KR20200008736A - Food composition for prevention and improvment of reflux esophagitis containing Cudrania tricuspidata extract as an effective factor - Google Patents

Food composition for prevention and improvment of reflux esophagitis containing Cudrania tricuspidata extract as an effective factor Download PDF

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KR20200008736A
KR20200008736A KR1020180082767A KR20180082767A KR20200008736A KR 20200008736 A KR20200008736 A KR 20200008736A KR 1020180082767 A KR1020180082767 A KR 1020180082767A KR 20180082767 A KR20180082767 A KR 20180082767A KR 20200008736 A KR20200008736 A KR 20200008736A
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reflux esophagitis
food composition
kkujippong
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김주연
정지용
심재중
장성식
이정열
심재헌
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주식회사한국야쿠르트
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    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23LFOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105Plant extracts, their artificial duplicates or their derivatives
    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23VINDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00Food compositions, function of food ingredients or processes for food or foodstuffs
    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23VINDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00Function of food ingredients
    • A23V2200/30Foods, ingredients or supplements having a functional effect on health
    • A23V2200/32Foods, ingredients or supplements having a functional effect on health having an effect on the health of the digestive tract

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Abstract

The present invention relates to a food composition for preventing and ameliorating reflux esophagitis, comprising a Cudrania tricuspidata ethanol extract having an activity of inhibiting a histamine H2 receptor as an active ingredient. The present invention can be used as a food composition, such as fermented milk, functional drinks, and functional health foods for preventing and ameliorating reflux esophagitis, comprising, as an active ingredient, a Cudrania tricuspidata alcohol extract which has an effect of reducing inflammatory mediators and damage of esophageal mucosa caused by gastric acid, and inhibiting the activity of the histamine H2 receptor which increases the acidity of gastric juice.

Description

꾸지뽕 주정추출물을 유효성분으로 하는 역류성 식도염 예방 및 개선용 식품조성물{Food composition for prevention and improvment of reflux esophagitis containing Cudrania tricuspidata extract as an effective factor}Food composition for prevention and improvment of reflux esophagitis containing Cudrania tricuspidata extract as an effective factor}

본 발명은 꾸지뽕 주정추출물을 유효성분으로 함유하는 역류성 식도염 예방 및 개선용 식품조성물에 관한 것으로서, 보다 상세하게는 히스타민 H2 수용체(histamie H2 receptor)를 억제하는 활성을 갖는 꾸지뽕 주정추출물을 유효성분으로 함유하는 역류성 식도염 예방 및 개선용 식품조성물에 관한 것이다.The present invention relates to a food composition for preventing and improving reflux esophagitis comprising courageous ethanol extract as an active ingredient, and more particularly, containing zigzag ethanol extract having an activity of inhibiting histamine H2 receptor as an active ingredient. It relates to a food composition for preventing and improving reflux esophagitis.

역류성 식도염은 위 내용물(주로 산과 펩신)이 식도로 역류하여 다양한 임상 증상과 점막의 변화를 가져오는 질환으로서 산에 노출되는 시간이 길수록 심한 병변을 보이며 만성적인 경과를 밟는다. 대개는 강한 산성의 위액이 역류하면서 식도를 자극하기 때문에 가슴 부위부터 목까지 타들어가는 듯 한 느낌을 받고, 이로 인해 가슴이 답답함과 더불어 호흡곤란까지 올 수 있으며, 심한 경우에는 통증이 느껴지기도 한다. 이를 속쓰림(Heartburn)이라고 한다. 음식을 삼킬 때 불편감이 느껴질 수도 있고 기침을 자주 한다거나 역류하는 분비물 때문에 입안에 쓴 맛이 계속 남아있고, 합병증으로 폐렴(aspiration pneumonia)이 발생하기도 한다(F.Rieder, P. Biancani, K. Harnett, L. Yerian, and G. W. Falk, "Inflammatory mediators in gastroesophageal reflux disease: impact on esophageal motility, fibrosis, and carcinogenesis,"American Journal of Physiology. Gastrointestinal and Liver Physiology, vol. 298, no. 5, pp. G571-G581, 2010).Reflux esophagitis is a disease in which gastric contents (mainly acids and pepsin) flow back into the esophagus, causing various clinical symptoms and mucosal changes. The longer the exposure to acid, the more severe the lesion and the chronic course. In general, strong acidic gastric fluid regurgitates the esophagus, causing a burning sensation from the chest to the neck, which can lead to chest tightness and shortness of breath, and in severe cases, pain. This is called heartburn. When swallowing food, discomfort may be felt, coughing or refluxing secretions may leave bitter taste in the mouth, and complications may cause pneumonia (F. Rieder, P. Biancani, K. Harnett). , L. Yerian, and GW Falk, "Inflammatory mediators in gastroesophageal reflux disease: impact on esophageal motility, fibrosis, and carcinogenesis," American Journal of Physiology.Gastrointestinal and Liver Physiology, vol. 298, no. 5, pp. G571-. G581, 2010).

역류성 식도염 원인으로 잘못된 식습관을 들 수 있다. 육식, 고지방, 인스턴트 식품 등은 위산 분비를 상승시키며, 과식이나 급하게 먹는 식습관은 위장내압을 상승시켜 위산의 중화를 어렵게 만든다. 위산분비가 증가하고 중화에 지장이 생기면 역류성 식도염 원인이 된다. 다른 역류성 식도염 원인으로는 식후에 눕는 습관이 있다. 식후에 눕거나 구부리는 자세를 취하면 위 내용물이 식도 가까이로 쏠려 역류가 더 일어나기 쉬워진다. 특히 과식 후에 바로 누워 자는 버릇은 역류성 식도염으로 직결되는 경우가 많다. 그 외에 담배를 피우거나, 커피·차·콜라·초콜릿·박하·오렌지 주스 등의 음식도 괄약근의 조이는 힘을 약화시켜 역류성 식도염을 유발한다. Reflux esophagitis is a common cause of poor eating habits. Meat, high fats, and instant foods increase the secretion of stomach acid, overeating or eating quickly to increase stomach pressure makes stomach acid difficult to neutralize. Increased gastric acid secretion and neutralization can cause reflux esophagitis. Another cause of reflux esophagitis is the habit of lying down after eating. Lying or bent after eating can cause gastric contents to get closer to the esophagus, making reflux more likely to occur. In particular, the habit of sleeping right after overeating often leads directly to reflux esophagitis. In addition to smoking, foods such as coffee, tea, cola, chocolate, peppermint and orange juice also weaken the tightening power of the sphincter, causing reflux esophagitis.

역류성 식도염이 계속 지속되다 보면 식도가 산성 자극을 계속 받게 되고, 식도는 스스로를 산성 자극에 버티게 하기 위해 상피를 식도점막이 아닌 위점막 같은 상피로 바꾸게 된다. 이 과정을 화생(Metaplasia)이라고 하며, 이 과정을 통해 상피의 종류가 변한 식도를 바렛 식도(Barrett's esophagus)라고 한다. 일반적인 식도에 비해 바렛식도는 식도암이 생길 위험이 훨씬 높아진다. 바렛 식도의 길이가 3cm 이상일 경우에는 특정 식도암의 발병률이 3~40배 높아진다. 바렛식도는 수술적으로 잘라내지 않는 이상 없어지는 것이 아니기 때문에 꾸준히 내시경으로 추적 관찰하면서 암으로 변이여부를 검사해야 한다(N. Vakil, S.V. van Zanten, P. Kahrilas et al., "The Montreal definition and classification of gastroesophageal reflux disease: a global evidence-based consensus," The American journal of gastroenterology, vol. 101, no. 8, pp. 1900-1920, 2006).If reflux esophagitis persists, the esophagus continues to have acidic irritation, and the esophagus replaces the epithelium with the epithelium, like the gastric mucosa, rather than the esophageal mucosa to sustain itself. This process is called metaplasia, and Barrett's esophagus is the esophagus that has changed the type of epithelium through this process. Compared to the general esophagus, Barrett's esophagus has a much higher risk of developing esophageal cancer. If Barrett's esophagus is more than 3 cm long, the incidence of certain esophageal cancers is 3 to 40 times higher. Barrett's esophagus does not disappear unless it is surgically cut off, so continual follow-up should be followed to examine for cancer (N. Vakil, SV van Zanten, P. Kahrilas et al., "The Montreal definition and classification of gastroesophageal reflux disease: a global evidence-based consensus, "The American journal of gastroenterology, vol. 101, no. 8, pp. 1900-1920, 2006).

일반적으로 히스타민 H2 수용체는 위 점막세포에 많이 존재하며 위산분비 신호를 조절하는 역할을 한다. 위장 상피세포에서 히스타민 H2 수용체가 활성화되면 cAMP가 증가하고 양성자 펌프(proton pump)가 활성화되어 위액 내에 수소이온(H+)의 방출이 증가하여 위액의 산도(acidity)가 증가하는데, 이는 위액에 더 많은 양의 염산이 존재한다는 의미이다. 역류성 식도염의 임상적 치료에 사용되는 약물은 대부분 양성자펌프 억제제(PPI)와 히스타민 H2 수용체 억제제(Histamine H2 receptor blocker)로 이러한 약물들은 위액의 산도를 낮추어 위액의 역류가 일어났을 때 식도점막이 위액에 자극을 덜 받도록 하는 역할을 한다. In general, histamine H2 receptors are present in gastric mucosal cells and regulate gastric acid secretion signals. The activation of histamine H2 receptors in gastrointestinal epithelial cells increases cAMP and activates the proton pump, which increases the release of hydrogen ions (H + ) in gastric juice, which increases acidity in the gastric juice. This means that large amounts of hydrochloric acid are present. Most of the drugs used for the clinical treatment of reflux esophagitis are proton pump inhibitors (PPIs) and histamine H2 receptor blockers. These drugs lower the acidity of the gastric juice, which causes the esophageal mucosa to enter the gastric juice when gastric reflux occurs. It serves to receive less stimulation.

특히, 역류성 식도염의 치료에는 위산의 분비를 억제하는 오메프라졸이나 라베프라졸과 같은 양성자펌프 억제제(PPI)가 사용되고 있다. 그러나, 양성자펌프 억제제를 사용하는 경우에도 재발률이 높으며, 장기 복용해야 하는 문제점이 있다. 실제로 8주 정도 복용하면 역류성 식도염은 치료되나, 약물 복용 중단 후 50~80% 환자가 1년 이내 재발한다고 보고되어 있다. In particular, a proton pump inhibitor (PPI) such as omeprazole or rabeprazole, which inhibits the secretion of gastric acid, is used for the treatment of reflux esophagitis. However, even when using a proton pump inhibitor, the recurrence rate is high, and there is a problem of taking a long term. In fact, reflux esophagitis is treated after 8 weeks, but it is reported that 50 to 80% of patients relapse within 1 year after discontinuation of the drug.

또한 역류성 식도염 치료를 위해 양성자펌프 억제제를 장기 복용할 경우에는 신경내분비 세포종양(neuroendocrine cell tumor)을 유발한다는 보고가 계속되고 있고, 2010년 미국 FDA에서는 양성자펌프 억제제(PPI)계 약물을 1년 이상 장기간 복용하거나 고용량으로 복용할 경우에 골절 위험이 증가할 수 있다고 보고하였다. 따라서, 역류성 식도염의 치료효과가 우수하고 장기 복용 시에도 인체에 안전한 역류성 식도염 치료제의 개발이 절실히 요구되고 있는 실정이다.In addition, long-term use of proton pump inhibitors for the treatment of reflux esophagitis has been reported to cause neuroendocrine cell tumors.In 2010, the US FDA reported that proton pump inhibitor (PPI) drugs were used for more than 1 year. Long-term or high doses have been reported to increase the risk of fractures. Therefore, there is an urgent need for the development of a reflux esophagitis therapeutic agent that is excellent in the effectiveness of the reflux esophagitis and is safe for the human body even when taken for a long time.

꾸지뽕나무(Cudrania tricuspidata)는 낙엽성 소교목으로, 한방에서 잎 부분을 자수경엽이라 하여 습진, 유행성 이하선염, 폐결핵, 타박상, 급성관절염 등을 치료하는데 사용되고 있으며, 목부, 근피 및 열매도 이용되고 있다. 그리고 우리나라 민간에서도 꾸지뽕나무를 다려서 마시면 간암 치료에 효과적인 것으로 전해져 내려오고 있다. Cudrania tricuspidata (Cudrania tricuspidata) is a deciduous arborescent, and the leaf part of the herb is called embroidered foliage, which is used to treat eczema, mumps, pulmonary tuberculosis, bruises, acute arthritis, and neck, root and fruit. In addition, the Korean civilian has been reported to be effective in treating liver cancer by ironing courageous mulberry trees.

대한민국 공개특허공보 제10-2013-0036939호(2013.04.15.)에는 꾸지뽕나무 추출물이 알콜로 유도된 위(胃) 손상에서 염증 및 위액분비 관련 유전자 발현을 억제하고, 위(胃) 보호물질인 뮤신(mucin) 합성을 증가시켜 알콜성 위장관 질환의 예방 및 치료효과를 가진다는 내용이 기술되어 있으나, 아직까지 꾸지뽕잎의 식도세포 보호 및 역류성 식도염 예방 및 치료 효과에 대한 기술은 전무한 실정이다. Korean Patent Laid-Open Publication No. 10-2013-0036939 (April 15, 2013) discloses that Koji mulberry extract inhibits gene expression related to inflammation and gastric juice secretion in alcohol-induced gastric injury. It has been described that it has an effect of preventing and treating alcoholic gastrointestinal diseases by increasing mucin (mucin) synthesis, but there is no description of the effects of esophageal cell protection and reflux esophagitis of coupe mulberry leaves.

이에 본 발명자들은 인체에 무해한 천연물질 유래의 역류성 식도염 예방 및 치료 효과를 갖는 물질을 검색하던 중 본 발명의 꾸지뽕 주정추출물이 위산의 주성분인 염산에 의한 식도세포의 손상 및 염증반응을 억제하여 역류성 식도염을 치료하는 효과가 있을 뿐 아니라 위액의 산도를 높이는 히스타민 H2 수용체의 활성을 억제하여 위액 역류시 위산에 대한 식도 자극을 줄여 역류성 식도염으로 인한 가슴쓰림(흉통) 및 바렛식도 등을 예방할 수 있는 것을 확인하여 본 발명을 완성하였다.The inventors of the present invention, while searching for a material having a prevention and treatment of reflux esophagitis derived from natural substances that are harmless to the human body, refractory esophagitis by inhibiting the damage and inflammatory reaction of esophageal cells by hydrochloric acid, which is the main component of gastric acid. In addition, it is effective in treating gastric acid and inhibiting the activity of histamine H2 receptor, which increases the acidity of gastric juice, thereby reducing esophageal irritation of gastric acid during gastric reflux, thereby preventing heartburn and chest pain caused by reflux esophagitis. The present invention was completed.

대한민국 공개특허공보 제10-2013-0036939호(2013.04.15.)Republic of Korea Patent Publication No. 10-2013-0036939 (2013.04.15.)

본 발명은 꾸지뽕 주정추출물을 유효성분으로 함유하는 역류성 식도염 예방 및 개선용 발효유, 기능성 음료, 건강기능식품 등 식품조성물을 제공하는 것을 목적으로 한다.An object of the present invention is to provide a food composition, such as reflux esophagitis prevention and improvement fermented milk, functional drinks, health functional foods containing kkujippong alcohol extract as an active ingredient.

상기 목적을 달성하기 위하여, 본 발명은 꾸지뽕 주정추출물을 유효성분으로 함유하는 역류성 식도염 예방 및 개선용 발효유, 기능성 음료, 건강기능식품 등 식품조성물을 제공하는 것을 특징으로 한다.In order to achieve the above object, the present invention is characterized by providing a food composition, such as reflux esophagitis preventive and improved fermented milk, functional drinks, health functional foods containing kkujippong alcohol extract as an active ingredient.

이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.

본 발명의 꾸지뽕 주정추출물은 꾸지뽕잎에서 추출된 것으로서 추출 용매는 C1 내지 C4의 알코올, 50% 내지 100% 주정(식용 에탄올)이 바람직하며, 70% 주정이 가장 바람직하다. Cudrania ethanol extract of the present invention is extracted from the jacuzzi leaf, the extraction solvent is C 1 to C 4 alcohol, 50% to 100% alcohol (edible ethanol) is preferred, 70% alcohol is most preferred.

건조된 꾸지뽕잎을 적당한 크기로 분쇄하여 추출용기에 넣는다. 꾸지뽕잎에 중량대비 1:2 내지 1:20, 바람직하게는 1:10의 주정을 첨가한다. 추출은 25℃ 내지 55℃ 바람직하게는 50℃에서 환류 또는 교반 추출하고, 상기 추출액은 1차 진동체 여과 후 2차 퍼라이트 여과한다. 추출시간은 3 내지 24시간이 바람직하며 6 내지 16시간이 더욱 바람직하다. 상기 여과된 추출액은 55℃ 내지 65℃에서 감압농축한 다음 고형분을 측정하여 고형분과 동일 분량의 덱스트린 내지 말토덱스트린(Dextrin, malto dextrin)을 혼합한다. 상기 말토덱스트린을 혼합한 농축액은 동결건조 혹은 감압건조 혹은 분무건조, 바람직하게는 동결건조한 후 분쇄하여 분말화 한다.The dried Cudrania leaf is ground to an appropriate size and put in an extraction container. Add ethanol of 1: 2 to 1:20, preferably 1:10, by weight to the cuddle leaves. The extraction is carried out at reflux or stirring at 25 ° C. to 55 ° C. and preferably at 50 ° C., and the extract is subjected to secondary perlite filtration after primary vibrating filtration. The extraction time is preferably 3 to 24 hours, more preferably 6 to 16 hours. The filtered extract is concentrated under reduced pressure at 55 ℃ to 65 ℃ and then measuring the solid content of the same amount of dextrin to maltodextrin (Dextrin, malto dextrin) is mixed. The concentrate mixed with the maltodextrin is lyophilized or vacuum dried or spray dried, preferably lyophilized and then ground to powder.

상기 제조된 꾸지뽕잎 추출물은 하기 화학식의 쿠드라잔톤 엘(cudraxanthone L)을 중량대비 0.5% 이상 함유하는 것을 특징으로 한다.The prepared Cudrania leaf extract is characterized in that it contains more than 0.5% by weight of the Cudraxanthone L (cudraxanthone L) of the formula.

Figure pat00001
Figure pat00001

본 발명의 꾸지뽕 주정추출물을 유효성분으로 함유하는 역류성 식도염 예방 및 개선용 식품조성물은 식품, 식품첨가제, 음료, 음료첨가제, 발효유, 건강기능식품 등으로 사용될 수 있다. 식품, 식품첨가제, 음료, 음료첨가제, 또는 건강기능식품으로 사용되는 경우, 각종 식품류, 발효유, 육류, 음료수, 초콜렛, 스넥류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류, 알코올 음료, 비타민 복합제, 주류 및 그 밖의 건강기능식품일 수 있으나, 이에 한정되는 것은 아니다.Reflux esophagitis preventing and improving food composition containing kkujippong alcohol extract of the present invention as an active ingredient can be used as food, food additives, beverages, beverage additives, fermented milk, health functional foods. When used as food, food additives, beverages, beverage additives, or health functional foods, various foods, fermented milk, meat, beverages, chocolate, snacks, confectionery, pizza, ramen, other noodles, gum, ice cream, alcoholic beverages, vitamins It may be a combination, alcohol, and other health functional food, but is not limited thereto.

특히, 꾸지뽕 주정추출물을 유효성분으로 함유하는 역류성 식도염 예방 및 개선용 발효유는 꾸지뽕 주정추출물, 유산균 배양액 및 혼합과즙시럽을 일정비율로 조합하여 150bar에서 균질한 후 10℃ 이하로 냉각한 후 용기에 포장하여 발효유를 제조한다.Especially, fermented milk for preventing and improving reflux esophagitis containing Cudrania alcohol extract as an active ingredient is homogenized at 150 bar by combining Cudrania alcohol extract, lactobacillus culture solution and mixed fruit juice syrup, and then cooled to below 10 ℃ and then packed in a container To prepare fermented milk.

또한, 꾸지뽕 주정추출물을 유효성분으로 함유하는 역류성 식도염 예방 및 개선용 기능성 음료는 혼합과즙시럽, 꾸지뽕 주정추출물 및 물을 일정한 비율로 조합하여 150bar에서 균질한 후 10℃ 이하로 냉각한 후 유리병, 패트병 등 소포장 용기에 포장하여 기능성 음료를 제조한다. In addition, reflux esophagitis preventing and improving functional beverages containing courageous ethanol extract as an active ingredient is a homogeneous at 150 bar by combining a mixed juice syrup, couji pontan alcohol extract and water in a constant ratio and then cooled to below 10 ℃ glass bottles, Functional drinks are prepared by packaging in small packaging containers such as plastic bottles.

또한, 꾸지뽕 주정추출물을 유효성분으로 함유하는 역류성 식도염 예방 및 개선용 건강기능식품은 상기 꾸지뽕 주정추출물을 포함하는 것 이외에 영양보조 성분으로 비타민 B1, B2, B5, B6, E 및 초산에스테르, 니코틴산 아미드, 올리고당 등이 첨가될 수 있으며 여타의 식품 첨가물이 첨가되어도 무방하다.In addition, reflux esophagitis preventing and improving health functional food containing kkujippong alcohol extract as an active ingredient, vitamin B 1 , B 2 , B 5 , B 6 , E and acetic acid as a nutritional supplement in addition to containing the kkujippong alcohol extract Esters, nicotinic acid amides, oligosaccharides, and the like may be added and other food additives may be added.

본 발명은 위산에 의한 식도점막의 손상 및 염증 매개물질을 감소시키며, 위액의 산도를 높이는 히스타민 H2 수용체의 활성을 억제하는 효능을 갖는 꾸지뽕 주정추출물을 유효성분으로 함유하는 역류성 식도염 예방 및 개선용 발효유, 기능성 음료, 건강기능식품 등 식품조성물로 이용될 수 있다.The present invention reduces the damage and inflammation mediators of the esophageal mucosa caused by gastric acid, and reflux esophagitis preventing and improving fermented milk containing courageous alcohol extract as an active ingredient having the effect of inhibiting the activity of the histamine H2 receptor to increase the acidity of gastric juice It can be used as a food composition, such as functional beverages, health functional foods.

도 1은 본 발명의 꾸지뽕 주정추출물이 위산의 주성분인 염산에 의해 유도되는 식도세포의 사멸을 억제하는 효과를 나타낸 그래프이다.
도 2는 본 발명의 꾸지뽕 주정추출물이 위산의 주성분인 염산에 의해 유도되는 IL-6의 상대적 유전자 발현 억제 효과를 농도별로 나타낸 그래프이다.
도 3은 본 발명의 꾸지뽕 주정추출물에 의한 히스타민 H2 수용체 활성 억제 효과를 농도별로 나타낸 그래프이다.
도 4는 본 발명의 꾸지뽕 주정추출물과 꾸지뽕잎 물가용 추출물 및 농도별 에탄올 추출물의 히스타민 H2 수용체 억제활성을 비교한 그래프이다.
도 5는 본 발명의 꾸지뽕 주정추출물과 꾸지뽕잎 물가용 추출물 및 농도별 에탄올 추출물의 쿠드라잔톤(cudraxanthone) 함량을 HPLC를 이용하여 비교한 것이다.
도 6은 본 발명의 꾸지뽕 주정추출물에 높게 존재하는 쿠드라잔톤(cudraxanthone)에 의한 히스타민 H2 수용체 활성 억제 효과를 농도별로 나타낸 그래프이다.
도 7은 본 발명의 꾸지뽕 주정추출물이 역류성 식도염을 억제함을 랫트의 급성 역류성 식도염 모델에서 확인한 사진이다.
도 8은 본 발명의 꾸지뽕 주정추출물에 의한 역류성 식도염 억제 정도를 랫트의 급성 역류성 식도염 모델에서 확인한 그래프이다.
도 9는 본 발명의 꾸지뽕 주정추출물이 위액의 총 산도(기초 산 분비량)를 감소시킴을 나타낸 그래프이다.
1 is a graph showing the effect of inhibiting the death of esophageal cells induced by hydrochloric acid, the main component of stomach acid kkujippong alcohol extract of the present invention.
Figure 2 is a graph showing the relative gene expression inhibitory effect of IL-6 induced by hydrochloric acid as the main component of stomach acid kkujippong alcohol extract of the present invention by concentration.
Figure 3 is a graph showing the inhibitory effect of histamine H2 receptor activity by concentration in the kkujippong alcoholic extract of the present invention.
Figure 4 is a graph comparing the histamine H2 receptor inhibitory activity of the kkujippong alcoholic extract, kkujippong leaf water-soluble extract and ethanol extract of each concentration of the present invention.
5 is a comparison of the Cudraxanthone (cudraxanthone) content of the Kudjippong alcoholic extract of the present invention and the Kudjippang leaf water-soluble extract and ethanol extract by concentration using HPLC.
Figure 6 is a graph showing the inhibitory effect of histamine H2 receptor activity by the concentration of cudraxanthone (cudraxanthone) present in the Kudjippong alcoholic extract of the present invention.
Figure 7 is a photograph confirming in the acute reflux esophagitis model of the rat kkujippong alcohol extract of the present invention inhibits reflux esophagitis.
8 is a graph confirming the degree of inhibition of reflux esophagitis by the kkujippong ethanol extract of the present invention in a rat model of acute reflux esophagitis.
9 is a graph showing that the kkujippong alcoholic extract of the present invention reduces the total acidity (base acid secretion) of gastric juice.

이하, 실시예를 통하여 본 발명을 보다 상세하게 설명한다. 그러나, 다음의 실시예는 본 발명의 범위를 한정하는 것은 아니며, 본 발명의 기술적 사상의 범위 내에서 당업자에 의한 통상적인 변화가 가능하다.Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples do not limit the scope of the present invention, and ordinary changes by those skilled in the art can be made within the scope of the technical idea of the present invention.

<실시예 1><Example 1>

꾸지뽕 주정추출물의 제조Preparation of Cudrania Alcoholic Extract

건조된 꾸지뽕잎을 적당한 크기로 분쇄하여 추출용기에 넣은 후, 꾸지뽕잎의 10배중량에 해당하는 70% 발효주정을 추출용기에 넣어 6시간 이상 50℃에서 환류 및 교반 추출하였다. 상기 꾸지뽕 주정추출물은 20메쉬(mesh)의 1차 진동체 여과 후 2차 퍼라이트로 흡착 여과하여 불용성 불순물을 제거하였다. 상기 여과된 꾸지봉 주정추출물은 60℃에서 감압농축한 다음, 고형분을 측정하여 고형분과 동일한 중량의 말토덱스트린(Malto dextrin)을 혼합하였다. 상기 말토덱스트린을 혼합한 꾸지뽕 주정추출물은 동결건조한 후 분쇄하여 분말화 하였다.The dried Cudrania leaf was crushed to an appropriate size and placed in an extraction container, and then 70% fermented alcohol corresponding to 10 times the weight of Cudrania leaf was put in the extraction vessel and refluxed and stirred at 50 ° C. for at least 6 hours. The zigzag ethanol extract was filtered by adsorption filtration with secondary perlite after filtration of the first 20 vibrating sieves (mesh) to remove insoluble impurities. The filtered Kudjibong alcohol extract was concentrated under reduced pressure at 60 ℃, and the solid content was measured to mix malto dextrin (Malto dextrin) of the same weight as the solid content. Kudjippong alcohol extract mixed with the maltodextrin was lyophilized and then ground to powder.

<실시예 2><Example 2>

꾸지뽕 주정추출물을 유효성분으로 함유하는 발효유의 제조Preparation of Fermented Milk Containing Cudrania Extract as an Active Ingredient

유산균 배양액과 본 발명의 꾸지뽕 주정추출물 및 혼합과즙시럽으로 구성된 발효유를 제조하는 방법은 다음과 같다.Method for producing a fermented milk consisting of lactic acid bacteria culture medium, kkujippong alcoholic extract and mixed juice syrup of the present invention is as follows.

먼저, 유산균 배양액은 원유 95.36중량%와 탈지분유(또는 혼합분유) 4.6중량%를 교반하여 15℃에서의 비중은 1.0473~1.0475, 적정산도는 0.200~0.220%, pH는 6.65~6.70, 20℃에서의 브릭스(Brixo)는 16.3~16.5% 정도가 되도록 혼합하였다. 혼합 후에 이를 UHT 열처리(135℃에서 2초간 살균)하고 40℃로 냉각한 뒤, 스트렙토코커스 써모필러스균과 유당분해효소(Valley laboratory, USA)를 각기 0.02중량%씩 첨가하고 6시간 동안 배양하여 BCP 배지에서의 총 유산균수가 1.0ⅹ109cfu/㎖ 이상, 적정산도가 0.89~0.91%, pH는 4.50~4.65가 되도록 하여 제조하였다.First, the lactic acid bacteria culture medium was stirred at 95.36% by weight of crude milk and 4.6% by weight of skim milk powder (or mixed milk powder), and the specific gravity at 15 ° C was 1.0473 to 1.0475, the titratable acidity was 0.200 to 0.220%, and the pH was 6.65 to 6.70, 20 ° C. Brix o was mixed to be about 16.3 ~ 16.5%. After mixing, it was UHT heat-treated (sterilized at 135 ° C for 2 seconds) and cooled to 40 ° C. Then, Streptococcus thermophilus and Lactobacillus (Valley laboratory, USA) were each added 0.02% by weight and incubated for 6 hours to incubate BCP. Total lactic acid bacteria in the medium was 1.0 × 10 9 cfu / ㎖ or more, the titratable acidity was 0.89 ~ 0.91%, pH was prepared to 4.50 ~ 4.65.

그런 다음, 혼합과즙시럽은 액상과당 13중량%, 백설탕 5중량%, 혼합과즙농축액 56Brixo 10.9중량%, 펙틴 1.0중량%, 후레쉬 후르츠 믹스 에센스 0.1중량% 및 정제수 70중량%를 35℃에서 교반하여 혼합한 후 UHT 열처리(135℃에서 2초간 살균)한 후 냉각하여 제조하였다.Then, the mixed fruit syrup is 13% by weight of liquid fructose, 5% by weight of white sugar, 10.9% by weight of mixed juice concentrate 56Brix o , 1.0% by weight pectin, 0.1% by weight of fresh fruit mix essence and 70% by weight of purified water at 35 ℃ After mixing UHT heat treatment (sterilization for 2 seconds at 135 ℃) and then prepared by cooling.

그런 다음, 상기 유산균배양액 69.5중량%와 상기 실시예 1의 꾸지뽕 주정추출물 0.1중량% 및 상기 혼합과즙시럽 30.4중량%를 조합하여 150bar에서 균질한 후 10℃ 이하로 냉각하여 본 발명의 꾸지뽕 주정추출물을 유효성분으로 함유하는 발효유를 제조하였다.Then, 69.5% by weight of the lactic acid bacteria culture solution, 0.1% by weight of the courageous drunk extract of Example 1 and 30.4% by weight of the mixed fruit juice syrup were homogenized at 150 bar, and then cooled to 10 ° C. or lower to make the zigzag alcohol extract of the present invention. Fermented milk containing as an active ingredient was prepared.

<실시예 3><Example 3>

꾸지뽕 주정추출물을 유효성분으로 함유하는 기능성 음료의 제조Preparation of Functional Drinks Containing Cudrania Alcoholic Extract as Active Ingredient

본 발명의 꾸지뽕 주정추출물 및 혼합과즙시럽으로 구성된 기능성 음료를 제조하는 방법은 다음과 같다.Method for producing a functional drink consisting of kkujippong alcohol extract and mixed fruit juice syrup of the present invention is as follows.

먼저, 혼합과즙시럽은 액상과당 13중량%, 백설탕 2.5중량%, 갈색설탕 2.5중량%, 혼합과즙농축액 56Brixo 10.9중량%, 펙틴 1.0중량%, 후레쉬 후르츠 믹스 에센스 0.1중량% 및 정제수 70중량%를 35℃에서 교반하여 혼합한 후 UHT 열처리(135℃에서 2초간 살균)한 후 냉각하여 제조하였다.First, mixed fruit syrup is 13% by weight liquid fructose, 2.5% by weight of white sugar, 2.5% by weight of brown sugar, 56% by weight of mixed juice concentrate 56Brix o , 1.0% by weight pectin, 0.1% by weight of fresh fruit mix essence and 70% by weight of purified water The mixture was stirred and mixed at 35 ° C., followed by UHT heat treatment (sterilized at 135 ° C. for 2 seconds), followed by cooling.

그리고 상기의 방법으로 제조된 혼합과즙시럽 30.4중량%와 상기 실시예 1의 꾸지뽕 주정추출물 0.1중량% 및 나머지 정제수 69.5중량%를 조합하여 150bar에서 균질한 후 10℃ 이하로 냉각한 후 이를 유리병, 패트병 등 소포장 용기에 포장하여 본 발명의 꾸지뽕 주정추출물을 유효성분으로 함유하는 기능성 음료를 제조하였다.And 30.4% by weight of the mixed juice syrup prepared by the above method, 0.1% by weight of the courageous liquor extract of Example 1 and 69.5% by weight of the remaining purified water were homogenized at 150 bar and then cooled to 10 ° C or less, and then it was a glass bottle, It was packaged in a small packaging container such as a plastic bottle to prepare a functional beverage containing the kkujippong alcoholic extract of the present invention as an active ingredient.

<실시예 4><Example 4>

꾸지뽕 주정추출물을 유효성분으로 함유하는 건강기능식품의 제조Preparation of Health Functional Foods Containing Cudrania Alcoholic Extract as Active Ingredient

상기 실시예 1의 꾸지뽕 주정추출물 0.1중량%에 영양보조성분(비타민 B1, B2, B5, B6, E 및 초산에스테르, 니코틴산 아미드) 및 올리고당을 상기의 실시예 1의 꾸지뽕 주정추출물 100중량부에 대하여 10중량부가 되도록 첨가하여 고속회전 혼합기에서 혼합하였다. 상기 혼합물에 멸균 정제수 10중량부를 첨가, 혼합하고 직경 1~2mm의 과립상으로 성형하였다. 상기 성형된 과립은 50℃의 진공건조기에서 건조시킨 후 12메쉬(mesh)를 통과시켜 균일하게 과립을 제조하였다. 상기와 같이 제조된 과립은 적당량씩 압출 성형되어 정제 또는 분말로 되거나 경질캡슐에 충전되어 경질캡슐제품으로 제조하였다.Nutritional supplements (vitamins B 1 , B 2 , B 5 , B 6 , E and acetate esters, nicotinic acid amide) and oligosaccharides in 0.1 weight% of the Cudrania ethanol extract of Example 1 100 It was added to 10 parts by weight with respect to parts by weight and mixed in a high speed rotary mixer. 10 parts by weight of sterile purified water was added to the mixture, mixed, and molded into granules having a diameter of 1 to 2 mm. The molded granules were dried in a vacuum dryer at 50 ° C., and then passed through 12 meshes to prepare granules uniformly. The granules prepared as described above were extruded by appropriate amounts into tablets or powders or filled into hard capsules to produce hard capsule products.

<비교예 1>Comparative Example 1

꾸지뽕잎 물 가용추출물의 제조Preparation of Cudrania Leaf Water Soluble Extract

꾸지뽕잎 물 가용추출물은 대한민국 공개특허공보 제10-2013-0036939호(2013.04.15)의 실시예 1과 같이 제조하였다.Kojippong leaf water soluble extract was prepared as in Example 1 of Republic of Korea Patent Publication No. 10-2013-0036939 (2013.04.15).

즉, 꾸지뽕나무의 잎 분말 50g에 1L의 증류수를 가하여 3시간 동안 250℃에서 환류(reflux)하여 추출을 수행하고 얻어진 추출물을 여과지(ADVANTEC FILTER PAPER 6, 150 mm)로 여과하여 여과액을 회전진공농축기(Sunil EYELA, RotatoryThat is, 1L of distilled water was added to 50 g of leaf powder of Cucumis japonica, followed by extraction at reflux at 250 ° C. for 3 hours, and the obtained extract was filtered through a filter paper (ADVANTEC FILTER PAPER 6, 150 mm) to filter the filtrate by rotating vacuum. Thickener (Sunil EYELA, Rotatory

evaporator, N-1N)로 감압 농축하여 동결건조하여 꾸지뽕잎 물 가용추출물을 제조하였다.evaporator, N-1N) was concentrated under reduced pressure, and lyophilized to prepare a soluble extract of Cudrania leaf water.

<비교예 2>Comparative Example 2

꾸지뽕잎 10% 에탄올 가용추출물의 추출물의 제조Preparation of Extracts of 10% Ethanol Soluble Extract of Cudrania Leaf

꾸지뽕잎 10% 에탄올 가용추출물은 대한민국 공개특허공보 제10-2013-0036939호(2013.04.15.)의 실시예 1과 같이 제조하였다.Cudrania leaf 10% ethanol soluble extract was prepared as in Example 1 of Republic of Korea Patent Publication No. 10-2013-0036939 (2013.04.15.).

즉, 꾸지뽕나무의 잎 분말 50g에 1L의 10% 에탄올을 가하여 3시간 동안 250℃에서 환류(reflux)하여 추출을 수행하고 얻어진 추출물을 여과지(ADVANTEC FILTER PAPER 6, 150 mm)로 여과하여 여과액을 회전진공농축기(Sunil EYELA, RotatoryIn other words, 1 g of 10% ethanol was added to 50 g of leaf powder of Cucumis japonica and refluxed at 250 ° C. for 3 hours to perform extraction, and the obtained extract was filtered through filter paper (ADVANTEC FILTER PAPER 6, 150 mm). Sunil EYELA, Rotatory

evaporator, N-1N)로 감압 농축하여 동결건조하여 꾸지뽕잎 10% 에탄올 가용추출물을 제조하였다.evaporator, N-1N) was concentrated under reduced pressure and lyophilized to prepare 10% ethanol soluble extract of Cudrania leaf.

<비교예 3>Comparative Example 3

꾸지뽕잎 30% 에탄올 가용추출물의 추출물의 제조Preparation of 30% Ethanol Soluble Extract of Cudrania Leaf

꾸지뽕잎 30% 에탄올 가용추출물은 대한민국 공개특허공보 제10-2013-0036939호(2013.04.15.)의 실시예 1과 같이 제조하였다.Kojippong leaf 30% ethanol soluble extract was prepared as in Example 1 of Republic of Korea Patent Publication No. 10-2013-0036939 (2013.04.15.).

즉, 꾸지뽕나무의 잎 분말 50g에 1L의 30% 에탄올을 가하여 3시간 동안 250℃에서 환류(reflux)하여 추출을 수행하고 얻어진 추출물을 여과지(ADVANTEC FILTER PAPER 6, 150 mm)로 여과하여 여과액을 회전진공농축기(Sunil EYELA, RotatoryThat is, 1L of 30% ethanol was added to 50 g of the leaves powder of Cucumis japonica, followed by extraction by reflux at 250 ° C. for 3 hours, and the obtained extract was filtered through filter paper (ADVANTEC FILTER PAPER 6, 150 mm). Sunil EYELA, Rotatory

evaporator, N-1N)로 감압 농축하여 동결건조하여 꾸지뽕잎 30% 에탄올 가용추출물을 제조하였다.evaporator, N-1N) was concentrated under reduced pressure and lyophilized to prepare 30% ethanol soluble extract of Cudrania leaf.

<시험예 1><Test Example 1>

본 발명의 꾸지뽕 주정추출물의 산에 의한 식도세포 손상 억제활성 측정Determination of Esophageal Cell Damage Inhibitory Activity by Acids of Cudrania ethanol Extract of the Present Invention

염산은 위산의 주성분으로 역류성 식도염은 위산 역류에 의한 식도세포의 손상에 따른 염증반응에 의해 발생한다. 따라서, 상기 실시예 1의 꾸지뽕 주정추출물이 염산에 의한 식도세포 손상을 억제하는지에 관한 시험관 시험을 실시하였다.Hydrochloric acid is the main component of gastric acid, and reflux esophagitis is caused by inflammatory reactions caused by gastric acid reflux. Therefore, an in vitro test was carried out to determine whether the Cudrania ethanol extract of Example 1 inhibits esophageal cell damage caused by hydrochloric acid.

먼저, 인간 식도 세포 Het-1a는 ATCC(American Type Culture Collection)에서 구입하여 BEGM™ 배지(Lonza)를 사용하여 37℃, 5% CO2 배양기에서 배양하였다. 상기 배양된 인간 식도 세포인 Het-1a 세포를 96 웰 플레이트(well plate)에 2×104cells/well로 분주하여 24시간 배양한 후, 상기 실시예 1의 꾸지뽕 주정추출물 10μg/mL와 염산을 최종농도가 각각 0mM, 5mM, 10mM, 15mM. 20mM이 되도록 처리하였다. 그런다음, 상기 염산처리 6시간 후, MTT 용액(SIGMA, M5655)을 넣고 1시간 더 배양한 다음 생성된 포르마잔(formazan)을 디메틸술폭시드(dimethyl sulfoxide; DMSO)를 첨가하여 용해시킨 후, 540nm에서 흡광도를 측정하여 세포생존율을 측정하였다.First, human esophageal cells Het-1a were purchased from the American Type Culture Collection (ATCC) and cultured in a 37 ° C., 5% CO 2 incubator using BEGM ™ medium (Lonza). Het-1a cells, the cultured human esophageal cells, were plated at 2 × 10 4 cells / well in a 96 well plate and cultured for 24 hours. After that, 10 μg / mL of Cudrania ethanol extract of Example 1 and hydrochloric acid were used. Final concentrations of 0mM, 5mM, 10mM and 15mM respectively. Treated to 20 mM. Then, after 6 hours of hydrochloric acid treatment, MTT solution (SIGMA, M5655) was added and incubated for 1 hour, and the resulting formazan was dissolved by adding dimethyl sulfoxide (DMSO), followed by 540 nm. Absorbance was measured at and cell viability was measured.

한편, 대조군에는 상기 실시예 1의 꾸지뽕 주정추출물 대신에 동량의 PBS(phosphate buffered saline)을 처리한 것 외에는 상기와 동일한 방법으로 실험하였다.On the other hand, the control group was tested in the same manner as described above except that treated with the same amount of PBS (phosphate buffered saline) in place of the kkujippong alcoholic extract of Example 1.

그 결과를 도 1에 나타내었다.The results are shown in FIG.

도 1에서 확인할 수 있는 바와 같이, 본 발명의 꾸지뽕잎 주정추출물을 처리하지 않은 대조군에서는 염산의 처리농도에 따라 최대 약 60%의 세포사멸이 이루어진 반면, 본 발명의 꾸지뽕 주정추출물을 처리하면 산에 의한 식도세포의 사멸이 크게 줄어드는 것을 확인할 수 있었다.As can be seen in Figure 1, the control group was not treated with kkujippong liquor extract of the present invention, while a maximum of about 60% cell death was made according to the concentration of hydrochloric acid, while treating the kkujippong ethanol extract of the present invention to the acid It was confirmed that the death of esophageal cells is greatly reduced.

<시험예 2><Test Example 2>

본 발명의 꾸지뽕 주정추출물의 산에 의한 식도세포 염증반응 억제활성 측정Determination of Inhibitory Activity of Esophageal Cell Inflammatory Response by Acid of Cudrania Extract Extract of the Present Invention

산에 의한 식도세포의 염증반응에는 염증성 싸이토카인인 IL-6가 중요한 인자로 보고되어 있다(Am J Physiol Gastrointest Liver Physiol. 2009 296: G388??G398. Effect of curcumin on acidic pH-induced expression of IL-6 and IL-8 in human esophageal epithelial cells (HET-1A): role of PKC, MAPKs, and NF-κB. Parvaneh Rafiee, Victoria M. Nelson.). 따라서, 상기 실시예 1의 꾸지뽕 주정추출물이 산에 의해 증가하는 IL-6를 억제하여 식도세포 염증반응을 억제하는지에 관한 시험관 시험을 실시하였다. An inflammatory cytokine, IL-6, has been reported as an important factor in acid-induced inflammatory responses (Am J Physiol Gastrointest Liver Physiol. 2009 296: G388 ?? G398. Effect of curcumin on acidic pH-induced expression of IL-). 6 and IL-8 in human esophageal epithelial cells (HET-1A): role of PKC, MAPKs, and NF-κB. Parvaneh Rafiee, Victoria M. Nelson.). Therefore, an in vitro test was performed to determine whether the Cudrania ethanol extract of Example 1 inhibits esophageal inflammatory response by inhibiting IL-6 increased by acid.

인간 식도 세포인 Het-1a 세포를 96 웰 플레이트(well plate)에 2×104cells/well로 분주하여 24시간 배양한 후, 상기 실시예 1의 꾸지뽕 주정추출물을 최종농도가 각각 0㎍/㎖, 2.5㎍/㎖, 5㎍/㎖, 10㎍/㎖. 15㎍/㎖이 되도록 처리하였다. 상기 실시예 1의 꾸지뽕 주정추출물의 처리 1시간 후 배지를 제거하고 염산을 첨가하여 배지의 pH를 7.2에서 4.5로 낮춘 배지로 교환하였다. pH4.5 배지 처리 1시간 후 다시 배지를 제거하고 pH7.2의 일반배지로 교환한 다음 6시간 동안 37℃, 5% CO2 배양기에서 세포를 배양하였다. 배양 6시간 후 세포를 PBS(phosphate buffered saline)로 3회 세척한 다음, trizol reagent(sigma)를 각 well에 처리하여 total RNA를 분리하였다. 분리된 total RNA는 정량 후 동량을 취하여 High Capacity cDNA Kit(thermofisher)을 이용하여 cDNA를 합성한 다음, SYBRㄾ Premix Ex Taq™(Takara) 및 IL-6 primer(Hs00174131_m1, thermofisher)를 사용하여 qPCR을 시행하여 IL-6의 상대적 발현량을 측정하였다(ABI PRISM 7500 Sequence Detection System).Het-1a cells, which are human esophageal cells, were cultured in a 96 well plate at 2 × 10 4 cells / well and incubated for 24 hours, and then the final concentration of Kudjippong alcoholic extract of Example 1 was 0 μg / ml, respectively. , 2.5 μg / ml, 5 μg / ml, 10 μg / ml. Treated to 15 μg / ml. After 1 hour of treatment with the Kudjippong alcoholic extract of Example 1, the medium was removed, and hydrochloric acid was added to replace the medium with a pH lowered from 7.2 to 4.5. After 1 hour of pH4.5 medium treatment, the medium was removed again, exchanged with a medium of pH 7.2, and the cells were cultured in a 37 ° C., 5% CO 2 incubator for 6 hours. After 6 hours of incubation, the cells were washed three times with PBS (phosphate buffered saline), and then trizol reagent (sigma) was treated to each well to separate total RNA. Total RNA isolated was quantified and synthesized cDNA using High Capacity cDNA Kit (thermofisher), and then qPCR using SYBR ㄾ Premix Ex Taq ™ (Takara) and IL-6 primer (Hs00174131_m1, thermofisher) The relative expression level of IL-6 was measured (ABI PRISM 7500 Sequence Detection System).

한편, 대조군은 pH4.5 배지를 처리하지 않고, 상기 실시예 1의 꾸지뽕 주정추출물을 처리하지 않은 것을 제외하고는 상기와 동일한 방법으로 실험하였다.On the other hand, the control group was not treated with pH4.5 medium, except that the treated jjippong alcohol extract of Example 1 was tested in the same manner as described above.

그 결과를 도 2에 나타내었다.The results are shown in FIG.

도 2에서 확인할 수 있는 바와 같이, 본 발명의 꾸지뽕 주정추출물을 처리하지 않은 경우에는 염산에 의해 IL-6의 발현이 크게 증가한 반면, 본 발명의 꾸지뽕 주정추출물을 처리한 경우에는 염산에 의한 IL-6의 발현이 본 발명의 꾸지뽕 주정추출물의 농도에 의존적으로 줄어든 것을 확인할 수 있었다. 이는 위산이 역류하여 식도세포를 자극했을 때 본 발명의 꾸지뽕 주정추출물이 위산에 의한 염증반응을 유전자 단계에서 억제할 수 있음을 나타낸다.As can be seen in Figure 2, the expression of IL-6 was significantly increased by hydrochloric acid when not treated with kkujippong ethanol extract of the present invention, while the treated kkujippong ethanol extract of IL- by hydrochloric acid It was confirmed that the expression of 6 is reduced depending on the concentration of kkujippong alcoholic extract of the present invention. This indicates that when acidic acid reflux stimulates esophageal cells, the courageous liquor extract of the present invention can inhibit the inflammatory response caused by gastric acid at the genetic stage.

<시험예 3><Test Example 3>

본 발명의 꾸지뽕 주정추출물의 히스타민 H2 수용체 억제활성 측정Measurement of the Histamine H2 Receptor Inhibitory Activity of Cudrania Alcohol Extract of the Present Invention

히스타민 H2 수용체의 활성을 억제하는 것은 역류성 식도염의 치료 및 바렛식도 예방에 밀접한 관계가 있으므로 상기 실시예 1의 꾸지뽕 주정추출물이 위액의 산도를 증가시키는 히스타민 H2 수용체(Histamine H2 receptor)의 활성을 억제하는지에 관한 시험을 실시하였다.Inhibiting the activity of the histamine H2 receptor is closely related to the treatment of reflux esophagitis and the prevention of Barrett's esophagus. Thus, if the Kudjippong ethanol extract of Example 1 inhibits the activity of the histamine H2 receptor that increases the acidity of gastric juice. Tests were conducted.

먼저, RPMI-1640 배지에 U937세포를 배양 후 원심분리하여 10μM RO-201723이 포함된 RPMI-1640 배지에 현탁하였다. 상기 세포 현탁액에 상기 실시예 1의 꾸지뽕 주정추출물을 최종농도 0㎍/㎖, 10㎍/㎖, 20㎍/㎖, 30㎍/㎖이 되도록 5분간 처리한 후 히스타민 H2 수용체 특이적 작용제(agonist)인 dimaprit 10μM을 처리하여 20분간 배양하였다. dimaprit 처리 20분 후 원심분리하여 회수한 U937세포에 cAMP assay cell lysis buffer를 처리한 다음, 곧바로 모든 세포를 동결하였다. 동결한 세포는 3번의 동결 및 해동을 반복하여 파쇄한 후 25,000rpm에서 10분간 원심분리 후 상등액을 취하여 cAMP 생성량을 측정하였다. cAMP의 측정은 R&D systems의 cAMP assay kit를 사용하였다.First, U937 cells were cultured in RPMI-1640 medium and centrifuged and suspended in RPMI-1640 medium containing 10 μM RO-201723. The cell suspension was treated for 5 minutes to the final concentration of the Kudjippong alcoholic extract of Example 1 to a final concentration of 0 ㎍ / ㎖, 10 ㎍ / ㎖, 20 ㎍ / ㎖, 30 ㎍ / ㎖ after histamine H2 receptor specific agonist (agonist) Dimaprit was treated with 10μM and incubated for 20 minutes. After 20 minutes of dimaprit treatment, U937 cells recovered by centrifugation were treated with cAMP assay cell lysis buffer, and all cells were immediately frozen. The frozen cells were crushed repeatedly by freezing and thawing three times, followed by centrifugation at 25,000 rpm for 10 minutes, and the supernatant was taken to measure cAMP production. cAMP was measured using the R & D systems cAMP assay kit.

한편, 대조군은 상기 실시예 1의 꾸지뽕 주정추출물과 dimaprit를 처리하지 않은 것을 제외하고는 상기와 동일한 방법으로 실험하였다.On the other hand, the control group was tested in the same manner as above, except that the jjippong alcohol extract of Example 1 and dimaprit were not treated.

그 결과를 도 3에 나타내었다.The results are shown in FIG.

도 3에서 확인할 수 있는 바와 같이, 본 발명의 꾸지뽕 주정추출물은 10~30㎍/㎖의 매우 낮은 농도에서도 히스타민 H2 수용체의 활성을 효과적으로 억제하는 것으로 나타났다.As can be seen in Figure 3, kkujippong alcoholic extract of the present invention was shown to effectively inhibit the activity of the histamine H2 receptor even at a very low concentration of 10 ~ 30㎍ / ㎖.

<시험예 4><Test Example 4>

본 발명의 꾸지뽕 주정추출물과 용매별 꾸지뽕잎 가용추출물의 히스타민 H2 수용체 억제활성 비교Comparison of Histamine-H2 Receptor Inhibitory Activity of Kojippong Alcoholic Extracts and Soluble Extracts of Kojippong Leaf by Solvents

상기 실시예 1의 꾸지뽕 주정추출물과 비교예 1 내지 3의 꾸지뽕잎 가용추출물의 히스타민 H2 수용체 억제활성을 비교하는 실험을 실시하였다.The experiment was performed to compare the histamine H2 receptor inhibitory activity of the Kudjippong alcohol extract of Example 1 and the Kudjippong soluble extract of Comparative Examples 1 to 3.

즉, RPMI-1640 배지에 U937세포를 배양 후 원심분리하여 10μM RO-201723이 포함된 RPMI-1640 배지에 현탁하였다. 상기 세포 현탁액에 상기 실시예 1의 꾸지뽕 주정추출물, 상기 비교예 1 내지 3의 꾸지뽕잎 가용추출물, 히스타민 H2 수용체 억제제(antagonist)인 라니티딘(ranitidine)을 각각 최종농도 10μg/㎖ 되도록 5분간 처리한 후 히스타민 H2 수용체 특이적 작용제(agonist)인 dimaprit 10μM을 처리하여 20분간 배양하였다. dimaprit 처리 20분 후 원심분리하여 회수한 U937세포에 cAMP assay cell lysis buffer를 처리한 다음, 곧바로 모든 세포를 동결하였다. 동결한 세포는 3번의 동결 및 해동을 반복하여 파쇄한 후 25,000rpm에서 10분간 원심분리 후 상등액을 취하여 cAMP 생성량을 측정하였다. cAMP의 측정은 R&D systems의 cAMP assay kit를 사용하였다.That is, the U937 cells were cultured in RPMI-1640 medium and centrifuged and suspended in RPMI-1640 medium containing 10 μM RO-201723. After treating the cell suspension for 5 minutes to the final concentration of 10 μg / ㎖ each of the courageous ethanol extract of Example 1, courageous leaf soluble extract of Comparative Examples 1 to 3, the histamine H2 receptor inhibitor (antagonist) ranitidine (ranitidine) respectively The histamine H2 receptor specific agonist dimaprit 10μM was treated for 20 minutes. After 20 minutes of dimaprit treatment, U937 cells recovered by centrifugation were treated with cAMP assay cell lysis buffer, and all cells were immediately frozen. The frozen cells were crushed repeatedly by freezing and thawing three times, followed by centrifugation at 25,000 rpm for 10 minutes, and the supernatant was taken to measure cAMP production. cAMP was measured using the R & D systems cAMP assay kit.

한편, 대조군은 상기 실시예 1의 꾸지뽕 주정추출물과 비교예 1 내지 3의 꾸지뽕잎 가용추출물 및 dimaprit를 처리하지 않은 것을 제외하고는 상기와 동일한 방법으로 실험하였다.On the other hand, the control group was tested in the same manner as in the above except that the jjippong drunk extract of Example 1 and the cooji mulberry leaves soluble extract and dimaprit of Comparative Examples 1 to 3.

그 결과를 도 4에 나타내었다.The results are shown in FIG.

도 4에서 확인할 수 있는 바와 같이, 실시예 1의 본 발명의 꾸지뽕 주정추출물은 10μg/㎖에서 히스타민 H2 수용체 억제제인 라니티딘(ranitidine)과 유사하게 매우 높은 히스타민 H2 수용체 억제활성을 갖는 반면, 비교예 1 내지 3의 꾸지뽕잎 가용추출물은 히스타민 H2 수용체 억제활성이 매우 낮은 것을 확인할 수 있었다. 특히, 비교예 1의 꾸지뽕잎 물 가용추출물과 비교예 2의 꾸지뽕잎 10% 에탄올 가용추출물은 히스타민 H2 수용체의 억제활성이 거의 없는 것으로 나타났다.As can be seen in Figure 4, the kkujippong alcohol extract of the present invention of Example 1 has a very high histamine H2 receptor inhibitory activity, similar to the histamine H2 receptor inhibitor ranitidine at 10μg / ㎖, Comparative Example 1 Soluble extract of Cudrania leaf to 3 was confirmed that the histamine H2 receptor inhibitory activity is very low. In particular, the soluble extract of Cudrania leaf water in Comparative Example 1 and the 10% ethanol-soluble extract of Cudrania leaf in Comparative Example 2 were found to have little inhibitory activity of the histamine H2 receptor.

<시험예 5><Test Example 5>

본 발명의 꾸지뽕 주정추출물과 용매별 꾸지뽕잎 가용추출물의 쿠드라잔톤 (cudraxanthone) 함량 분석Analysis of the Cudraxanthone Content of the Cudrania Liquor Soluble Extract of Cudrania Alcohol Extract and Solvents of the Present Invention

상기 실시예 1의 꾸지뽕 주정추출물과 상기 비교예 1 내지 3의 꾸지뽕잎 가용추출물의 쿠드라잔톤(cudraxanthone) 함량을 HPLC 분석법을 통하여 분석하였다.The Cudraxanthone content of the Cudrania ethanol extract of Example 1 and the Cudrania leaf soluble extract of Comparative Examples 1 to 3 were analyzed by HPLC analysis.

즉, 꾸지뽕 근피 및 수피에는 6종의 잔톤(xanthone)계 화합물인 쿠드라잔톤(cudraxanthone)이 존재하는 것으로 알려져 있다. 쿠드라잔톤(cudraxanthone)의 HPLC분석은 Supelco Discovery C18(5μm, 250×4.6mm, CA, USA) 칼럼을 사용하였으며, 칼럼 온도는 35℃로 설정하였다. 이동상으로는 멸균정제수에 희석한 0.05% 인산 용액(A)과 100% 아세토니트릴(B)을 사용하여 1.0ml/min의 유속으로 시료 5㎕를 주입하여 Gradient condition으로 분석하였다. 표준화합물은 ChemFaces (#CFN99422)에서 구매하였으며 276nm에서 피크가 확인되었다. 상기 실시예 1의 꾸지뽕 주정추출물과 상기 비교예 1 내지 3의 꾸지뽕잎 가용추출물에 포함된 쿠드라잔톤(cudraxanthone)의 함량은 표준화합물의 검정선으로부터 함유량을 계산하였다. 예상 retention time(RT)은 13분이었으며 실제 retention time은 13.13분으로 확인되었다.That is, it is known that cudraxanthone, which is a six kinds of xanthone-based compounds, exists in the zigzag root and bark. HPLC analysis of cudraxanthone was performed using a Supelco Discovery C18 (5 μm, 250 × 4.6 mm, CA, USA) column, and the column temperature was set to 35 ° C. As a mobile phase, 5 µl of the sample was injected at a flow rate of 1.0 ml / min using 0.05% phosphoric acid solution (A) and 100% acetonitrile (B) diluted in sterile purified water and analyzed under Gradient conditions. The standard compound was purchased from ChemFaces (# CFN99422) and peaked at 276 nm. The content of the Cudraxanthone contained in the Cudrania ethanol extract of Example 1 and the Cudrania leaf soluble extract of Comparative Examples 1 to 3 was calculated from the calibration curve of the standard compound. The expected retention time (RT) was 13 minutes and the actual retention time was found to be 13.13 minutes.

그 결과를 도 5에 나타내었다.The results are shown in FIG.

도 5에서 확인할 수 있는 바와 같이, 실시예 1의 본 발명의 꾸지뽕 주정추출물에는 쿠드라잔톤(cudraxanthone)이 25mg/g으로 매우 높게 존재하는 반면, 비교예 1 내지 3의 꾸지뽕잎 가용추출물에는 쿠드라잔톤(cudraxanthone)이 0.09mg/g, 0.12mg/g, 0.14mg/g으로 매우 낮게 존재하는 것을 확인할 수 있었다. 이는 쿠드라잔톤(cudraxanthone)이 본 발명의 꾸지뽕 주정추출물에 특이적으로 존재하는 화합물로 높은 쿠드라잔톤 함량이 본 발명의 꾸지뽕 주정추출물을 특정 짓는 지표성분으로 사용될 수 있음을 뜻한다.As can be seen in Figure 5, the Cudraxanthone extract of the present invention of Example 1 in the Kudraxanthone (cudraxanthone) is present very high as 25mg / g, whereas in the Cudrania leaves soluble extract of Comparative Examples 1 to 3 Kudra Xanthone (cudraxanthone) was found to be very low as 0.09mg / g, 0.12mg / g, 0.14mg / g. This means that cudraxanthone is a compound specifically present in the courageous drunk extract of the present invention, which means that a high kudraxanthone content can be used as an index component for specifying the courageous drunk extract of the present invention.

<시험예 6><Test Example 6>

쿠드라잔톤 (cudraxanthone)의 히스톤 H2 수용체 억제활성Histone H2 Receptor Inhibitory Activity of Cudraxanthone

상기 시험예 5에서와 같이 본 발명의 꾸지뽕 주정추출물에 특이적으로 높게 존재하는 쿠드라잔톤(cudraxanthone)이 히스톤 H2 수용체 억제활성이 있는지를 측정하였다.As in Test Example 5, it was determined whether the cudraxanthone present in the courageous drunk extract of the present invention has a high histone H2 receptor inhibitory activity.

(1)RPMI-1640 배지에 U937세포를 배양 후 원심분리하여 10μM RO-201723이 포함된 RPMI-1640 배지에 현탁하였다. 상기 세포 현탁액에 상기 실시예 1의 꾸지뽕 주정추출물 10μg/㎖를 5분간 처리한 후 히스타민 H2 수용체 특이적 작용제(agonist)인 dimaprit 10μM을 처리하여 20분간 배양 하였다. dimaprit 처리 20분 후 원심분리하여 회수한 U937세포에 cAMP assay cell lysis buffer를 처리한 다음, 곧바로 모든 세포를 동결하였다. 동결한 세포는 3번의 동결 및 해동을 반복하여 파쇄한 후 25,000rpm에서 10분간 원심분리 후 상등액을 취하여 cAMP 생성량을 측정하였다. cAMP의 측정은 R&D systems의 cAMP assay kit를 사용하였다.(1) U937 cells were cultured in RPMI-1640 medium and centrifuged and suspended in RPMI-1640 medium containing 10 μM RO-201723. The cell suspension was treated with 10 μg / ml of courageous drunk extract of Example 1 for 5 minutes, and then incubated for 20 minutes by treating 10 μM of dimaprit, which is a histamine H2 receptor-specific agonist. After 20 minutes of dimaprit treatment, U937 cells recovered by centrifugation were treated with cAMP assay cell lysis buffer, and all cells were immediately frozen. The frozen cells were crushed repeatedly by freezing and thawing three times, followed by centrifugation at 25,000 rpm for 10 minutes, and the supernatant was taken to measure cAMP production. cAMP was measured using the R & D systems cAMP assay kit.

(2)상기 실시예 1의 꾸지뽕 주정추출물 대신에 쿠드라잔톤(cudraxanthone)은 ChemFaces(#CFN99422)의 표준화합물을 DMSO에 희석하여 0.06μg/mL, 0.12μg/mL, 0.25μg/mL, 0.5μg/mL로 처리한 것을 제외하고는 상기 (1)과 동일한 방법으로 실험하였다.(2) Cudraxanthone (cudraxanthone) in place of the Cudrania ethanol extract of Example 1 was diluted by diluting the standard compound of ChemFaces (# CFN99422) in DMSO 0.06μg / mL, 0.12μg / mL, 0.25μg / mL, 0.5μg The experiment was conducted in the same manner as in (1), except that the treatment was performed at / mL.

(3)상기 실시예 1의 꾸지뽕 주정추출물을 처리하지 않은 것을 제외하고 상기 (1)과 동일한 방법으로 실험하였다.(3) The experiment was carried out in the same manner as in (1), except that the jjippong drunk extract of Example 1 was not treated.

(4)상기 실시예 1의 꾸지뽕 주정추출물, dimaprit, 쿠드라잔톤(cudraxanthone)을 처리하지 않은 것을 제외하고는 상기 (1)과 동일한 방법으로 실험하였다.(4) The experiment was carried out in the same manner as in (1), except that the jjippong drunk extract of Example 1, dimaprit, and cudraxanthone were not treated.

그 결과를 도 6에 나타내었다.The results are shown in FIG.

도 6에서 확인할 수 있는 바와 같이, 실시예 1의 본 발명의 꾸지뽕 주정추출물에 높게 존재하는 쿠드라잔톤(cudraxanthone)은 농도 의존적으로 히스톤 H2 수용체의 활성을 억제하는 것으로 나타났다.As can be seen in Figure 6, it was found that the high concentration of cudraxanthone (cudraxanthone) present in the courageous ethanol extract of the present invention of Example 1 inhibits the activity of histone H2 receptor in a concentration-dependent manner.

<시험예 7><Test Example 7>

본 발명의 꾸지뽕 주정추출물의 역류성 식도염 치료 효과Treatment Effect of Reflux Esophagitis of Cudrania Alcohol Extract of the Present Invention

상기 실시예 1의 꾸지뽕 주정추출물이 실제 역류성 식도염을 억제할 수 있는지에 대한 동물효능평가를 하였다.Animal efficacy evaluation was performed to determine whether the Kudjippong alcoholic extract of Example 1 can actually suppress reflux esophagitis.

즉, 6주령 스프라그 도울리(Spraugue-Dawley: SD)계 수컷 흰쥐를 24시간 동안 절식시키고, 물은 충분한 양을 공급하였다. 체중을 측정한 다음 상기 실시예 1의 꾸지뽕 주정추출물 20㎎/㎏과 50㎎/㎏을 각각 0.5% CMC(carboxymethyl cellulose) 용액에 현탁하여 역류를 유도하기 1시간 전에 경구 투여하였다.That is, 6-week-old Spraugue-Dawley (SD) male rats were fasted for 24 hours, and water was supplied in a sufficient amount. After measuring the body weight, 20 mg / kg and 50 mg / kg of Kojippong ethanol extract of Example 1 were suspended orally in 0.5% CMC (carboxymethyl cellulose) solution, respectively, 1 hour before induction of reflux.

상기 실시예 1의 꾸지뽕 주정추출물 투여 1시간 후 틸레타민(tiletamine)/졸라제팜(zolazepam)(10mg/kg; Zoletil 50, Virbac Laboratories, France)과 2% 자일라진 하이드로클로라아드(xylazine hydrochloride)(2mg/kg, Rumpun, Byely Co., Korea)를 복강 주사하여 랫드를 마취하였다. 마취된 랫드의 복부의 털을 깍고 포비돈으로 소독한 다음 4~5cm 정도로 중앙선을 따라 절개하여 개복을 실시하였다. 유문부위를 silk(3-0, B.Braun Surgical S.A., Spain)을 이용하여 결찰하였고, 순차적으로 전위와 분문 사이의 limiting ridge부분을 동일한 방법으로 결찰하였다. 유문 및 전위부 결찰 직후 복벽은 3/0 흡수성 봉합사(3/0 Surgisorb, Samyang, Seoul, Korea)로 폐쇄하였고, 피부는 silk(3-0, B.Braun Surgical S.A., Spain)을 이용하여 결찰하였다. 위의 방법은 위의 용적을 줄여 자연스러운 위액의 역류를 유도하는 수술적 역류성 식도염 유도 방법으로 염산, 알코올 및 소염제 등 자극성 약물을 사용하는 방법 보다 실제에 가까운 역류성 식도염을 유발할 수 있는 방법이다.1 hour after administration of the Kudjippong alcoholic extract of Example 1, tiletamine / zolazepam (10 mg / kg; Zoletil 50, Virbac Laboratories, France) and 2% xylazine hydrochloride Rats were anesthetized by intraperitoneal injection (2 mg / kg, Rumpun, Byely Co., Korea). The anesthetized rat's abdomen was shaved, sterilized with povidone, and incision was performed along the midline about 4-5 cm to open. The pyloric region was ligated using silk (3-0, B.Braun Surgical S.A., Spain), and the limiting ridge between the dislocation and the branch was ligated in the same manner. Immediately after the pyloric and dislocation ligation, the abdominal wall was closed with 3/0 absorbable suture (3/0 Surgisorb, Samyang, Seoul, Korea), and the skin was ligation using silk (3-0, B.Braun Surgical SA, Spain). . The above method is a surgical reflux esophagitis method that induces natural gastric reflux by reducing the volume of the stomach, and is a method that can cause more reflux esophagitis than a method of using irritant drugs such as hydrochloric acid, alcohol, and anti-inflammatory agents.

유문 및 전위부 결찰 수술 8시간 후, 모든 동물은 CO2 가스로 계획 치사한 후 개복하여 위와 식도를 적출한 후 위의 대만부와 식도를 수술용 가위를 이용하여 세로로 절단한 뒤 PBS(phosphate buffer saline)로 위내부와 식도주위 혈액을 세척하였다. 절개된 위(胃)와 식도는 깨끗한 종이 위에 전개하였으며 지정된 장소에서 200~300lux 실광 하에 디지털 카메라(Nikon, Coolpix P5100, Japan)로 촬영을 실시하였다. 촬영시 개체 표식과 scale bar(mm 단위)를 두고 카메라와 검체와의 거리는 동일하게 하며 촬영을 실시하였다. 얻어진 이미지는 Image J program(Wayne Rasband, National Institutes of Health, Bethesada, MD, USA)을 이용하여 식도점막상에 발생된 손상의 면적(mm3)을 측정하고 식도 전체의 면적(mm3)대비 손상면적에 대한 상대비율(%)을 산출하였다.After 8 hours of pyloric and dislocation ligation surgery, all animals were planed to death with CO 2 gas, and then opened and removed, and the stomach and esophagus were cut vertically with surgical scissors, followed by PBS (phosphate buffer). saline) to wash the blood in the stomach and the surrounding esophagus. The incised stomach and esophagus were developed on clean paper and photographed with a digital camera (Nikon, Coolpix P5100, Japan) at 200-300 lux room light at the designated place. Photographing was performed with the object marker and scale bar (mm units) at the same distance from the camera and the specimen. The obtained image was measured using the Image J program (Wayne Rasband, National Institutes of Health, Bethesada, MD, USA) to measure the area of injury (mm 3 ) on the esophageal mucosa and to compare the area of the entire esophagus (mm 3 ). The relative ratio (%) to area was calculated.

모든 시험 결과는 평균치±표준오차로 표시하고 SPSS(version 20, IBM SPSS Statistics, USA)를 사용하여 검정하였다.All test results were expressed as mean ± standard error and tested using SPSS (version 20, IBM SPSS Statistics, USA).

모든 자료들에 대해 분산의 동질성을 비교하기 위한 Levene's test를 실시하며, 분산이 동질성을 갖는 경우 one-way analysis of variance(ANOVA)를 실시하여 유의성을 분석하였다.Levene's test was performed to compare the homogeneity of variances for all data, and if variances were homogeneous, one-way analysis of variance (ANOVA) was performed to analyze the significance.

음성대조군에는 0.5% CMC(carboxymethyl cellulose)만을 투여한 것을 제외하고는 상기 실험방법과 동일하게 실험하였다.The negative control group was tested in the same manner as the test method except that only 0.5% CMC (carboxymethyl cellulose) was administered.

양성대조군에는 상기 실시예 1의 꾸지뽕 주정추출물 대신에 라니티딘(ranitidine; Sigma-Aldrich) 7.7㎎/㎏을 투여한 것을 제외하고는 상기 실험방법과 동일하게 실험하였다.The positive control group was tested in the same manner as in the above experimental method, except that 7.7 mg / kg of ranitidine (ranitidine; Sigma-Aldrich) was used instead of the Cudrania alcohol extract of Example 1.

그 결과를 도 7 및 도 8에 나타내었다.The results are shown in FIGS. 7 and 8.

도 7 및 도 8에서 확인할 수 있는 바와 같이, 부형제인 CMC(carboxymethyl cellulose)만을 경구투여한 음성대조군은 식도 점막층이 위산에 의해 대부분 손상을 받아 심한 발적과 충혈이 발생되었으며 식도점막의 손상부위 비율이 57.72±14.59%로 가장 높은 수치로 확인되었다. 이에 비하여 라니티딘(ranitidine) 7.7mg/kg을 경구투여한 양성대조군은 29.90±17.82%로 유의성 있게 식도 손상이 감소되었으며(p<0.01), 실험군인 본 발명의 꾸지뽕 주정추출물을 20mg/kg 경구투여한 경우에는 식도 손상비율이 26.58±22.32%, 본 발명의 꾸지뽕 주정추출물을 50mg/kg 경구투여한 경우에는 25.18±21.40%로 양성 대조군과 비교하여 유의성 있게 식도 손상 정도가 감소됨이 확인되었다(p<0.01).As can be seen in Figures 7 and 8, the negative control group orally administered only the excipient CMC (carboxymethyl cellulose), the esophageal mucosa is most damaged by gastric acid caused severe redness and hyperemia and the rate of damage to the esophageal mucosa The highest value was 57.72 ± 14.59%. In contrast, the positive control group that received oral ranitidine 7.7mg / kg significantly reduced the esophageal damage to 29.90 ± 17.82% ( p <0.01), and 20mg / kg oral administration of the zigzag alcohol extract of the present invention as an experimental group. In the case of esophageal damage ratio of 26.58 ± 22.32%, 50 mg / kg oral administration of courageous extract of the present invention was 25.18 ± 21.40% significantly reduced the degree of esophageal damage compared to the positive control ( p <0.01 ).

이는 본 발명의 꾸지뽕 주정추출물이 실제 임상에서 역류성 식도염 치료제로 사용되고 있는 약물인 라니티딘(ranitidine)과 동등한 역류성 식도염 치료효과가 있다는 것으로서 천연물 추출물로서는 매우 우수한 역류성 식도염 치료 효과가 있음을 시사한다.This suggests that kkujippong alcoholic extract of the present invention is effective in treating reflux esophagitis equivalent to ranitidine, a drug that is used as a reflux esophagitis treatment in actual clinical practice, and thus has very good reflux treatment effect as a natural extract.

<시험예 8><Test Example 8>

본 발명의 꾸지뽕 주정추출물의 위액 산도 조절 효과Gastric acidity control effect of Kudjippong alcoholic extract of the present invention

역류된 위액에 의한 식도점막의 자극은 식도세포의 변형을 일으켜 식도세포를 암 발생률이 높은 바렛식도로 변형시킬 뿐만 아니라 역류성 식도염의 약화 및 제발에 영향을 준다. 또한 역류성 식도염이 있는 상황에서 강한 산성을 지니는 위산이 역류하게 되면 심한 가슴쓰림(흉통)을 유발한다. 이에 위액의 총 산도를 낮추는 것은 역류성 식도염으로 인한 고통을 줄이는 데에도 매우 중요하다. 이에 본 발명의 꾸지뽕 주정추출물이 위액의 총 산도를 낮추는지 알아보기 위하여 SD계열 5주령 랫트를 1주일 간 순치 후 시험물질 투여 전 약 24시간 정도 절식 시켰다. 시험물질 투여 당일날 오전에 시험물인 상기 실시예 1의 꾸지뽕 주정추출물 5mg/kg, 10mg/kg, 15mg/kg, 20mg/kg을 각각 경구투여하고 1시간 후에 동물을 아이소플루레인(isoflurane)으로 마취한 다음, 유문부를 결찰하였다. 유문부 결찰 8시간 후에 각 군당 각각 2마리씩 CO2 가스로 계획 치사한 후 위를 유분부와 분문부를 각각 결찰하여 적출하였다. 적출한 후 위액 10㎖를 주사기를 이용하여 수집하였다. 수집한 위액은 3,000rpm에서 10분간 원심분리 후 상등액만 취하여 위액량(㎖), pH 및 위산도를 측정하였다.Stimulation of the esophageal mucosa by refluxed gastric juice causes the esophageal cell to deform, transforming the esophageal cell into Barrett's esophagus with a high incidence of cancer, as well as weakening and reflux of reflux esophagitis. In addition, reflux of strong acid in the presence of reflux esophagitis causes severe heartburn (chest pain). Lowering the total acidity of gastric juice is also very important in reducing the pain caused by reflux esophagitis. In order to determine whether the Kudjippong alcoholic extract of the present invention lowers the total acidity of gastric juice, the SD-based 5-week-old rats were fasted for about 24 hours before the administration of the test substance after a week-long treatment. On the morning of the day of administration of the test substance, oral administration of 5 mg / kg, 10 mg / kg, 15 mg / kg, and 20 mg / kg of the Cudrania extract extract of Example 1, which is the test substance, was anesthetized with isoflurane after 1 hour. Then, the pyloric portion was ligated. After 8 hours of ligation of the pyloric larvae, two animals of each group were planned to be killed with CO 2 gas, and the stomach was ligated and the torso were extracted. After extraction, 10 ml of gastric juice was collected using a syringe. The collected gastric juice was centrifuged at 3,000 rpm for 10 minutes, and only the supernatant was taken to measure the amount of gastric juice (ml), pH, and gastric acidity.

위산도 측정은 원심분리한 위액 중 1㎖를 튜브에 분주한 후 0.1N NaOH를 사용하여 pH 7.0으로 적정하며, 적정에 사용된 0.1N NaOH의 양을 측정하였다. Gastric acidity was measured by dispensing 1ml of centrifuged gastric juice into a tube, titrating to pH 7.0 using 0.1N NaOH, and measuring the amount of 0.1N NaOH used for titration.

총 산도 산출 공식은 '총산도={적정된 NaOH의 양(㎖) x 총 위액량(㎖) x 0.1N (NaOH의 보정가) x 50(산도배수)}/결찰시간(h)' 이다. The formula for calculating total acidity is 'total acidity = {quantified amount of NaOH (ml) x total gastric juice (ml) x 0.1 N (corrected value of NaOH) x 50 (acidity multiple)) / ligation time (h)'.

측정결과는 SPSS statistics 22 for medical science를 사용하여, 대조군과 시험물질 투여군의 비교는 모수적인 다중비교(parametric multiple comparison procedures)인 ONE-WAY ANOVA를 통하여 군간 비교하였으며 P<0.05인 경우 통계학적으로 유의하다고 판정하였다.The results were measured using SPSS statistics 22 for medical science, and the comparison between control group and test substance administration group was compared between groups using ONE-WAY ANOVA, a parametric multiple comparison procedure, and statistically significant at P <0.05. It was determined that.

음성대조군에는 상기 실시예 1의 꾸지뽕 주정추출물 대신 부형제인 0.5% CMC(carboxymethyl cellulose)만을 경구투여한 것을 제외하고는 상기 실험방법과 동일하게 실험하였다.The negative control group was tested in the same manner as in the above experimental method, except that 0.5% CMC (carboxymethyl cellulose), an excipient, was orally administered instead of the Cudrania alcohol extract of Example 1.

그 결과를 도 9에 나타내었다.The results are shown in FIG.

도 9에서 확인할 수 있는 바와 같이, 본 발명의 꾸지뽕 주정추출물은 위액의 총 산도를 음성대조군에 비해 유의적으로 낮추었으며, 이러한 효과는 꾸지뽕 주정추출물의 농도에 의존적으로 나타났다. 이는 본 발명의 꾸지뽕 주정추출물이 직접적인 식도세포의 손상억제 및 염증억제 뿐만이 아니라 계속된 위산 자극에 의한 가슴쓰림과 식도의 바렛식도화를 예방 및 치료할 수 있다는 것을 시사한다.As can be seen in Figure 9, the kkujippong alcohol extract of the present invention significantly lowered the total acidity of gastric juice compared to the negative control group, this effect appeared dependent on the concentration of kkujippong alcohol extract. This suggests that kkujippong alcoholic extract of the present invention can prevent and treat not only direct inhibition of esophageal cell damage and inflammation, but also prevention and treatment of heartburn and esophageal Barrett's esophagus due to continued gastric stimulation.

Claims (9)

꾸지뽕 주정추출물을 유효성분으로 함유하는 역류성 식도염 예방 및 개선용 식품조성물.
Food composition for preventing and improving reflux esophagitis, containing Cudrania alcohol extract as an active ingredient.
제1항에 있어서,
상기 꾸지뽕 주정추출물은 히스타민 H2 수용체(histamie H2 receptor)를 억제하는 것을 특징으로 역류성 식도염 예방 및 개선용 식품조성물.
The method of claim 1,
The kkujippong alcohol extract is a food composition for preventing and improving reflux esophagitis characterized in that it inhibits the histamine H2 receptor (histamie H2 receptor).
제1항 또는 제2항에 있어서,
상기 꾸지뽕 주정추출물은 꾸지뽕잎에서 추출된 것을 특징으로 하는 역류성 식도염 예방 및 개선용 식품조성물.
The method according to claim 1 or 2,
The kkujippong alcohol extract is a food composition for preventing and improving reflux esophagitis, characterized in that extracted from kkujippong leaves.
제1항 또는 제2항에 있어서,
상기 꾸지뽕 주정추출물의 추출용매는 70% 주정(酒精)인 것을 특징으로 하는 역류성 식도염 예방 및 개선용 식품조성물.
The method according to claim 1 or 2,
The extraction solvent of the kkujippong alcohol extract is a food composition for preventing and improving reflux esophagitis, characterized in that 70% alcohol (酒精).
제3항에 있어서,
상기 꾸지뽕 주정추출물의 추출용매는 70% 주정(酒精)인 것을 특징으로 하는 역류성 식도염 예방 및 개선용 식품조성물.
The method of claim 3,
The extraction solvent of the kkujippong alcohol extract is a food composition for preventing and improving reflux esophagitis, characterized in that 70% alcohol (酒精).
제1항 또는 제2항에 있어서,
상기 식품 조성물은 기능성 음료, 건강기능식품, 발효유 중 어느 하나인 것을 특징으로 하는 꾸지뽕 주정추출물을 유효성분으로 함유하는 역류성 식도염 예방 및 개선용 식품조성물.
The method according to claim 1 or 2,
The food composition is a food composition for preventing and improving reflux esophagitis containing kkujippong alcoholic extract as an active ingredient, characterized in that any one of the functional beverage, health functional food, fermented milk.
제3항에 있어서,
상기 식품 조성물은 기능성 음료, 건강기능식품, 발효유 중 어느 하나인 것을 특징으로 하는 꾸지뽕 주정추출물을 유효성분으로 함유하는 역류성 식도염 예방 및 개선용 식품조성물.
The method of claim 3,
The food composition is a food composition for preventing and improving reflux esophagitis containing kkujippong alcoholic extract as an active ingredient, characterized in that any one of the functional beverage, health functional food, fermented milk.
제4항에 있어서,
상기 식품 조성물은 기능성 음료, 건강기능식품, 발효유 중 어느 하나인 것을 특징으로 하는 꾸지뽕 주정추출물을 유효성분으로 함유하는 역류성 식도염 예방 및 개선용 식품조성물.
The method of claim 4, wherein
The food composition is a food composition for preventing and improving reflux esophagitis containing kkujippong alcoholic extract as an active ingredient, characterized in that any one of the functional beverage, health functional food, fermented milk.
제5항에 있어서,
상기 식품 조성물은 기능성 음료, 건강기능식품, 발효유 중 어느 하나인 것을 특징으로 하는 꾸지뽕 주정추출물을 유효성분으로 함유하는 역류성 식도염 예방 및 개선용 식품조성물.
The method of claim 5,
The food composition is a food composition for preventing and improving reflux esophagitis containing kkujippong alcoholic extract as an active ingredient, characterized in that any one of the functional beverage, health functional food, fermented milk.
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