Alnus Sibirica Extracts Suppress the Expression of Inflammatory Cytokines Induced by Lipopolysaccharides, Tumor Necrosis Factor-α, and Interferon-γ in Human Dermal Fibroblasts.

The effects of Alnus sibirica (AS) extracts on cytokine expression induced by inflammatory stimulants were examined in human dermal fibroblasts (HDFs) and RAW264.7 cells. The anti-oxidative effect and effect on cell viability of AS extracts were evaluated, and four extracts with the highest anti-oxidative effects were selected. HDFs and RAW264.7 cells were treated with inflammatory stimulants, and the expression of cytokines involved in acute (IL-6 and IL-10) and chronic (IL-18) inflammation, the initiation of the immune response (IL-33), and non-specific immune responses (IL-1ß, IL-8, and TNF-α) were determined using a reverse-transcription polymerase chain reaction. LPS increased the expression of all the cytokines, except for IL-18; however, AS extracts, particularly AS2 and AS4, reduced this increase, and TNF-α treatment markedly increased the expression of cytokines related to non-specific immune responses. IFN-γ treatment induced no significant changes, except for increased IL-33 expression in HDFs. AS extracts inhibited the increase in the expression of IL-33 and other cytokines in HDFs. Thus, the exposure of HDFs and RAW264.7 cells to inflammatory stimulants increased the expression of cytokines related to all the inflammatory processes. HDFs are involved not only in simple tissue regeneration but also in inflammatory reactions in the skin. AS2 and AS4 may offer effective therapy for related conditions.

Topical application of Rosa multiflora root extract improves atopic dermatitis-like skin lesions induced by mite antigen in NC/Nga mice.

The roots of Rosa multiflora THUNB. (RM) has been used in oriental traditional medicines as remedies for scabies, rheumatic arthralgia and stomatitis which were practicably related with today's inflammatory and allergic diseases. In the present study, we evaluated whether RM root extract (RME) and its major constituent, 2-(3,4-dihydroxyphenyl)-6-(4-hydroxyphenyl)-8-(2,4-dihydroxyphenyl)-2,3-trans-6,7-cis-7,8-trans-3,4,7,8-tetrahydro-2H,6H-pyrano[2,3-f] chromene-3,7,9-triol (RM-3) belongs to condensed tannins, improve atopic dermatitis (AD)-like skin lesions in NC/Nga mice induced by mite antigen. Topical application of RME as well as RM-3 improved skin severity and suppressed mRNA levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) on skin tissues, in addition, significantly reduced T helper 2 (Th2) immune responses via interleukin 10 (IL-10) up-regulation. Thus, RME, contains lots of condensed tannins such as RM-3 which possesses potent anti-inflammtory and immune-modulatory effects, may be useful for treatment of skin allergies and can be developed as new alternative herbal therapy against AD.

Efficacy of oregonin investigated by non-invasive evaluation in a B16 mouse melanoma model.

Oregonin has been reported to act as a mediator of antibiosis, a liver-protective agent, an antioxidant, an anti-inflammatory agent, and to prevent cancer outbreaks. B16 melanoma cells were separated with trypsin-ethylenediaminetetraacetic acid, resuspended in 50 µl of phosphate-buffered saline and transplanted into the backs of 6- to 8-week-old male Balb/c nude mice through subcutaneous injection. Treatment doses of oregonin were administered three times weekly, for 30 days from the 11th day after transplantation of the melanoma cells, in each group. The study consisted of a control group, a dacarbazine group, an oregonin group and a dacarbazine + oregonin group. Measurements were taken before treatment and on the 5th, 7th, 10th and 15th days after treatment for each group. Based on survival rates after transplantation, the control group showed less than 50% survival after 20 days, while the treatment groups showed at least 50% survival up to the 41st day.

Topical application of two condensed tannins from the root of Rosa multiflora Thunberg for the treatment of atopic dermatitis (AD) in NC/Nga mice.

Recently, the isolation of several condensed tannins from the roots of Rosa multiflora Thunberg, a traditional herbal therapy in oriental medicine for rheumatoid arthritis and scabies, was described. Two of the major condensed tannins - procyanidin B-3 (ProB3) and ent-guibourtinidol-(4ß â†’ 6)-catechin (RM-1) - were then applied topically to atopic dermatitis-like skin lesions on NC/Nga mice in order to assess their immunomodulatory properties. Both ProB3 and RM-1 significantly reduced the serum levels of eosinophils, IgE and certain Th2 cytokines (IL-4, 5 and 13) (p < 0.05 or 0.01). Additionally, ProB3 and RM-1 significantly reduced both the mRNA and protein expression of COX-2 and iNOS in mouse skin tissues (p < 0.01). Such results strongly suggest that ProB3 and RM-1 may be useful in the treatment allergic skin conditions, most notably atopic dermatitis.

Design, synthesis, bioconversion, and pharmacokinetics evaluation of new ester prodrugs of olmesartan.

Synthesis of new ester prodrugs of olmesartan is described. Their in vitro stabilities in simulated gastric juice, rat plasma, and rat liver microsomes were tested. And the pharmacokinetic parameters for olmesartan after their oral administration were also estimated and compared with those in case of olmesartan medoxomil. Compounds 13 and 14 demonstrated high stability in simulated gastric juice and were rapidly metabolized to olmesartan in rat liver microsomes and rat plasma in vitro. In addition, C(max) and AUC(last) parameters were significantly increased in case of compounds 13 and 14 compared with olmesartan medoxomil. These results indicate that compounds 13 and 14 with cyclohexylcarboxyethyl and adamantylcarboxymethyl promoieties, respectively, are promising prodrugs of olmesartan with markedly increased oral bioavailability.

Effect of Alnus japonica extract on a model of atopic dermatitis in NC/Nga mice.

AIM OF THE STUDY: The bark of Alnus species has long been used in traditional oriental medicine in the treatment of many pathological conditions, including fever, hemorrhage, diarrhea, alcoholism, various skin diseases (e.g. chronic herpes, eczema and prurigo), and inflammation. In order to assess the immunomodulatory efficacy of a novel herbal medicine in treating atopic dermatitis, we measured serum levels of several allergic and inflammatory biomarkers in NC/Nga mice before and after treatment with this experimental agent. MATERIALS AND METHODS: Gene and protein expression analyses of iNOS and COX-2 were quantified by real time PCR and Western blot analysis and serum levels of IL-4, -5 and -13 were also measured by ELISA, all of which were reduced after treatment with the experimental agent. Additionally, serum concentrations of IgE and blood eosinophil counts were reduced in treated mice. RESULTS: The topical application of leaf and bark extract from Alnus japonica suppressed the development of AD-like skin lesions. The percent of blood eosinophils was decreased after treatment with leaf and bark extract from Alnus japonica. The serum IgE and Th2-related cytokine levels were decreased after treatment with leaf and bark extract from Alnus japonica compared with those treated with base cream (vehicle treated AD group). The IL-4, IL-5 and IL-13 were lower than those of vehicle treated AD group. CONCLUSIONS: We contend that leaf and bark extract from Alnus japonica may prove useful in the treatment of atopic dermatitis and other allergic skin diseases, although more in-depth clinical studies are necessary before clinical implementation.

Facilitated skin permeation of oregonin by elastic liposomal formulations and suppression of atopic dermatitis in NC/Nga mice.

To develop an external preparation of oregonin (ORG) for the treatment of atopic dermatitis (AD), conventional creams (CC) and elastic liposomes (EL) containing ORG have been formulated and examined for their in vitro skin permeation properties and in vivo therapeutic efficacy assessments. EL, consisting of soybean phosphatidylcholine and Tween 80 (85 : 15 w/w %), were of flexible nanocarriers: they were about 130 nm in size and had a 4-fold greater deformability index than conventional liposomes. In a skin permeation study using a Franz diffusion cell mounted with depilated mouse skin, liposomal systems were superior to cream, revealing greater flux values. Both CC and EL were diversified with the addition of Trans-activating transcriptional activator (Tat) peptide, a sort of cell penetrating peptide, and subjected to in vivo efficacy evaluations in NC/Nga mice with AD-like lesions. On clinical observation for skin severity, rapid and profound improvement was observed in the treatment group with Tat-added liposomes (EL/T), showing a significant difference (p<0.05) versus Tat-added cream. The results indicated that EL/T treatment is effective for normalizing the immune-related responses and alleviating AD, evaluated as changes in the levels of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin (IL)-4, immunoglobulin E (IgE), and eosinophils in skin or blood.

Effect of taxifolin glycoside on atopic dermatitis-like skin lesions in NC/Nga mice.

Increased levels of eosinphils, IgE, IL-4, 5, and 13 and pro-inflammatory factors (COX-2, iNOS) are observed in patients with atopic dermatitis (AD). Taxifolin 3-O-beta-D-glucopyranoside (TAX) from the roots of Rhododendron mucronulatum (RM) was examined to determine whether its immunomodulatory effect was applicable for treating atopic dermatitis.A total of 7 groups of NC/Nga mice with AD were treated by topical application or intraperitoneal injection of TAX for 4 weeks. Follow-up evaluations were done to assess the changes in clinical observations, eosinophil counts, and levels of IgE, cytokines, COX-2 and iNOS.In the clinical observation during the experimental period, TAX treatment significantly reduced the severity of AD-like lesions induced in NC/Nga mice. Eosinophil and IgE levels decreased after treatment of the animals with TAX. TAX may thus be associated with improvement of eosinophil-related allergic diseases. The expression of cytokines (IL-4, 5 and 13) was significantly inhibited in the TAX-treated group, suggesting that TAX might play an immunoregulatory role associated with AD. In RT-PCR, iNOS and COX-2 expression levels were reduced in the TAX-treated group. In western blotting, the expression levels of iNOS and COX-2 were also reduced in the TAX-treated group.These findings suggest that TAX is effective for the treatment of AD by preventing the production of inflammatory cytokines and by reducing skin inflammation.

Effect of topical application and intraperitoneal injection of oregonin on atopic dermatitis in NC/Nga mice.

The diarylheptanoid, oregonin (ORE), which was isolated from the bark of Alnus japonica Steudel that grows natively in Korea, has been known to exert antioxidative, anti-inflammatory, anti-cancer and immune response inhibitory effects. The antioxidative effect of ORE was observed on the superoxide and 1,1-diphenyl-2-picrylhydrazyl radical, as well as on the expression of inducible nitric oxide synthase and cyclooxygenase-2 in lipopolysaccharide-treated RAW264.7 macrophages. The statistically significant inhibitory action of ORE against production of cytokines induced by bacterial products or by interleukin (IL)-1beta, free radicals and nitrogen species, and a corresponding increase in cellular calcium concentration because of ORE were confirmed in bone marrow and spleen dendritic cells that are known to play important functions in the development and advancement of atopic dermatitis (AD). It was thus expected that ORE would exert a beneficial effect in the treatment of AD. A study on the pharmaceutical benefits of ORE against AD has not yet been conducted in vivo. We therefore used an in vivo AD animal model, namely the NC/Nga mice, and by applying ORE onto the animals through skin application as well as intraperitoneal injection, we attempted to evaluate the benefits of ORE in this system. Evaluation of ORE was conducted by following the SCORE method to score the effect, as well as by measuring the Th2 cytokines IL-4, IL-5 and IL-13 levels from serum and lymphocytes, and IgE and eosinophil levels from serum. Additionally, the expression of mRNA and protein levels was estimated using real-time polymerase chain reaction and Western blotting analysis. The following categories of clinical evaluation, Th2 cytokines IL-4, IL-5 and IL-13 values, serum IgE levels, serum eosinophil levels, and mRNA and protein expression levels of iNOS and COX-2, were evaluated from topical application and intraperitoneal injection groups of ORE. The effects of ORE on AD in NC/Nga mice were confirmed as being similar to the positive control group, while a significant difference with the negative control group was observed. The results presented in this report suggest that ORE might be beneficial in the treatment of AD.

Diarylheptanoid hirsutenone prevents tumor necrosis factor-alpha-stimulated production of inflammatory mediators in human keratinocytes through NF-kappaB inhibition.

Keratinocytes may play an important role in the pathogenesis of skin disease in atopic dermatitis. Diarylheptanoids such as oregonin and hirstanonol are demonstrated to have anti-inflammatory and anti-oxidant effects. The present study was to investigate the effect of hirsutenone, one of the diarylheptanoids, against tumor necrosis factor (TNF)-alpha-stimulated responses in human keratinocytes. Hirsutenone attenuated the TNF-alpha-induced production of cytokine IL-8, prostaglandin E(2) and chemokine CCL27, and the formation of reactive oxygen/nitrogen species in keratinocytes. Immunosuppressants (dexamethasone and cyclosporin A) inhibited the TNF-alpha-elicited formation of IL-8, prostaglandin E(2) and CCL27, but did not affect formation of reactive species. Bay 11-7085 (an inhibitor of NF-kappaB activation) and anti-oxidant N-acetylcysteine attenuated the TNF-alpha-induced formation of inflammatory mediators and reactive species. Hirsutenone, dexamethasone, cyclosporin A and Bay 11-7085 inhibited the TNF-alpha-induced phosphorylation of inhibitory kappaB and the activation of nuclear factor (NF)-kappaB. The results show that hirsutenone seems to reduce the TNF-alpha-stimulated production of inflammatory mediators in keratinocytes by suppressing the activation of NF-kappaB that may be mediated by reactive oxygen species. The findings suggest that hirsutenone may exert an inhibitory effect against the pro-inflammatory mediator-induced skin disease.

Comparison of oral bioavailability of genistein and genistin in rats.

Genistein (GT) is an isoflavone from Leguminosae and has received much attention as a phytoestrogen. Genistin is a glycoside form of GT (genistein-7-O-beta-D-glucopyranoside, GT-glu) is mainly found in soy-derived foods. In this study, we examined the pharmacokinetic properties and bioavailability of GT in rats and compared with those of GT-glu. In order to characterize and compare the pharmacokinetics of GT and GT-glu, these compounds were administered intravenously and orally. The plasma concentration of GT was determined by HPLC after enzymatic hydrolysis. After oral administration of GT with various doses (4, 20, 40 mg/kg), the bioavailability of GT was 38.58, 24.34 and 30.75%, respectively. The T(max), C(max) and AUC(0-infinity) of GT after oral administration of GT (40 mg/kg), were 2h, 4876.19 ng/ml, 31,269.66 ng h/ml, respectively. When smaller amount of GT was administered, the faster T(max) was observed. Oral administration of GT-glu resulted in longer T(max), lower C(max), and greater bioavailability than that of GT. The pharmacokinetic parameters of GT following oral administration of GT-glu (64 mg/kg as GT-glu, 40 mg/kg as GT) were obtained as follows: 8h (T(max)), 3763.96 ng/ml (C(max)), 51,221.08 ng h/ml (AUC(0-infinity)) and 48.66% (absolute bioavailability), respectively. These results indicate that the oral bioavailability of GT-glu is greater than that of GT.

Bone loss preventing effect of Sophorae Fructus on ovariectomized rats.

The preventive effects of Sophorae Fructus extracts (I: hot water extract and II: combination product using I) on bone loss in ovariectomized (OVX) rats were investigated. Sophorae Fructus extracts were orally administrated to OVX rats for 9 weeks. Ovariectomy caused the increase of body weight and deoxypyridinoline (Dpd: bone resorption marker) and decrease of calcium (Ca: bone formation marker) level in serum. Dpd level were significantly decreased and Ca levels were elevated at 9 weeks in Sophorae Fructus extracts administered groups after ovariectomy at a dose of 0.556 g/kg/day compared with control group. In administered groups, trabecular bone area (TBA) in the tibia and lumbar were also increased compared with control group in histomorphological analysis. The preventive or treatment effects of Sophorae Fructus extracts on bone loss in OVX rats appears to be due to suppression of bone turnover.

Inhibition of IL-1beta and IL-6 in osteoblast-like cell by isoflavones extracted from Sophorae fructus.

Osteoporosis is recognized as one of the major hormonal deficiency diseases, especially in menopausal women and the elderly. When estrogen is reduced in the body, local factors such as IL-1beta and IL-6, which are known to be related with bone resorption, are increased and promote osteoclastogenesis, which is responsible for bone resorption. In the present study, we investigated whether glucosidic isoflavones (Isocal, PIII) extracted from Sophorae fructus affect the proliferation of osteoblasts and prevent osteoclastogenesis in vitro by attenuating upstream cytokines such as IL-1beta and IL-6 in a human osteoblastic cell line (MG-63) and in a primary osteoblastic culture from SD rat femurs. Interestingly, IL-1beta and IL-6 mRNA were significantly suppressed in osteoblast-like cells treated with 17beta-estradiol (E2) and PIII when compared to positive control (SDB), and this suppression was more effective at 10(-8)% than at the highest concentration of 10(-4)%. In addition, these were confirmed in protein levels using ELISA assay. In the cell line, the cells showed that E2 was the most effective in osteoblastic proliferation over the whole range of concentration (10(-4)%-10(-12)%), even though PIII also showed the second greatest effectiveness at 10(-8)%. Nitric oxide (NO) was significantly (p<0.05) upregulated in PIII and E2 over the concentration range 10(-6)% to 10(-8)% when compared to SDB, without showing any dose dependency. In bone marrow primary culture, we found by TRAP assay that PIII effectively suppressed osteoclastogenesis next to E2 in comparison with SDB and culture media (control). In conclusion, these results suggest that local bone-resorbing cytokines can be regulated by PIII at lower concentrations and that, therefore, PIII may preferentially induce anti-osteoporosis response by attenuating osteoclastic differentiation and by upregulating NO.