KR100452128B1 - Hovenodulinol, an active compound extracted from Hovenia dulcis THUNB, a process for preparing the same, and an functional food for alleviating lingering intoxication containing the same - Google Patents

Hovenodulinol, an active compound extracted from Hovenia dulcis THUNB, a process for preparing the same, and an functional food for alleviating lingering intoxication containing the same Download PDF

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KR100452128B1
KR100452128B1 KR10-2000-0055068A KR20000055068A KR100452128B1 KR 100452128 B1 KR100452128 B1 KR 100452128B1 KR 20000055068 A KR20000055068 A KR 20000055068A KR 100452128 B1 KR100452128 B1 KR 100452128B1
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alcohol
functional food
hovenodulinol
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현 용 이
김호상
박영식
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김호상
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
    • C07D311/22—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
    • C07D311/26—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3
    • C07D311/28—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 2 only
    • C07D311/32—2,3-Dihydro derivatives, e.g. flavanones
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, 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
    • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
    • A23L2/38—Other non-alcoholic beverages
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, 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
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A23V2200/334—Foods, ingredients or supplements having a functional effect on health treating the effects of consuming alcohol, narcotics or other addictive behavior, e.g. treating hangover or reducing blood alcohol levels
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2250/00—Food ingredients
    • A23V2250/20—Natural extracts
    • A23V2250/21—Plant extracts
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2300/00—Processes
    • A23V2300/14—Extraction

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  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Nutrition Science (AREA)
  • Engineering & Computer Science (AREA)
  • Food Science & Technology (AREA)
  • Polymers & Plastics (AREA)
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  • Mycology (AREA)
  • Medicines Containing Plant Substances (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

본 발명은 지구자 열매에서 추출한 활성 성분인 하기 화학식 1로 표시되는 호베노둘리놀(Hovenodulinol), 그의 제조 방법, 및 그를 함유하는 숙취해소를 위한 기능성식품에 관한 것이다.The present invention relates to Hobenodulinol represented by the following formula (1) which is an active ingredient extracted from the earth fruit, a preparation method thereof, and a functional food for hangover relief containing the same.

[화학식 1][Formula 1]

Description

지구자 열매의 활성 성분인 호베노둘리놀, 그의 제조 방법, 및 그를 함유하는 숙취해소를 위한 기능성식품 {Hovenodulinol, an active compound extracted from Hovenia dulcis THUNB, a process for preparing the same, and an functional food for alleviating lingering intoxication containing the same}Hovenodulinol, an active compound extracted from Hovenia dulcis THUNB, a process for preparing the same, and an functional food for alleviating lingering intoxication containing the same}

본 발명은 지구자 열매에서 추출한 활성 성분인 하기 화학식 1로 표시되는 호베노둘리놀, 그의 제조 방법, 및 그를 함유하는 숙취해소를 위한 기능성식품에 관한 것이다.The present invention relates to a hobenodulinol represented by the following formula (1), which is an active ingredient extracted from the earth fruit, a preparation method thereof, and a functional food for hangover relief containing the same.

[화학식 1] 호베노둘리놀[Formula 1] Hobenodulinol

최근 숙취 해소에 대한 일반인들의 요구가 증가하면서, 숙취 해소를 위한 다양한 처방이 시도되고 있으며, 특히 종전부터 우리 선조들이 사용해 오던 식물을 원료로 사용하여 숙취 해소를 위한 식품 또는 의약품을 개발하는 추세이다.Recently, as the public's demand for hangovers has been increasing, various prescriptions for hangovers have been attempted, and in particular, there is a trend to develop foods or medicines for hangovers by using the plants used by our ancestors as raw materials.

이러한 숙취 해소 기능을 갖고 있다고 알려진 여러 식물 중 지구자 나무(학명:Hovenia dulcisTHUNB)의 효능에 대해 관심이 증가되고 있다(김일식 역, 본초강목, 1992, 청담출판사; 김필홍, 두산세계대백과사전, 1989, 두산출판사).Among various plants known to have a hangover-relieving function, there is increasing interest in the efficacy of the earth tree ( Hovenia dulcis THUNB). , Doosan Publisher).

지구자 나무는 헛개나무 또는 괴조(拐棗)라고도 하는 갈매나무과의 교목으로, 높이는 10-17m이고, 수피는 흑회색이며, 열매는 갈색이 돌고 지름이 8 mm 정도의 크기로 닭의 발톱 모양이다. 과실을 가지고 있는 과병부는 초겨울에 수확하는데, 열매는 은은한 향기가 있고, 단맛이 있어 먹을 수 있으며(생식 가능) 음식의 맛을 돋군다(박상훈 역, 동의보감, 1991, 아카데미출판사).The earthen tree is a tree of the sea buckthorn tree, also known as a liana or zombia, 10-17m high, bark is black gray, the fruit is brown, 8mm in diameter, and looks like a chicken claw. . The fruit-bearing department with fruits is harvested in early winter, and the fruit has a subtle fragrance, sweetness, which can be eaten (reproducible), and enhances the taste of food (Park Sang-hoon, Dong-bo-gam, 1991, Academy Press).

헛개나무는 전통적으로 과병(果柄)을 달여서 복용하면 주독을 해독하고, 구토를 멎게 하는 작용이 있다고 전해지고 있다. 고대 한방에서는 이 지구자 나무가 술을 썩히는 작용이 있다고 하며, 생즙은 술독을 풀고 구역질을 멎게 한다고 하였다. 전통적으로 종자는 주정중독(酒精中毒), 소변불리(小便不利) 및 구토에, 과경은 건위(健胃), 자양보혈(慈養補血)에 효과가 있다고 전해진다.The bark tree is traditionally known to have a decoction when it is used to detoxify poisoning and stop vomiting. In ancient Chinese medicine, the earth's tree is said to have a decaying effect, and the juice is said to detoxify and relieve nausea. Traditionally, seeds are known for alcohol poisoning, urinary deficiency, and vomiting, and hyperplasia is effective for healthy stomach and nourishment blood.

지구자 나무의 잎, 줄기, 열매 등의 추출물들이 숙취 해소 및 혈중 알코올 분해 촉진 기능 및 간 기능 활성에 효과가 있다는 결과들이 생체외 실험, 생체내 실험 및 임상 실험 등을 통해 발표되고 있다(Yoshikawa, M., et al., Chem. Pharm. Bull., 43(1995), 532-534); Yoshikawa et al., Chem. Pharm. Bull. 44(1996),1736-1743; Yoshikawa et al., Chem. Pharm. Bull., 40(1992), 2287-2291).Extracts such as leaves, stems, and fruits of the earth tree have been shown to be effective in relieving hangover and promoting blood alcohol degradation and liver function in vitro, in vivo, and in clinical trials (Yoshikawa, M., et al., Chem. Pharm. Bull., 43 (1995), 532-534); Yoshikawa et al., Chem. Pharm. Bull. 44 (1996), 1736-1743; Yoshikawa et al., Chem. Pharm. Bull., 40 (1992), 2287-2291).

그 밖에도, 지구자 추출물이 항돌연변이, 항암, 항고혈압 기능의 다양한 생리 활성을 갖는다는 연구가 발표되고 있으나(Kennedy, L.M. et al., Chem. Senses 13(1988), 529-543; Lee, M.K. et al., Kor. J. Med. Crop. Sci. 7(1999), 185-192), 현재까지의 연구 결과들은 하나의 단일 물질이 아니라, 물, 에탄올, 메탄올 등과 같은 용매의 조 추출물(crude extracts) 또는 1,2차 분획을 통해 얻어진 복합 분리물을 가지고 활성 연구를 수행한 결과들이었다. 지구자 추출물을 여러 단계에 걸쳐 분리함으로써 단일 물질임을 확인한 경우도 있는데, 이들은 후라보노이드 계통이나 긴 사슬의 당을 포함한 다당 구조물로서, 호베노틴(Hovenotin), 호베노이둘로사이드(Hovenidulocioside) 등으로 명명되었다(Khetwal K.S. et al., Plant. Med. 54(1988), 89-90; Ondo M., et al., J. Nat. Prod. 52(1989), 1100-1106).In addition, studies have suggested that spermatozoon extracts have various physiological activities such as antimutagenic, anticancer and antihypertensive functions (Kennedy, LM et al., Chem. Senses 13 (1988), 529-543; Lee, MK). et al., Kor. J. Med. Crop. Sci. 7 (1999), 185-192), to date, studies are not a single substance, but crude extracts of solvents such as water, ethanol, methanol, etc. The results of the activity studies were performed with extracts) or composite isolates obtained from the first and second fractions. Some isolates have been identified as single substances by separating the spermatozoon extract in several stages. These are polysaccharide constructs containing flavonoid strains or long-chain sugars, which are named hobennotin and hovenidulocioside. (Khetwal KS et al., Plant. Med. 54 (1988), 89-90; Ondo M., et al., J. Nat. Prod. 52 (1989), 1100-1106).

지구자 나무를 재료로 한 연구는 근피에서 펩티드 알칼로이드를 분리하고, 잎, 뿌리에서 사포닌을 분리하여 구조를 결정하는 데까지 이르렀으나, 알코올을 흡수한 생체에 미치는 헛개나무 투여의 영향에 대해서는 과학적인 연구 결과가 없다. 또한, 지구자에 존재하는 단일 물질에 대한 연구는 상대적으로 미흡하였으며, 일부 단일 물질의 구조가 밝혀져 이들의 작용 기작 또는 기능에 대한 결과들이 문헌에 개시된 바 있지만(Gaydou E.M. et al., Ann. Chem. 2(1977), 303-308; Lee, Y.J. et al., Planta. Med. 59(1993), 17-19; Yoshikawa, M. et al., Yakugaku Zasshi 117(1997), 108-118), 이들 연구도 정확한 구조와 작용 기작에 대한 완전한 결과를 제시하지 못하였다.Studies on earth-based materials have led to the separation of peptide alkaloids from the root bark and the determination of their structure by the separation of saponins from the roots and roots, but the scientific study of the effects of hut tree administration on alcohol-absorbed organisms. There is no result. In addition, studies of single substances present in the Earth have been relatively inadequate, and the structure of some single substances has been revealed, and the results of their mechanism of action or function have been disclosed in the literature (Gaydou EM et al., Ann. Chem). 2 (1977), 303-308; Lee, YJ et al., Planta.Med. 59 (1993), 17-19; Yoshikawa, M. et al., Yakugaku Zasshi 117 (1997), 108-118), These studies did not provide complete results on the exact structure and mechanism of action.

본 발명의 신규 물질인 호베노둘리놀과 구조가 유사한 물질로서 다음 화합물들이 알려진 바 있으나, 이들의 알코올 분해능이나 숙취 해소 기능에 대하여는 보고된 바가 없다.Although the following compounds are known as structures having a similar structure to the novel substance of hobenodulinol, the alcohol degrading ability and hangover resolution function have not been reported.

특히, 이들 물질을 식품이나 의약에 적용하기 위해서는 국내 식품법에 적용을 받아야 하는데, 국내 식품 공전에는 지구자의 경우 열매만이 식용으로 사용이 가능하도록 규정하고 있으므로, 잎이나 줄기와 같은 다른 부위의 활용에는 제약이 있다.In particular, in order to apply these substances to foods or medicines, they must be applied to the domestic food law.In the Korean Food Code, only the fruit of the earth can be used for food, so the use of other parts such as leaves and stems There is a restriction.

따라서, 본 발명자들은 국내에서 식용으로 사용가능한 지구자 열매에 존재하는 활성 물질을 단일 물질로 순수하게 분리해내어 이 물질의 완전한 분자 구조를 규명하고자 연구노력하였다. 그 결과, 지구자 열매의 추출물로부터 이제까지 보고된 바 없는 신규의 화합물을 발견하였으며, 이 화합물이 알코올 분해와 숙취 해소에 미치는 영향을 실험을 통하여 확인하여 본 발명을 완성하였다.Therefore, the present inventors have made efforts to identify the complete molecular structure of the active substance by purely separating the active substance present in the earth fruit that can be used for food in Korea as a single substance. As a result, a novel compound has not been reported so far from the extract of the fruit of the earth, and the effect of the compound on alcohol decomposition and hangover relief was confirmed through experiments to complete the present invention.

도 1은 본 발명에 따른 호베노둘리놀의 세포독성 실험 결과를 나타낸 것이다(실시예 6).Figure 1 shows the cytotoxicity test results of hobenodulinol according to the present invention (Example 6).

본 발명은 숙취의 주원인인 체내 알코올 및 알데하이드의 분해에 직접적으로 관여하는 성분으로서, 지구자 열매에 존재하는 호베노둘리놀(화학식 1)을 최초로 분리하고, 그 화학적 구조를 밝혀 신규 물질로 제공한다.The present invention is a component directly involved in the decomposition of alcohol and aldehydes, which are the main causes of hangovers, and is the first to separate Hobenoldolinol (Formula 1) present in the fruit of the earth, revealing its chemical structure and providing it as a novel substance. .

[화학식 1] 호베노둘리놀[Formula 1] Hobenodulinol

본 발명에 따르면, 호베노둘리놀은 음건된 지구자 열매를 분쇄하고, 이를 물과 에탄올로 추출, 농축시킨 후 동결건조시켜, 이를 부탄올로 다시 추출하고 정제함으로써 수득된다.According to the present invention, hobenodulinol is obtained by pulverizing the dried global fruit, extracting it with water and ethanol, concentrating and lyophilizing, extracting it again with butanol and purifying it.

수득된 호베노둘리놀의 알코올 분해 능력은 동물 실험 및 임상 실험에 의해 입증된다.The alcohol degrading ability of the obtained hobenodilinol is demonstrated by animal experiments and clinical trials.

이하, 본 발명을 실시예를 들어 상세히 설명하나, 본 발명이 하기 실시예로만 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited only to the following Examples.

실시예 1 : 지구자 열매로부터 호베노둘리놀의 단리Example 1 Isolation of Hobenodulinol from Fermented Berries

지구자 나무에서 채취해 잘 씻은 후 음건된 열매 5 Kg을 분쇄기로 갈아 분말을 만든다. 이 분말에 물과 에탄올 혼합용매(1:1 부피비)를 5배 부피로 넣어 85℃에서 12 시간 동안 두 번 추출한다. 이 추출액을 진공 강압 농축기에서 12 시간 이상 농축시킨 후 동결 건조기에서 건조해 분말 상태의 추출물을 얻는다.After harvesting from the earthen tree, wash well and grind 5 Kg of dried fruit to grinder to make powder. Water and ethanol mixed solvent (1: 1 volume ratio) were added to this powder in a 5-fold volume and extracted twice at 85 ° C. for 12 hours. The extract is concentrated in a vacuum step-down concentrator for at least 12 hours and then dried in a lyophilizer to obtain an extract in powder form.

이 추출물을 약간의 증류수에 녹인 후, 증류수로 포화된 부탄올에 섞어 20℃ 실온에서 3일간 분획 깔대기에서 분획을 실시한다. 부탄올 층만을 분리해 이 액을 실리카겔 60 칼럼 (크기 5x70 cm, 0.1ml/min) 에 0-50% 메탄올 농도구배를 갖는 용액으로 용리시켜 분리한다. 이러한 과정을 거쳐 5개의 구획(fraction)이 분리되는데 그 중 활성이 가장 높은 구획인 구획 3을 세파덱스(Sephadex) G-70 (Pharmacia) 컬럼으로 분리하여, 얻어진 4개의 구획 중 활성이 가장 높은 4 번째 구획을 정제(preparative) HPLC (Waters, Delta prep. 4000, C1815μ60A )로 분리하고, 이렇게 하여 얻어진 3개의 구획 중 2 번째 구획을 다시 정제 HPLC 로 분리해 동결 건조시킴으로써 매우 연한 담황색의 순수 물질 분말을 얻었다. 이 물질은 하기의 분석결과, (2R,3R)-5,7,5'-트리히드록시-3',4'-디메톡시디히드로플라보놀로 밝혀졌고, 본 발명자들은 이를 호베노둘리놀 (Hovenodulinol)이라 명명했다.The extract is dissolved in a little distilled water, mixed with butanol saturated with distilled water, and fractionated in a separatory funnel at 20 ° C for 3 days. Only the butanol layer is separated and the solution is separated by elution with a solution having a 0-50% methanol concentration gradient in a silica gel 60 column (size 5x70 cm, 0.1 ml / min). Through this process, five fractions are separated, and partition 3, which is the highest activity, is separated by Sephadex G-70 (Pharmacia) column. second refining compartments (preparative) HPLC (Waters, Delta prep. 4000, C 18 15 μ 60A) to separate and, thus a very pale buff again separated by preparative HPLC of the second compartment of the three compartments obtained by freeze-drying Pure material powder was obtained. This material was found to be (2R, 3R) -5,7,5'-trihydroxy-3 ', 4'-dimethoxydihydroflavonol, as determined by the following analysis. Hovenodulinol).

실시예 2 : 호베노둘리놀의 알코올 분해능 측정 (동물 실험)Example 2 Determination of Alcohol Degradation of Hobenodulinol (Animal Experiment)

체중 300g 내외의 스프라그-다울리 (Sprague-Dawley, CD strain) 계통의 쥐를 알코올이나 순수 물질 어느 것도 주지 않은 대조군, 정상 쥐에 알코올만 먹인 동물군 및 지구자 분리물 섭취군(피실험군)으로 각각 10 구씩 실험에 이용했다. 사육 기간 중에는 쥐 고형 사료와 증류수를 자유롭게 취하도록 하였고, 실험 전 후 24 시간부터는 절식했다. 실험 개시 30 분전에 300 mg/Kg의 우레탄을 복강 투여해 마취시켰다(Dool, R. et al., J. Natl. Cancer Inst. 66(1981), 1192-1198; Doyle, A. et al., Cell ＆ Tissue Culture: Laboratory Procedures (1993)). 대조군은 증류수 5ml를, 피실험군은 증류수 5ml 에 1mg/체중kg 으로 하여 본 발명의 화합물을 용해한 용액을 알코올 투여 15분 전에 주사기를 이용해 경구 투여하였다. 순수 알코올만 투여하는 쥐에게는 20%(w/v) 농도로 조절한 알코올 용액을 1g/kg체중이 되게 경구 투여하였다.Sprague-Dawley (CD strain) rats weighing around 300g and no alcohol or pure substances were given to the control group, normal rats were fed alcohol only, and rats were fed the aliquots (test group). 10 spheres each were used for the experiment. During the breeding period, rats were fed with solid feed and distilled water freely, and fasted 24 hours after the experiment. Anesthesia was administered by intraperitoneal administration of 300 mg / Kg of urethane 30 minutes before the start of the experiment (Dool, R. et al., J. Natl. Cancer Inst. 66 (1981), 1192-1198; Doyle, A. et al., Cell & Tissue Culture: Laboratory Procedures (1993). The control group was 5 ml of distilled water, and the test group was 1 mg / kg body weight in 5 ml of distilled water. The solution of the compound of the present invention was orally administered 15 minutes before alcohol administration. Rats receiving pure alcohol only were orally administered with an alcohol solution adjusted to a concentration of 20% (w / v) to 1 g / kg body weight.

상기 방법으로 알코올 투여 후 1 시간 간격으로 쥐의 안와 정맥에서 1ml 씩 채혈하여 에탄올 분석 키트(Sigma, USA)로 혈액 내 알코올 양을 측정했다. 마지막 채혈 후 즉시 간을 적출 해 4℃에서 5 배의 0.25 M 자당에 균질화시켜 혈중 아세트알데하이드(acetaldehyde) 농도 측정 실험 때까지 -20℃에서 보관했다. 혈중 아세트알데하이드를 정량하기 위하여 채취된 혈액 1ml에 2mM n-프로판올 0.2ml를 넣은 후 밀폐된 용기 내에서 65℃에서 15분간 가온하여 증발된 부분의 가스 1m를 주사기로 취해 가스크로마토그래피를 이용하여 아세트알데하이드를 측정하였다.In the above method, 1 ml of rats were collected from the orbital vein of the rats at 1 hour intervals after alcohol administration, and the amount of alcohol in the blood was measured using an ethanol analysis kit (Sigma, USA). Immediately after the last blood collection, the livers were extracted and homogenized to 5 times 0.25 M sucrose at 4 ° C. and stored at −20 ° C. until the acetaldehyde concentration test was performed. In order to quantify the acetaldehyde in blood, 1 ml of 2 mM n-propanol was added to 1 ml of collected blood and warmed at 65 ° C. for 15 minutes in an airtight container. Aldehyde was measured.

결과는 표 1에 나타낸다.The results are shown in Table 1.

표 1. 쥐를 대상으로 호베노둘리놀을 투여한 경우와 투여하지 않은 경우, 알코올 투여 후 시간에 따른 혈중 알데하이드 및 알코올 농도 측정 결과Table 1. Results of measurement of blood aldehyde and alcohol concentrations over time after alcohol administration in mice with and without hobenodulinol

실시예 3 : 인체의 호기 알코올 농도 측정Example 3 Measurement of Aerobic Alcohol Concentration in Human Body

건강한 성인 남자 30명(18-50세, 음주 경력 4-25년)의 지원자를 피험자로 선30 volunteers (18-50 years old, 4-25 years of drinking experience) were selected as subjects.

정하였다. 실험 전 후에는 24 시간 동안 음주를 금지하고 실험 당일은 공복으로 실험에 참여하였다. 호기 중의 알코올 농도(mg/L)의 측정은 반 유체식 가스 센서를 내장한 알코스캔(Alcoscan; model AL-2000)이란 장치를 이용하였다. 상기 30 명 중 10 명은 대조군으로 각각 100ml의 증류수를 투여하고 기계를 이용해 호기 중 알코올이 검출되지 않는 것을 확인한 후, 증류수 투여 30분 후에 0.5g/체중 kg 의 알코올(알코올 도수, 40% v/v)이 되도록 조제하여 10분 이내에 전량을 마시도록 하였다. 30분 경과 후, 한시간 간격으로 알코올 섭취 후 6 시간까지 호기 중 알코올 농도를 측정했다. 다른 20 명에게는 알코올 섭취 실험을 위해 지구자에서 분리된 본 발명의 물질을 5mg/체중Kg의 양으로 용해시킨 증류수를 마시게 한 후, 30 분 동안 가만히 있게 한다. 이 후 0.5g/체중kg의 알코올(알코올 도수, 40% v/v)이 되도록 조제하여 10분 이내에 전량을 마시도록 하였다. 30분 경과 후, 한시간 간격으로 알코올 섭취 후 6 시간까지 호기 중 알코올 농도를 측정했다. 이같이 측정한 수치의 평균치를 산출해 피시험군의 호기 중 알코올 농도로 정했다. 또한, 아세트알데하이드의 양을 측정하기 위해 호기 알코올 측정 시 나오는 타액 1g을 취해 상기 쥐의 혈중 아세트알데하이드 측정 방법과 같은 방법으로 측정했다.Decided. Before and after the experiment, alcohol was prohibited for 24 hours, and the experiment was taken on an empty stomach on the day of the experiment. The alcohol concentration (mg / L) in the exhalation was measured using an apparatus called Alcoscan (model AL-2000) incorporating a semi-fluid gas sensor. Ten of the 30 people were each administered 100ml of distilled water as a control, and after confirming that no alcohol in the expiration was detected using a machine, 0.5g / kg of body weight alcohol (alcohol water, 40% v / v) after 30 minutes of distilled water administration. ) To drink the whole amount within 10 minutes. After 30 minutes, the inhalation alcohol concentration was measured at one hour intervals up to 6 hours after alcohol intake. The other 20 were allowed to drink distilled water dissolved in the amount of 5 mg / kg body weight of the substance isolated from the earth for alcohol intake experiments, and then left for 30 minutes. Thereafter, 0.5 g / kg body weight of alcohol (alcohol frequency, 40% v / v) was prepared to drink the whole amount within 10 minutes. After 30 minutes, the inhalation alcohol concentration was measured at one hour intervals up to 6 hours after alcohol intake. The average value of the measured values was calculated and determined as the alcohol concentration in the breath of the test group. In addition, in order to measure the amount of acetaldehyde, 1 g of saliva from the measurement of aerobic alcohol was taken and measured in the same manner as the method of measuring acetaldehyde in the rat.

결과는 다음 표에 나타낸다.The results are shown in the following table.

표 2. 인체에 호베노둘리놀을 투여한 경우와 투여하지 않은 경우, 시간에 따른 혈중 알코올 및 타액 알데하이드 농도의 측정 결과Table 2. Measurement results of blood alcohol and saliva aldehyde concentrations over time with and without Hobenodulinol in humans

실시예 4 : 체내 알코올 분해에 직접적으로 영향을 주는 효소인 알코올 분해효소(Alcohol dehydrogenase, ADH) 와 아세트알데하이드 분해효소(Acetaldehyde dehydrogenase, ALDH)의 활성 증진 측정 실험Example 4 Activity Enhancement Test of Alcohol Dehydrogenase (ADH) and Acetaldehyde Dehydrogenase (ALDH), Enzymes That Directly Affect Alcohol Degradation in the Body

실시예 2의 실험에서 마지막 채혈 후 즉시 간을 적출하여 7 배의 0.25M 설탕 용액에 섞어 4℃에서 호모제나이저를 이용해 균질화시켰다. 이것을 1000 rpm 속도로 15 분간 원심분리 후, 다시 40000 rpm에서 1 시간 원심 분리하여 침전물인 간의 사이토솔(cytosol) 부분을 분리해 ADH와 ALDH의 효소원으로 사용했다.In the experiment of Example 2, immediately after the last blood collection, livers were extracted, mixed with 7 times 0.25M sugar solution, and homogenized using a homogenizer at 4 ° C. This was centrifuged at 1000 rpm for 15 minutes, and then centrifuged at 40000 rpm for 1 hour to separate the precipitated cytosol portion of the liver and used as an enzyme source of ADH and ALDH.

ADH 활성은 이 효소원 0.1 ml를 0.5M 세미카바자이드 0.02ml, 0.1M NAD 가 함유된 미리 항온처리된 반응액 2.0 ml 에 혼합해 37℃에서 반응시켜 340 nm에서 10 분간 흡광도를 연속적으로 측정했다. 이 흡광도와 정상적인 쥐에서 적출한 간의 ADH인 대조군의 비로부터 ADH 의 활성을 계산했다(Lebsack M.E. et al., Biochem. Pharmacol. 26 (1976), 1151-1159).ADH activity was measured by measuring 0.1 ml of this enzyme source in 0.02 ml of 0.5 M semicarbazide and 2.0 ml of a pre-incubated reaction solution containing 0.1 M NAD and reacting at 37 ° C. for 10 minutes at 340 nm. . The activity of ADH was calculated from this absorbance and the ratio of the control group, which is the liver of ADH taken from normal rats (Lebsack M. E. et al., Biochem. Pharmacol. 26 (1976), 1151-1159).

ALDH 활성 측정을 위해, 0.2M 에탄올 0.1 ml에 0.5M 세미카바자이드 0.02ml를 첨가한 후, 이 액에 다시 0.1M NAD 0.02ml와 0.1M Tris 완충용액(산도 8.5) 2.0 ml를 첨가해 섞은 후 30℃에서 10 분간 정치해 둔다. 이 액에 상기 효소원 0.1 ml를 첨가해 37℃에서 340 nm 흡광도를 측정해 에탄올을 넣지 않은 대조군과 비교해 효소 활성 증진 정도를 다음 식에 의해 계산했다(Choi, S.Y. et al., Kor. J. Biochem. 25(1992), 452-458).To measure ALDH activity, add 0.02 ml of 0.5 M semicarbazide to 0.1 ml of 0.2 M ethanol, and then add 0.02 ml of 0.1 M NAD and 2.0 ml of 0.1 M Tris buffer (pH 8.5). Leave at 30 ° C for 10 minutes. 0.1 ml of the enzyme source was added to the solution, and the absorbance was measured at 37 ° C., and the degree of enzymatic activity enhancement was calculated by the following equation compared with the control without ethanol (Choi, SY et al., Kor. J. Biochem. 25 (1992), 452-458).

효소활성율 (%) = [(대조군 흡광도 값- 실험군 흡광도 값)/대조군 흡광도값]×100Enzyme activity rate (%) = [(control absorbance value-experimental absorbance value) / control absorbance value] × 100

인체에 대한 실험에서는 알로스캔을 사용하여 호기 중 알코올 양을 측정하는방법을 사용하였다.In the experiments on the human body using an alloscan method to measure the amount of alcohol in the exhalation.

결과를 다음 표 3에 나타낸다.The results are shown in Table 3 below.

표 3 : 호베노둘리놀을 투여한 경우와 투여하지 않은 경우 알코올 분해 관련 효소의 활성증가율 비교표 Table 3: Comparison of the rate of increase in activity of alcohol-degrading enzymes with and without hobenodulinol

실시예 5 : 간 해독 효소 활성 증진 기능Example 5 Liver Detoxification Enzyme Activity Enhancement Function

간의 해독 작용에 가장 중요하게 작용하는 효소의 하나인 GST (Glutathion-S-Transferase)의 활성을 측정하였다(Mohn, 1981). 조제된 반응 시약에 호베노둘리놀이 제외된 반응액을 대조구로 하였으며, 호베노둘리놀을 농도별로 첨가하여 37℃에서 5분간 반응시킨 다음, 기질로서 1-클로로-2,4-디니트로벤젠을 첨가한 후 다시 37℃에서 2분간 반응시켰다. 반응후 20% TCA를 가하여 반응을 종결시키고 원심분리한 후 상등액을 340nm에서 흡광도를 측정한 뒤 다음과 같이 GST의 활성도를 계산하였다(Mohn G.R., Mut. Res. 87(1981), 191-195).The activity of GST (Glutathion-S-Transferase), one of the enzymes most important for liver detoxification, was measured (Mohn, 1981). To the prepared reaction reagent, the reaction solution excluding hobenodilinol was used as a control. The reaction was carried out at 37 ° C. for 5 minutes by addition of hobenudolinol, and 1-chloro-2,4-dinitrobenzene was used as a substrate. After the addition, the mixture was reacted at 37 ° C. for 2 minutes. After the reaction, 20% TCA was added to terminate the reaction, centrifugation, and the supernatant was measured for absorbance at 340 nm. The activity of GST was calculated as follows (Mohn GR, Mut. Res. 87 (1981), 191-195). .

총 활성 (단위) = A340/9.6×희석배수×3ml/0.1×조 추출물 부피(ml)Total activity (units) = A340 / 9.6 x dilution factor x 3 ml / 0.1 x crude extract volume (ml)

특이적 활성 (단위/mg 단백질) = 총 활성/ 총 단백질Specific activity (unit / mg protein) = total activity / total protein

활성율 (%) = [특이적 활성실험군/ 특이적 활성대조군]×100% Activity = [specific activity experimental group / specific activity control group] × 100

결과를 다음 표에 나타낸다.The results are shown in the following table.

표 4 : 호베노둘리놀의 간 해독효소인 GST 의 활성 증진 정도 비교표 Table 4: Comparison table for the degree of activity enhancement of the liver detoxifying enzyme, GST, of hobenodulinol

실시예 6 : 세포 독성 실험Example 6: Cytotoxicity Experiment

세포 독성실험은 인간 정상 간세포인 WRL68 (인체 태아간)을 사용하여 실험하였으며, 이 세포에 사용된 배지는 DMEM과 10% 가열-비활성화된 FBS(소 태아 혈청)로 적응시켜 배양하였다. 또한 세포독성 측정방법으로는 SRB(술포르호다민 B)라는 시약을 사용해 세포의 증식이나 독성을 측정하였다.Cytotoxicity experiments were performed using human normal hepatocytes, WRL68 (human fetal liver), and the medium used for these cells was incubated with DMEM and 10% heat-inactivated FBS (fetal bovine serum). In addition, as a cytotoxicity measurement method, the cell proliferation and toxicity were measured using a reagent called SRB (sulfohodamine B).

우선, 실험 대상 세포인 WRL68(10% FBS, DMEM 배지)의 농도를 4×104내지 5×104세포/ml으로 96웰 플레이트의 각 웰에 100??l 씩 첨가하여 24 시간 동안First, the concentration of WRL68 (10% FBS, DMEM medium), the cells to be tested, was added to each well of a 96 well plate at 4 × 10 4 to 5 × 10 4 cells / ml for 100 hours.

배양(37℃, 5% CO2)한 후 각각의 시료를 최종 농도 0.2, 0.4, 0.6, 0.8, 1.0 mg/ml로 100??l 씩 첨가하여 간 배양하였다. 배양이 완료된 후에 상등액을 제거하고 차가운 10%(w/v) TCA(트리클로로아세트산) 100??l를 가하여 4℃에서 1시간 동안 방치한 후 증류수로 4-5회 세척하여 TCA를 제거하고 실온에서 플레이트를 건조한 뒤 각 웰에 1%(v/v) 아세트산에 녹인 0.4%(w/v) SRB 용액을 100??l 씩 첨가하고 상온에서 30분 동안 염색시켰다. 결합되지 않은 SRB 염색액은 1% 아세트산으로 4-5회 정도 세척, 건조시킨 후에 10 mM 트리스 완충액 100??l를 첨가하여 염색액을 녹여낸 후 540 nm에서 마이크로플레이트 탐독기를 이용하여 흡광도를 측정하였다.After incubation (37 ° C., 5% CO 2 ), each sample was incubated by adding 100 μl at a final concentration of 0.2, 0.4, 0.6, 0.8, 1.0 mg / ml. After the incubation was completed, the supernatant was removed, cold 100% (w / v) TCA (trichloroacetic acid) was added to 100 ?? l, and left at 4 ° C. for 1 hour, followed by 4-5 washes with distilled water to remove TCA. After drying the plate in each well was added 0.4% (w / v) SRB solution dissolved in 1% (v / v) acetic acid 100 ~ l each and stained for 30 minutes at room temperature. Unbound SRB stain was washed 4-5 times with 1% acetic acid and dried, and then 100 mM of 10 mM Tris buffer was added to dissolve the stain, and the absorbance was measured at 540 nm using a microplate reader. .

결과는 도 1과 같다.The results are shown in FIG.

이상의 실시예에서 나타난 바와 같이, 지구자 열매에서 추출한 본 발명에 따른 화학식 1의 호베노둘리놀은 알코올 및 아세트알데하이드 분해능력이 우수하여, 숙취 해소에 탁월한 효과를 나타내며, 체내의 알코올 분해에 직접적으로 영향을 주는 효소인 ADH 및 ALDH의 활성을 증진시키고, 간의 해독 작용에 주요하게 작용하는 효소인 GST의 활성을 촉진하는 효능을 가진 화합물로서, 세포에 독성이 없다. 따라서, 본 발명에 의해 규명된 신규 물질인 호베노둘리놀은 숙취 해소를 위한 기능성 식품, 음료나 알코올 분해 및 숙취 회복을 위한 의약품의 유효 성분으로 유용하다.As shown in the above examples, Hobenodulinol of the formula (1) according to the present invention extracted from the fruit of the earth is excellent in the ability to decompose alcohol and acetaldehyde, has an excellent effect on resolving hangover, and directly to the decomposition of alcohol in the body It is a compound having the effect of promoting the activity of the enzymes affecting ADH and ALDH, and promoting the activity of GST, an enzyme that plays a major role in liver detoxification, and is nontoxic to cells. Therefore, the novel substance hobenodulinol identified by the present invention is useful as an active ingredient of a functional food for eliminating hangovers, a beverage or alcohol, and a medicine for recovering from hangovers.

Claims (5)

하기 화학식 1로 표시되는 화합물.A compound represented by the following formula (1). [화학식 1][Formula 1] 지구자 열매를 분쇄하고, 이를 물과 혼합하여 에탄올로 추출, 농축시킨 후 동결건조시키고, 수득된 물질에 다시 물과 부탄올을 가하여 다시 추출한 다음, 얻어진 부탄올층의 유기물을 정제하는 것을 특징으로 하는, 하기 화학식 1로 표시되는 화합물의 제조방법.After crushing the fruit of the earth, it is mixed with water, extracted with ethanol, concentrated and lyophilized, and extracted again by adding water and butanol to the obtained material, and then purified the organic material of the obtained butanol layer, Method for producing a compound represented by the following formula (1). [화학식 1][Formula 1] 삭제delete 삭제delete 하기 화학식 1로 표시되는 화합물을 유효 성분으로 포함하는 숙취 해소를 위한 기능성 식품.Functional food for hangover relief comprising the compound represented by the following formula (1) as an active ingredient. [화학식 1][Formula 1]
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JPH04282318A (en) * 1991-03-08 1992-10-07 Suntory Ltd Food and drink for ameliorating drunken sickness
US5360915A (en) * 1991-10-17 1994-11-01 Rutgerswerke Aktiengesellschaft Ag Modified huminates and their preparation
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KR19990046190A (en) * 1998-09-29 1999-07-05 천형식 Drink for removing hangover using barn wood and its manufacturing method
KR19990064643A (en) * 1999-04-22 1999-08-05 천형식 Method of making use material of foods from Hovenia dulcis thunb, Extracts.
WO2002024678A1 (en) * 2000-09-19 2002-03-28 Hyeon Yong Lee Hovenodulinol, an active compound extracted from hovenia dulcis thunb, a process for preparing the same, and an alcohol decomposing agent or an agent for allevating lingering intoxication containing the same

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* Cited by examiner, † Cited by third party
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JPH04282318A (en) * 1991-03-08 1992-10-07 Suntory Ltd Food and drink for ameliorating drunken sickness
US5360915A (en) * 1991-10-17 1994-11-01 Rutgerswerke Aktiengesellschaft Ag Modified huminates and their preparation
KR970073403A (en) * 1996-05-20 1997-12-10 구형우 Beverage composition containing Hovenia dulcis extract and method for its production
KR19990046190A (en) * 1998-09-29 1999-07-05 천형식 Drink for removing hangover using barn wood and its manufacturing method
KR19990064643A (en) * 1999-04-22 1999-08-05 천형식 Method of making use material of foods from Hovenia dulcis thunb, Extracts.
WO2002024678A1 (en) * 2000-09-19 2002-03-28 Hyeon Yong Lee Hovenodulinol, an active compound extracted from hovenia dulcis thunb, a process for preparing the same, and an alcohol decomposing agent or an agent for allevating lingering intoxication containing the same

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