Letter pubs.acs.org/JAFC
Anti-hyaluronidase Activity in Vitro and Amelioration of Mouse Experimental Dermatitis by Tomato Saponin, Esculeoside A ABSTRACT: The increasing incidence of atopic dermatitis during recent decades has prompted the development of safe and effective agents for prevention of atopic diseases. Esculeoside A, a glycoside of spirosolane type, is identified as a major component in ripe tomato fruits. The present study investigated the effects of esculeoside A and its aglycon esculeogenin A on hyaluronidase activity in vitro and antiallergy in experimental dermatitis mice. Esculeogenin A/esculeoside A (esculeogenin A equivalent) with an IC50 of about 2 μM/9 μM dose-dependently inhibited hyaluronidase activity measured by a modified Morgan−Elson method. Oral treatment with esculeoside A 10 mg/kg of experimental dermatitis mice for 4 weeks significantly decreased the skin clinical score to 2.5 without any detectable side effects compared with 6.75 of the control. The scratching frequency of esculeoside A 100 mg/kg application was decreased significantly as 107.5 times compared with 296.67 times of the control. Thus, the present study showed that esculeoside A/esculeogenin A significantly blocks hyaluronidase activity in vitro and that esculeoside A ameliorates mouse experimental dermatitis. KEYWORDS: experimental atopic dermatitis, anti-hyaluronidase, tomato, esculeoside A, esculeogenin A
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fruits was calculated as about 0.043%. The chemical structure of esculeoside A is shown in Figure 1A. Esculeogenin A, the aglycon of esculeoside A, was treated with 2 N HCl for 1 h. Hyaluronidase Inhibitory Assay. The inhibitory activity on hyaluronidase was measured according to a modified Morgan−Elson method as described previously.10 Briefly, samples prepared in 0.1 M acetate buffer (pH 4) and hyaluronidase (type IV-S: from bovine testes, Sigma, St. Louis, MO, USA) in buffer with a final concentration of 5 mg/mL were incubated at 37 °C. Then, compound 48/80 (Sigma) in buffer with a final concentration of 0.5 mg/mL was added and incubated. After hyaluronic acid sodium salt (from rooster comb, Wako, Osaka, Japan) in buffer with a final concentration of 0.4 mg/mL had been included, the mixture was incubated. p-Dimethylaminobenzaldehyde (Wako) acetate solution was then added and incubated. Then the absorbance was determined at 585 nm, and the enzyme inhibitory activity (%) was quantified. Esculeoside A or esculeogenin A as a stock solution (2.8 mg/mL) was prepared with 0.7% DMSO and diluted with 0.1 M acetate buffer. Chromoglycate (Wako) was prepared with water as a stock solution of 10 mg/mL. In Vivo Experimental Protocols. The animal experimental protocol is illustrated in Figure 2A. All animal experiments were performed under the Guidelines of the Japanese Pharmacological Society for the Care and Use of Laboratory Animals. Female BALB/c mice, 7 weeks old, were purchased from Kyudo Co., Ltd. (Fukuoka, Japan). Food and water were available ad libitum. First, mouse hind paws were implanted with magnets for the following scratch analysis. After experimental dermatitis had been induced by DNCB on the dorsal skin, esculeoside A (10, 50, and 100 mg/kg of body weight), dexamethasone (Nichiiko, Toyama, Japan) (0.1 mg/kg of body weight), or saline was given orally every day for 4 weeks, respectively. In the period of treatment, the skin disorders were evaluated. The scratch behavior were measured after 4 weeks. In the end the dorsal skin was collected for histopathological analysis. Induction of Experimental Dermatitis. For induction of experimental dermatitis, mouse skin was treated with DNCB (Wako) as described by Lee et al.11 Briefly, after dorsal hairs in an area of approximately 8 cm2 were completely removed, 0.1 mL of 1% DNCB
INTRODUCTION Atopic dermatitis is a pruritic inflammatory skin disorder, and hyaluronidase, an enzyme for hyaluronic acid (HA) degradation, is well-known to be related to inflammation and allergic responses.1 It has been reported that during inflammation and tissue damage, hyaluronidase cleaves polysaccharide HA in low molecular weight HA fragments,2 which induce pro-inflammatory immune responses.3 Moreover, the skin inflammation induced by sensitizers such as 2,4,6-trinitrochlorobenzene was completely prevented by a hyaluronidase inhibitor.4,5 Therefore, the inhibitory effect on the activity of hyaluronidase may play an essential role during the pathological processes of atopic dermatitis. Antiallergic agents such as steroids and antihistamines are useful for treating atopic dermatitis. Nohara and co-workers have succeeded in isolating esculeoside A, a glycoside of spirosolane type, from fresh tomato fruit (Lycopersicon esculentum).6 Esculeoside A is identified as a major component in ripe tomatoes, at 4-fold the content of lycopene, the main tomato carotenoid.7 Growth inhibition on human breast adenocarcinoma cell line and antiatherosclerosis effects by esculeoside A have been reported.6,8 As a plant steroid glycoalkaloid, there has been no study to evaluate the possible effects of esculeoside A in ameliorating atopic dermatitis. Therefore, this study investigated the effects of esculeoside A and its aglycon on hyaluronidase activity in vitro and on mice demonstrating experimental dermatitis induced by 2,4-dinitrochlorobenzene (DNCB). The results showed that esculeoside A inhibited hyaluronidase activity in vitro and ameliorated experimental dermatitis in vivo.
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MATERIALS AND METHODS
Preparation of Esculeoside A and Esculeogenin A. Esculeoside A and its aglycon esculeogenin A were extracted as previously described.6,9 Briefly, fresh ripe cherry tomato fruits were smashed and then filtered. The supernatant was applied to column chromatography over a porous polystyrene gel (Diaion HP-20) and washed with water; then the methanol extract was passed through a reverse silica gel column chromatography. Finally, the 60% methanol eluate gave esculeoside A. The average of esculeoside A yield from ripe tomato © 2015 American Chemical Society
Received: Revised: Accepted: Published: 403
November 10, 2015 December 30, 2015 December 30, 2015 December 30, 2015 DOI: 10.1021/acs.jafc.5b05320 J. Agric. Food Chem. 2016, 64, 403−408
Letter
Journal of Agricultural and Food Chemistry
Figure 1. Dose-dependence of esculeoside A/esculeogenin A on the inhibition of hyaluronidase activity. (A) Chemical structure of esculeoside A. Glc, glucose; Gal, galactose; Xyl, xylose. (B) Concentration of esculeoside A calculated as the equivalent of esculeogenin A. The molecular weight of esculeoside A is 1270.38 g/mol. Each value was the average of triplicates, and each bar indicates the mean ± SEM (n = 5). EsA, esculeoside A; Esg A, esculeogenin A. as a sensitizer was applied to the dorsal skin twice every 3 days. In the next week, 0.1 mL of 0.2% DNCB for challenge was applied on the dorsal skin twice every 3 days. DNCB was prepared in acetone. After the challenge was completed, sample was applied. Clinical Skin Score. Skin lesions were measured with four symptoms: erythema/hemorrhage, edema, excoriation/erosion, and scaling/dryness. Each symptom was graded from 0 to 3 (none, 0; mild, 1; moderate, 2; severe, 3). Clinical skin score was calculated as the sum of the individual scores as previously reported.12 Histological Analysis. After the mice were killed, the dorsal skins were collected and kept in 10% neutral formalin. The skin samples were prepared in paraffin by a conventional method. The cross sections of skin were prepared and stained with hematoxylin and eosin (H&E) and examined for histological evaluation. The epidermal and dermal thickening and the infiltration of inflammatory cells were evaluated respectively following the four-grade system: 0 (none), 1 (mild), 2 (moderate), and 3 (severe). Evaluation of Scratching Behavior. To detect and evaluate the scratching behavior from the hind toes of mice, Microact (Neuroscience, Tokyo, Japan) was used as previously reported.13,14 Briefly, under ether anesthesia, the dorsal side of both hind paws of the mice was implanted subcutaneously with a small Teflon-coated magnet (1 mm in diameter, 3 mm long) on the day before DNCB challenge. The magnet was checked to remain in situ throughout the whole experimental period. The mouse with magnets was put in an observation chamber, 11 cm in diameter and 18 cm high, surrounded by a round coil. An electric current in the coil, which was induced by movement of the hind paws with the implanted magnets, was
amplified and recorded by the MicroAct software. The recording time for a mouse was 30 min. The measurement was performed on the 29th day after esculeoside A application. The following parameters were used to register scratch events: threshold (V) 0.05, event gap (s) 0.05, max freq (Hz) 20, min freq (Hz) 5, min duration (s) 0.25. Statistical Analysis. All data are the mean ± SEM. Comparisons between the two groups were performed with a Student’s t test. Multiple comparisons were carried out using the Dunnet test. Probability (p) values
