Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by elevated immunoglobulin E (IgE), mast cell infiltration and skin lesions including pruritus, erythema and eczema. Cudrania tricuspidata extracts have been clinically administered for a long time in the East Asia including Korean and China as a home-remedy to diminish the inflammation of gastritis and hepatitis. To examine whether it works on AD or not, an AD-like animal model was experimented in this study. AD was induced by applying Dermatophagoides farinae ( D. farinae ) extract to the backs of 9-week old NC/Nga mice for 21 days. Following this, an ethanol extract of C. tricuspidata stems (EECT) was applied topically for 14 days to the sensitized skin, while distilled water was used as a control (EECT0 mice). Anti-AD effects of EECT were evaluated using scores for AD-like skin lesions, serum IgE levels and mast cell counts in the skin dermal layers to assess inflammation. Topically applied ethanol extract of Cudrania tricuspidata stems (EECT 7.5, 25 and 75 mg/mL) markedly reduced AD-like skin lesions after 4 days (by 30.1%, 31.4% and 38.5%, respectively) and also after 14 days (by 63.6%, 66.1% and 49.6%, respectively), while distilled water improved AD by 17.8% and 38.7%, respectively (p < 0.05). Serum IgE production was reduced in the EECT7.5, EECT25 and EECT75 groups after 4 days (by 57.6%, 65.9% and 59.3%, respectively) and after 14 days of the treatment (by 82.0%, 79.6% and 75.3%, respectively), while distilled water decreased it by 38.8% and 62.3% (p = 0.0001 and p = 0.0001, respectively). Mast cell counts increased after sensitization by D. farinae extract (p = 0.003) and EECT attenuated the mast cell overproduction, and reduced mast cell degranulation markedly. Attenuation was most obvious in the early stage of EECT treatment when the AD was most acute.
Atopic dermatitis (AD) is a chronic inflammatory skin disease accompanied by representative symptoms such as itch, erythema, excoriation, edema, dryness and scaling [
We examined a natural material with anti-inflammatory effects as a new drug candidate for AD treatment. The selected natural material was extract of Cudrania tricuspidata (C. tricuspidata), a kind of mulberry tree which is native to East Asian countries such as Korea, China and Japan [
Dried stems of C. tricuspidata were finely powdered, and biologically active compounds were extracted with 95% ethanol (1:10, v/v) for 24 h, in a 60˚C water bath, then filtered through filter paper (ADVANTEC No.2, 150 mm) under vacuum. The filtrate was concentrated by evaporation at 45˚C, then deep-frozen at −70˚C after reconstitution in 70% ethanol in a concentration range of 100 - 200 mg/mL. The frozen filtrate was diluted appropriately after thawing to room temperature. Three concentrations of the ethanol extract (EECT) namely 7.5, 25 and 75 mg/mL, were prepared by diluting with distilled water, and tested topically.
Twenty-eight NC/Nga mice (9 week-old, male) were purchased from Central Exp. Animals Co. Ltd. (South Korea), and all animal procedures were approved by the Institutional Animal Care and Use Committee of Hanyang University (No: HY-IACUC-10-062). After one-week of adaptation, the mice were anesthetized with ether and their heads, necks and shoulders shaved with razors and waxing cream. Then twenty-four of the mice were topically administered 100mg of D. farinae (house mite) extract-ointment (Biostar AD ointment, Japan) on their shaved skin regions six times over 21 days to induce AD-like skin lesions [
On the 21st day EECT was administered to three experimental groups of 4 mice receiving 7.5 mg/mL, 25 mg/ml and 75 mg/mL EECT, respectively, for 14 days. The EECT was topically applied with a small flat brush once a day on all the treated skin areas. Distilled water was administered to the control group (EECT0, n = 4) in the same manner, while the “normal” group (normal, n = 4) received no treatment.
The changes of the AD-like skin lesions were assessed visually by two trained investigators on the 21st day of sensitization, and after the 4th, 10th and 14th day of EECT treatment. The severity was scored as the sum of the AD scores for each category, namely itch, edema, erythema/hemorrhage, excoriation/erosion and dryness/ scaling, graded as 0 (no symptoms), 1 (mild symptoms), 2 (moderate symptoms) and 3 (severe symptoms) [
Orbit blood was collected under ether anesthesia on the final day of AD induction, then on the 4th, 10th and 14th days of treatment. Sera were deep-frozen at −70˚C after centrifugation of blood at 3000 rpm, 4˚C for 20 min. Serum IgE levels were determined using a Mouse IgE ELISA Assay kit (XpressBio Life Science Products, Catalog No: 595-700, Express Biotech International, Thurmont, USA) at 450 nm expressed as {(serum IgE level at baseline − serum IgE level on the 4th, 10th or 14th day of EECT treatment)/serum IgE level at baseline} × 100.
After the 14 days of EECT treatment, all the mice were killed under ether anesthesia after evaluation of the AD-like skin lesions and orbit blood sampling, and the skin was removed from the affected regions of the animals’ heads, necks and shoulders. The skin was fixed with 10% formaldehyde, embedded in paraffin, and thin sections were made. The sections were stained with toluidine blue O, and microscopic examination was carried out at ×200; the total numbers of mast cells including granulated and degranulated forms were counted at five sites (400 μm2) chosen at random in each skin sample [
The measurements on the five groups were compared by one-way ANOVA and by post-hoc Duncan or Scheffe test at p < 0.05 using SPSS ver. 21.0. Eight mice died due to excess anesthesia during the AD induction period.
The changes of AD-like skin lesions from the baseline at the end of AD induction to the 14 days after the EECT treatment are presented in
± 5.4% (4 days) to 38.7% ± 9.9% (14 days)) in the EECT0 group. In spite of the higher AD reduction in the EECT-treated groups, no significant differences were found between the EECT0 and EECT-treated groups after 14 days of EECT treatment. Importantly, the AD-like skin lesions were improved most when the mice were treated with EECT 25 mg/mL for 14 days, and the therapeutic effect of EECT 25 mg/mL was significantly greater than that seen in the EECT0 group in the early stage of treatment.
The changes of serum IgE levels during the EECT treatment for 14 days are presented in
Numbers of mast cells (both granulated and degranulated forms) in the skin dermis layers stained with toluidine BlueO were examined by optical microscopy (200×) (
the EECT-treated groups, especially the EECT7.5 group, than in the distilled water-treated (EECT0) group. This tendency was particularly evident in the skin dermis of heads and necks. The granulated and degranulated forms of mast cells were also counted separately, and only the latter increased significantly after AD induction (p = 0.0001; normal (45.0 ± 8.1) vs. EECT0 (484.8 ± 60.8)) while degranulated mast cells increased significantly less when treated with EECT (EECT7.5 (315.0 ± 36.6), EECT25 (289.0 ± 19.6) and EECT75 (362.0 ± 18.2)) than with distilled water (
In the current study, topically applied EECT had a significant effect on the severity of AD-like skin lesions, serum IgE levels and on mast cell counts in the skin dermis. The anti-AD effects of EECT were detectible when EECT 7.5, 25 and 75 mg/mL were applied topically for 14 days to the mice (
AD appears to be associated with inflammatory immune dysregulation of the skin, impaired skin barrier function, and IgE-mediated sensitization to food and environmental allergens such as microbial pathogens [
Many skin disorders like AD seem to be related to infiltration and activation of mast cells, resulting in skin inflammation [
The current study has a few limitations. Thus, the considerable variability of one group resulted in limited statistical power of the comparison with the other groups. It may have been caused by the small sample size of the groups. If the sample size had been larger, the anti-AD effect of EECT 7.5, 25 and 75 mg/mL might have been more significant at 14 days of EECT treatment than that of distilled water.
In spite of the above limitation, the current study’s strengths include the fact that the EECT was applied topically to the AD-like skin lesions as an ointment. Most traditional oriental natural substances, such as mulberry tree extract and ginseng are orally administered. Secondly, NC/Nga mice develop AD-like skin lesions spontaneously under normal conditions, and they are a suitable model for some aspects of human AD, with IgE hyperproduction which is influenced by environmental factors, in part, due to interleukin-4 and -5 released by mast cells of the affected skin [
Ethanol extract of C. tricuspidata stems (EECT) administered topically to an NC/Nga mouse AD model induced by D. farinae extract decreased serum IgE levels and degranulated mast cell counts in the dermis of the skin, and consequently improved the AD-like skin lesions. The anti-AD effect was especially significant when EECT 7.5 and 25 mg/mL were applied for 14 days, and the therapeutic effect was more obvious in the early stage of EECT treatment when AD was strongly induced than later in the treatment.
Yoo-Sin Park,Shin-Hee Kim,Sang-Yeon Kim,Gae-Myoung Koh,Ju-Hwan Suh,Ju-Seop Kang, (2016) Topical Application of Cudrania tricuspidata Stem Extract Inhibits Atopic Dermatitis-Like Skin Lesions in an NC/Nga Mouse Model: An Experimental Animal Study. Pharmacology & Pharmacy,07,358-367. doi: 10.4236/pp.2016.78044