KR20220109786A - A composition comprising coptidis rhizoma and evodiae fructus extract for preventing or treating chronic acid reflux esophagitis - Google Patents

Abstract

The present invention has studied an effect of chronic reflux esophagitis according to a mixing ratio of Coptis japonica and Tetradium ruticarpum in a chronic reflux esophagitis animal model by mixing Coptis japonica and Tetradium ruticarpum, which are effective for reflux esophagitis. In addition, by analyzing the degree of damage to the esophageal mucosa, the expression of proteins related to inflammation and esophageal function, the present invention is to suggest a specific combination of a complex extract of Coptis japonica and Tetradium ruticarpum that exhibits ideal pharmacological effects for the prevention or treatment of chronic reflux esophagitis.

Description

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to a composition for the prevention or treatment of chronic reflux esophagitis, and more particularly, to a composition for preventing or treating chronic reflux esophagitis comprising a complex extract of H.

Gastroesophageal reflux disease (GERD) is one of the diseases of the gastrointestinal tract that causes morphological changes in the esophagus or causes clinical symptoms due to reflux of gastric acid into the esophagus. generated (refer to Non-Patent Document 1). Reflux esophagitis (RE) is a case in which lesions such as ulcers or inflammation occur in the esophagus due to the reflux of gastric contents into the esophagus among gastroesophageal reflux diseases. Among them, lower esophageal sphincter function is considered to be the biggest cause (see Non-Patent Document 2). Gastroesophageal reflux disease (GERD) was a disease that occurred mainly in the West, but in Korea, it increased by 35% in 2015 compared to 2010. Although the prevalence is low compared to the West, once diagnosed, it is difficult to cure and requires lifelong treatment. and management are deepening (refer to Non-Patent Document 3).

The most basic method for the treatment of reflux esophagitis is to refrain from taking calcium channel antagonists and sleeping pills, and to control lifestyle such as avoiding overeating and oily food. There are H ₂ receptor antagonists (H ₂ RA) and proton pump inhibitors (PPIs) that inhibit gastric acid secretion. In this case, the recurrence rate reaches 50-80% (see Non-Patent Documents 4 and 5). Therefore, there is a need to develop a new therapeutic agent that can effectively treat reflux esophagitis with few side effects even if taken for a long period of time.

Coptidis Rhizoma (黃蓮) is a dried rhizome of a perennial plant belonging to the ranunculaceae family. Yellow rhubarb has anti-inflammatory and anti-inflammatory effects in modern pharmacology, as it has detoxifying, detoxifying, and detoxifying effects. In addition, the effects of antibacterial, antioxidant, anti-atopic, etc. were found through many studies, and it was confirmed in previous studies that it was effective in the treatment of chronic reflux esophagitis (see Non-Patent Documents 6 to 9).

Osuyu (Evodiae Fructus, 吳茱萸) refers to the fruit of osuyu belonging to the Rutaceae family, and has been used in oriental medicine for diarrhea, abdominal pain and indigestion (see Non-Patent Document 10). As the pharmacological action of Osuyu, antibacterial effect, neuroprotective effect, anti-inflammatory effect, etc. are known (see Non-Patent Documents 11 to 13). A recent study was known to have an effect of protecting the esophageal mucosa in acute reflux esophagitis, and previous studies confirmed that it is also effective in chronic reflux esophagitis (see Non-Patent Documents 4 and 14).

[Prior literature]

- Non-Patent Document 1: Lee, S. H. and Baik, T. H. (2019) A comparative study on the effects of Pinellia ternata, Zingiber officinale and Sobanhatang on reflux esophagitis. J. Korean Med. 40: 17-34.

- Non-Patent Document 2: Song, C. H. and Baek, T. H. (2018) A comparative study of Sepiae os, Arcae concha, Ostreae concha and esomeprazole in a mouse model of reflux esophagitis. J. Korean Med. 39: 92-105.

- Non-Patent Document 3: Park, C. S., Kim, D. H. and Lee, K. J. (2017) Evaluation of the effectiveness of alkaline mineral water on reflux esophagitis. J. Korean Soc. Water 4: 20-26.

- Non-Patent Document 4: Lee, J. A., Park, H. J., Kim, S. H., Kim, M. J., Kim, K. J. Shin, M. R. and Roh, S. S. (2019) Evaluation of Evodiae fructus extract on the chronic acid reflux esophagitis in rats. Kor. J. Herbol. 34: 15-23.

- Non-Patent Document 5: Lee, S. H., Lee, J. A., Shin, M. R., Lee, J. H. and Roh, S. S. (2020) The protective effect of water extract of Phellodendri cortex in chronic reflux esophagitis-induced rats. Kor. J. Herbol. 35: 25-36.

- Non-Patent Document 6. Kim, M. W., Seo, S. J., Park, G. R. and Lee, Y. S. (2018) Anti-wrinkle and antioxidant activity of 1,3-butylene glycol mixed extracts from Coptidis rhizoma. J. Invest. Cosmetol. 14: 267-276.

- Non-Patent Document 7: Jung, A. R., Ahn, S. H., Jeong, H. S. and Kim, K. B. (2019) Effect of Coptidis rhizoma extract on atopic dermatitis-like skin lesions in NC/Nga mice. J. Physiol & Pathol. Korean Med. 33: 102-108.

- Non-Patent Document 8: Lee, S. H. and Kim, M. J. (2019) Antimicrobial effect of natural plant extracts against periodontopathic bacteria. J. K. C. A. 19: 242-255.

- Non-Patent Document 9: Kim, S. H., Roh. S. S., Lee, J. A., Shin, M. R. Lee, A. R., Koo, J. S. and Park, H. J. (2019) Improving effects on rats with chronic acid reflux esophagitis treated of Coptidis rhizoma extract. Kor. J. Herbol. 34: 117-124.

- Non-Patent Document 10: Yang, J. Y., Lee, P. J. and Kim, J. B. (2019) Effect of Evodiae fructus methanol extract on virulence-related genes’ expression of Helicobacter pylori. Korean J. Clin. Lab. Sci. 51: 316-322.

- Non-Patent Document 11: Jeon, E. S., Han, M. D. and Kim, H. D. (2003) Antimicrobial activity of Streptococcus mutans by Schizandrae fructus and Evodiae fructus extracts. J. Dent. Hyg. Sci. 3: 39-44.

- Non-Patent Document 12: Kim, S. T., An, S. H., Kim, J. D. and Kim, Y. G. (2003) Protective effect of MeOH extract of Evodia officinalis on cyanide-induced neurotoxicity in cultured neuroblastoma cells. Kor. J. Pharmacogn. 34: 282-287.

- Non-Patent Document 13: Yun, H. J., Heo, S. K., Lee, Y. T., Park, W. H. and Park, S. D. (2008) Anti-inflammatory effect of Evodia officinalis DODE in mouse macrophage and human vascular endotherial cells. Kor. J. Herbol. 23: 29-38.

- Non-Patent Document 14: Kim, D. J. and Roh, S. S. (2012) Effect on acute reflux esophagitis by Evodiae fructus aquous extract. Kor. J. Herbol. 27: 51-58.

- Non-Patent Document 15: Blois, M. S. (1958) Antioxidant determinations by the use of a stable free radical. Nature 181: 1199-1200. 16. Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M. and Rice-Evans, C. (1999) Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radic. Biol. Med. 26: 1231-1237.

- Non-Patent Document 17: Omura, N., Kashiwagi, H., Chen, G., Suzuki, Y., Yano, F. and Aoki, T. (1999) Establishment of surgically induced chronic acid reflux esophagitis in rats. Scand. J. Gastroenterol. 34:948-953.

- Non-Patent Document 18: Kim, Y. H., Kim, S. Y. and Hwang, M. W. (2011) A case study of taeumin patient with gastro-esophageal reflux disease (GERD) who treated successfully with Yeoldahansotang (Reduohanshao-tang). J. S. C. M. 23: 132-138.

- Non-Patent Document 19: Lee, S. K. and Lim, S. W. (2016) The administration of Jeungmiyijin-tang to rats with induced gastro reflux esophagitis. J. Int. Korean Med. 37: 1030-1041.

- Non-Patent Document 20: Kim, D. G., Shin, J. H. and Kang, M. J. (2018) Antioxidant and anti-inflammatory activities of water extracts and ethanol extracts from Portulaca oleracea L. Korean J. Food Preserv. 25: 96-106.

- Non-Patent Document 21: Nam, J. W. and Lee, Y. S. (2012) Role of NADPH oxidase in the mechanism of arachidonic acid-induced apoptosis in HepG2 human hepatoblastoma cells. Yakhak Hoeji 56: 80-85.

- Non-Patent Document 22: Nam, J. J. and Kim Y. J. (2015) Fractionated Trapa japonica extracts inhibit ROS-induced skin inflammation in HaCaT keratinocytes. J. Soc. Cosmet. Scientists Korea 41: 45-55.

- Non-Patent Document 23: Kim, C. Y., Kang B. B., Hwang, J. S. and Choi, H. S. (2018) Red ginseng-derived saponin fraction inhibits lipid accumulation and reactive oxygen species production by activating nuclear factor (erythroid-derived 2)-like 2 (Nrf2)/Kelch-like ECH-associated protein 1 (Keap1) pathway. Korean J. Food Sci. Technol. 50: 688-696.

- Non-Patent Document 24: Choi, D. H., Cho, U. M. and Hwang, H. S. (2018) Antiinflammation effect of rebaudioside A by inhibition of the MAPK and NF-κB signal pathway in RAW264.7 macrophage. J. Appl. Biol. Chem. 61: 205-211.

- Non-Patent Document 25: Kim, M. J., Bae, N. Y., Kim, K. B. W. R., Park, S. H., Jang, M. R., Im, M. H. and Ahn, D. H. (2016) Anti-inflammatory activity of ethanol extract of Sargassum miyabei Yendo via inhibition of NF-κB and MAPK activation. Microbiol. Biotechnol. 44: 442-451.

- Non-Patent Document 26: Shin, D. H. and Choi, Y. H. (2013) Glutamine deprivation inhibits invasion of human prostate carcinoma LnCap cells through inactivation of matrix metalloproteinases and modulation of tight junctions. J. Korean Soc. Food Sci. Nutr. 42: 1167-1174.

- Non-Patent Document 27: Jeong, J. W. and Choi, Y. H. (2014) Cordycepin inhibits migration and invasion of HCT116 human colorectal carcinoma cells by tightening of tight junctions and inhibition of matrix metalloproteinase activity. J. Korean Soc. Food Sci. Nutr. 43: 86-92.

- Non-Patent Document 28: Nam, H. H., Seo, Y. S., Lee, J. H., Seo, Y. H., Moon, B. C., Kim, W. J., Nan, L., Choo, B. K. and Kim, J. S. (2020) Effect on rat model of reflux esophagitis treated with Charybdis japonica extract. Kor. J. Herbol. 35: 17-23.

- Non-Patent Document 29: Chen, H., Fang, Y., Li, W., Orlando, R. C., Shaheen N. and Chen, X. L. (2013) NFkB and Nrf2 in esophageal epithelial barrier function. Tissue Barriers 1: e27463.

In the present invention, the effect on reflux esophagitis according to the mixing ratio of Hwanglily and Osuyu was studied in an animal model of chronic gastroesophageal reflux disease by mixing H. By analyzing the expression of , we present a specific combination of Hwangryeon and Osuyu complex extract that exerts an ideal effect for the prevention or treatment of chronic reflux esophagitis.

As an aspect for solving the above problems, the present invention provides a pharmaceutical composition for preventing or treating chronic reflux esophagitis, comprising a complex extract of H.

In addition, the complex extract provides a pharmaceutical composition, characterized in that the weight ratio of hwangryeon and osuyu is 1:0.8 to 1:2.5.

In addition, the complex extract provides a pharmaceutical composition, characterized in that the hot water extract.

As another aspect for solving the above problems, the present invention provides a health functional food for preventing chronic reflux esophagitis comprising a complex extract of yellow liana and osuyu as an active ingredient.

According to the present invention, the effect on reflux esophagitis according to the mixing ratio of the yellow lotus lotus and Osuyu was studied in an animal model of chronic gastroesophageal reflux disease by combining H. As a result of analyzing the expression of related proteins, significant results were confirmed in a specific combination of Hwangryeon and Osuyu complex extract, so the composition according to the present invention can be very effectively used for the prevention or treatment of chronic reflux esophagitis.

1 is a graph showing the results of measuring the DPPH radical scavenging activity of the Hwangryeon-Osuyu complex extract in an experimental example of the present invention.
2 is a graph showing the results of measuring the ABTS radical scavenging activity of the Hwangryeon-Osuyu complex extract in the experimental example of the present invention.
Figure 3 is a photograph showing whether the esophageal mucosal ulceration after surgical induction of CARE (chronic acid reflux esophagitis) in the experimental example of the present invention;
4 is a photograph and graph showing the results of NADPH Oxidase expression in the esophageal tissue in the experimental example of the present invention.
5 is a photograph and graph showing the expression results of Nrf2 and Keap-1 in the esophageal tissue in the experimental example of the present invention.
6 is a photograph and graph showing the results of MAPKs protein expression in the esophageal tissue in the experimental example of the present invention.
7 is a photograph and graph showing the results of the expression of inflammatory protein in the esophageal tissue in the experimental example of the present invention.
8 is a photograph and graph showing the expression results of Proinflammatory Enzymes and Cytokines in the esophageal tissue in the experimental example of the present invention.
9 is a photograph and graph showing the expression results of MMP-2 and MMP-8 in the esophageal tissue in the experimental example of the present invention.
10 is a photograph and graph showing the results of Tight Junction Protein expression in the esophageal tissue in an experimental example of the present invention.

Hereinafter, the present invention will be described in detail through preferred embodiments. Prior to this, the terms or words used in the present specification and claims should not be construed as being limited to conventional or dictionary meanings, and the inventor should properly understand the concept of the term in order to best describe his invention. Based on the principle that it can be defined, it should be interpreted as meaning and concept consistent with the technical idea of the present invention. Accordingly, since the configuration of the embodiments described in this specification is only the most preferred embodiment of the present invention and does not represent all the technical spirit of the present invention, various equivalents and modifications that can be substituted for them at the time of the present application It should be understood that there may be In addition, throughout the specification, when a part "includes" a certain component, it means that other components may be further included, rather than excluding other components, unless otherwise stated.

In the present invention, by oral administration of complex extracts of yellow lily and oysterwort in different compounding ratios, the results of analysis of the expression of proteins related to the degree of damage to the esophageal mucosa, inflammation and esophageal function in an animal model of chronic reflux esophagitis showed significant results at specific compounding ratios. .

Accordingly, the present invention provides a pharmaceutical composition for the prevention or treatment of chronic reflux esophagitis comprising a complex extract of H. rhododendron and Osuyu as an active ingredient, and a health functional food for preventing chronic reflux esophagitis comprising a complex extract of H. start

There is no limitation on the form or properties of the complex extract, and it may be a solution or a concentrate, and may be a solid or powder obtained by removing the solvent used to prepare the extract.

The complex extract may be prepared using a conventional extraction method known in the art, for example, a solvent extraction method. The extraction solvent used in preparing the extract using the solvent extraction method is water, a lower alcohol having 1 to 4 carbon atoms (eg, methanol, ethanol, propanol and butanol) or a mixture thereof, which is a hydrous lower alcohol, propylene glycol, 1,3 -Butylene glycol, glycerin, acetone, diethyl ether, ethyl acetate, butyl acetate, dichloromethane, chloroform, may be selected from the group consisting of hexane and mixtures thereof, preferably hot water extraction method may be used.

In the present invention, the compounding ratio of the complex extract is preferably 1:0.8 to 1:2.5 by weight, more preferably 1:1.5 to 1:2.5, most preferably, based on the content added during solvent extraction. may be 1:1.8 to 1:2.2, and it was confirmed as the most ideal combination condition when comprehensively considering the degree of decrease in the expression factor of chronic reflux esophagitis in the mixing ratio.

The composition according to the present invention is provided in the form of a pharmaceutical composition or health functional food, and when provided as a pharmaceutical composition, one or more pharmaceutically acceptable carriers, excipients or diluents in addition to the extract of the present invention may be additionally contained. have. As used herein, "pharmaceutically acceptable" means a non-toxic composition that is physiologically acceptable and does not inhibit the action of the active ingredient when administered to humans and does not normally cause allergic reactions such as gastrointestinal disorders, dizziness, or similar reactions. say

The pharmaceutically acceptable carrier may further include, for example, a carrier for oral administration or a carrier for parenteral administration. Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. In addition, various drug delivery materials used for oral administration of the peptide formulation may be included. In addition, the carrier for parenteral administration may include water, a suitable oil, saline, aqueous glucose and glycol, and the like, and may further include a stabilizer and a preservative. Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, and the like, in addition to the above components. For other pharmaceutically acceptable carriers and agents, reference may be made to those described in Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).

The composition according to the present invention may be administered to mammals including humans by any method. For example, it may be administered orally or parenterally. Parenteral administration methods include, but are not limited to, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual or rectal administration. can be

The pharmaceutical composition according to the present invention may be formulated as a formulation for oral administration or parenteral administration according to the administration route as described above.

In the case of a formulation for oral administration, the composition of the present invention may be formulated as a powder, granule, tablet, pill, dragee, capsule, liquid, gel, syrup, slurry, suspension, etc. using methods known in the art. can For example, oral preparations can be obtained by mixing the active ingredient with a solid excipient, pulverizing it, adding a suitable adjuvant, and processing it into a granule mixture to obtain tablets or dragees. Examples of suitable excipients include sugars including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol and maltitol, and starches, including corn starch, wheat starch, rice starch and potato starch, cellulose, Cellulose, including methyl cellulose, sodium carboxymethylcellulose and hydroxypropylmethyl-cellulose, and the like, fillers such as gelatin, polyvinylpyrrolidone, and the like may be included. In addition, cross-linked polyvinylpyrrolidone, agar, alginic acid or sodium alginate may be added as a disintegrant if necessary. Furthermore, the pharmaceutical composition of the present invention may further include an anti-aggregating agent, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent, and an antiseptic agent.

Formulations for parenteral administration may be formulated in the form of injections, creams, lotions, external ointments, oils, moisturizers, gels, aerosols and nasal inhalants by methods known in the art. These formulations are described in Remington's Pharmaceutical Science, 19th ed., Mack Publishing Company, Easton, PA, 1995, which is a commonly known recipe for all pharmaceutical chemistry.

The total effective amount of the composition of the present invention may be administered to a patient as a single dose, or may be administered by a fractionated treatment protocol in which multiple doses are administered for a long period of time. The pharmaceutical composition of the present invention may vary the content of the active ingredient depending on the severity of the disease. Preferably, the preferred total dose of the pharmaceutical composition of the present invention may be about 0.01 μg to 10,000 mg, most preferably 0.1 μg to 1,000 mg per kg of body weight of the patient per day. However, the dosage of the pharmaceutical composition is determined by considering various factors such as the formulation method, administration route and number of treatments, as well as the patient's age, weight, health status, sex, severity of disease, diet and excretion rate, etc., the effective dosage for the patient is determined. Therefore, considering this point, those of ordinary skill in the art will be able to determine an appropriate effective dosage of the composition of the present invention. The pharmaceutical composition according to the present invention is not particularly limited in its formulation, administration route and administration method as long as the effect of the present invention is exhibited.

On the other hand, the term 'prevention' used in the present invention means any action of inhibiting or delaying a disease by administration of a composition comprising the mixture of the present invention. In addition, the term 'treatment' used in the present invention refers to any action in which symptoms of a disease are improved or cured by administration of a composition comprising the mixture of the present invention.

Hereinafter, the present invention will be described in more detail with reference to specific experimental examples.

Manufacture of complex extracts of Hwangnyeon and Osuyu

100 g of Hwangryeon and Osuyu (purchased from Onggi Pharmacy (Daegu, Korea) and only those that meet the pharmacopoeial standards after sensory testing according to the herbal medicine standard, respectively) and 100 g, 200 g and 50 of Osuyu, respectively. After pulverizing g, 1 L of distilled water was added, followed by extraction for 2 hours in a hot water extractor. The obtained extract was concentrated with a reduced pressure concentrator, and then completely dried using a freeze dryer to obtain a powder (yield: 14% of yellow lily and 15.66% of Osuyu), and stored at -80°C for use. Hwangryeon-Osuyu 1:1 mixture was denoted as CE, Hwangryeon-Osuyu 1:2 mixture was denoted as CEE, and Hwangryeon-Osuyu 2:1 mixture was denoted as CCE.

test substance

The reagents used in this experimental example are as follows.

2-Diphenyl-1-picrylhydrazyl (DPPH), 7 mM 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), potassium phosphate monobasic, potassium phosphate dibasic from Sigma-Aldrich Co. (St (Louis, MO, USA) was purchased and used. Nitrocellulose membranes were purchased from Amersham GE Healthcare (Chicago, IL, USA), NADPH oxidase 4 (NOX4), p47 ^phox , nuclear factor erythroid 2-related factor 2 (Nrf2), kelch like ECH associated protein 1 (Keap-1) , phospho-p38 MAPK (p-p38), phospho-extracellular signal-regulated kinase (p-ERK), phospho-c-Jun N-terminal Kinase (p-JNK), phosphorylation of nuclear factor-kappa B p65 (NF- κBp65), inhibitor of nuclear factor kappa B alpha (IκBα), phosphorylation inhibitor of nuclear factor kappa B alpha (p-IκBα), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-8 (MMP-8), zonula occludens-1 (ZO-1) occludin, claudin-1, claudin-4, β-actin, and histone were purchased from Santa Cruz Biotechnology (Dallas, CA, USA), and c-Fos and c-Jun were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). The secondary antibody was purchased from GeneTex, Inc. (Irvine, LA, USA) and used. Protease inhibitor mixture, ethylenediaminetetraacetic acid (EDTA), was purchased from Wako Pure Chemical Industries, Ltd. (Osaka. Japan). 2',7'-Dichlorofluorescein diacetate (DCFH-DA) and dihydrorhodamine 123 (DHR123) were purchased from Molecular Probes (Eugene, OR, USA), and ECL western blotting detection reagents were purchased from GE Healthcare. A BCA protein assay kit for protein quantification was purchased from Thermo Scientific (Waltham, MA, USA).

Experimental method

Measurement of DPPH and ABTS Radical Scavenging Activity

To measure the antioxidant efficacy of the sample, the radical scavenging activity was measured using the DPPH free radical scavenging method (see Non-Patent Document 15). 100 μl of a 60 mM DPPH solution and 100 μl of a sample solution diluted by concentration were mixed and reacted in the dark for 30 minutes, and then absorbance was measured at 540 nm. L-ascorbic acid was used as a control, and the concentration of the sample that reduced radicals by 50% was expressed as an IC ₅₀ value.

ABTS radical scavenging activity was measured according to the method of Re et al. (see Non-Patent Document 16). 7 mM ABTS solution and 2.4 mM potassium persulfate were mixed and left in the dark at room temperature for about 16 hours to form ABTS+, and then diluted with ethanol so that the absorbance (30°C, 415 nm) value was 0.70±0.02. 95 μl of the diluted ABTS solution and 5 μl of the sample solution diluted by concentration were mixed, reacted for 15 minutes, and absorbance was measured at 30° C. and 415 nm. L-ascorbic acid was used as a control, and the concentration of the sample that reduced radicals by 50% was expressed as an IC ₅₀ value.

The radical scavenging activity was calculated according to Equation 1 below.

[Equation 1]

DPPH and ABTS Radical scavenging activity (%)

= (1 - OD _sample / OD _blank ) × 100

OD _sample : Absorbance when sample is added

OD _blank : Absorbance without sample (control)

laboratory animal

Five-week-old SD rats were purchased from Daehan Bio (Eumseong, Korea) and acclimatized to the breeding room environment for one week before the experiment was carried out. The conditions of the animal breeding room were conventional system, temperature 22±2, humidity 50±5%, and light:dark cycle were controlled at a 12-hour cycle. In addition, feed (crude protein 18% or more, crude fat 5.0% or more, crude fiber 5.0% or less, crude fiber 8.0% or less, calcium 1.0% or more, phosphorus 0.85% or more, potassium 0.55% or more, sodium 0.25% or more, magnesium 0.15% or more, NIH-41, Zeigler Bros, Inc., USA) and water were sufficiently supplied. This experiment was conducted with the approval (approval number: 2020-053) of the Institutional Animal Care and Use Committee (IACUC) of Daegu Haany University for ethical and scientific feasibility review and efficient management of animal experiments.

Chronic reflux esophagitis induction and animal treatment

Before surgery, after fasting for 18 hours, Zoletil (Virbac, France) was injected intraperitoneally to anesthetize and the operation was performed. Abdominal hairs are removed with an epilator, and the mid-abdominal region is opened 2 cm. The fundus is tied with black silk (2-0) thread, and the latex ring (18-Fr Nelaton catheter, 2 mm in thickness) is attached to the pylorus. ) was inserted and tied with nylon (5-0) thread, and the peritoneum and skin were sutured (see Non-Patent Document 17). To reduce infection, dilute antibiotic (gentamicin sulfate) and anti-inflammatory agent (Dexamethasone) were injected subcutaneously for 3 days, and water was supplied 24 hours after the operation, and feed was supplied from 48 hours after the operation. After a one-week recovery period, the normal group without any treatment (Nor), the control group administered with distilled water after induction of chronic reflux esophagitis (Veh), and after induction of chronic reflux esophagitis, Hwangnyeon-Osuyu (1:1) mixture 200 mg/kg was randomized into 4 groups (CE) and a group administered with 200 mg/kg of Hwangryeon-Osuyu (1:2) mixture (CEE) after induction of chronic gastroesophageal reflux disease (CEE). Feed intake was measured and orally administered once a day for 14 days.

Esophageal Tissue Western Blotting

In the esophageal tissue, 100 mM Tris-HCl (pH 7.4), 5 mM Tris-HCl (pH 7.5), 2 mM MgCl ₂ ,

15 mM CaCl ₂ , 1.5 M sucrose, 0.1 M DTT, buffer A with protease inhibitor cocktail added, and ground with a tissue grinder (Biospec Product, Bartlesville, OK, USA), 10% NP-40 solution added. After standing on ice for 30 minutes, centrifugation was performed at 12,000 rpm for 2 minutes to separate the supernatant containing the cytoplasm. Then, rinse twice in buffer A with 10% NP-40 and 100 μl of buffer C (50 mM HEPES, 50 mM KCl, 0.3 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 0.1 mM PMSF, 10% glycerol) was added and resuspended, and then vortexed 3 times every 10 minutes. After centrifugation at 12,000 rpm at 4°C for 10 minutes, the supernatant containing nuclei was obtained and stored frozen at -80°C, respectively. NOX4, p47 ^phox , Keap-1, p-p38, p-ERK, p-JNK, IκBα, p-IκBα, iNOS, COX-2, TNF-α, IL-6, MMP-2, 10-12 to measure the expression of MMP-8, ZO-1, occludin, claudin-1, claudin-4, β-actin proteins and Nrf2, c-Fos, c-Jun, NF-κBp65, and histone proteins in the nucleus 10-μg protein

After electrophoresis using 12% SDS polyacrylamide gel, the acrylamide gel was transferred to a nitrocellulose membrane. Each primary antibody was treated on the prepared membrane and incubated at 4°C overnight, then washed 5 times every 6 minutes with PBS-T, and the secondary antibody used for each treated primary antibody (PBS-T 1:3000 with diluted and used) for 1 hour at room temperature, and then washed 5 times every 6 minutes with PBS-T. After exposing the membrane to an enhanced chemiluminescence (ECL) solution, sensitized it to Sensi-Q2000 Chemidoc (Lugen Sci Co., Ltd., Seoul, Korea) to confirm protein expression, and then the corresponding band ATTO Densitograph Software (ATTO Corporation, Tokyo) , Japan) was quantified using the program. The protein levels of each group are shown relative to the normal group (indicated by 1).

statistical analysis

In vitro values were expressed as mean±SEM and in vivo values were expressed as mean±SD. One-way analysis of variance (ANOVA) test was performed using SPSS (Version 25.0, IBM, Armonk, NY, USA). Afterwards, the least-significant differences (LSD) test was conducted to verify the statistical significance of the mean difference in each group at ^* p <0.05, ^** p <0.01, and ^*** p <0.001.

Experiment result

Measurement of DPPH and ABTS Radical Scavenging Activity

DPPH and ABTS radical scavenging activity were measured to evaluate the antioxidant activity of the Hwangryeon-Osuyu complex extract, and the results are shown in FIGS. 1 and 2, respectively. The measurement results were expressed as IC ₅₀ values.

Referring to FIG. 1, as a result of measuring DPPH radical scavenging activity, CE 22.62±1.01 μg/ml, CEE 27.90±0.32 μg/ml and CCE 36.70±0.98 μg/ml, CE showed the most excellent DPPH radical scavenging activity, Referring to FIG. 2 , as a result of measuring ABTS radical scavenging activity, CE 35.65±0.28 μg/ml, CEE 36.01±0.31 μg/ml, and CCE 38.64±0.73 μg/ml, CE showed the best ABTS radical scavenging activity.

Check for damage to the esophageal mucosa

3 is a photograph showing whether the esophageal mucosa is ulcerated after surgical induction of CARE (chronic acid reflux esophagitis).

Referring to Figure 3, when the degree of damage to the esophageal mucosa was visually observed, no damage or ulcer was found in the esophageal mucosa of the Nor group without any treatment, but in the Veh group, an ulcer was found in the middle or lower esophagus, Compared with the Nor group, the length of the esophagus was reduced and the thickness was increased. On the other hand, in the CE and CEE groups, damage and ulceration of the esophagus were significantly reduced.

Analysis of NADPH Oxidase Expression Level in Esophageal Tissue

4 is a photograph and graph showing the results of NADPH Oxidase expression in the esophageal tissue.

4, as a result of confirming the expression of NOX4 and p47 ^phox , which are NADPH oxidases, in the esophageal tissue, the Veh group significantly increased compared to the Nor group, and the CE group compared to the Veh group showed a significant increase in NOX4 26% and p47 ^phox 16%. In the CEE group, NOX4 26% and p47 ^phox 24% were significantly reduced. In particular, the expression of p47 ^phox was further reduced by 11% in the CEE group compared to CE.

Analysis of Nrf2 and Keap-1 Expression Levels in Esophageal Tissues

5 is a photograph and graph showing the results of Nrf2 and Keap-1 expression in esophageal tissue.

Referring to FIG. 5 , the expression of Nrf2 in the esophageal tissue was decreased by 34% in the Veh group compared to the Nor group, and significantly increased by 38% in the CE group and the CEE group compared to the Veh group. In addition, the expression of Keap-1 increased by 15% in the Veh group compared to the Nor group, and significantly increased by 15% in the CE group and 17% in the CEE group compared to the Veh group.

Analysis of MAPKs protein expression in esophageal tissue

6 is a photograph and graph showing the results of MAPKs protein expression in esophageal tissue.

Referring to FIG. 6 , as a result of confirming the expression of c-Fos, c-Jun, p-p38, p-ERK and p-JNK, which are MAPKs proteins in the esophageal tissue, the expression of c-Fos and c-Jun was higher than that of the Nor group. While the Veh group increased by 24% and 19%, respectively, the expression was significantly decreased by about 20% in the CEE group compared to the Veh group. In particular, the expression of c-Jun was significantly decreased in the CEE group compared to the CE group. In addition, the expression of p-p38 increased by 27% in the Veh group compared to the Nor group, whereas the expression decreased to the same level as the Nor group in the CE group. The expression of p-ERK increased by 27% in the Veh group compared to the Nor group, Compared to the Veh group, the CEE group was significantly reduced by 29%, and the expression was reduced by 10% compared to the Nor group. The expression of p-JNK was increased by 15% in the Veh group compared to the Nor group, and decreased by 11% in the CE group and 13% in the CEE group compared to the Veh group.

Analysis of Inflammatory Protein Expression in Esophageal Tissue

7 is a photograph and graph showing the results of the expression of inflammatory proteins in the esophageal tissue.

Referring to FIG. 7 , as a result of confirming the expression of inflammatory proteins, NF-κBp65 and p-IκBα, in the esophageal tissue, NF-κBp65 and p-IκBα were significantly increased by 21% and p-IκBα by 31% in the Veh group compared to the Nor group, and CE compared to the Veh group. In the group, the expression of NFκBp65 decreased by 18%, showing the same expression level as in the Nor group, and in the CEE group, it was significantly decreased by 21%. In addition, the expression of p-IκBα was decreased by 18% in the CEE group, indicating an expression level similar to that of the Nor group.

Analysis of Proinflammatory Enzymes and Cytokines in Esophageal Tissue

8 is a photograph and graph showing the expression results of Proinflammatory Enzymes and Cytokines in the esophageal tissue.

Referring to FIG. 8, as a result of confirming the expression of proinflammatory enzymes iNOS, COX-2 and cytokines TNF-α, IL-6 in the esophageal tissue, the expression of iNOS and COX-2 was increased by 25% in the Veh group compared to the Nor group. , iNOS 21% and COX-2 13% were significantly reduced in the CEE group compared to the Veh group. In addition, TNF-α 22% and IL-6 18% increased significantly in the Veh group compared to the Nor group, and significantly decreased by 20% or more in the CEE group compared to the Veh group, respectively.

Analysis of MMP-2 and MMP-8 Expression Levels in Esophageal Tissues

9 is a photograph and graph showing the results of MMP-2 and MMP-8 expression in the esophageal tissue.

9, as a result of confirming the expression of MMP-2 and MMP-8 in the esophageal tissue, the expression of MMP-2 and MMP-8 increased significantly by more than 20% in the Veh group compared to the Nor group, whereas the CE group compared to the Veh group MMP-2 11% and MMP-8 19% were significantly decreased in the CEE group, and both MMP-2 and MMP-8 were significantly decreased by about 20% in the CEE group.

Analysis of Tight Junction Protein Expression in Esophageal Tissue

10 is a photograph and graph showing the results of Tight Junction Protein expression in esophageal tissue.

Referring to FIG. 10, as a result of confirming the expression of ZO-1, occludin, claudin-1 and claudin-4, which are tight junction proteins, in the esophageal tissue, ZO-1, occludin, claudin-1 and claudin- in the Veh group compared to the Nor group. All 4 increased significantly by about 20%. On the other hand, the expression of ZO-1, occludin and claudin-4 was significantly decreased by about 20% in the CEE group compared to the Veh group. The expression of claudin-1 was decreased by 17% in the CE group and 13% in the CEE group, and it was confirmed that the expression of claudin-1 was significantly increased in the CE group than in the CEE group.

Gastroesophageal reflux disease (GERD) includes symptoms such as heartburn, chest pain, sore throat, and chronic cough as well as histological lesions in the form of ulcers or erosions due to the reflux of gastric acid into the esophagus (non-patented). See document 2). GERD is on the rise due to the aging of the population and changes in eating habits, leading to chronic diseases if not properly managed. Currently, suppression of gastric acid secretion by H ₂ receptor blockers and PPIs is used as the most important treatment for GERD. It is known that 80% relapse, so long-term treatment is required to prevent recurrence (see Non-Patent Documents 18 and 19). In order to solve this problem, many studies are being made for the development of a new therapeutic agent without side effects, and in the present invention, to develop a new material for the treatment of reflux esophagitis, Hwang-ryeon and Osuyu, which are known to be effective for reflux esophagitis, are blended in a compounding ratio. Efficacy for reflux esophagitis was evaluated.

First, the antioxidant efficacy of three compounding ratios of CE (Hwangryeon-Osuyu 1:1 weight ratio), CEE (Hwangryeon-Osuyu 1:2 weight ratio), and CCE (Hwangryeon-Osuyu 2:1 weight ratio) was investigated. All living things generate reactive oxygen species (ROS) in the process of generating the energy necessary to sustain life. In normal cells, reactive oxygen species exist in balance with antioxidant factors, and deficiency of antioxidant factors causes tissue damage by destroying cellular components, resulting in various diseases ranging from chronic inflammation and malignant tumors (Non-Patent Document 20). Reference). As a result of confirming the scavenging activity of DPPH and ABTS radicals in the present invention, excellent antioxidant effects were shown in CE and CEE, and animal experiments were conducted using two mixing ratios of CE and CEE (see FIGS. 1 and 2).

After inducing chronic reflux esophagitis through surgery, CE and CEE were orally administered for 14 days, and blood and esophageal tissues were collected at an autopsy on the 15th day. As a result of visual observation of the degree of damage to the esophageal tissue, ulcers and erosions were not observed in the esophageal mucosa in the normal group, but ulcers in the middle and lower esophagus were confirmed in the control group. It was confirmed that the tissue was swollen and thickened. On the other hand, it was confirmed that the damage to the esophageal tissue was significantly reduced in the CE group and the CEE group compared to the control group (see FIG. 3 ).

NADPH oxidase generates reactive oxygen species from neutrophils. Reactive oxygen species generated by NADPH oxidase are known to induce oxidative stress in the body and cause protein damage and DNA damage, and further induce cell death (see Non-Patent Documents 21 and 22). Oxidative stress generated in the body is recognized as a risk factor involved in the onset and progression of various diseases such as inflammatory diseases, obesity, and cancer. have. In a normal state, Nrf2 forms a complex with Keap-1 in the cytoplasm, and Keap-1 is degraded by stimuli such as oxidative stress, and Nrf2 enters the nucleus to protect cells through cytotoxicity and ROS regulation (non-patented). See document 23). As a result of confirming the expression of NOX4 and p47 ^phox , which are NADPH oxidases, in the present invention, it was confirmed that the expression of NOX4 and p47 ^phox was significantly reduced in the CE group and the CEE group administered with the Hwangryeon-Osuyu complex extract. In particular, it was confirmed that the expression of p47 ^phox was greatly reduced in the CEE group (see FIG. 4 ). In addition, it was confirmed that the administration of CE and CEE regulates the expression of Nrf2/Keap-1 (see FIG. 5 ).

Mitogen-activated protein kinase (MAPK) plays an important role in regulating extracellular signaling and cell division and proliferation. MAPK refers to proteins such as p38, ERK, and JNK, and is activated by LPS or growth factors to induce the activation of NF-κB and induces the expression of several pro-inflammatory proteins such as iNOS and COX-2 and inflammatory cytokines. (See Non-Patent Documents 24 and 25). As a result of confirming the expression of MAPKs and NF-κB related proteins in the present invention. The expression of MAPKs including c-Fos, c-Jun, p-p38, p-ERK and p-JNK was significantly reduced in the CEE group, and the expression of NF-κBp65 and p-IκBα in the CE and CEE groups. It also decreased significantly (see FIGS. 6 and 7). In addition, CEE administration significantly reduced the expression of pro-inflammatory proteins and inflammatory cytokines, and the expression of inflammatory cytokines was also significantly reduced in the CE administration group (see FIG. 8). It is judged that the yellow lotus-wosuyu complex extract reduces damage to the esophageal mucosa through the MAPKs and NF-κB pathways in an animal model of chronic reflux esophagitis.

Numerous studies have revealed that NF-κB is activated in epithelial cells of GERD patients, and NF-κB is known to regulate the expression of tight junction (TJ) proteins and matrix metalloproteinases (MMPs). MMPs are known to play a key role in destroying inflammatory tissues as proteolytic enzymes, and are known to regulate the homeostasis of connective tissue in the body, but are known to cause degradation and reorganization of the extracellular matrix if the regulatory function is disrupted. (See Non-Patent Documents 26 and 27). In addition, the TJ protein is a major protein that connects the cell space, and there are ZO-1, occludin, claudins, etc., and when the esophageal mucosa is damaged by GERD, it induces a decrease in TJ in the connective tissue (see Non-Patent Documents 27 to 29). ). As a result of the experiment in the present invention, the expression of MMPs protein and TJ protein was significantly regulated in the CE group and the CEE group, and in particular, the CEE group showed a more excellent effect. This indicates that the Hwangryeon-Osuyu complex extract protected epithelial cell connective tissue damage, and the effect was more pronounced when the compounding ratio of Osuyu was relatively high.

As described above, in the present invention, the esophageal mucosal protective effect of the Hwangryeon-Osuyu complex extract with different mixing ratios in chronic reflux esophagitis was evaluated. Antioxidant efficacy was confirmed by varying the mixing ratio of Hwangryeon-Osuyu, and animal experiments were conducted using 1:1 and 1:2 mixing ratios, which had excellent effects. In the chronic reflux esophagitis model, it was confirmed that CEE had a better esophageal mucosal protective effect than CE. It was confirmed that this esophageal mucosal protective effect relieves inflammation by inhibiting the MAPKs and NF-κB pathways to suppress the expression of inflammatory factors such as iNOS, COX-2, TNF-α and IL-6, as well as MMPs and tight By regulating the expression of the junction protein, damage in the connective tissue of the esophagus was suppressed. According to the above results, it is considered that the Hwangryeon-Osuyu mixture can be developed as a new pharmaceutical composition or health functional food for the treatment of reflux esophagitis.

Hereinafter, an example of the application of the pharmaceutical composition for preventing or treating reflux esophagitis containing the complex extract of H. rhododendron and Osuyu as an active ingredient according to the present invention will be described, but the present invention is not limited thereto.

Formulation Example 1: Preparation of powder

Table 1 below exemplifies the composition of the powder containing the complex extract of Hwangryeon-ryeon and Osuyu according to the present invention described above as an active ingredient. The powder may be prepared by mixing the following ingredients and filling the airtight bag.

ingredient content Hwangnyeon and Osuyu Complex Extract 200 mg lactose 100 mg talc 10 mg

Formulation Example 2: Tablet Preparation

Table 2 below exemplifies the tablet composition containing the complex extract of Hwangryeon-ryeon and Osuyu according to the present invention described above as an active ingredient. Tablets can be prepared by mixing the following ingredients and then tableting according to a conventional tablet manufacturing method.

ingredient content Hwangnyeon and Osuyu Complex Extract 200 mg corn starch 100 mg lactose 100 mg magnesium stearate 2 mg

Formulation Example 3: Capsule preparation

Table 3 below exemplifies the composition of capsules containing the complex extract of Hwangryeon-ryeon and Osuyu according to the present invention described above as an active ingredient. Capsules can be prepared by mixing the following ingredients according to a conventional capsule preparation method and filling the gelatin capsules.

ingredient content Hwangnyeon and Osuyu Complex Extract 200 mg crystalline cellulose 3 mg lactose 14.8 mg magnesium stearate 0.2 mg

Formulation Example 4: Preparation of Injections

Table 4 below exemplifies the composition of the injection containing the complex extract of Hwangryeon-ryeon and Osuyu according to the present invention described above as an active ingredient. Injections can be prepared with the following component contents per ampoule (2 ml) according to a conventional injection preparation method.

ingredient content Hwangnyeon and Osuyu Complex Extract 200mg mannitol 3mg Sterile distilled water for injection 14.8mg Na ₂ HPO ₄ 12H ₂ O 0.2mg

Formulation Example 5: Preparation of liquid preparation

Table 5 below exemplifies the composition of the liquid formulation containing the complex extract of Hwangryeonryeon and Osuyu according to the present invention described above as an active ingredient. The liquid preparation is prepared by adding and dissolving each of the following components in purified water according to a conventional liquid preparation method, adding an appropriate amount of lemon flavor, mixing the following components, adding purified water to adjust the total to 100 ml, and filling and sterilizing a brown bottle. can

ingredient content Hwangnyeon and Osuyu Complex Extract 200 mg Lee Seonghwadang 10 g mannitol 5 g Purified water appropriate amount

Formulation Example 6: Health functional food production

Table 6 below shows the composition of a health functional food containing the complex extract of yellow lily and Osuyu according to the present invention described above as an active ingredient. In the following composition, the composition ratio of the vitamin and mineral mixture indicates that a component suitable for a relatively healthy functional food is mixed as a preferred formulation example, but the mixing ratio may be arbitrarily modified, and each component according to a conventional health functional food manufacturing method After mixing, granules are prepared and can be used in the preparation of health functional food compositions according to a conventional method.

ingredient content ingredient content Hwangnyeon and Osuyu Complex Extract 1000 mg folic acid 50 μg vitamin mixture appropriate amount Calcium Pantothenate 0.5 mg vitamin A acetate 70 μg mineral mixture appropriate amount vitamin E 1.0 mg ferrous sulfate 1.75 mg vitamin B1 0.13 mg zinc oxide 0.82 mg vitamin B2 0.15 mg magnesium carbonate 25.3 mg vitamin B6 0.5 mg monobasic potassium phosphate 15 mg vitamin B12 0.2 μg Dibasic Potassium Phosphate 55 mg vitamin C 10 mg Potassium Citrate 90 mg biotin 10 μg calcium carbonate 100 mg nicotinic acid amide 1.7 mg magnesium chloride 24.8 mg

Formulation Example 7: Health Beverage Preparation

Table 7 below shows the composition of a functional health drink containing the complex extract of Hwangryeon-ryeon and Osuyu according to the present invention as described above as an active ingredient. For health drink production, for example, the following ingredients are mixed according to a conventional health drink manufacturing method, and then stirred and heated at 85° C. for about 1 hour, then the resulting solution is filtered and obtained in a sterilized 2L container, sealed and sterilized, and then refrigerated. It can be stored and used. The following composition ratio is composed of ingredients suitable for relatively favorite beverages in a preferred embodiment, but the mixing ratio may be arbitrarily modified according to regional and national preferences such as demand class, demanding country, and use.

ingredient content Hwangnyeon and Osuyu Complex Extract 1000 mg citric acid 1000 mg oligosaccharide 100 g Plum Concentrate 2 g Taurine 1 g Purified water mix 900 ml whole

Preferred embodiments of the present invention described above are disclosed to solve the technical problem, and various modifications, changes, additions, etc. will be possible within the spirit and scope of the present invention by those skilled in the art to which the present invention pertains. , such modifications and changes should be regarded as belonging to the following claims.

Claims (4)

A pharmaceutical composition for the prevention or treatment of chronic reflux esophagitis, comprising a complex extract of H. The method of claim 1,
The complex extract is a pharmaceutical composition, characterized in that the weight ratio of hwangryeon and osuyu is 1:0.8 to 1:2.5. The method of claim 1,
The complex extract is a pharmaceutical composition, characterized in that it is a hot water extract. A health functional food for the prevention of chronic reflux esophagitis, containing a complex extract of yellow lily and oyster oil as an active ingredient.

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