KR20160121696A - Pharmaceutical Composition for Treatment or Prevention of Inflammation and Allergic Diseases Comprising Pine Leaf and Smilax China Extracts - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating inflammation and allergosis, containing a composite extract of pine needle and Smilacis Rhizoma as an active ingredient. According to the present invention, the composition containing the composite extract of pine needle and Smilacis Rhizoma shows anti-inflammatory effects by inhibiting production of LTC4 and b-HEX secreted from mast cells as well as production of NO, TNF-alpha, and IL-6 cytonkines secresed from macrophage after stimulation by LPS. Thus, the composition can be used in pharmaceutical compositions for preventing and treating inflammatory disease and allergosis.

Description

TECHNICAL FIELD The present invention relates to a pharmaceutical composition for the treatment or prevention of inflammation and allergic diseases,

The present invention relates to a pharmaceutical composition for the prevention or treatment of inflammation and allergic diseases, which comprises a combined extract of Bryophyta and Bombyxiflorum as an active ingredient.

Macrophages play an important role in specific and nonspecific immune responses to microbial and viral infections. Macrophages activated by external stimuli produce and release various inflammatory mediators such as NO, PGE ₂ , and pro-inflammatory cytokines, including TNF-a, IL-1 and IL-6 (Ritchlin CT, et al., Mechanisms of TNF-alpha and RANKL-mediated osteoclastogenesis and bone resorption in psoriatic arthritis J Clin Invest 2003 , 111: 821-31).

Mast cells are derived from bone marrow cells and are matured in tissues and then activated by antigen-antibody and various cytokines, and are involved in inflammation and allergic reactions (Puxeddu I, et al., Mast cells in allergy and beyond Int J Biochem Cell B , 2003 , 35: 1601-1607). Activated mast cells are already known to increase the allergic response by biosynthesizing eosanoids such as histamine, lipid mediators prostaglandines and leukotriens, which have already been produced in the granules (Prussin C, et al. , IgE, mast cells, basophils, and eosinophils. J Allergy Clin Immunol, 2006, 117: S450- S456).

The key mediators that drive inflammatory and allergic diseases prostaglandin acids (prostaglandines), leukotriene acids (leukotriens), platelet activating factor (PAF), etc. Phospholipase A ₂ (phospholipase A _2), cyclooxygenase (cyclooxygenase) And arachidonic acid, a precursor by lipoxygenase (Harizi H, et al., Arachidonic-acid-derived eicosanoids: roles in biology and immunopathology. Trends Mol Med , 2008 , 14 (10): 461-469).

Leukotrienes constitute a local functional hormone group produced in vivo from arachidonic acid and important leukotrienes include leukotriene B ₄ (LTB ₄ ), leukotriene C ₄ (LTC ₄ ), leukotriene D ₄ (LTD ₄ ) and leukotriene E ₄ LTE ₄ ). The biosynthesis of these leukotrienes is initiated by the enzyme 5-lipoxygenase acting on arachidonic acid to produce an epoxide known as leukotriene A ₄ , which can be converted to other leukotrienes (LTB ₄ , LTC ₄ , LTD ₄ , LTE ₄ ). Leukotriene is involved in allergic and allergic reactions such as pulmonary artery diseases such as asthma, chronic bronchitis and related obstructive airways diseases, allergic rhinitis, contact dermatitis, allergic conjunctivitis, inflammation such as arthritis or inflammatory bowel disease, pain . Recently, drugs that are attracting attention as an allergic asthma treatment drug have histamine release inhibition, inhibition of leukotriene C ₄ formation, and inhibition of platelet activating factor production (Dahlen SE, Treatment of asthma with antileukotrienes: first line or last resort therapy Eur J Pharmacol , 2006 , 533: 40-56; Kitamura S, Leukotriene (B4, C4, D4, E4), Nippon Rinsho , 2005 , Sl8: 28-33).

The perennial shrubs of Pinus tabulaeformis CORR. = P. densiflora S. et Z.) is a foliage of Manchuria pine, Pinus densiflora, and Pinus densiflora, and leaves contain essential oils such as α-pinene, β-pinene, and camphene, flavonoids and resins, It is well known that it is known to be a medicinal herb .

It is a perennial of the vine shrub belonging to the lily family, Smilax china L. = Coprosmanthus japonicus KUNTH) is a rhizome of rhizomes and rhizomes, and rhizome contains various saponins composed of diosgenin and diosgenin, containing alkaloids, phenols, amino acids, organic acids, saccharides, and 11.2% there are known to be contained 39.1% linoleic acid, oleic acid 48.4 % ( jeongboseop and sinmingyo low, illustrated hyangyak (herbal) Dictionary, Younglim four, pp 185-186, 1998).

1. Korean Patent Publication No. 10-2014-0040184 2. Korean Patent Publication No. 10-2014-0040183 3. Korean Patent Registration No. 10-1361626 4. Korean Patent Publication No. 10-2012-0077889

It is an object of the present invention to provide a therapeutic agent for inflammatory and allergic diseases which is effective by using a combined extract of Bryophyta and Bombyxiflorum. To this end, the inventors of the present invention found that the combined extracts of Bryophyta and Bombyx mori produced NO, TNF-a and IL-6 cytokines secreted from macrophages after LPS stimulation, b-HEX and LTC ₄ secreted from mast cells And confirming the anti-inflammatory effect inhibiting the production, thereby completing the present invention.

In the present invention,

There is provided a pharmaceutical composition for the treatment or prevention of an inflammatory and allergic disease which comprises an extract of Bokwibae and Seokjung rye as an active ingredient.

Compound extracts can be obtained using water, C1-C4 alcohols and other organic solvents as extraction solvents. Preferably, the complex extract may be obtained by repeating the process of immersing the bamboo shoots and the soil bamboo shoots in ethanol and then repeating the step of obtaining the extract solution one to five times. At this time, ethanol is not particularly limited, but preferably 50% or more of ethanol may be used, more preferably 70 to 75% ethanol may be used, and particularly preferably 75% ethanol may be used.

The extraction method of the complex extract is not particularly limited, but when alcohol is used as the extraction solvent, it is preferably carried out at a temperature of 90 ° C or lower. More preferably, the complex extract of the present invention can be obtained by repeating the process of immersing in ethanol at 50 to 70 ° C for 18 to 30 hours and then extracting the extract at room temperature when ethanol is used as the extraction solvent.

The extracts of the present invention containing the combined extracts of P. japonica and Bombyx mori revealed that the NO, TNF-a and IL-6 cytokine secretion secreted by macrophages after LPS stimulation, as evidenced by the examples, b-HEX and LTC _4, and thus can be used as an effective drug for the prevention and treatment of inflammatory diseases or allergic diseases.

FIG. 1 is a graph showing the results of inhibition of nitrite formation (IC ₅₀ = 16.8 μM) of the composition of the present invention containing a combined extract of Bovine Leaf and Sea Bream.
Fig. 2 shows the results of inhibition of TNF-a production by the composition of the present invention.
Fig. 3 shows the experimental results of inhibition of IL-6 production by the composition of the present invention.
4 is a graph showing the results of inhibition of b-HEX formation (IC ₅₀ = 3.2 μg / ml) of the composition of the present invention.
Figure 5 is the LTC ₄ Inhibitory experiments is (IC ₅₀ = 13.1μg / ml) present composition.

Hereinafter, the present invention will be described in more detail with reference to specific examples. However, these embodiments are only for describing the present invention more specifically, and the scope of the present invention is not limited by these embodiments.

[ Example ]

Example  1. Preparation of complex extract

(40 g) and bamboo shoot (60 g) purchased from Human Hub Co., Ltd. (http://www.humanherb.co.kr/) were immersed in 1,500 ml of 75% ethanol at 60 ° C for 24 hours, The extract was filtered twice through a Vaccum rotary evaporator (VR-205c, Nihon Seiko, Japan), and the filtrate was concentrated under reduced pressure. , 10.8 g (yield: 10.8%) of a 75% ethanol extract of the complex extract was obtained through concentration and drying under reduced pressure to evaporate the solvent.

[ Experimental Example ]

Experimental material

Cell culture reagents such as RPMI-1640, Modifed Eagle Medium (MEM), non-essential amino acids solution, fetal bovine serum (FBS), streptomycin and penicillin were purchased from Hyclone (Logan, UT, USA). Of the reagent used in the experiment SCF (Stem cell factor, STEMCELL, Vancouver, Canada), LPS (Lipopolysaccharides, Sigma, St. Louis, MO, USA), IL-10 (Recombinant Mouse IL-10, BD Pharmingen TM, San Jose , CA, USA) were purchased and used. LTC ₄ Measurement kit was purchased from Cayman (Ann Arbor, MI, USA) and IL kit was purchased from R & D System (Minneapolis, MN, USA).

Cell culture

Mouse macrophage RAW 264.7 cells were cultured in DMEM medium containing 10% FBS, 100 U / ml penicillin, and 100 μg / ml streptomycin at 37 ° C and 5% CO ₂ .

Bone marrow cells were isolated from the femur of BALB / C mice and cultured in RPMI-1640 (Invitrogen) containing 10% FBS, 100 U / ml penicillin, 100 μg / ml streptomycine The medium was cultured for about 3 weeks with a final volume of 20% containing PWM-SCM containing IL-3 as the source of IL-3, resulting in more than 90% homogeneous BMMC. Cells were cultured at 37 ° C in a 5% CO ₂ incubator, and BMMCs were plated in 96-well plates at a constant number of cells.

Sample Preparation

As a sample to be used for the experiment, the combined extracts of Bryophyta and Bombyx mori were dissolved in DMSO solvent and diluted to a concentration of 6.25, 12.5, 25, 50, 100 μg / ml.

Experimental Example  1. Nitric oxide (NO) and Proinflammatory  Measurement of cytokine production

In order to examine the inhibitory effect on the amount of nitric oxide (NO) and proinflammatory cytokine IL-6 produced from the sample obtained in the above example, experiments were carried out as follows (Yang JH et al. , Anti-inflammatory activity of ethylacetate fraction of Cliona celata. Immunopharmacol Immunotoxicol 2011, 33 (2): 373-379).

RAW 264.7 cells (1.5 × 10 ⁵ cells / well) were pre-cultured on a 24-well plate and DMEM medium was added. After the complex herb extract was pretreated for 1 hour at a concentration of 200 ng / ℃, and cultured at 5% CO ₂ condition. After incubation for 24 hours, the supernatant was collected and reacted with 100 μl of the same amount of Griess reagent (G4410, Sigma), and the absorbance was measured at 570 nm using an ELISA reader (Sunrise-Basic Tecan, TECAN) The amount of NO produced is shown graphically using a standard curve. TNF-a and IL-6 cytokines were measured by ELISA kit (R & D System, USA).

As shown in FIG. 1, in order to examine the inhibitory effect of NO and NO production on the extracts of Bryophyta japonica and Bombyx mori, the extracts were pretreated, and the amounts of nitrite produced at concentrations of 12.5 μg / ml, 25 μg / ml and 50 μg / Showed inhibitory effects at concentrations of 15 μM, 9 μM and 2 μM, respectively, and the IC ₅₀ for NO production was 16.8 μM.

As shown in Fig. 2, cytokine was significantly increased in the cell supernatants treated with LPS to 34.43 ng / ml in the case of TNF-a, but the extracts of 25 .mu.g / ml, 50 .mu.g / / Ml), 27.77 ng / ml, 24.65 ng / ml, and 22.72 ng / ml, respectively, in the cell supernatants pretreated with TNF-α.

As shown in FIG. 3, IL-6 production was significantly increased to 3032 pg / ml in the cell supernatants treated with LPS. However, IL-6 production was inhibited at 1259 pg / ml, 721 pg / ml and 98 pg / ml in the cell supernatants pretreated with IL-6, 25 μg / ml and 50 μg / ml.

Experimental Example  2. b- hexaminidase (b-Hex) emission suppression measurement

In order to confirm the degree of degranulation through the measurement of β-Hex enzyme activity, which is a marker substance of BMMC obtained from the above-mentioned BMMC, an experiment was conducted as follows (Son JK et al, Ginkgetin, a Biflavone from Ginko biloba leaves, inhibits cyclooxygenases-2 and 5-lipoxygenase in mouse bone marrow-derived mast cells. Biol Pharm Bull 2005, 28 (12): 2181-84).

After culturing for 30 min at 37 ° C and 5% CO ₂ , KL (100 ng / ml) (c-kit ligand, STEMCELL Technologies, Inc.) was preincubated with 2 × 10 ⁵ cells / Vancouver, Canada) and incubated for 15 minutes and centrifuged at 3000 rpm and 4 ° C for 5 minutes. The supernatant β-hex substrate [100mM citrate buffer (citric acid 0.955 %, sodium citrate dihydrate 1.478%, pH 4.5), 1.3mg / ㎖ p -nitrophenyl-N-acetyl-bD-glucosaminide] and 1: 2 (w / v The reaction was terminated with 0.2 M glycine (pH 10.7) (Sigma) at 37 ° C for 1 hour, and the reaction was terminated at 405 nm wavelength using an ELISA reader (Tecan System, San Jose, Calif., USA) Absorbance was measured and the measured value was converted into a minute ratio (release%).

As shown in FIG. 4, 57%, 64%, and 72% of the extracts of P. japonica and Bombyx mori were inhibited at concentrations of 6.25 μg / ml, 12.5 μg / ml and 25 μg / IC ₅₀ was about 3.2 mg / ㎖.

Leukotriene ( Leukotriene , LT ) Production amount measurement

To investigate the inhibitory effect on the amount of leukotriene (LT) produced from the sample obtained in the above example, the following experiment was conducted using the method described in the existing literature (Hua JM et al., 14.5-Methoxy-8- (2-hydroxy-3-butoxy-3-methylbutyloxy) -psoralen isolated from Angelica dahurica inhibits cyclooxygenase-2 and 5-lipoxygenase in mouse bone marrow-derived mast cells. Arch Pharm Res 2008 , 31: 617-621).

To confirm the production of LT, the combined extracts of Bryophyte and Bombyx mori were treated with 1 × 10 ⁶ cells / ml BMMC at 37 ° C and 5% CO ₂ for 1 hour. SCF was stimulated with 50 ng / The supernatant was separated by centrifugation (Centrifuge 5415R, Eppendorf) at rpm, 4 ° C for 5 minutes. The LTC ₄ content in the supernatant was measured using the LTC ₄ -EIA kit (Cayman).

As shown in FIG. 5, in the cells stimulated by SCF, the amount of LTC ₄ production increased sharply (76.74 ng / ml). Pine and sat bokryeong After a pre-treatment to determine the LTC ₄ production-inhibiting effect of the compound extract, production of LTC ₄ is 6.25㎍ / ㎖, 12.5㎍ / ㎖, 25㎍ / ㎖ three concentrations in each production 47.57 ng / ㎖ , 40.96 ng / ml and 28.25 ng / ml, respectively, and the IC ₅₀ for the production of LTC ₄ was 13.1 μg / ml.

Claims (3)

A pharmaceutical composition for the treatment or prevention of an inflammatory and allergic disease which comprises an extract of Bokwibae and Tochigi korea as an active ingredient. [Claim 2] The pharmaceutical composition according to claim 1, wherein the complex extract is obtained by repeating the process of immersing the bamboo shoots and the soil bamboo shoots in ethanol and repeating the step of obtaining the extract solution one to five times. [Claim 3] The pharmaceutical composition according to claim 2, wherein the complex extract is obtained by immersing the extract in ethanol at 50 to 70 [deg.] C for 18 to 30 hours, and then extracting the extract at room temperature.

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