suppressions of inammatory cytokines and IgE Fig. 6 Effects of maltol on dermal thicknesses and on serum IgE and IL-4 concentrations in DNCB-induced atopic hairless mice. Epidermal thicknesses (a), mast cell densities in dermis (b), total serum IgE levels (c), and total serum IL-4 levels (d). CON: vehicle controls, DNCB: DNCBcontrols, DNCB-maltol: DNCB plus 0.5% maltol treated mice, and DNCB-Elidel: DNCB plus 1% Elidel treated mice. Results are presented as the means AE standard errors (n ¼ 7) of two independent experiments performed in triplicate. # p < 0.05 vs. vehicle controls; *p < 0.05 vs. DNCB controls.
synthesis. In particular, IL-4 is a key proinammatory cytokine in the context of allergic diseases and plays an important role in the induction of IgE synthesis. 29 Immunosuppressive drugs, tacrolimus, and pimecrolimus selectively target mast cells and inhibit IL-4 release and mast cell degranulation. 5,30 In contrast to corticosteroids, these topical calcineurin inhibitors were developed as maintenance therapies for AD and do not induce adverse effects, such as, steroid-induced skin atrophy, secondary infections, and acne. 30,31 Taken together, our ndings indicate that the strong IL-4 inhibitory effect of maltol means it has potential use for the treatment of AD.
In conclusion, a new compound, maltol 3-O-(4 0 -O-cis-p-coumaroyl)-b-Dglucopyranoside (isosoyamaloside I) and two known compounds (maltol and soyamaloside I) were isolated from the aerial parts of S. chamaejasme. Maltol most potently inhibited IL-4 levels in RBL-2H3 cells and suppressed the development of atopic dermatitis-like symptoms in our DNCBinduced mouse model of AD. Furthermore, DNCB-induced increases in TEWL and skin pH values were reduced by applying maltol to skin. We believe the observed anti-atopic effect of maltol was probably responsible for the observed enhancements of epidermal barrier functions. Accordingly, we suggest that maltol be considered a developmental candidate for novel anti-inammatory or immunomodulatory drugs.

Conflicts of interest
There are no conicts of interest to declare.