KR102140797B1 - Anti-inflammatory composition comprising crude venom isolated from Bracon hebetor - Google Patents

Abstract

The present invention relates to an anti-inflammatory composition comprising a natural poison derived from Kochi bee, wherein the natural poison derived from Kochi bee has the effect of inhibiting the production of nitrogen oxides, suppressing the expression of iNOS, COX-2, and inflammatory cytokines and inflammatory reaction pathways. Includes the ability to inhibit the activity of. The composition may be used as a pharmaceutical composition, food composition or animal feed composition.

Description

Anti-inflammatory composition comprising crude venom isolated from Bracon hebetor}

The present invention relates to a composition having an anti-inflammatory effect comprising a natural poison derived from Kochi bee, the pharmaceutical use of the composition and a food or animal feed containing the same.

Parasitoids are important biological resources for pest control because they spawn and multiply on pests such as moths to kill them. One type of parasitic bee, the barley moth beetle (Bracon hebetor), is used as a natural resource for biological control of the stored grain pest Plodia interpunctella. It is known that such parasitic bees not only paralyze host insects by injecting toxins composed of various biomaterials, but also exhibit various physiological functions such as developmental inhibition and antibacterial action.

Any type of poison usually consists of a mixture of proteins that interfere with the host's nest, but some poisons, such as bee venom, are also considered a panacea for human disease. The host-parasite relationship between P. interpontella and B. hebeta has been extensively studied in the past, but no studies have highlighted the effects of poison in any other context so far. Therefore, current research on BHV has been conceptualized based on the effects of established bee venom on mammalian diseases including inflammation and cancer.

Intrusion of foreign substances into the body was countered by a natural defense mechanism called inflation (inflammatory reaction). Inflammation is a phenomenon in which various chemicals in cells are organized to fight invading substances. While inflammation occurs inside the body, signs such as edema, antagonism, pain, and reddening of the skin become visible. And inside the cell, nitrogen oxide (NO) is released when L-arginine is converted to L-citrueline. Subsequently, a series of pro-inflammatory mediators and cytokines are released that serve to trigger an inflammatory process in relation to the removal of foreign antigens. These include cyclic oxygenase-2 (cyclooxygenase, COX-2), interleukin, that is, IL-1β and TNF-α, which inhale IL-1β and other inflammatory chemicals and fill the gap. All these mediators and cytokines activate the traditional inflammatory pathways, NF-κB and MAPK pathways. Activation of these pathways regulates the systemic inflammatory response to neutralize the intruder. Inflammation is the body's natural defense mechanism, while uncontrolled and generalized inflammatory reactions have devastating effects on the body. Therefore, inflammation must be managed in a timely manner through the body itself or through external substances.

Therefore, as an effective substance for inflammation, a substance derived from nature is capable of effective inflammation management with few side effects, and thus research and development on it are actively conducted, and effects on various substances have been verified.

The present inventors have found that while trying to search for substances which may have an anti-inflammatory effect in mammals, barley moth known that a control effect on pests turned cocoon bee (Bracon hebetor), including humans, results of various experiments for a venom derived from , The present invention was completed by confirming that the poison has an anti-inflammatory effect.

U.S. Patent No. 6,156,539 Patent Publication No. 10-2013-0031773

The object of the present invention, the cocoon bee ( Bracon It is to provide a composition for anti-inflammatory, including bee venom derived from hebetor ).

Another object of the present invention, the cocoon bee ( Bracon It is to provide an anti-inflammatory pharmaceutical composition comprising a bee venom derived from hebetor , food, health food or functional feed composition for animals.

In order to achieve the object of the present invention as described above, the present invention is a composition for anti-inflammatory comprising bee venom derived from high bee punishment, suppressing nitrogen oxide production, suppressing iNOS or COX-2 expression, inflammatory cytokines, or inflammatory reactions. It provides a composition having an effect of inhibiting the activation of the pathway.

The cocoon bee of the present invention is a parasitic wasp having a length of 2 to 3 mm, and is an insect belonging to the hymeniptera and the cocoon family (Braconidea), and is known as a type of parasitic bee that lays eggs and hatches the larvae of the galang moth. have. The cocoon bee of the present invention is a Bracon hebetor, and the Korean name is also called barley moth flesh. In the text and claims of the present specification, bee venom derived from Kochi bee is interpreted to mean Bracon hebetor Venom (BHV).

The bee venom derived from the Kochi bee was separated from the venom gland of the Kochi bee. In one embodiment of the present invention, the poison secreting gland of the Kochi bee was separated, stirred in distilled water (DDW), and centrifuged to obtain the supernatant. It was used separately. However, since the bee venom derived from Kochi bee of the present invention can be subjected to an appropriate detoxification and purification process for use as an anti-inflammatory composition, it is not limited to being interpreted as being only natural.

Toxic substances of the conventional Kochi bee paralyze the host over a long period of time, and interfering with the developmental process or immune process, are considered to have dangerous toxicity and are known to be inflammatory substances. It was confirmed that the derived bee venom (BHV) contains an anti-inflammatory peptide having excellent anti-inflammatory, anti-tumor and immune-enhancing functions, and on the basis of this, the present invention has been completed for an anti-inflammatory composition. Although several types of proteins exist in BHV, they are composed of three major amino acids having a molecular weight of about 73 KDa, and when administered to mammals at appropriate doses, it was confirmed that they have no cytotoxicity and have anti-inflammatory effects. .

As used herein, “anti-inflammatory” or “inflammation suppression” refers to inflammatory reactions harmful to the human body, such as chronic inflammation that progresses to a disease state, inflammatory reactions caused by external factors, and inflammatory reactions caused by autoimmune reactions such as asthma or rheumatoid arthritis. It is a concept that includes restraint.

The anti-inflammatory composition of the present invention may be administered in an amount sufficient to suppress inflammation, and the method may be administered orally or parenterally depending on the purpose. In addition, the present invention is not limited thereto, but is preferably 0.0001 mg/kg to 100 mg/kg, more preferably 0.01 mg/kg to 10 mg/kg. The dosage may vary depending on the patient's weight, age, sex, health status, diet, method of administration, removal rate, and severity of the disease.

In order to confirm the anti-inflammatory effect of the composition containing bee venom derived from Kochi bee (BHV) of the present invention, inhibition of nitrogen oxide (NO) production, inducible nitrogen oxide synthase (iNOS) or Evaluation experiments were performed on COX-2 expression inhibition, inflammatory cytokine IL-1β, IL-6, and TNF-α expression inhibitory effects, and inflammatory pathways, NF-κB or MAPK pathway activation inhibitory effects.

As a result of confirming through the experimental examples, it was confirmed that the BHV of the present invention has an effect of inhibiting nitrogen oxide production, iNOS or COX-2 production, and has an effect of inhibiting inflammatory cytokine expression and activation of the inflammatory reaction pathway, and corresponding It was confirmed that there was no cytotoxicity at the concentration having the effect.

In order to achieve another object of the present invention, the present invention provides an anti-inflammatory pharmaceutical composition, food/health food composition or animal functional feed composition comprising bee venom derived from the Kochi bee.

The pharmaceutical composition of the present invention may be various oral or parenteral formulations. In the case of formulation, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations include at least one excipient in one or more compounds such as starch, calcium carbonate, sucrose or lactose ( lactose) and gelatin. Also, in addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspending agents, intravenous solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used as diluents, various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, can be included. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents, suspension solvents may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin butter, and glycerogelatin may be used.

The pharmaceutical dosage form of the composition of the present invention may also be used in the form of their pharmaceutically acceptable salts, and may also be used alone or in combination with other pharmaceutically active compounds, as well as in a suitable collection. The salt is not particularly limited as long as it is pharmaceutically acceptable, for example, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrofluoric acid, hydrobromic acid, formic acid, tartaric acid, lactic acid, citric acid, fumaric acid, maleic acid, succinic acid, methanesulfonic acid , Benzenesulfonic acid, toluenesulfonic acid, naphthalenesulfonic acid, and the like.

The pharmaceutical composition according to the present invention, oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, ointments, creams, external preparations, suppositories and sterile injectable solutions, etc. It can be formulated and used in any form suitable for pharmaceutical preparations.

The composition according to the present invention can be administered to mammals such as rats, mice, livestock, and humans by various routes, such as parenteral and oral, and all modes of administration can be expected. For example, oral, rectal Alternatively, it may be administered by intravenous, intramuscular, subcutaneous, intrauterine dura mater, or intracerebroventricular injection.

In addition, the food composition of the present invention may have an effect of preventing or improving inflammation, various foods such as chewing gum, caramel products, candies, ice cream, confectionery, beverage products such as soft drinks, mineral water, alcoholic beverages, vitamins or It may be a health functional food including minerals.

Drinks containing bee venom derived from Kochi bee of the present invention are essential ingredients in the indicated proportions, and there are no particular limitations on other ingredients other than those containing bee venom derived from Kochi bee, and various flavors or natural carbohydrates, etc. are added as in conventional beverages. It can contain as an ingredient.

In addition to the above, the functional food composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, colorants and enhancers (cheese, chocolate, etc.), pectic acid and salts thereof, alginic acid And salts, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonic acid used in carbonated beverages, and the like. In addition, the functional food compositions of the present invention may contain natural fruit juice and fruit juice for the production of fruit juice beverages and vegetable beverages. These ingredients can be used independently or in combination. The proportion of these additives is not so critical, but is generally selected from 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

In addition, the functional feed composition for animals of the present invention can prevent inflammation by steadily ingesting poultry, mammals, fish, and the like, and may have a therapeutic effect on previously occurring inflammation. The functional feed composition may be included as an active ingredient in the feed itself, or may be added as an additive to the basic feed.

Since the composition containing bee venom derived from Kochi bee of the present invention has an excellent anti-inflammatory effect, it can be usefully used in anti-inflammatory pharmaceutical compositions, health functional foods, or animal feed compositions.

Figure 1 shows the effect of the BHV of the present invention to inhibit nitrogen oxides mediated by LPS and toxicity evaluation results at the corresponding concentration. 1(a) and 1(b) show the results of cell experiments, and FIGS. 1(c) and 1(d) show the results of animal experiments, respectively.
2 is a result of confirming the effect of BHV on the expression of iNOS and COX-2, and analyzed total RNA and protein amounts from cells, lungs, and testicular tissues. 2(a) and 2(b) are graphs comparing the expression and expression levels of RNA expression for each treatment group through PCR using GAPDH and β-actin as control in RAW 264.7 cells. , Figure 2 (c) and Figure 2 (d) is a result of displaying the expression level and protein expression ratio of RNA in the lung and testis, respectively. Each experiment was performed three times independently, and the bar values of *p <0.01, **p<0.005 and ***p<0.001 were considered statistically significant. (BL = basal lung; BT = basal testis; LL = LPS lung; LT = LPS testis; LV = lung venom; TV= testis venom)
Figure 3 shows the results confirming the expression level of the inflammatory cytokine mRNA (IL-1 β , IL-6, and TNF- α) of BHV. FIG. 3(a) shows the cytokine expression level in the cell, FIG. 3(b) shows the cytokine expression level in the lung, and FIG. 3(c) shows the cytokine expression level in the testis. will be. The value of the bar graph is the mean±standard error of 4 independent experiments. *** p <0.001 and ** p <0.005 values compared to the LPS treatment group.
4 is a result of measuring the change in TNF-α and MDA levels in serum by BHV. Figure 4 (a) is a TNF-α level, Figure 4 (b) is the result of measuring the MDA level. The value of the bar graph is the mean±standard error of 3 independent experiments.*** p <0.001 was considered statistically significant. 4(c) to (h) are the results of lung tissue (c to e) and testicular tissue (f to h) through H&E staining, and the scale bar is 100 μm.
5 is a result confirming the effect of BHV on NF- κ B and MAPK pathways, FIG. 5(a) is a result of confirming the nuclear and cytoplasmic protein expression levels from RAW 264.7 cells, and FIG. 5(b) is a lung and It is the result confirmed from the testimony. Figure 5 (c) is a result confirming the expression of the protein related to the MAPK pathway. Data are means ± standard error (n = 3); ** p <0.005 and *** p <0 001. β-actin was used as a control.
FIG. 6 shows the effect of BHV on NF- κ B and AP-1 transcriptional activity, and is a result of luciferase analysis for cells stimulated with PMA. Bar graphs are the mean ± standard error of three experiments. *** p <0 001 and ** p <005 were considered statistically significant.

Hereinafter, the present invention will be described in more detail by examples. These examples are only for explaining the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

<Experiment Method and Materials>

1. Substances and reagents

Dulbecco's Modified Eagle's medium (DMEM) (Daegu, Korea), fetal bovine serum (FBS) (Wellsen, Daejeon, Korea), streptomycin and penicillin (Lonza, MD, USA), TRIzol® reagents (Invitrogen, Carlsbad, CA, USA), oligo dT (Bionica, Daejeon, Korea), iNOS, COX-2, TNF-α, IL-6 and IL-1β primers (Bionica, Daejeon, Korea), Lipopolysaccharide (LPS) (Escherichia coli 055 : B5) and 3- (4,5- dimethylthiazol-2-yl) -2,5 diphenyltetrazoliumbromide (MTT) is sigma-were purchased from Aldrich (St. Louis, MO, USA) . All forms of IRAK1, TAK1, IKK α / β , IκB, NF-κB p65, iNOS, COX-2, β-actin -specific antibody and anti rabbit horse peroxidase- coupled secondary antibody is all Cell Signaling Technology (Danvers in , MA, USA). All other reagents and chemicals were purchased from Sigma-Aldrich.

2. B. hebetor Natural poison collection from female wasps

The collection, purification and characterization of natural poisons was done according to Quistad et al. Moreau and Asgari. In short, B. hebetor Was specially bred and selected during laboratory culture and fixed on ice. The wasps of each female wasp were then dissected under a microscope and transferred to distilled water. At the same time, 10 glands were decomposed, transferred to 50 μL of distilled water, homogenized manually, and centrifuged at 12,000 rpm for 5 minutes at 4°C. Then, the supernatant containing natural poison was stored at -20°C for future experiments. The experimental bee venom protein was quantified according to Bradford Assay.

3. Ethical standards

All animal care and laboratory procedures were performed in strict accordance with internationally recognized guidelines for the use of laboratory animals (IACUC), and this protocol was approved by the Animal Care Committee of Kyungpook National University in 2017 (Permit No. 2017-36). As acute LPS treatment, rats were observed for a total of 96 hours after administration and mortality was followed every 12 hours. After 96 hours, the remaining mice in the survival study were euthanized by CO ₂ inhalation. For the study of chronic LPS treatment, we monitored mice for a total of 24 hours and generally 12 hours for mortality or other pathology.

4. Animal experiment

Male ICR mice (26-28 g), 6-8 weeks of age, were purchased from Orient Bio (Gyeonggi-do, Korea). Rats were housed at a light and dark cycle of 12 hours at a relative humidity of 60±10% and a temperature of 21±2°C. Food and water were provided freely. Rats were divided into 3 groups, and survival experiments were performed at 30 mg/kg LPS (n=10 per group), and clinical studies (n=6 per group) were performed at 20 mg/kg LPS. For survival and clinical studies, bee venom ( B. hebetor venom, BHV) was administered intraperitoneally daily for 5 days prior to LPS treatment. Group 1 is basal (vehicle treatment), group 2 is LPS-administered group (30 mg/kg or 20 mg/kg), group 3 is BHV (30 μg/kg ) + LPS group.

5. Cell culture

The mouse macrophage cell line RAW 264.7 derived from ATCC, TIB-71 was cultured in Dulbecco's Modified Eagle Medium (DMEM) medium supplemented with 5% fetal bovine serum (FBS) (Wellsen, Daejeon), and supplementally penicillin 100 IU/mL and 100 μg/mL streptomycin sulfuric acid (Lonza, MD, USA) were added. The cells were cultured in a humidified 5% CO ₂ incubator at 37°C. This cell line was obtained directly from the company.

6. Measurement of nitrogen oxides (NO)

The Griess reaction is the principle of measuring NO production. That is, RAW 264.7 cells were seeded in a 96-well plate at a density of 2 x 10 ⁴ cell/well and 18 with or without LPS (0.1 μg/mL) in the absence or presence of BHV (0.1-0.4 μg/mL). Incubated for an hour. Subsequently, the same amount of distilled water was mixed with the same amount of Griess reagent (0.2% naphthylethylenediamine dihydrochloride and 2% sulfanylamide in 5% phosphoric acid) and incubated at room temperature for 5 minutes (25°C). The absorbance of each well was analyzed at 540 nm through a microplate reader (VersaMax, Molecular Devices, LLC, California, USA).

7. Cell viability (MTT) analysis

The cytotoxic effect of BHV was studied by measuring cell viability through MTT analysis.

Briefly, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide phosphorus MTT reagent was added to the culture medium in a 96-well plate with a final concentration of 0.1 mg/mL. After incubation at 37°C and 5% CO ₂ for 4 hours, the purple colored crystals were homogenized in 100 μL/well of dimethyl sulfoxide (DMSO) and absorbance was measured at 560 nm.

8. RNA extraction and qRT-PCR

For transcription studies, RAW 264.7 cells were pretreated with BHV at a given concentration for 30 minutes, then stimulated with LPS (0.1 μg/mL) for 18 hours. TRIzol reagents were harvested for total RNA extraction from both cells and tissues according to the manufacturer's instructions (Invitrogen, Carlsbad, California, USA). Subsequent steps were performed as described above. Table 1 shows the quantitative sequence of the primers used for PCR.

9. Western blot analysis

As direct evidence of the signal pathway according to BHV, RAW264.7 cells with and without LPS (0.1 μg/mL) were treated with BHV (0.1-0.4 μg/mL). Using the NE-PER nuclear and cytoplasmic extraction reagent ^® (Thermo Fisher Scientific Inc., Korea) were isolated nuclear and cytoplasmic fraction from cells and tissues, the preceding steps were conducted according to described in Saba like.

10. TNFα, NO and MDA analysis

After euthanizing the mice used in the experiment, plasma was collected from septic shock mice, and then TNF-α ELISA (R & D systems), NO, and MDA (Abcam) were applied to various commercial kits according to the manufacturer's instructions.

11. H&E Staining

After euthanizing the mice, lung and testicular tissue was collected in 10% neutral buffered formalin, and then the tissues were treated for basic H&E staining according to established protocols.

12. Transient transfection and luciferase analysis

Luciferase activity of HEK 293T cells (ATCC, CRL 1573) was measured by the calcium-phosphate method. Briefly, cells were dispensed at 60 mm at a density of 5 x 10 ⁵ cells/plate, incubated for 24 hours, and then transfected with TK Renilla (pRL-TK) and NF-κB firefly luciferase (pNF-κBLuc) constructs. The transfected medium was replaced with normal FBS supplementation medium after 6 hours, and then cultured for 18 hours. The next day, the cells were seeded in 24-well plates at a density of 5×10 ⁴ cells/well and incubated again overnight. Then, after pre-treatment with BHV (0.1-0.4 μg/mL) for 30 minutes, PMA stimulation was received for 6 hours. The next day, the luciferase activity of the cells was quantified using Promega's Dual-Glo luciferase assay kit (Promega Corporation, Wisconsin, USA) according to the manufacturer's instructions. Luciferase activity was measured using a GloMax Luminometer (Promega). Luciferase activity was normalized to TK Renilla activity. The transcriptional activity of AP1 was also studied in a similar way.

13. Statistical analysis

Data were statistically analyzed using one-way analysis of variance (ANOVA) and Dunnett test. Values are expressed as mean±standard error. **p<0.05 and ***p<0 001 were considered statistically significant.

<Experiment results>

1. Inflammation inhibitory effect by BHV

The gram-negative bacterial cell wall, a common inflammatory factor, consists of lipopolysaccharide (LPS). Therefore, they are used in both in vitro and in vivo studies as inflammatory agents. Therefore, in our study, we investigated whether BHV affects NO production by LPS in RAW 264.7 cells and septic shock mouse models. As a result, as shown in Fig. 1(a), BHV strongly inhibited LPS-induced inflammation without showing any cytotoxic effect on the concentration range used in the present invention (Fig. 1(b)). Furthermore, NO was suppressed from the plasma of mice receiving chronic LPS (Fig. 1(c)), and the survival rate of 100% in the BHV-treated group was also shown in mice receiving lethal LPS (Fig. 1(d)).

2. BHV inhibitory effect of pro-inflammatory mediator expression

Pro-inflammatory mediators are components that cause or mediate inflammation within cells. The most common and rapid inflammatory mediators are iNOS and COX-2. Therefore, we analyzed the effect of BHV on the expression of pre-inflammatory mediators. As demonstrated by Figures 2 (a), 2 (b), 2 (c), and 2 (d), BHV was obtained from mice receiving chronic LPS doses by PCR and immunoblot analysis in both cells and lungs. It can be clearly confirmed that the transcription and expression levels of iNOS and COX-2 in the testis tissue are suppressed.

3. BHV's inflammatory cytokine reduction effect

Pre-inflammatory cytokines are chemicals that are released in response to inflammation caused as a result of external invaders. They serve to increase the size of inflammation and, if uncontrolled, can cause severe septic shock. 3(a), 3(b), and 3(c) show that BHV is a rapid dose-dependent form of mRNA expression levels of IL-1β, IL-6 and TNF-α in both cells and tissues. It is the result of confirming the decrease in cells and tissues. In addition, we analyzed TNF-α and MDA levels in the serum of the septic shock mouse model, and found that all of them were strongly inhibited in the BHV-treated group (FIGS. 4A and 4B ). We also identified lung and testicular tissues more vulnerable to LPS shock. Figure 4(c) shows the clear alveolar space and normal tissue of the lung without infiltrating inflammatory cells. However, Figure 4(d) shows a septic shock rat with alveoli with inflammatory cells. However, as shown in Fig. 4(e), it was confirmed that BHV treatment was performed on the alveoli containing inflammatory cells to return to almost normal. For testicular tissue, Figure 4(f) shows the normal organization of the testicles with proper alignment of the cells and formation of sperm in the vas deferens. However, in Fig. 4(g), the vas deferens are regressed due to LPS shock, and it can be seen that BHV is restored to normal form (Fig. 4(h)).

4. NF-κB and MAPK pathways, the main factors of BHV's anti-inflammatory activity

The NF-κB pathway is a universal cascade of transcription factors, followed by a substance to elicit anti-inflammatory properties. The NF-κB pathway is activated when Toll-like receptor 4 (TLR-4) binds LPS to trigger downstream activation of events. Figure 5(a) shows how BHV reduced phosphorylation of interleukin receptor-related kinase 1 (IRAK1) and transforming growth factor beta-activated kinase 1 (P-TAK-1), the most important starting factors of the NF-κB pathway. Indicates BHV then inhibits phosphorylation of IKKα/β, which breaks down IKB/α. IκB/α removes NF-κB from adjacent subunits and translocates to a nucleus where phosphorylation by BHV is strongly inhibited. In addition, NF-κB in lung and testicular tissues was strongly inhibited when treated with BHV (Figure 5(b)).

The MAPK pathway is also an inflammatory pathway that is very commonly activated when stressing cells, consisting of extracellular signal regulatory kinase (ERK), c-Jun N terminal kinase (JNK) and p38. These factors increase the expression of active protein 1 (AP-1) in the nucleus, which regulates the production of various genes related to inflammation and immunity. The results showed that BHV dependently inhibited the phosphorylation level of all three basic components of the MAPK pathway, and showed that not only NF-κB but also MAPK was affected by the BHV protein of the present invention (Figure 5 ( c)).

5.BHV inhibits transcriptional activity of NF-κB and AP-1

To confirm direct evidence of BHV signal transduction through the NF-κB and MAPK pathways, HEK 293T cells were transfected with each plasmid and expression levels were confirmed by luciferase analysis. As shown in Figure 6, BHV reduces the expression level of NF-κB and AP-1 (an activation transcription factor of MAPK in the nucleus) in a concentration-dependent manner through the NF-κB and MAPK pathways, so that the BHV poison actually has a strong anti-inflammatory effect. It proved that it mediates.

So far, the present invention has been focused on the preferred embodiments. Those skilled in the art to which the present invention pertains will understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered in terms of explanation, not limitation. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the equivalent range should be interpreted as being included in the present invention.

Claims (8)

In an anti-inflammatory composition comprising 0.1 to 0.4 μg/mL of natural poison derived from brocon hebetor as an active ingredient,
The natural poison derived from the cocoon bee is characterized in that it exhibits a nitrogen oxide (NO) inhibitory effect,
The natural poison derived from the Kochi bee is characterized by exhibiting an inhibitory effect on the expression of inducible nitrogen oxide (iNOS) or COX-2, which is a mediator of the inflammatory reaction,
The natural poison derived from the Kochi bee is characterized by exhibiting an inhibitory effect on the expression of inflammatory cytokines, interleukin-1β, interleukin-6 or TNF-α,
The natural poison derived from Kochi bee is characterized in that it inhibits the activation of the NF-κB or MAPK pathway, anti-inflammatory composition.
In vitro,
A method of inhibiting nitrogen oxide (NO) by treating the composition of claim 1.
In vitro,
A method of inhibiting the expression of inducible nitrogen oxide (iNOS) or COX-2, which is a mediator of the inflammatory response, by treating the composition of claim 1.
In vitro,
A method for inhibiting the expression of the inflammatory cytokine of interleukin-1β, interleukin-6 or TNF-α by treating the composition of claim 1.
In vitro,
A method of inhibiting activation of the NF-κB or MAPK pathway by treating the composition of claim 1.
An anti-inflammatory pharmaceutical composition comprising the anti-inflammatory composition of claim 1 as an active ingredient.
A composition for preventing or improving inflammation, comprising the anti-inflammatory composition of claim 1 as an active ingredient. A functional animal feed composition comprising the anti-inflammatory composition of claim 1 as an active ingredient.

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