KR20190121696A - Treating agent for insect bite containing glechoma grandis extract - Google Patents

Abstract

The present invention is directed to an insect bite treatment agent containing an extract of Glechoma hederacea. The insect bite treatment agent containing the extract of Glechoma hederacea according to an embodiment of the present invention can contain medicinal herbs and can improve stability thereof. Furthermore, the insect bite treatment agent can enhance immune characteristics, improve insect bite treatment characteristics, suppress inflammation, relieve itching, and have improved stability against temperature. In addition, the insect bite treatment agent can inhibit NO activity, inhibit expression of iNOS and COX-2, improve electron donating ability, improve ABTS scavenging ability, and enhance cell viability of the extract of Glechoma hederacea.

Description

Insect bite treatment containing gingiva flower extract {TREATING AGENT FOR INSECT BITE CONTAINING GLECHOMA GRANDIS EXTRACT}

The present invention relates to an insect bite treatment containing a medicinal herb extract.

Natural medicines (hereinafter referred to as 'medicinal herbs') have been used effectively for many diseases through pharmacological activities such as anti-inflammatory and analgesic for a long time. The active ingredients in some herbal medicines have a great influence on the homeostasis of the human body by activating the complement system of the immune system of the human body to reinforce the biological defense system against diseases.

Glechoma grandis (A. Gray) Kuprian, distributed in Korea, is a perennial herb found in Jeonnam, Gyeongnam, Gyeonggi, and north of the Yellow Sea, and used as a substitute for Geumjeoncho in Korea. 'Geumchocho' mentioned in the Chinese medicine literature is overworked in primrose; Lysimachia It is an outpost of christinae hance, and it is used to treat hepatobiliary diseases and stones of the urinary system because it has the effects of detoxification, canning, and small size.

Skin itching caused by insect bites, such as mosquitoes in the summer, is difficult to withstand for 7 to 10 days after the bite. In order to suppress this, we use medicines related to insect bites, but this is mainly composed of compounds that do not contain herbal medicines or products using natural synthetic synthetic chemicals in existing non-natural compound products, which may not be suitable for those with weak skin. Can be.

Itching by insect bites is an inflammation caused by a substance contained in the insect's saliva. Immunity is a kind of protective action for the body to protect itself from the invasion of microorganisms from the outside, where cells, molecules and tissues protect organs against infectious agents. In the innate immune system, macrophages play a very important role in the immune system as part of the host's defense mechanism. Nitric oxide (NO), phosphaglandin E ₂ (PGE ₂ ) It can improve the production of cytokines and inhibit the growth of cancer cells and various harmful bacteria. However, persistent and excessive chronic inflammatory reactions cause tissue damage in the body and play an important role as mediators of various diseases including endotoxin stimulation due to inflammatory cytokines or reactive oxygen species.

As a representative example of the inflammatory response, a heterodimer of toll like receptor 4 (TLR4) is converted to lipopolysaccharide (LPS), one of the inflammatory mediators in macrophages, when pathogens penetrate into our bodies. ) And induce the activation of nuclear factor-κB (NF-κB), an intracellular transcription. When NF-κB moves to the nucleus, it induces gene expression of inflammatory derivative nitric oxide (iNOS, inducible Nitric Oxide Synthase), cyclooxygenase-2 (COX-2, Cyclooxygenase-2), and cytokines. Nitric oxide synthase (NOS) is used to synthesize NO as an indicator during L-arginine. NO is absolutely produced by iNOS in NOS. Excessive NO production promotes inflammatory responses, promotes biosynthesis of inflammatory mediators, and intensifies inflammation, resulting in tissue damage, genetic variation, and nerve damage.

The study of cyclooxygenase (COX) was conducted in the early 1990s and is divided into COX-1 and COX-2. COX-1 is expressed in most tissues and produces prostaglandin to regulate physiological functions such as regulating blood flow in the kidneys or protecting cells in the stomach. COX-2 is expressed in macrophages in response to infection or damage caused by microorganisms and stress of various factors. In other words, iNOS and COX-2 expression, NO, and PGE ₂ are representative inflammatory factors of immune cells, and other inflammatory factors are tumor necrosis factor-alpha (TNF-α, Tumor necrosis factor), which is a pro-inflammatory cytokine. -α), monocyte chemoattractant protein-1 (MCP-1), interleukin-1beta (IL-1β, Interleukin-1β), interleukin-6 (IL-6, Interleukin-6), and the like. .

Previous studies on the gingival flower herb include the chemical composition analysis of the gingival flower of Jo, the study on the electron donating ability and the nitrite removal activity, and the herbicidal activity of the essential oil of the gingival flower Kim, Park A study was conducted to compare essential oils by fertilizer treatment when cultivation of gingiva larvae. There was a study on the effect of Lee's nectar extract on streptozotocin-induced diabetes. Lee also concentration-dependently inhibited NO production induced by lipopolysaccharide (LPS) and recombinant interferon-gamma (rIFN-γ) by Geumchocho hot-water extract, and cells such as IL-12, TNF-α and IL-6. It has been reported to increase the production of active substances.

However, studies on inflammation-relaxing action and insect bite of gingiva flower extract using ethanol extract have not been carried out before the present inventors.

Insect bite treatment agent containing a medicinal herb extract according to an embodiment of the present invention is to include a herbal medicine.

The insect bite treatment agent containing the gingiva flower extract according to an embodiment of the present invention is to improve the stability of the therapeutic agent.

Insect bite treatment containing a medicinal herb extract according to an embodiment of the present invention is to improve the immune properties.

Insect bite treatment containing a gingiva flower extract according to an embodiment of the present invention is to improve the therapeutic properties.

Insect bite treatment containing a gingiva flower extract according to an embodiment of the present invention is to suppress inflammation and to relieve itching.

Insect bite treatment containing a medicinal herb extract according to an embodiment of the present invention is to improve the stability to temperature.

Insect bite treatment agent containing a gingiva flower extract according to an embodiment of the present invention is to inhibit the production of NO.

The insect bite treatment agent containing the gingiva flower extract according to an embodiment of the present invention is to suppress the expression of iNOS and COX-2.

Insect bite treatment agent containing a gingiva flower extract according to an embodiment of the present invention is to improve the electron donating ability.

Insect bite treatment agent containing a medicinal herb extract according to an embodiment of the present invention is to improve the ABTS scavenging properties.

The insect bite treatment agent containing the gingiva flower extract according to an embodiment of the present invention is to improve cell viability of the gingiva flower extract.

In addition to the above object, embodiments according to the present invention can be used to achieve other objects not specifically mentioned.

Insect bite treatment agent containing a gingiva flower pool extract according to an embodiment of the present invention ( Glechoma) grandis (A. Gray) Kuprian) extract as an active ingredient.

The pH of the therapeutic agent may be 5 to 8, preferably the pH may be 5 to 5.5.

The therapeutic agent may reduce the diameter of the swollen skin within 15 to 30% within 5 to 10 minutes after the insect bites.

The gingiva flower extract can inhibit the protein expression of COX-2 and the mRNA expression of COX-2.

According to an embodiment of the present invention, a method for preparing insect bite treatment includes preparing a solution in which distilled water or cypress water is dispersed by dipping water with hycel (Hycel, Hydroxyethylcellulose), aloe moist and vegetable glycerin in the dispersed solution. (Glycerin) mixing to prepare a first solution, to prepare a gingkole powder, to prepare a second solution by adding a kerba powder to the first solution, and to add oil to the second solution And then mixing to prepare a medicinal herb.

Distilled water or white water may be included in the 45 to 55 parts by weight, the high cell 0.1 to 2 parts by weight.

Aloe moist may contain 10 to 30 parts by weight, and vegetable glycerin (Glycerin) may be included in 1 to 3 parts by weight.

Long bottle powder may be included 1 to 5 parts by weight.

In the preparing of the first solution, the method may further include heating the first solution.

In the preparing of the third solution, the method may further include adding at least one of menthol or allantoin.

In the preparing of the third solution, the method may further include mixing in an Agi mixer for 1 to 5 minutes so that no layer is formed in the third solution.

In preparing the medicinal herbaceous medicine, oil may be additionally included. The oil may include at least one or more of lime essential oil, neroli essential oil, rosemary essential oil and eucalyptus essential oil, and may further include an emulsifier such as olive liquid, if necessary.

Method for producing gingival pulverulent powder, extracting stems and leaves of gingiflora, drying and grinding gingiflora, adding 8 times to 12 times the weight of the pulverized gingiva It may include the step of obtaining the gingiva flower extract, filtering the gingko blotch extract and then concentrating under reduced pressure to remove the solvent, and lyophilizing the kerba extract from which the solvent has been removed.

The gingiva flower extract may be extracted using an alcohol having 1 to 5 carbon atoms or a mixture thereof.

In the step of obtaining alcohol extract of the gingiva flower, the gingiva flower extract may be obtained by repeating the step of separating the supernatant and the precipitate after immersing the pulverized gingiva flower sample in alcohol at room temperature for 20 to 30 hours. have.

Insect bite treatment agent containing a medicinal herb extract according to an embodiment of the present invention may include a herbal medicine, it may improve the stability of the therapeutic agent. In addition, it is possible to improve the immune characteristics, to improve the insect bite treatment characteristics, to suppress inflammation, relieve itching, and improve the stability to temperature. In addition, it is possible to inhibit the activity of NO, inhibit the expression of iNOS and COX-2, improve the electron donating ability properties, improve the ABTS scavenging properties, and improve the cell viability of the gingiva flower extract.

1 is an image showing a insect bite treatment containing a medicinal herb extract of the present invention.
Figure 2 is an image showing the manufacturing process of the insect bite treatment containing a medicinal herb extract of the present invention.
Figure 3 is an image showing a clinical experiment of insect bites containing medicinal herbs extract according to the present invention.
Figure 4 is a graph showing the average value of skin response change of the experimenter according to Comparative Examples 1 to 3, Example 1.
Figure 5 is a graph showing the electron donating ability of the gingiva flower extract according to Example 2.
Figure 6 is a graph showing the ABTS scavenging ability of the gingiva flower extract according to Example 2.
Figure 7 is a graph showing the pH measurement results of the emulsion of 3% of the gingiva flower extract according to Example 2.
Figure 8 shows the cell viability of the gingiva ethanol extract of the present invention on macrophages.
Figure 9 is a graph showing the measurement results of NO production inhibition of the ethanol extract of Longfloweria paniculata of the present invention.
FIG. 10 is a graph illustrating a result of analyzing protein expression of a gingiva flower extract according to Example 2. FIG.
Figure 11 is a graph showing the iNOS and COX-2 mRNA expression inhibition results of the gingiva ethanol extract of the present invention in RAW264.7 cells.

DETAILED DESCRIPTION Embodiments of the present invention will be described in detail with reference to the accompanying drawings so that those skilled in the art may easily practice the present invention. As those skilled in the art would realize, the described embodiments may be modified in various different ways, all without departing from the spirit or scope of the present invention. In addition, in the case of well-known technology, a detailed description thereof will be omitted.

Throughout the specification, when a part is said to "include" a certain component, it means that it can further include other components, without excluding other components unless specifically stated otherwise.

With reference to FIGS. 1 and 2, a method of preparing an insect bite treatment containing a medicinal herb extract according to an embodiment will be described.

Insects may include, but are not limited to, for example, mosquitoes, bees, vampire insects, bed bugs, spiders, mites, fleas, teeth, ants, mites, scabies, and the like.

First, 0.1 to 2 parts by weight of distilled water (DI water) or Cyclic water is added to 45 to 55 parts by weight of Hycel (Hycel, Hydroxyethylcellulose) and dispersed. A first solution is prepared by mixing 10 to 30 parts by weight of Aloe Moist and 1 to 3 parts by weight of vegetable glycerin (Glycerin) to this solution. In this case, the first solution may include the step of heating by transferring to a water bath (waterbath).

Next, 1 to 5 parts by weight of gingiva flower powder and 10 to 20 parts by weight of vegetable ethanol are mixed to prepare a second solution. At this time, the magnetic bar can be stirred into the second solution. For example, when the gingiva flower powder is 1 part by weight or less, the effect of the insect bite treatment may not appear, and when 5 parts by weight or more, the gingiva flower powder may not be dissolved in ethanol and residues may remain.

Subsequently, a second solution is added to the first solution to prepare a third solution. The third solution can be mixed for about 1 to 5 minutes with an Agi mixer so that no layer is formed. At this stage it is possible to add menthol and allantoin as minor additives. 1 to 10 parts by weight of menthol and 5 to 15 parts by weight of allantoin are weighed and prepared, followed by addition of the third solution when mixing with an azi mixer.

Next, a fourth solution is prepared by adding vegetable oils to the third solution to maintain skin texture and moisture retention when using the product. Vegetable oils that can be added include 0.1 to 5 parts by weight of lime essential oil, 0.1 to 5 parts by weight of neroli essential oil, 0.1 to 5 parts by weight of rosemary essential oil, and 0.1 to 5 parts by weight of eucalyptus essential oil, followed by mixing to prepare a fourth solution. do. The vegetable oil to add may be selected in whole or in part. If necessary, an emulsifier such as olive liquid may be additionally included, and may be included in an amount of 0.1 to 5 parts by weight. In this case, the fourth solution may be prepared in an emulsion form, but is not limited thereto. The fourth solution prepared refers to an insect bite treatment containing a medicinal herb extract. Insect bites containing gingiva flower extract can relieve itching and reduce the diameter of swollen skin when bitten by insects. At this time, the diameter may be reduced by about 15 to 40% compared to the state of bulging most within 3 to 20 minutes after being bitten by insects, and on average, the diameter may be reduced by more than 30% within 10 minutes, but is not limited thereto. Do not.

The insect bite treatment agent containing the gingiva flower extract according to the embodiment may exhibit a pH of about 5 to 8, preferably about 5 to 5.5 in pH.

In addition, at temperatures between 0 ° C and 50 ° C, chemical and physical changes such as solidification, precipitation, turbidity (transparency), and crystallization separation are stable at relatively low temperatures, and the appearance of discoloration, discoloration, and discoloration at a relatively high temperature is stable. , Viscosity, evaporation, suspension, precipitation, turbidity (transparency), and separation properties can be maintained.

Herbal extract according to the embodiment may be extracted by ethanol, preferably may be extracted by 70% (v / v) of ethanol.

First, the stem and leaves of the gingiflora are collected and hot-air dried (or natural shade dried or lyophilized) and then ground to prepare a sample. An alcohol corresponding to 8 to 12 times the weight of the ground sample is prepared, added to the ground sample, and immersed at room temperature for about 20 to 30 hours to separate the supernatant from the precipitate. In this case, the alcohol may be an alcohol having 1 to 5 carbon atoms or a mixture thereof, but is not limited thereto. This separation can be repeated three times in the same manner. Each sample extract may be filtered and concentrated under reduced pressure to remove the solvent and then freeze-dried, and the sample may be stored at about -20 ° C. At this time, the freeze-dried sample refers to the extract of Longflower.

For the medicinal herb extract according to the example, the anti-inflammatory effect of the medicinal herb extract was induced after inducing an inflammatory response using LPS in RAW264.7 cells.

Figure 7 is a graph showing the cell viability of the gingiva ethanol extract against macrophages according to an embodiment.

Referring to Figure 7, RAW264.7 cells do not have a significant effect on the cell death of the ethanol extract of the cedar flower, it is effective in the growth (1 × 10 ⁵ cells / well) of RAW264.7 cells. At this time, the cytotoxicity may be measured by MTT assay of M. ethanol extract. RAW264.7 cells can be used by treatment with dimethylsulfoxide (DMSO) at constant concentration for about 24 hours.

There are three types of NOS in mammals, Type I, II, and III, which are divided into homologous enzymes according to their chemical properties. Unlike neuronal NOS of type I and endothelial NOS of type II, which are always present in cells, type III iNOS is only induced by stimuli such as cytokines, LPS and bacterial toxins in some cells. Can be expressed. COX is a facilitating enzyme for the conversion of arachidonic acid to prostaglandin. COX-1 is involved in the maintenance of body homeostasis, such as gastrointestinal protection and renal function regulation in most tissues. It is divided into COX-2, which increases intracellular expression at the site of inflammation by cleavage factor or cytokine.

70% (v / v) ethanol 70% (v / v) extract according to the embodiment shows a cell viability of about 95.8% at a concentration of about 500 μg / ml in macrophages (Raw264.7), particularly at a concentration of about 500 μg / ml The inhibition rate of nitric oxide production was about 37.4%. In experiments using Western blot and RT-PCR, mRNA expressions of iNOS and COX-2 may be inhibited in the gingiva extract as compared to vitamin C (Vit. C), which is a control group. Thus, Echinacea extract can be used as an anti-inflammatory substance. In addition, the medicinal herbaceous extract can be used as an immune enhancer to enhance the immune response. At this time, the anti-inflammatory activity may show a NO production inhibitory effect. This test was derived from the Griess assay of LPS-stimulated RAW 264.7 cells.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the following Examples are merely examples of the present invention, and the present invention is not limited to the following Examples.

In embodiments, the electron donating ability was performed by modifying the (EDA:: Electron donating abilities) Measurement experiment method of Blois (Blois MS 1199-1120 1958. Antioxidant determination by the use of a stable free radical Nature 26...) . In addition, the antioxidant capacity measurement using ABTS radical is ABTS ⁺ cation decolorization assay method (Roberta R, Nicoletta P, Anna P, Ananth P, Min Y, Catherine RE.Antioxidant acitivity applying an improved ABTS radical cation decolorization assay.Free radical biology & Medicine (1999), 26. 1231-1237. Stability testing with temperature was performed with reference to Wilkinson et al. (Wilkinson JB, Moore RJ (Eds.). 1982. Harry's cosmeticology. Chemical Publishing Co, Inc. New York. 749.). In addition, cell culture was performed using DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin (100 ml / ml), 37 ℃, 5% CO2 incubator (incubator) ) Were subcultured. RAW264.7 cells, the cell lines used in the experiment, were purchased from Korea Cell Line Bank (Dulbecco's modified eagle's medium), FBS (Fetal bovine serum), PBS (Phosphate buffered saline) and penicillin. Penicillin / Streptomycin and Trypsin were purchased from Thermo Scientific Hyclone (USA) and Gibco BRL Co. (Grand Island, USA). 3- [4,5-dimethylthiazol] -2-yl] -2,5-diphenyl-tetrazolium bromide (MTT) used for cytotoxicity measurements was purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA), DMSO (dimethyl sulfoxide) was purchased from BioShop (Canada). Lipopolysaccaride (LPS), grease reagent (gris regent), etc., which are used for anti-inflammatory measurement, are described in Sigma Chemical Co. (St. Louis, MO, USA). Western blots were purchased from Santa Cruz (CA, USA), with primary antibodies such as β-actin, iNOS, and COX-2 as primary antibodies and goat anti-rabbit. The GoScript ™ Reverse Transcription System for reverse transcription-polymerase chain reaction (RT-PCR) was purchased from Promega Corporation (Madison, Wisconsin, USA). Rotary vacuum evaporator (EYELA, Japan), microcentrifuge (gyrozen, Korea), microscope (Olympus, Japan), CO2 incubator (Vision Scientific, Korea), microplate reader (Tecan, Austria), PCR (ASTEC Co, Japan), Davinch-Chemi ™ imager CAS-400SM system (Davinch-K Co, Korea), UV transilluminator (BioTop, Switzerland), mini-PROTEAN®Tetra Cell (Bio-Rad, USA), mini Trans-Blot® Cell (Bio-Rad, USA) was used.

Example 1 Preparation of Insect Bites Containing Herbal Extract

1 part by weight of Hycel (Hycel, Hydroxyethyl Cellulose) is dispersed in 50 parts by weight of baekbaek water. The solution is mixed with 20 parts by weight of Aloe Moist and 2 parts by weight of vegetable glycerin (Glycerin), and then transferred to a water bath to prepare a first solution. Subsequently, 3 parts by weight of echinacea powder and 16 parts by weight of ethanol 70% (v / v) were mixed to prepare a second solution. Put a magnetic bar in the second solution and stir.

Next, a second solution is added to the first solution, 4 parts by weight of menthol and 10 parts by weight of allantoin are added, followed by stirring while mixing to prepare a third solution.

Then, a fourth solution is prepared by adding 0.1 parts by weight of lime essential oil, 0.1 parts by weight of neroli essential oil, 0.4 parts by weight of rosemary essential oil, and 0.4 parts by weight of eucalyptus essential oil to 2 parts by weight of olive liquid.

A fourth solution is added to the third solution and mixed with slow stirring to prepare an insect bite treatment containing a gingiva flower extract.

Experimental Example 1-Clinical Trial of Therapeutics

An insect bite treatment agent according to Example 1, an insect bite treatment agent of another product was prepared, and an experiment was performed to verify whether the treatment agent showed efficacy in humans.

Date: 2018.05.31. 10:00 to 14:00

Experimenter: 2 persons

Preliminary Results: The developed long-flowered mosquitoes verified the product through the stability test after the production of the product and then proved the effect of 'reducing itching and swelling' when mosquito bites in the living environment. However, human verification experiments were conducted to compare the results with the exact improvement values and comparisons with other products and comparative examples.

Methods: 120 rats of `` no-virus, aseptic white-grown mosquitoes '' were distributed in the laboratory of Professor Lee Dong-gyu of Busan Kosin University. Each tester had four conditional test mouths on both arms (Comparative Example 1: No treatment, Comparative Example 2: Experiment site that scratched the bite after biting, Comparative Example 3: Treatment product sold by another company after biting, Example 1: After biting Insect bite treatment treatment containing the gingiva flower extract developed), and put the arm into the cage to perform a mosquito bite for 3 minutes. After 3 minutes, the initial swelling diameter was measured. After treating the conditions for each test group, the result was measured again after 20 minutes.

Tables 1 and 2 are tables showing the skin reactions of the test subjects treated with insect bite treatment containing the gingiva flower extract after mosquito bites according to the embodiment. The diameter of the swollen skin was measured when 2 minutes of mosquito bites and 20 minutes after the bite were performed on 2 persons in the experimenter 1 group and 4 persons in the experimenter 2 group. At this time, the first and second groups of experimenters divided only the groups, and the experimenters experimented under the same conditions.

Referring to Figures 3, Table 1 and 2, the experimenters treated the insect bite treatment agent according to Example 1 after the bite mosquito bite in the experiment group 1 when 20 minutes after the bite compared to 3 minutes after the bite On average, the diameter was reduced by -0.285 mm, and in the Experimental Group 2, the diameter was reduced by -2.965 mm on average.

Example 1 (Experiment 1 group) One 2 Standard Deviation 3 minutes after bite 11.15 12.22 20 minutes after bite 11.07 11.73 Change value -0.08 -0.49 Change value mean -0.285 0.290

Example 1 (Experiment 2 group) One 2 3 4 Standard Deviation 3 minutes after bite 11.27 20.87 12.81 13.43 20 minutes after bite 7.55 17.35 12.88 8.74 Change value -3.72 -3.52 0.07 -4.69 Change value mean -2.965 2.087

Comparative example  One - No treatment group  experimenter

We prepared 120 virus-free mosquitoes grown in a virus-free and sterile form, divided them into groups 1 and 2 and put the arms in the cage to bite mosquitoes for 3 minutes. This is shown in Table 3 and Table 4. At this time, the first group and the second group of the experimenter only divided the group, the experimenters experimented under the same conditions.

Tables 3 and 4 are tables showing the skin reactions of the experimenters who did not undergo any treatment after being bitten by mosquitoes according to Comparative Example 1. Four people in group 1 and 4 groups in group 2 were measured for 3 minutes after mosquito bites and 20 minutes after bites.

Referring to Tables 3 and 4, in the experiment group 1, the test subjects who did not process any mosquito bites increased the diameter by 14.825 mm on average 20 minutes after the bite after 3 minutes after the bite. In the experimenter group 2, the diameter increases by 4.348 mm on average.

Comparative Example 1 (Group 1) One 2 3 4 Standard Deviation 3 minutes after bite 6.39 6.05 8.00 12.35 20 minutes after bite 17.86 21.21 31.53 21.49 Change value 11.47 15.16 23.53 9.14 Change value mean 14.825 6.310

Comparative Example 1 (Experiment 2 group) One 2 3 4 Standard Deviation 3 minutes after bite 4.56 10.05 7.39 9.79 20 minutes after bite 8.84 13.04 14.28 13.02 Change value 4.28 2.99 6.89 3.23 Change value mean 4.348 1.785

Comparative Example 2-Experimenter who performed scratching after being bitten by insects

Prepared 120 virus-free mosquitoes grown in a virus-free and sterile form, and divided them into a group 1 and a group 2 and put the arms in the cage to examine the skin reactions of the scratched test subjects after 3 minutes of mosquito bites And 6. At this time, the first and second groups of experimenters divided only the groups, and the experimenters experimented under the same conditions.

Tables 5 and 6 are tables showing skin reactions of test subjects scratched after mosquito bites according to Comparative Example 2. Five subjects in group 1 and 2 groups in group 2 were measured for the diameter of swollen skin when 3 minutes passed after mosquito bite and 20 minutes after bite.

Referring to Tables 5 and 6, the experimenters who scratched after mosquito bites in the experiment group 1 had an average diameter increase by 10.898 mm when 20 minutes had passed after being bitten, compared to 3 minutes after mosquito bites. In the group, the diameter increased by 2.850 mm on average.

Comparative Example 2 (Experimental Group 1) One 2 3 4 5 Standard Deviation 3 minutes after bite 9.49 12.44 10.64 9.75 8.23 20 minutes after bite 24.16 26.17 16.00 24.96 13.75 Change value 14.67 13.73 5.36 15.21 5.52 Change value mean 10.898 5.011

Comparative Example 2 (Experimental Group 2) One 2 Standard Deviation 3 minutes after bite 11.60 10.71 20 minutes after bite 15.62 12.39 Change value 4.02 1.68 Change value mean 2.850 1.655

Comparative Example 3-Experimenter who treated another company's insect bite treatment

Prepared 120 virus-free mosquitoes grown in virus-free and sterile form, divided into 1 group and 2 groups of test subjects, put arm in cage, bite mosquito for 3 minutes, and then treated skin reactions of other insect bites. It was examined and shown in Tables 7 and 8. At this time, the first and second groups of experimenters divided only the groups, and the experimenters experimented under the same conditions.

Tables 7 and 8 are tables showing skin reactions of test subjects treated with other companies' insect bite treatments after being bitten by mosquitoes according to Comparative Example 3. Three people in group 1 and 3 groups in group 2 were measured for 3 minutes after mosquito bites and 20 minutes after bites.

Referring to Tables 7 and 8, in the experiment group 1, the test subjects treated with other company's insect bites after mosquito bites were 15.550 mm on average 20 minutes after being bitten compared to 3 minutes after the bite. Increasing the diameter, in the experiment group 2 it was confirmed that the diameter increases by 1.600 mm on average.

Comparative Example 3 (Group 1) One 2 3 Standard Deviation 3 minutes after bite 11.08 10.31 11.36 20 minutes after bite 25.92 21.06 32.42 Change value 14.84 10.75 21.06 Change value mean 15.550 5.192

Comparative Example 3 (Experimental Group 2) One 2 3 Standard Deviation 3 minutes after bite 13.70 9.03 7.55 20 minutes after bite 13.77 11.66 9.65 Change value 0.07 2.63 2.10 Change value mean 1.600 1.351

Experimental Example  2-experimenter mean and standard deviation

The average and standard deviation were calculated by combining the data of the experimenters according to Example 1 and Comparative Examples 1 to 3, which are shown in Tables 9 to 13 and FIG. 4.

Table 9 is a table showing the average value of skin response change of the experimenter according to Comparative Example 1.

Table 10 is a table showing the average skin response change value of the experimenter according to Comparative Example 2.

Table 11 is a table showing the average value of skin response change of the experimenter according to Comparative Example 3.

Table 12 is a table showing the average skin response change value of the experimenter according to Example 1.

Table 13 is a table showing the average of the skin response change value of the experimenters according to Comparative Examples 1 to 3, Example 1 calculated using the following formula 1.

<Equation 1>

Referring to Tables 9 to 12, Comparative Example 1 showed an increase in diameter by 9.586 mm on average, Comparative Example 2 showed an increase in diameter by 8.599 mm on average, and Comparative Example 3 by 8.575 mm on average Although the increase in diameter was shown, the average decrease in diameter of Example 1 was -2.072 mm.

Referring to Table 13 and Figure 4, compared to Comparative Example 1, in Comparative Example 2 was observed to increase the diameter width 25 to 30% swelling than the initial measurement and itching continued. In Comparative Example 3, itching was partially alleviated, but there was no change in diameter. When the therapeutic agent according to Example 1 was used, the itch was relieved, and an effect of reducing the swelling diameter of about 20 to 25% was observed.

Comparative Example 1 (Experiment 1 group + Experiment 2 groups) One 2 3 4 5 6 7 8 Standard Deviation 3 minutes after bite 6.39 6.05 8.00 12.35 4.56 10.05 7.39 9.79 20 minutes after bite 17.86 21.21 31.53 21.49 8.84 13.04 14.28 13.02 Change value 11.47 15.16 23.53 9.14 4.28 2.99 6.89 3.23 Change value mean 9.586 7.057

Comparative Example 2 (Experiment 1 group + Experiment 2 groups) One 2 3 4 5 6 7 Standard Deviation 3 minutes after bite 9.49 12.44 10.64 9.75 8.23 11.60 10.71 20 minutes after bite 24.16 26.17 16.00 24.96 13.75 15.62 12.39 Change value 14.67 13.73 5.36 15.21 5.52 4.02 1.68 Change value mean 8.599 5.711

Comparative Example 3 (Experiment 1 group + Experiment 2 groups) One 2 3 4 5 6 Standard Deviation 3 minutes after bite 11.08 10.31 11.36 13.70 9.03 7.55 20 minutes after bite 25.92 21.06 32.42 13.77 11.66 9.65 Change value 14.84 10.75 21.06 0.07 2.63 2.10 Change value mean 8.575 8.360

Example 1 (Experiment 1 group + Experiment 2 groups) One 2 3 4 5 6 Standard Deviation 3 minutes after bite 11.15 12.22 11.27 20.87 12.81 13.43 20 minutes after bite 11.07 11.73 7.55 17.35 12.88 8.74 Change value -0.08 -0.49 -3.72 -3.52 0.07 -4.69 Change value mean -2.072 2.132

Comparative Example 1 Comparative Example 2 Comparative Example 3 Example 1 Experimenter 1 group 14.825 10.898 15.550 0.285 Experimenter 2 group 4.348 2.850 1.600 -2.965 Experimenter Average 9.586 8.599 8.575 -2.072 Standard Deviation 7.057 5.711 8.360 2.132

Example 2-Preparation of Longflower Flower Extract

5kg of stems and leaves of gingiflora were collected and ground after hot air drying or natural shade drying or freeze drying. 70% (v / v) of ethanol in the amount of 10 times the weight of the sample was added to the ground sample, soaked at room temperature for about 24 hours, and the supernatant and the precipitate were separated and extracted three times in the same manner. Each sample extract was filtered using a filter paper (Whatman No. 2), concentrated under reduced pressure with an EYELA evaporator to remove the solvent, and lyophilized, and the sample was stored at -20 ° C. At this time, the long bottle flower may be collected from the Osan member.

Experimental Example 3 Measurement of Electron Donating Ability

After preparing a sample according to Example 2, 60μ 0.2 mM DPPH solution dissolved in ethanol and 120μ prepared by concentration were added to a 96well plate and reacted at room temperature for 15 minutes, and the absorbance at 517 nm was measured using a microplate reader. This is shown in FIG. 5. At this time, the electron donating ability is expressed as the absorbance decrease rate of the sample addition group and the no addition group.

Referring to Figure 5, the gingiva flower extract showed an electron donating ability of about 20% at 500μ, it can be seen that the electron donating ability of about 50% at 1000μ.

Experimental Example 4-ABTs ⁺ Measurement of cation radical scavenging activity assay

Prepare a sample according to Example 2 and prepare 7 mM 2,2'-azino-bis (3-ethyl-benzothiazoline-6-sulfonic acid) (ABTS, 2,2-azino-bis- (3-ethyl-benzo thiazoline) -6-sulfonic acid) and 2.45 mM potassium persulfate were mixed and left at room temperature for 24 hours to form ABTS ⁺ . Thereafter, diluted with ethanol, 100 μs of the sample was added to ABTS ⁺ 100 μ, and the absorbance was measured at 700 nm.

Referring to FIG. 6, ABTS scavenging activity began to exhibit ABTS scavenging activity (Inhibitation Rate,%) at 50 μ concentration, showing an scavenging activity of about 20% or less at 100 μ concentration, and about 80% at 500 μ concentration It can be confirmed that it exhibits about 90% or more of erasing ability at 1000 µ.

Experimental Example 5-Stability Test According to Temperature

The sample according to Example 2 was prepared, and the sample was stored at predetermined temperature conditions (0, 25, and 40 ° C) according to the stability test method according to the temperature, and the state change according to the change over time was evaluated under the conditions of Table 14. .

Temperature Date range Check points 0 ℃ One Separation
Precipitation
Discoloration
pH
etc 3 25 ℃ 5 7 40 ℃ 15 30

The pH measurement results of the emulsion of 3% of the gingiva flower extract according to Example 2 are shown in FIG. 7.

Referring to Figure 7, the average pH was found to be a distribution of 5.17, showing a stable pH without significant change in the value for 30 days.

In addition, temperature (Incubation) stability evaluation was performed to determine the chemical and physical changes of the emulsion of 3% of the gingiva flower extract according to Example 2, which is shown in Table 15.

Referring to Table 15, it can be seen that the emulsion added with the kerba flower extract stored at 0, 25, 40 ℃ temperature shows stability under all temperature conditions for 30 days. At this time, the appearance rancidity, discoloration, discoloration, viscosity, evaporation, flotation, precipitation, turbidity (transparency), separation, etc. were observed at high temperature (40 ℃), and coagulation, precipitation, turbidity (transparency), crystal precipitation separation, etc. were observed at low temperature. The chemical and physical changes of were observed.

0 ℃ 25 ℃ 40 ℃ 1 day stability stability stability 3 days stability stability stability 5 days stability stability stability 7 days stability stability stability 15th stability stability stability 30 days stability stability stability

Experimental Example 6-Cell Viability Analysis by MTT Assay

A medicinal herb extract according to Example 2 was prepared, and cytotoxicity was confirmed, which is shown in FIG. 7.

The MTT assay is a test method for measuring the amount of formazan produced by the respiratory chain of mitochondria using a yellow water-soluble substance MTT.

Referring to FIG. 8, the cytotoxicity was measured using the MTT assay. As a result, when the gingiva flower extract was treated in RAW 264.7, the survival rate was measured as 95.8% in the concentration of 500 μg / ml. It was confirmed that the toxicity was very low in RAW 264.7 cells. Therefore, the experiment was performed by setting 50, 100, 500 μg / ml, which does not affect cytotoxicity. At this time, cell viability was measured according to Carmichael's method. Dispense the RAW 264.7 cells into a 96 well plate at 0.18 ml to 5 × 10 ⁴ cells / well, prepare samples by concentration, add 0.02 ml, and then incubate at 37 ° C and 5% CO _2. Incubated for 24 hours. The control group was incubated under the same conditions by adding the same amount of distilled water. After incubation for 4 hours with addition of 0.04 ml of MTT solution prepared at 2.5 mg / ml concentration, the culture medium was removed, 0.15 ml of DMSO per well was added, and reacted at room temperature for 30 minutes, followed by ELISA reader at 540 nm. Absorbance was measured. Cell viability was measured by the absorbance decrease rate of the sample solution addition group and no addition group.

Experimental Example 7-NO Production Inhibition Activity Assay

Preparation of the medicinal herbaceous extract according to Example 2, and confirmed the effect of the medicinal herbaceous extract on the production of NO known to play an important role in causing inflammation, which is shown in FIG.

9 is a graph showing the cell viability of the gingiva ethanol extract for macrophages. The effect of the gingiva flower extract on NO production in Raw264.7 cells was confirmed, and NO production was confirmed in LPS-induced Raw264.7 cells. At this time, RAW264.7 cells (2 × 10 ⁵ cells / well) were treated with 70% ethanol (v / v) extract and LPS (1 μg / ml) for about 24 hours. NO production is determined as the media supernatant by Griess reagent.

Referring to FIG. 9, the LPS-treated group showed a higher NO expression level than the LPS-untreated group, and the group treated with the Medicinal Herb extract was found to reduce NO expression. In particular, it was confirmed that the inhibitory rate of 41.2% at a concentration of 500 μg / ml, overexpressing inflammation with LPS in macrophage cell line, inhibiting the inflammation with excellent results, ethanol extract of longflower plant is effective in inhibiting inflammation Could confirm. At this time, the amount of NO generated from RAW264.7 cells was measured in the form of NO ₂ present in the cell culture solution using a grease reagent according to the method of Green et al. Raw 264.7 cells were dispensed at 1 × 10 ⁵ cells / ml in 6 well plates. After 24 hours of incubation at 37 ° C. in a CO ₂ incubator, wash twice with 1 × PBS. After treatment with LPS 10ug / ml except for the normal experimental group, after 2 hours, the sample solution prepared for each concentration was incubated for 24 hours, the supernatant was obtained, and then reacted for 10 minutes in a 96 well plate by adding the same amount of griess reagent. Absorbance at 540 nm was measured. The NO inhibitory activity was measured by the absorbance reduction rate of the sample addition group and no addition group.

Experimental Example 8-Protein Expression Analysis by Western Blot

A medicinal herb extract according to Example 2 was prepared and analyzed for protein expression, which is shown in FIG. 10. In this case, western blot was tested to examine the effect of the extract of Longfloweria on the protein expression of iNOS and COX-2 factors. In this case, beta-actin (β-actine), a housekeeping gene, was used as a positive control in order to compare the amount of protein expression, and vitamin C (vitamin C) was used as a control.

Figure 10 is a graph showing the iNOS and COX-2 expression rate of the Centella ethanol extract in RAW264.7 cells. The left graph (A) shows the iNOS protein expression rate of the zinnia ethanol extract, and the right graph (B) shows the COX-2 protein expression rate of the zinnia ethanol extract. RAW264.7 cells (1 × 10 ⁶ cells / well) were treated with serpentine free medium (SFM) for 1 hour and then treated with 50, 100 and 500 μg / ml for the kerba extract for 24 hours. Each value is represented by the mean ± SD of each of the average three experiments.

Referring to FIG. 10, iNOS and COX-2 showed 92.73% and 26.90% expression levels at 500 μg / ml, which was the highest concentration of the gingiva flower extract, in the cell group treated with gingiva flower extract. When compared with vitamin C, which is a control group of iNOS and COX-2, it was confirmed that the protein expression and significant results of iNOS were shown, and COX-2 was superior to the control group.

To check the activity of iNOS and COX-2, cell line RAW 264.7 was seeded at 1 × 10 ⁶ cells / well in a 100 mm tissue culture dish for 24 hours after cell seeding. The cells were stabilized. After removing the medium, the LPS was treated at 1 ug / ml for 2 hours, and then the extract was incubated with the concentration-treated medium for 24 to 48 hours, and then the medium was removed again and washed twice with PBS. Complete mini 1 tab was dissolved in 10 ml of RIPA (radio-immunoprecipitation assay) buffer at 100 ul and centrifuged at 4, 13,200 rpm for 20 minutes. The supernatant obtained by centrifugation was quantified by a BCA protein assay kit, and 20 ul of protein was separated by electrophoresis on a 10% acrylamide gel. The separated protein was transferred to a polyvinylidene fluoride (PVDF) membrane using a transfer instrument (BIORAD) and incubated for 1 hour in a blocking buffer (5% skim milk in TBST) at room temperature. Primary antibodies of iNOS, COX-2, and β-actin were diluted and left overnight at 4 ° C., and washed three times with tris-buffered saline and tween 20 (TBST) at 10 minute intervals. Secondary antibodies of iNOS and COX-2 were anti-rabbit IgG-HRP, and β-actin secondary antibody was diluted to 1: 1,000 using anti-mouse IgG-HRP and room temperature. Incubated for 2 hours. After washing three times, the band was identified and quantified using an image quant LAS 4,000 (GE Healthcare Bio-Sciences AB, Sweden) instrument.

Experimental Example 9-Total RNA Isolation and cDNA Synthesis

A medicinal herb extract according to Example 2 was prepared, and total RNA isolation and cDNA synthesis analysis were performed.

Cell seeding at 1 × 10 ⁶ cells / well in a 100 mm culture dish and incubated for 24 hours, then treated with LPS at 1 ug / ml for 2 hours, followed by extracts. Incubated for hours. After removing the supernatant of the medium, 1 ml of trizol lysis buffer was dispensed into a well to dissolve the cells, and 200 ul of chloroform was dispensed and shaken up and down for 20 seconds. After centrifugation at 4, 13,200 rpm for 20 minutes, the supernatant was transferred to a tube containing 500 ul of isopropanol. After centrifugation at 13,200 rpm for 20 minutes, the supernatant was removed, and 1 ml of 75% DEPC water (EtOH-diethylpyrocarbonate water) was dispensed into each tube, followed by centrifugation at 4 ° C and 13,200 rpm for 5 minutes. After removal it was dried at room temperature. Dissolve the DEPC-treated water by dispensing 50 ul of DEPC-treated water and add 5 ul of RNA solution and 195 ul of sterile water to a 96 well plate to measure the absorbance at 260 nm and 280 nm. Total RNA amount was measured. 1 ul of Oligo (dT) 15 primer (500 ug / ml), 10 ul of extracted RNA (2 ug) and nuclease free water, and reacted at 75 ° C for 5 minutes, followed by 5X reaction buffer ( reaction buffer, MgCl2, PCR nucleotide mix, riboninase inhibitor, reverse transcriptase, and nuclease free water were added for 5 minutes at 25 ° C and 42 ° C. CDNA was synthesized by reacting at 60 ° C. for 15 minutes at 70 ° C.

Experimental Example 10 Reverse transcription-PCR

A medicinal plant extract according to Example 2 was prepared, and PCR was performed to determine mRNA expression of iNOS and COX-2, which are shown in FIGS. 11 and 16. At this time, in order to examine the effect of the gingival bee extract on the mRNA expression of iNOS and COX-2 factors, the gingival bee extract was treated with 50, 100, and 500 μg / ml by concentration to perform RT-PCR. Confirmed. In this case, GAPDH, a housekeeping gene, was used as a positive control group and vitamin C was used as a control group to compare mRNA expression levels.

FIG. 11 is a graph showing iNOS and COX-2 mRNA expression rates of Euphorbia ethanol extract in RAW264.7 cells. The left graph (A) shows the iNOS mRNA expression rate of the zinnia ethanol extract, and the right graph (B) shows the COX-2 mRNA expression rate of the zinnia ethanol extract. RAW264.7 cells (1 × 10 ⁶ cells / well) were treated for 1 hour in serum free medium (SFM) medium and then treated with 50, 100 and 500 μg / ml for the kerba extract for 24 hours. Each value is represented by the mean ± SD of each of the average three experiments.

Table 16 shows the sequence of primers via RT-PCR.

Referring to FIG. 11 and Table 16, mRNA expressions of iNOS and COX-2 were decreased in the cell group treated with C. eryngii extract, showing 82.08% and 84.61% inhibition at 500 μg / ml concentration, respectively. When compared with vitamin C, the expression of iNOS was significant in the expression of M. zinnia herb and the control group, and COX-2 was found to be excellent in expression. At this time, PCR was performed as follows. First, 5X buffer (green Gotaq flexi buffer), MgCl2, PCR nucleotide mix (10mM), primer, GoTaq DNA polymerase, nuclease free water, synthesis One cDNA was added and mixed well, followed by PCR. Afterwards, GAPDH and iNOS were 2 minutes at 96 ° C, 10 seconds at 96 ° C, 30 seconds at 64 ° C, 1 minute at 72 ° C, 10 minutes at 72 ° C (40 cycles), and COX-2 at 2 ° C at 96 ° C. , 10 seconds at 94 ℃, 30 seconds at 51 ℃, 1 minute at 72 ℃, 10 minutes (72 cycles) at 72 ℃. Subsequently, 1.5% agarose gel with 0.002% ethidium bromide added was electrophoresed at 100 ° C for 40 minutes, and then quantified by LAS 4,000. .

Gene Primer Sequence (5 '→ 3') GAPDH sense TGA AGG TCG GTG TGA ACG GAT TTG GC anti-sense CAT GTA GGC CAT GAG GTC CAC CAC COX-2 sense GGA GAG ACT ATC AAG ATA GT anti-sense ATG GTC AGT AGA CTT TTA CA iNOS sense AAT GGC AAC ATC AGG TCG GCC ATC ACT anti-sense GCT GTG TGT CAC AGA AGT CTC GAA CTC

Although the preferred embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto, and various modifications and improvements of the present invention using the basic concepts of the present invention defined in the following claims are also provided. It belongs to the scope of the invention.

Claims (6)

Long Bottle Flower { Glechoma grandis (A. Gray) Kuprian} An insect bite treatment containing as an active ingredient.
According to claim 1, wherein the pH of the therapeutic agent is insect bites therapeutic agent of 5 to 8.
According to claim 1, wherein the therapeutic agent is insect bites therapeutic agent to reduce the diameter of the swollen skin 15 to 30% within 5 to 10 minutes after the insect bites.
The method of claim 1, wherein the medicinal plant extracts inhibit insect bite treatment of COX-2 protein expression and COX-2 mRNA expression.
Preparing a solution by dispersing hydroxyethyl cellulose in distilled water or cypress water;
Preparing a first solution by mixing aloe moist and vegetable glycerin in the dispersed solution,
Preparing gingiva flower powder,
Preparing a second solution by adding the gingiva flower powder to the first solution,
Preparing a third solution by adding the second solution and menthol and allantoin;
Preparing a fourth solution by adding an emulsifier to vegetable oils, and
Preparing a fourth solution by adding an emulsifier to vegetable oils, and
Mixing the third solution with the fourth solution
How to prepare insect bites.
The manufacturing method of the long bottle flower powder in claim 5,
Harvesting stems and leaves of the gingiva flower,
Pulverizing after drying the long bottle flower hot air,
Adding ethanol to the pulverized gingiva flower plant to obtain gingiva flower extract;
Filtering the medicinal herbaceous extract and concentrating under reduced pressure to remove the solvent, and lyophilizing the medicinal herbaceous extract from which the solvent has been removed.
How to prepare insect bites.

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