KR20140093414A

KR20140093414A - The active components in the extract of Rhus verniciflua bark that are effective in treating or improving contact dermatitis symptoms.

Info

Publication number: KR20140093414A
Application number: KR1020130005701A
Authority: KR
Inventors: 이양기
Original assignee: 이양기
Priority date: 2013-01-18
Filing date: 2013-01-18
Publication date: 2014-07-28

Abstract

In this study, antiinflammatory activity of Rhus verniciflua var. Bark was investigated using 2,4-dinitroflurorobenzene (DNFB) -induced inflammation-induced mice and activated macrophages. As a result of the study, the lacquer bark not only reduced the increase of vascular permeability, which is characteristic of dermatitis, which has clear histological morphological changes of the dermis and epidermis, but also reduced dilatation of the ear tissue and inflammation of the ear tissue. Toluidine blue staining showed reduced mast cell activity in the group treated with extracts of Rhus verniciflua. Rhus verniciflua extract significantly reduced inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) expression in activated macrophages during translation. In addition, the production of tumor necrosis factor-a (TNF-a) and interleukin 6 (IL-6) was significantly reduced when lycopersan bark extracts were treated with activated macrophages. This suggests that inhibition of iNOS, COX-2 and proinflammatory cytokines may occur through NFkB signaling pathways. In conclusion, extracts of Rhus verniciflua can be used variously as pharmaceutical compositions and health functional foods for treating dermatitis (seborrheic, atopic, contact dermatitis).

Description

TECHNICAL FIELD The present invention relates to a composition for improving and treating dermatitis comprising extracts of Rhus verniciflua (Rhus verniciflua).

Description of the Prior Art

(1) atopic dermatitis (atopic dermatitis) is an eczema-like skin lesion that occurs in people with atopic dermatitis. It is also called endogenous eczema and benign edema. There is a genetic tendency but the cause is unknown. When the symptoms become worse, the head changes to the same skin, and the skin of the whole body is also flared. The entire skin becomes rough and blue-white (especially on the side of the elbows and knees of the knuckles), papules and ovaries are formed and fused to form psychedelia. When symptoms are severe, use ointments (antihistamines and steroids) to relieve symptoms.

(2) Contact dermatitis (contact dermatitis): Skin inflammation caused by contact with external substances. It is characterized by itching that the so-called skin is hung. It has the same symptoms as acute eczema, but it occurs as a reaction to certain substances acting externally. Antihistamines and corticosteroids (steroids) are used as anti-inflammatory drugs to alleviate them.

(3) seborrhoic dermatitis (seborrhoic dermatitis): hair, forehead, armpit and sebaceous secretions are often occur in many areas of the dermatitis. Also called seborrheic eczema. It is dermatitis mainly caused by erythema and thin scales (dandruff). Unlike normal eczema, it is caused by abnormality of the constitution or sebaceous glands. The lightest type is dandruff, dry ring. It is sensitive to sunlight and heat, and there are many things that are worse in spring and fall, and it is easy to recur even better. Avoid oily foods, and do not irritate your head with nails. Use an antihistamine and corticosteroids as underpants to relieve underwear.

Treatment of contact dermatitis should be treated according to the general principles of eczema. The characteristic symptoms of contact dermatitis are erythema, edema blisters and dirt, and cold wrinkles are used to dry the watery lesions and use moist cream and lotion. Chronic contact dermatitis, characterized by keratin and folliculitis, is a temporary, effective treatment of oily ointments or cream. When systemic lesions are spread or localized effects are less effective, systemic antihistamines and corticosteroids (steroids) Use medication to relieve symptoms. But due to side effects. Camouflage. Kidney function deterioration is accompanied. Various types of infection and expression in the in vivo defense system against irritants in metabolites in vivo, and various chemical mediators are involved in the mechanism of expression of inflammation, and the pathogenesis thereof is very complicated. Conventional antiinflammatory agents are classified into steroidal and nonsteroidal antiinflammatory agents. Most of synthetic antiinflammatory agents have antiinflammatory effects in natural products which have side effects such as long-term use and long-term side effects, It is a desperate situation.

An object of the present invention is to provide a pharmacological composition and a health functional food raw material using a natural substance free from the side effects of prevention and treatment of dermatitis containing Rhus verniciflua Bark extract as an active ingredient.

One) DNFB Induced by Figure In dermatitis mice Lacquer tree bark Efficacy of vascular permeability and efferent expansion of extract

The pharmacological efficacy of Rhus verniciflua extract has been reported so far. In order to explain the therapeutic role of Rhus verniciflua extract in vivo in skin inflammation related diseases, we performed experiments using a mouse model of dermatitis. In order to confirm the inhibitory activity of the dermatophyte extract of the lacquer tree, an Evans blue solution was intravenously injected into the tail of the anesthetized mouse 1 hour before sacrificing the mouse. Mice in which DNFB (2,4-dinitrofluorobenzene) was repeatedly treated showed leakage of Evans blue solution. Evans blue solution leakage in the ear tissues was significantly reduced ( p <0.01) (Fig. 1 (a)) compared to the DNFB treated group in the extract from lacquer wood extract. The amount of Evans blue solution leaking from the ear tissue indicates the degree of vascular permeability change reflecting ear swelling (Figure 1 (b)).

Figure 1 shows that the extracts of Rhus verniciflua have a noticeably reduced ear swelling. The average glomerular thickness of the DNFB treated group was 0.437 .007 mm, but the average thickness of the ear treated group was 0.2730.006 mm (Fig. 1 (c)).

2) The efficacy of DNFB-induced extracts of Rhus verniciflua on histological changes in ear dermatitis mice

DNFB-induced dermatitis (Fig. 2) is associated with edema of the epidermis and skin lesions called hyperkeratosis and epidermal spongiformis, which increase the number of inflammatory cells in the dermis. For histopathological analysis, on the 11th day after DNFB treatment, ear tissue sections were prepared and hematoxylin and eosin staining were performed. The skin epidermis and dermis of DNFB treated group showed edema, hyperkeratosis, and epidermal sponging, and the number of inflammatory cells was increased. However, in the group treated with extracts of Rhus verniciflua, the number of inflammatory cells in epidermis and dermis was remarkably decreased, and the symptoms of hyperkeratosis were also decreased (Fig. 3 (a)). In addition, the epidermis in the ear tissue of the lacquer bark extract-treated group showed a 1.4-fold decrease in ear thickness compared to the DNFB-treated group (Fig. 3 (b)). It was confirmed that the thickness of epidermis and dermis decreased in comparison with that of DNFB treatment group in the case of lacquer bark extract treatment. These results indicate that the extracts of Rhus verniciflua are associated with anti - inflammatory activity.

3) Efficacy of bark extract of Rhus verniciflua on the infiltration of mast cells in dermatitis treated mice treated with DNFB.

Mast cells are one of the inflammatory cells that penetrate the skin of dermatitis patients. These cells secrete a granulocyte that induces allergies, such as prostaglandins, leukotrienes, and various precursor inflammatory cytokines, through IgE receptors through IgE receptors with high affinity for cell surfaces to activate allergies. Therefore, we examined the effect of extracts of Rhus verniciflua on the penetration and degranulation of mast cells through toluidine blue staining.

The number of mast cells infiltrating and degranulating in the dermis layer of the lacquer bark extract group was significantly reduced compared to the DNFB-treated group (Figure 4).

4) Effect of lacquer tree bark on iNOS and COX-2 in RAW264.7 cells.

Since lacquer bark extracts in ruminants have reduced the symptoms of contact dermatitis in animal models, we further investigated whether lacquer bark extract inhibits the production of nitrite and PGE2-mediated precursor inflammatory mediators in LPS-treated RAW264.7 cells. Macrophages accounting for one part of the cells in the skin are useful therapeutic targets for allergic contact dermatitis (ACD). ACD has been reported to significantly induce the expression of NO and PGE2 as precursor inflammatory mediators. During inflammatory reactions, LPS induces activity along with morphological changes in RAW 264.7 cells. We investigated whether extracts of Rhus verniciflua can inhibit macrophage activity and reduce inflammatory mediators and cytokine production. As a result, LPS significantly increased NO and iNOS production in RAW264.7 cells (Figure 5). The extracts of Rhus verniciflua inhibited the secretion of NO induced by LPS treatment and iNOS protein expression in a concentration-dependent manner (Figure 5). In addition, extracts of lacquer bark did not affect cell viability as measured by CCK-8 assay. This suggests that the inhibitory effect of NO is not directly related to cytotoxicity.

COX-2, which promotes the production of prostaglandins such as PGE2, plays an important role in inflammation. We analyzed COX-2 protein levels in RAW264.7 cells. Immunotoxicity analysis showed that COX-2 expression decreased in the group treated with extracts of lacquer tree bark (Figure 5). PGE ₂ is abundantly produced in the skin during the induction phase of contact dermatitis. Rhus verniciflua extract treated before LPS stimulation inhibited the production of PGE ₂ (Figure 5).

5) Effect of lacquer tree bark on bulb inflammation cytokine in RAW264.7 cells

The activated macrophages showed a high amount of tumor necrosis factor (TNF-). We have investigated whether lacquer bark can lower the level of TNF- elevated by LPS stimulation. The extracts of Rhus verniciflua significantly reduced TNF-a mRNA levels (Figure 6A). Together with TNF-a, interleukin (IL) -6 plays an important role in skin inflammation (eg, contact dermatitis) and hypersensitivity reactions. IL-6 mRNA levels were significantly lower in cells treated with LPS than in LPS-only cells ( p <0.001) (Figure 6B). These results suggest that the extracts of Rhus verniciflua can reduce the proinflammatory cytokines and mediators, which may have anti-inflammatory effects.

6) Efficacy of lacquer tree bark on the movement of NF-B

In order to further elucidate the mechanism of action of lacquer bark extracts on macrophage activity and dermatitis, we examined the effect of extracts of Lacquer tree extract on LPS-induced NF-B activity. The activity of NF-B plays an important role in the inflammatory response of RAW264.7 cells. IB phosphorylation and subsequent degradation is an important process in NF-KB activation by various stimuli. In this study, cell proteins were isolated from nuclear and cytoplasmic proteins. An equivalent amount of protein extracted from the nucleus was used to determine if the lacquer tree bark could affect the translocation of NF-κВ in the nucleus and the cytoplasmic protein to determine how the lacquer bark would act on the degradation of IB. We found that the extracts of Rhus verniciflua bark strongly inhibited IB degradation in the nucleus as well as the NF-B p65 subunit (Fig. 7). In addition to the intracellular migration of the NF-B p65 subunit, extracts of Rhus verniciflua significantly reduced phosphorylation of IB-. These results were also confirmed by intracellular NF-B p65 subunit immunohistochemistry in the ear tissue of DNFB-induced figure dermatitis (Figure 7). A low amount of NF-kB p65 was found in the control and lacquer bark extract treated groups. On the other hand, a remarkable amount of NFkB was found in the tissues of DNFB treated mice. These results indicate that extracts of Rhus verniciflua inhibit allergic dermatitis by inhibiting NF-B function and inhibit macrophage activity.

7) HPLC analysis of components containing extract of bark of Rhus verniciflua and its antiinflammatory activity

Many studies have shown that flavonoids, such as picetine and quercetin, are active ingredients of Rhus verniciflua extracts that exhibit anti-inflammatory activity. Of these components, picetin was analyzed by uriciol - free extracts of lacquer wood extracts. In the HPLC condition of lacquer bark extract, picetine was found at the retention time of 18 minutes in the chromatogram. The lacquer bark was prepared at a concentration of 17.4 mg / mL. Pisetin was 0.391% in the lacquer tree bark. Found. Picetine significantly reduced LPS production of proinflammatory cytokines such as TNF- and IL-6 as well as iNOS in RAW264.7 cells (Figure 8).

Expected effect

Since the extract of Rhus verniciflua of the present invention exhibits the effect of prevention and treatment in an animal model of dermatitis, it can be used variously as a pharmaceutical composition for improving and treating dermatitis which does not cause side effects by using natural products and as a raw material for health functional food.

FIGURE 1: Experiments with mice
FIGURE 2: Effect of reducing the edema caused by dermatitis in the lacquer tree bark
FIGURE 3: Reduced edema and thinning of the epidermis by 1.4 times compared with DNFB treatment group, suggesting that it suppresses the formation of hyperkeratosis and sponge formation
FIGURE 4: Confirmation of the penetration and degranulation of mast cells into the skin using the toluidine blue staining method showed that the number of mast cells increased by DNFB decreased when the lacquer bark was treated
FIGURE 5: It was confirmed that the amount of PGE2 significantly increased by concentration of Rhus verniciflua
FIGURE 6: The expression of IL-6 and TNF-alpha genes induced by LPS was significantly reduced by lacquer tree bark
FIGURE7: Rhus verniciflua extract inhibits allergic dermatitis by inhibiting NF-B function and inhibits macrophage activity
FIGURE 8: Picetine significantly reduced LPS production of proinflammatory cytokines such as TNF- and IL-6 as well as iNOS in RAW 264.7 cells
FIGURE9: The cell survival rate of Raw264.7 treated with the lacquer sample was not significantly different from the cell survival rate of Raw264.7 (control group)

1) Preparation of rush extract.

The lacquer tree bark, certified by the KFDA, was collected from 10 - year - old lacquer bark, which lives around the western part of Korea, and used as a basic sample. 400 g of dried lacquer bark was thinly sliced, and uriciol, an allergen component, was removed from drinking water using the drinking water at the Chungdang Geunjung Institute (http://ckadhc.com) (Figure 9). Toxicity elimination has been demonstrated by the KFDA (No. 204074). The lacquer bark was dried at 25 ~ 35 ℃ for 10 days in the absence of direct sunlight and then pulverized with powder. The lacquer crushed powder was concentrated to 100 ℃ in water and the extract was vacuum / dry evaporator. The yield was 6.993% (w / w).

2) An experimental animal.

Six-week old female C57BL6 / N mice were purchased from Orient Bio (Eumsung, Korea). Mice were protected at an isolation facility where temperature (23 ± 2 ºC), humidity (35-60%) and 12-h light (12-h darkness cycle) were controlled and maintained. Experiments were conducted at Konkuk University (Seoul, Korea) in accordance with the guidelines of the Animal Ethics Committee (IACUC). The authentication code number is ku10009.

3) Induced dermatitis (ACD) induced by 2,4-dinitrofluorobenzene (DNFB).

Figure dermatitis was induced by repeated treatment of shaved embryos and ear surface of C57BL / 6N mice (n = 5). After shaking the C57BL / 6N mouse (n = 5), dissolve DNFB to 0.5% in a mixture of acetone and olive oil in a ratio of 4: 1, and then add 100 μl of 0.5% DNFB to the mice for 1, 2 and 3 days Lt; / RTI > Thereafter, 25 μl of a 0.2% DNFB solution was applied to the mouse ear skin on the mouse embryos on the fifth and seventh days and on the ninth day to induce allergic contact dermatitis. In the normal group, only a mixture of acetone and olive oil (4: 1. v / v) was administered, and the lacquer bark extract was orally administered at a concentration of 500 mg / kg for 11 days.

4) Histological study.

The ear tissues fixed with formalin were made into paraffin blocks to prepare slice slices with a thickness of 5 μm. The slides were stained (H & E staining) with hematoxylin (BBC Biochemical, Mount Vernon, Washington, USA) and eosin (Shimakyu's Pure Chemicals, Osaka, Japan) and observed under an optical microscope. Digital microscope photographs were taken at a fixed 200x magnification of the microscope.

5) Ear thickness measurement.

For ear thickness measurements, Axiovision image analysis 4.7 software (Carl Zeiss Vision GmbH, Munchen, Germany) was used in tissue stained with H & E staining. The dyed ear tissue was photographed with a digital microscope camera and the ear thickness was measured using software (Figure 2).

6) Biological permeability analysis.

The mice were sacrificed 1 hour after intravenous injection of 1% Sigma-Aldrich staining solution into the tail of the mice. Evan 's blue stained ear tissues were separated and then immersed in. (Sigma - Aldrich, St. Louis, Mo., USA), and the Evans blue solution was extracted at 60 ° C for more than half a day. The Evans blue solution from the ear tissue was measured at an optical density of 600 nm using a spectrophotometer, and the concentration of the Evans blue solution was calculated based on the standard curve of the Evans Blue solution quantification.

7) Toluidine blue staining.

To observe mast cells, cut sections of the ear tissues were stained with toluidine blue (Sigma-Aldrich, St. Louis, Mo., USA). After deparaffinization and hydration, the sections were stained in toluidine blue solution for 2-3 minutes, dehydrated and clarified with alcohol and xylene. The sections of the dyed ear tissues were observed under an optical microscope and mast cells were identified at a magnification of 400.

8) Tissue immunochemical staining .

The immunocruz staining system kit (Santa Cruz Biotechnology, Santa Cruz, Calif., USA) was used to confirm the expression of NF-B in the ear tissues. The 5 m- The sections were incubated with NF-KB p65 antibody (Santa Cruz, Santa Cruz, CA, USA, 1: 600) for 1 hour and then washed with PBS. (DAB chromogen kit, Vector Laboratories, Burlingame, Calif., USA) for 30 min, and then incubated for 30 min with HRP-streptavidin conjugate for 30 min. , And then stained for 1 minute with 1% methyl green solution.

9) Cell culture.

RAW264.7 macrophages were purchased from the American Type Culture Collection (Manassas, Va., USA) and cultured in DMEM supplemented with 1% penicillin and 10% FBS (Gibco BRL, Grand Island, NY, USA) (Invitrogen Co., Carlsbad, Calif., USA). The cells were cultured in a humid incubator of 5% CO ₂ , 37C.

10) Cell viability.

The survival rate of RAW264.7 cells was measured using the EZ-CyTox kit (Daelillab service CO, Korea). RAW 264.7 cells were cultured in 96-well plates (2 × 10 ⁴ cells / well) and cultured for 24 hours with 0, 25, 50, 100, and 200 g / ml ricin bark extracts. 10 L of EZ-CyTox reagent was added to each well, reacted at 37 ° C for 2 hours, and absorbance was measured at 450 nm using an ELISA Multi-Detection Reader (Tecan, Mannedorf, Switzerland).

11) NO and PGE ₂ .

The concentration of nitrite, an indicator of NO production, was measured in the same manner as in the previous paper. PGE ₂ inhibition of lacquer tree bark was measured by the method described in the manual using prostaglandin E ₂ EIA kit (Cayman Chemical Company, Ann Arbor, MI, USA).

12) Immunoblot analysis.

Immunomodal analysis was performed in the same manner as in the previous paper. The total protein (20 mg) was electrophoresed on a 12% SDS-PAGE polyacrylamide gel and the proteins separated by molecular weight were electrophoresed on polyvinylidene fluoride membranes (PVDF) (Bio-Rad Laboratories, Berkeley, California) . The PVDF membrane is immersed in 5% skim milk solution and reacted with the primary antibody. At the end of the reaction, the membrane is washed with 1 PBS-T buffer, and reacted with HRP-conjugated secondary antibody (1: 5,000) for 1-2 hours before washing. Immunoreactive bands were measured using a western blot kit.

13) RNA extraction, cDNA synthesis and real-time PCR (real-time PCR).

RNA extraction and cDNA synthesis were carried out using a total RNA isolation kit (Macherey-Nagel GmbH & Co., Dren, Germany) and a first strand cDNA synthesis kit (Fermentas, USA) PCR Master Mix and 2 ml each of 10x QuantiTect Primer (Qiagen, Valencia, CA, USA). Real-time PCR was performed using an ABI500 thermal cycler (Applied Biosystems, Foster City, CA, USA). The results were corrected by the amount of glyceraldehydes-3-phosphated dehydrogenase (GAPDH) mRNA. Expression levels of iNOS, IL-6 and TNF-alpha mRNA were expressed by the Delta Ct value calculation method. Primers for iNOS (QT00100275), IL-6 (QT00098875), TNF-a QT00104006 and GAPDH (QT01658692) were designed using the Primer Express program (Applied Biosystems).

14) HPLC analysis.

High performance liquid chromatography (HPLC) experiments were performed on Agilent 1260 (Luna C18, 250 4.6 mm, 5 m diameter, Phenomenex, Torrance, Calif.) To analyze the components contained in the extracts. Infinity HPLC system (Santa Clara, Calif.) Was used. Flow rate and injection volume were 1.2 mL / min and 5 20 mL, respectively. The chromatogram was measured at 260 nm and collected at 30C. Fisetin was purchased from Sigma-Aldrich (St. Louis, Mo., 99%) and used without further purification. The mobile phase for the ethanol extract of Rhus verniciflua was 8% water - soluble methanol.

Statistical analysis.

Results are expressed as mean standard error (n = 5 per group). Student's t- test or One-way ANOVA / Dunnett's t- test was used to compare the significance between the control group and the sampled group. Statistical analysis was performed using SPSS, version 12 (SPSS Inc., Chicago, IL, USA).

Claims

Contact dermatitis. A pharmaceutical composition for improving and treating dermatitis comprising seborrhoeic dermatitis, atopic dermatitis, and symptomatic lacquer bark extract as active ingredients.

Health functional foods containing extracts of Rhus verniciflua inhibiting mast cells as active ingredients

A pharmaceutical composition for the improvement and treatment of dermatitis containing an extract of Lacquer Tree Bark as an active ingredient Functional cosmetic ingredients

An anti-inflammatory food additive containing lycopene bark extract picetine as an active ingredient