KR20180134481A - Method for producing mixed extract of Dendropanax morbifera, Hovenia dulcis, Coriolus versicolor, Artemisia capillaris and Oenanthe javanica using response surface methodology - Google Patents

Method for producing mixed extract of Dendropanax morbifera, Hovenia dulcis, Coriolus versicolor, Artemisia capillaris and Oenanthe javanica using response surface methodology Download PDF

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KR20180134481A
KR20180134481A KR1020170072234A KR20170072234A KR20180134481A KR 20180134481 A KR20180134481 A KR 20180134481A KR 1020170072234 A KR1020170072234 A KR 1020170072234A KR 20170072234 A KR20170072234 A KR 20170072234A KR 20180134481 A KR20180134481 A KR 20180134481A
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radical scavenging
polyphenol content
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홍재희
홍성은
홍성민
윤지혜
이현주
오준석
김성연
장보윤
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동부생약 영농조합법인
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L19/00—Products from fruits or vegetables; Preparation or treatment thereof
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Preparation or treatment thereof
    • A23L2/38—Other non-alcoholic beverages
    • A—HUMAN NECESSITIES
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    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • 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
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    • 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
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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    • 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
    • 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
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    • A23V2300/24—Heat, thermal treatment

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Abstract

본 발명은 반응표면분석법을 이용한 황칠나무, 헛개나무, 운지버섯, 인진호 및 미나리 혼합 추출물의 제조 방법에 관한 것으로, 보다 상세하게는 반응표면분석법을 통해 추출온도, 추출시간 및 추출용매:시료 비율에 따른 황칠나무, 헛개나무, 운지버섯, 인진호 및 미나리 혼합 추출물의 추출수율, DPPH 라디칼 소거능, 총 폴리페놀 함량 및 ROS 생성 억제활성이 증가된 혼합 추출물의 추출 최적조건을 확립하였으며, 이를 이용하여 추출수율, DPPH 라디칼 소거능, 총 폴리페놀 함량 및 ROS 생성 억제활성이 증가된 황칠나무, 헛개나무, 운지버섯, 인진호 및 미나리 혼합 추출물을 효율적으로 수득할 수 있으므로, 상기 혼합 추출물을 포함하는 음료 조성물을 제조하는 데에 유용하게 사용될 수 있다. More particularly, the present invention relates to a method for preparing a mixed extract of Hwangchil, Hovenia dulcis, Fengji mushroom, Injinhwa, and Minori using a reaction surface analysis method. More particularly, Extraction yield, DPPH radical scavenging ability, total polyphenol content and ROS production inhibitory activity of mixed extracts of Hwangchujang, Hovenia dulcis, Echinacea, Inchinho, and Minari were determined. , DPPH radical scavenging ability, total polyphenol content, and ROS production inhibitory activity can be efficiently obtained, so that a beverage composition containing the mixed extract can be produced It can be used to advantage.

Description

반응표면분석법을 이용한 황칠나무, 헛개나무, 운지버섯, 인진호 및 미나리 혼합 추출물의 제조 방법{Method for producing mixed extract of Dendropanax morbifera, Hovenia dulcis, Coriolus versicolor, Artemisia capillaris and Oenanthe javanica using response surface methodology}TECHNICAL FIELD The present invention relates to a method for producing mixed extracts of Hwangchil, Hovenia japonica, Fenugumi mushroom, Inchinho and Minari using a reaction surface method,

본 발명은 반응표면분석법을 이용한 황칠나무, 헛개나무, 운지버섯, 인진호 및 미나리 혼합 추출물의 제조 방법에 관한 것이다.The present invention relates to a method for preparing a mixed extract of Hwangchujang, Hovenia dulcis, Fengji mushroom, Injinjeol and Minari mixed by using a reaction surface analysis method.

RSM(response surface methodology)은 최소의 실험횟수로부터 최대의 정보를 얻을 수 있는 실험계획법으로, 이를 통해 얻어진 실험 자료를 분석하여 복잡한 시스템의 성능을 이해하고, 반응변수에 영향을 주는 유의한 요인들을 찾고 최적화하는데 사용된다. 여러 개의 인자 중 하나씩 각각 변화시켜 실험을 진행하는 전통적인 실험법인 1회 1인자(one factor at a time) 실험법은 최적의 실험 조건을 찾기 어렵고 복합 공정에서 중요한 인자 간에 상호작용을 고려하지 못하고, 실험영역 전체를 균형 있게 판단하지 못하기 때문에 국지적 최적점을 찾게 되는 문제점이 있다. RSM은 전형적인 최적화 방법으로 일반적으로 여러 변수를 사용하여 최적 조건을 찾는 시스템으로 하나의 변수와 다른 변수들과의 상호작용으로 인한 효과를 측정하여 변수들의 최적값을 확인할 수 있다.The response surface methodology (RSM) is an experimental design method that obtains the maximum information from the minimum number of experiments. By analyzing the experimental data obtained through it, we understand the performance of the complex system and find the significant factors influencing the response variables It is used to optimize. One factor at a time experiment, which is one of the traditional experimental methods to conduct the experiment by changing one of several factors, is difficult to find the optimal experimental conditions and does not consider interaction among important factors in complex process, There is a problem that a local optimal point is found because the whole can not be judged in a balanced manner. RSM is a typical optimization method. In general, it is a system to find optimum condition by using several variables. It can check the optimum value of variables by measuring the effect of interaction between one variable and other variables.

황칠나무(Dendropanax morbifera Lev.)는 두릅나무과 오갈피속의 상록활엽교목으로, 높이 15m 이상까지 자라는 한국 고유의 토종나무이다. 어린 가지는 녹색이며 광택이 있고, 꽃은 6월에 피며, 길이 7~19mm의 열매가 검게 익는다. 최저기온이 영하 2℃ 이상 및 연평균기온이 12~15℃ 이상인 지역에서 자라는 난대성 식물이다. 세계적으로 황칠을 분비하는 황칠나무는 한반도 서남부 내륙과 해안, 도서, 그리고 제주도에서만 자라는 희귀종이며, 현재는 그 수량이 극히 제한되어 있다. 현재 도서 지역으로 신안, 진도, 완도 등지와 제주도가 있고, 해안지역으로는 해남, 장흥, 강진, 고흥, 승주, 광양, 여수 등지에 소규모의 자생지가 있다. Dendropanax morbifera Lev.) is an evergreen broad-leaved arborescens in Araliaceae and Ogalli , native to South Korea native to 15m or higher. The young branches are green and lustrous, the flowers bloom in June, and the fruit 7 to 19 mm long is black. It is a mild vegetation that grows in areas with a minimum temperature of minus 2 ° C or higher and an annual average temperature of 12-15 ° C or higher. Hwangchil is a rare species that grows only inland in the southwestern part of the Korean peninsula, the coast, books, and Jeju Island. Currently, there are Shinan, Jindo, Wando and Jeju Island in the present area. There are small habitats in Haenam, Jangheung, Kangjin, Goheung, Seungju, Gwangyang and Yeosu.

헛개나무(Hovenia dulcis var. koreana Nakai)는 갈매나무과의 낙엽활엽교목으로, 수고 20m 및 흉고직경 80cm까지 자란다. 일본, 중국 등 동북아시아 지역에서도 동속의 Hovenia dulcis 및 Hovenia tomentosa가 자생하고 있으나 과경의 크기, 종자, 꽃색 등에서 다르게 나타나 우리나라의 특산종으로 기록되고 있다. 또한, 헛개나무는 내한성과 내음성이 강하고 맹아력이 강한 수종으로 우리나라의 경기, 강원 이남의 고도 70~900m의 사면이나 계속 부위의 비옥한 임지에서 잘 자라며 예로부터 주독해독, 정혈, 이뇨, 갈증해소, 해독작용 등에 효과가 있는 것으로 알려져 있다. Hovenia There is dulcis . koreana Nakai) is a deciduous broad-leaved arboreous tree with a height of 20m and a diameter of 80cm. Hovenia in the East Asia, including Japan and China dulcis And Hovenia tomentosa is indigenous, but its size, seed, flower color, In addition, the horny tree is a strong and cold-temperate species with strong cold and vigor. It grows well in Korea's Gyeonggi Province, at the altitude of 70 ~ 900m in the south of Gangwon, and fertile forest in the continental region. And is known to have an effect on detoxification.

운지버섯(Coriolus versicolor)은 담자균류의 민주름버섯목 구멍장이 버섯과에 속하는 버섯으로 균모는 얇고 단단한 가죽질이며 반원형이고, 침엽수나 활엽수의 고목에 군생하는 일년생이며, 한국, 일본, 중국 등의 산악지대에 주로 자생하고 있다. 운지버섯은 일명 구름버섯이라고도 불리며 전통적으로 건강보조식품이나 민간 치료약으로 이용되었으며, 운지버섯의 일반 성분은 수분 5.62%, 단백질 4.20%, 탄수화물 65.09%, 섬유질 23.24%, 회분 6.37% 및 지방질 1.10%를 함유하고 있다. Coriolus versicolor is a mushroom belonging to the mushroom family, which is thin and hard leather and semicircular, and is a year-old who grows in coniferous or broad-leaved trees and is a member of Korea, Japan, China It grows mainly in the mountainous region. Ungmy mushroom, also known as cloud mushroom, is traditionally used as a health supplement or a private remedy. The common components of mushroom are water 5.62%, protein 4.20%, carbohydrate 65.09%, fiber 23.24%, ash 6.37% and fat 1.10% .

인진호(Artemisia capillaris)는 국화과(Compositae)에 속하는 사철쑥 및 동속 근연식물의 어린 줄기와 잎으로서 그의 성분으로는 카필라리신(capillarisin), 스코파론(scoparone), 카페인산(caffeic acid), 정유성분인 β-피넨(pinene), 카필린(capillin), 카필렌(capillene), 카필라린(capillarin), 지방산인 스테아린산(stearic acid), 팔미틴산(palmitic acid), 무기성분인 산화칼슘 등이 존재하는 것으로 알려져 있다. Artemisia capillaris is a young stem and leaf of a black mosquito and related plants belonging to the family Asteraceae (Compositae), and its components are capillarisin, scoparone, caffeic acid, It is known to exist in the form of pinene, capillin, capillene, capillarin, fatty acid stearic acid, palmitic acid and inorganic calcium, .

미나리(Oenanthe javanica Dc.)는 주로 한국, 일본, 중국, 타이완, 말레이시아, 일본 등지의 습지에서 서식하는 크기가 20∼50cm인 미나리과의 여러해살이 풀 로서 그 전초에 플라보노이드(flavonods) 및 콜린(cholin) 정유가 포함되어 있고, 퀴놀린(quinoline)계의 알칼로이드(alkaloid)로 루타민(rutamin)과 r-파가린(r-pagarin), 쿠마린(cumarin) 등도 함유하고 있다. Oenanthe javanica Dc.) is a perennial herb that lives in wetlands such as Korea, Japan, China, Taiwan, Malaysia, Japan, and has a size of 20 to 50cm. It contains flavonods and cholin essential oil And quinoline alkaloids such as rutamin, r-pagarin, cumarin, and the like.

한편, 한국공개특허 제2006-0081014호는 헛개나무 추출물의 제조 방법을 개시하고 있으며, 한국공개특허 제2015-0047814호는 반응표면분석법을 이용한 머위로부터 페타신 함유 추출물을 제조하는 방법을 개시하고 있다. 하지만, 본 발명의 반응표면분석법을 이용한 황칠나무, 헛개나무, 운지버섯, 인진호 및 미나리 혼합 추출물의 제조 방법에 대해 아직까지 개시된 바가 없다.Korean Patent Laid-Open Publication No. 2006-0081014 discloses a method for preparing Hovenia dulcis extract, and Korean Laid-open Patent Application No. 2015-0047814 discloses a method for preparing a petacin-containing extract from a hull using a reaction surface analysis method . However, no method for preparing the mixed extract of Hwangchil, Hovenia dulcis, Fengji mushroom, Injinjeol and Minari mixed by using the reaction surface analysis method of the present invention has been disclosed yet.

본 발명은 상기와 같은 요구에 의해 도출된 것으로서, 본 발명에서는 반응표면분석법을 이용하여 황칠나무, 헛개나무, 운지버섯, 인진호 및 미나리 혼합 추출물의 최적 추출조건을 확립하였다. 구체적으로, 독립변수로서 추출온도, 추출시간 및 추출용매:시료 비율에 대하여, 5단계의 -2, -1, 0, 1 및 2로 코드화하여 중심합성계획(central composite design)에 따라 16구간으로 설정하여 추출 실험을 실시하였으며, 상기 독립변수에 의해 영향을 받는 종속변수로서 추출수율, DPPH 라디칼 소거능(DPPH radical scavenging activity), 총 폴리페놀 함량(TPC; total polyphenol content) 및 ROS(reactive oxygen species) 생성 억제활성을 3회 반복측정하여 평균값을 계산한 후, 회귀분석을 실시하였다. 그 결과, 추출수율은 추출온도, DPPH 라디칼 소거능은 추출시간, 총 폴리페놀 함량은 추출용매:시료 비율 및 ROS 생성 억제활성은 추출온도에 큰 영향을 받는 것을 알 수 있었으며, 이때의 최적 추출조건을 확인함으로써, 본 발명을 완성하였다. The present invention has been made in view of the above-mentioned needs. In the present invention, optimal extraction conditions of Hwangchilgwa, Hwanggi, Yunji mushroom, Injinjeol and Minari mixed extracts were established using the reaction surface analysis method. Specifically, the extraction temperature, the extraction time, and the extraction solvent: sample ratio as independent variables were coded as 5, -2, -1, 0, 1, and 2 and were divided into 16 sections according to the central composite design (DPPH radical scavenging activity, total polyphenol content (TPC), and reactive oxygen species (ROS)) as the dependent variables influenced by the independent variables. The production inhibitory activity was measured three times repeatedly, and the average value was calculated and regression analysis was performed. As a result, it was found that the extraction yield, the DPPH radical scavenging ability, the extracting time, the total polyphenol content, the extraction solvent, the sample ratio and the ROS production inhibitory activity were greatly influenced by the extraction temperature. By confirming, the present invention has been completed.

상기 과제를 해결하기 위하여, 본 발명은 1) 물, C1~C4의 저급알코올 또는 이들의 혼합 용매를 추출용매로 사용하고, 80~120℃의 추출온도, 2~10시간의 추출시간 및 5~15:1의 추출용매(㎖):시료(g)의 비율의 추출 조건으로, 황칠나무, 헛개나무, 운지버섯, 인진호 및 미나리 혼합물을 추출하는 단계; 2) 상기 단계 1)에서 추출한 황칠나무, 헛개나무, 운지버섯, 인진호 및 미나리 혼합 추출물을 여과하는 단계; 및 3) 상기 단계 2)의 여과물을 감압농축하는 단계;를 포함하며, 추출수율, DPPH 라디칼 소거능, 총 폴리페놀 함량 및 ROS(reactive oxygen species) 생성 억제활성이 증진된 황칠나무, 헛개나무, 운지버섯, 인진호 및 미나리 혼합 추출물의 제조 방법을 제공한다.In order to solve the above-mentioned problems, the present invention provides a process for producing a polyvinyl alcohol, comprising the steps of: 1) using water, a C 1 -C 4 lower alcohol or a mixed solvent thereof as an extraction solvent and extracting at 80 to 120 ° C, 5 to 15: 1 extracting solvent (ml): extracting a mixture of woodworm, hornblende, fenugreek, starch, and parsley as an extraction condition of a ratio of sample (g); 2) filtering out the mixed extract of Hwangchil, Hovenia dulcis, Echinacea, Injinho and Minnaris extracted in step 1); And 3) concentrating the filtrate of step 2) under reduced pressure, wherein the extract yield, DPPH radical scavenging activity, total polyphenol content and ROS (reactive oxygen species) And a method for producing a mixed extract of Ungji mushroom, Injin-ho and Minori.

또한, 본 발명은 상기 제조 방법으로 제조된 추출수율, DPPH 라디칼 소거능, 총 폴리페놀 함량 및 ROS(reactive oxygen species) 생성 억제활성이 증진된 황칠나무, 헛개나무, 운지버섯, 인진호 및 미나리 혼합 추출물을 제공한다.In addition, the present invention provides a method for producing an extract of Hwangchil, Houttuynia japonica, Fungus mushroom, Injinho, and Minari, enhanced by the extraction yield, DPPH radical scavenging activity, total polyphenol content and ROS (reactive oxygen species) to provide.

또한, 본 발명은 상기 추출수율, DPPH 라디칼 소거능, 총 폴리페놀 함량 및 ROS(reactive oxygen species) 생성 억제활성이 증진된 황칠나무, 헛개나무, 운지버섯, 인진호 및 미나리 혼합 추출물을 포함하는 음료 조성물을 제공한다.The present invention also relates to a beverage composition comprising a mixture of Hwangchil, Hovenia dulcis, Echinacea, and Inchinho, which has enhanced the extraction yield, DPPH radical scavenging activity, total polyphenol content and ROS (reactive oxygen species) to provide.

본 발명은 반응표면분석법을 이용한 황칠나무, 헛개나무, 운지버섯, 인진호 및 미나리 혼합 추출물의 제조 방법에 관한 것으로, 보다 상세하게는 반응표면분석법을 통해 추출온도, 추출시간 및 추출용매:시료 비율에 따른 황칠나무, 헛개나무, 운지버섯, 인진호 및 미나리 혼합 추출물의 추출수율, DPPH 라디칼 소거능, 총 폴리페놀 함량 및 ROS 생성 억제활성이 증가된 혼합 추출물의 추출 최적조건을 확립하였으며, 이를 이용하여 추출수율, DPPH 라디칼 소거능, 총 폴리페놀 함량 및 ROS 생성 억제활성이 증가된 황칠나무, 헛개나무, 운지버섯, 인진호 및 미나리 혼합 추출물을 효율적으로 수득할 수 있으므로, 상기 혼합 추출물을 포함하는 음료 조성물을 제조하는 데에 유용하게 사용될 수 있다. More particularly, the present invention relates to a method for preparing a mixed extract of Hwangchil, Hovenia dulcis, Fengji mushroom, Injinhwa, and Minori using a reaction surface analysis method. More particularly, Extraction yield, DPPH radical scavenging ability, total polyphenol content and ROS production inhibitory activity of mixed extracts of Hwangchujang, Hovenia dulcis, Echinacea, Inchinho, and Minari were determined. , DPPH radical scavenging ability, total polyphenol content, and ROS production inhibitory activity can be efficiently obtained, so that a beverage composition containing the mixed extract can be produced It can be used to advantage.

도 1은 추출온도 및 추출시간(A), 추출온도 및 시료에 대한 추출용매비(B) 및 추출시간 및 시료에 대한 추출용매비(C)에 따른 본 발명의 황칠나무, 헛개나무, 운지버섯, 인진호 및 미나리 혼합 추출물의 추출수율에 대한 반응표면 분석 결과이다.
도 2는 추출온도 및 추출시간(A), 추출온도 및 시료에 대한 추출용매비(B) 및 추출시간 및 시료에 대한 추출용매비(C)에 따른 본 발명의 황칠나무, 헛개나무, 운지버섯, 인진호 및 미나리 혼합 추출물의 DPPH 라디칼 소거능에 대한 반응표면 분석 결과이다.
도 3은 추출온도 및 추출시간(A), 추출온도 및 시료에 대한 추출용매비(B) 및 추출시간 및 시료에 대한 추출용매비(C)에 따른 본 발명의 황칠나무, 헛개나무, 운지버섯, 인진호 및 미나리 혼합 추출물의 총 폴리페놀 함량(TPC)에 대한 반응표면 분석 결과이다.
도 4는 추출온도 및 추출시간(A), 추출온도 및 시료에 대한 추출용매비(B) 및 추출시간 및 시료에 대한 추출용매비(C)에 따른 본 발명의 황칠나무, 헛개나무, 운지버섯, 인진호 및 미나리 혼합 추출물의 ROS 생성 억제활성에 대한 반응표면 분석 결과이다. FIG. 1 is a graph showing the relationship between the extraction temperature and the extraction time (A), the extraction temperature and the extraction solvent ratio (B) for the sample and the extraction solvent ratio (C) , And the extractive yield of mixed extracts of Injin - ho and.
FIG. 2 is a graph showing the effect of the extract of the present invention on the extracting temperature and extraction time (A), the extraction temperature and the extraction solvent ratio (B) to the sample and the extraction solvent ratio (C) , And the DPPH radical scavenging ability of the mixed extracts of Injin and Minari.
FIG. 3 is a graph showing the relationship between the extracting temperature and the extracting time (A), the extraction temperature and the extraction solvent ratio (B) for the sample and the extraction solvent ratio (C) , And the total polyphenol content (TPC) of the mixed extracts of Injinho and Misaria.
FIG. 4 is a graph showing the relationship between the extracting temperature and the extracting time (A), the extraction temperature and the extraction solvent ratio (B) for the sample and the extraction solvent ratio (C) , And the inhibitory activity of ROS in the mixed extracts of Inchinho and Minari, respectively.

본 발명의 목적을 달성하기 위하여, 본 발명은 In order to achieve the object of the present invention,

1) 물, C1~C4의 저급알코올 또는 이들의 혼합 용매를 추출용매로 사용하고, 80~120℃의 추출온도, 2~10시간의 추출시간 및 5~15:1의 추출용매(㎖):시료(g)의 비율의 추출 조건으로, 황칠나무, 헛개나무, 운지버섯, 인진호 및 미나리 혼합물을 추출하는 단계; 1) water, a C 1 -C 4 lower alcohol or a mixed solvent thereof is used as an extraction solvent, and an extraction temperature of 80 to 120 ° C, an extraction time of 2 to 10 hours and an extraction solvent of 5 to 15: 1 ): Extracting a mixture of Hwangchujang, Hovenia dulcis, Fungus mushroom, Injinhwa and Muller mixture as the extraction condition of the ratio of the sample (g);

2) 상기 단계 1)에서 추출한 황칠나무, 헛개나무, 운지버섯, 인진호 및 미나리 혼합 추출물을 여과하는 단계; 및2) filtering out the mixed extract of Hwangchil, Hovenia dulcis, Echinacea, Injinho and Minnaris extracted in step 1); And

3) 상기 단계 2)의 여과물을 감압농축하는 단계;를 포함하며, 추출수율, DPPH 라디칼 소거능, 총 폴리페놀 함량 및 ROS(reactive oxygen species) 생성 억제활성이 증진된 황칠나무, 헛개나무, 운지버섯, 인진호 및 미나리 혼합 추출물의 제조 방법을 제공한다.3) The step of concentrating the filtrate of step 2) under reduced pressure, wherein the extraction yield, the DPPH radical scavenging activity, the total polyphenol content and the reactive oxygen species (ROS) Mushroom, Injin-ho, and parsley mixed extract.

본 발명의 일 구현 예에 따른 제조방법에서, 상기 혼합 추출물은 물, 탄소수 1 내지 4의 저급 알코올 및 이들의 혼합용매로 구성되는 군으로부터 선택되는 용매, 바람직하게는 물을 용매로 이용하여 추출한 것이다.In the production method according to an embodiment of the present invention, the mixed extract is extracted with a solvent, preferably water, as a solvent selected from the group consisting of water, a lower alcohol having 1 to 4 carbon atoms, and a mixed solvent thereof .

또한, 상기 혼합 추출물은 추출처리에 의해 얻어지는 추출물, 추출물의 희석액 또는 농축액, 추출물을 건조하여 얻어지는 건조물, 조정제물 또는 정제물 중 어느 하나를 포함하는 것으로 한다. 추출방법은 특별히 제한되지 않고, 예를 들어 열수 추출, 냉침 추출, 초음파 추출, 및 환류 추출 등이 있다. 추출물을 농축할 경우에는, 감압농축, 역삼투압 농축 등의 방법이 사용될 수 있다. 농축 후 건조 단계는 동결건조, 진공건조, 열풍건조, 분무건조, 감압건조, 포말건조, 고주파건조, 적외선건조 등을 포함하나 이에 제한되지 않는다.In addition, the mixed extract may include any one of the extract obtained by the extraction treatment, the diluted or concentrated liquid of the extract, the dried product obtained by drying the extract, the adjusted product, or the purified product. The extraction method is not particularly limited, and examples thereof include hot water extraction, cold extraction, ultrasonic extraction, and reflux extraction. When the extract is concentrated, methods such as concentration under reduced pressure and reverse osmosis can be used. The post-concentration drying step includes, but is not limited to, freeze drying, vacuum drying, hot air drying, spray drying, vacuum drying, foam drying, high frequency drying, infrared drying and the like.

또한, 상기 황칠나무, 헛개나무, 운지버섯, 인진호 및 미나리 혼합 추출물은 황칠나무, 헛개나무, 운지버섯, 인진호 및 미나리를 1~10:1~10:1~10:1~10:1~10의 중량비로 혼합하여 추출한 것일 수 있으며, 바람직하게는 황칠나무, 헛개나무, 운지버섯, 인진호 및 미나리를 1:1:1:1:1의 중량비로 혼합하여 추출한 것일 수 있으나, 이에 제한되지 않는다.The mixed extracts of Hwigyeolgwa, Hwagwon, Yunji mushroom, Injinho, and Buttercup can be used in an amount of 1 to 10: 1 to 10: 1 to 10: 1 to 10: And the mixture may be extracted by mixing the mixture at a weight ratio of 1: 1: 1: 1: 1, but the present invention is not limited thereto.

또한, 상기 단계 1)의 추출 조건은,Further, the extraction condition of the step 1)

(a) 박스 벤켄 계획법(BBD, Box-Behnken design)으로 추출온도(X1), 추출시간(X2), 추출용매:시료의 비율(X3)에 대하여, 5단계의 -2, -1, 0, 1 및 2로 코드화하여 실험범위를 설계하는 단계;(a) For the extraction temperature (X 1 ), extraction time (X 2 ), and extraction solvent: sample ratio (X 3 ) in the Box-Bencken design method (BBD) , 0, 1, and 2 to design the experimental range;

(b) 상기 단계 (a)의 설계된 실험범위로, 상기 황칠나무, 헛개나무, 운지버섯, 인진호 및 미나리 혼합물을 추출하여 추출수율, DPPH 라디칼 소거능, 총 폴리페놀 함량 또는 ROS 생성 억제활성에 대한 실험값을 획득하는 단계;(b) extracting the Hwangbokgol, Hwanggi mushroom, Yinjinho and Mung bean mixture from the experimental range of the step (a) to determine the extract yield, DPPH radical scavenging ability, total polyphenol content or ROS production inhibitory activity ;

(c) 상기 단계 (b)의 실험값을 이용하여 하기 수학식 1~4로 표시되는 이차 회귀식 모델을 도출하는 단계; 및(c) deriving a secondary regression model expressed by the following equations (1) to (4) using the experimental value of the step (b); And

(d) 상기 단계 (c)에서 도출된 수학식 1~4로 표시되는 이차 회귀식 모델을 변랑분석(ANOVA)하여 신뢰도를 입증하는 단계;를 포함하는 것일 수 있으나, 이에 제한되지 않는다.(d) proving reliability by performing a diagonal analysis (ANOVA) on the secondary regression model expressed by Equations 1 to 4 derived in the step (c).

[수학식 1][Equation 1]

Y1 = 92.45 - 1.62X1 + 10.44X2 + 2.21X3 + 0.01X1 2 + 0.13X1X2 - 0.19X2 2 - 0.001X1X3 + 0.1X2X3 - 0.1X3 2 Y 1 = 92.45 - 1.62X 1 + 10.44X 2 + 2.21X 3 + 0.01X 1 2 + 0.13X 1 X 2 - 0.19X 2 2 - 0.001X 1 X 3 + 0.1X 2 X 3 - 0.1X 3 2

[수학식 2]&Quot; (2) "

Y2 = -105.07 + 2.97X1 + 8.77X2 + 1.94X3 - 0.02X1 2 - 0.02X1X2 - 0.13X2 2 + 0.06X1X3 - 0.68X2X3 - 0.17X3 2 Y 2 = -105.07 + 2.97X 1 + 8.77X 2 + 1.94X 3 - 0.02X 1 2 0.02 X 1 X 2 - 0.13X 2 2 + 0.06 X 1 X 3 - 0.68 X 2 X 3 - 0.17 X 3 2

[수학식 3]&Quot; (3) "

Y3 = -32.19 + 1.02X1 + 6.73X2 - 0.36X3 - 0.01X1 2 - 0.05X1X2 + 0.17X2 2 + 0.02X1X3 - 0.44X2X3 + 0.10X3 2 Y 3 = -32.19 + 1.02X 1 + 6.73X 2 - 0.36X 3 - 0.01X 1 2 - 0.05X 1 X 2 + 0.17X 2 2 + 0.02 X 1 X 3 - 0.44 X 2 X 3 + 0.10 X 3 2

[수학식 4]&Quot; (4) "

Y4 = 344.52 - 3.54X1 + 5.77X2 + 4.09X3 + 0.02X1 2 - 0.04X1X2 + 0.04X2 2 + 0.01X1X3 - 0.38X2X3 - 0.07X3 2 Y 4 = 344.52 - 3.54X 1 + 5.77X 2 + 4.09X 3 + 0.02X 1 2 - 0.04X 1 X 2 + 0.04X 2 2 + 0.01X 1 X 3 - 0.38X 2 X 3 - 0.07X 3 2

(상기 수학식 1 내지 4에 있어서, Y1는 추출수율의 예측값(%), Y2는 DPPH 라디칼 소거능의 예측값(%), Y3는 총 폴리페놀 함량의 예측값(mg/g), Y4는 ROS 생성 억제활성의 예측값(%), X1은 추출온도(코드 단위), X2는 추출시간(코드단위) 및 X3는 추출용매:시료 비율(코드단위)를 의미한다).(In the equation 1 to 4, Y 1 is the predicted value (%) of the extraction yield, Y 2 are predicted values (%) of DPPH radical scavenging activity, Y 3 is the predicted value (mg / g) of the total polyphenol content, Y 4 X 1 is the extraction temperature (code unit), X 2 is the extraction time (code unit), and X 3 is the extraction solvent: sample ratio (code unit).

또한, 본 발명은 상기 제조 방법으로 제조된 추출수율, DPPH 라디칼 소거능, 총 폴리페놀 함량 및 ROS(reactive oxygen species) 생성 억제활성이 증진된 황칠나무, 헛개나무, 운지버섯, 인진호 및 미나리 혼합 추출물을 제공한다.In addition, the present invention provides a method for producing an extract of Hwangchil, Houttuynia japonica, Fungus mushroom, Injinho, and Minari, enhanced by the extraction yield, DPPH radical scavenging activity, total polyphenol content and ROS (reactive oxygen species) to provide.

또한, 본 발명은 상기 추출수율, DPPH 라디칼 소거능, 총 폴리페놀 함량 및 ROS(reactive oxygen species) 생성 억제활성이 증진된 황칠나무, 헛개나무, 운지버섯, 인진호 및 미나리 혼합 추출물을 포함하는 음료 조성물을 제공한다.
The present invention also relates to a beverage composition comprising a mixture of Hwangchil, Hovenia dulcis, Echinacea, and Inchinho, which has enhanced the extraction yield, DPPH radical scavenging activity, total polyphenol content and ROS (reactive oxygen species) to provide.

이하, 실시예를 이용하여 본 발명을 더욱 상세하게 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로 본 발명의 범위가 이들에 의해 제한되지 않는다는 것은 당해 기술분야에서 통상의 지식을 가진 자에게 있어 자명한 것이다.
Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not limited thereto.

재료 및 방법Materials and methods

1. 황칠나무, 1. Yellowwood, 헛개나무Hinoki , , 운지버섯Finger Mushroom , , 인진호In Jin-ho 및 미나리 준비 And buttercup preparation

본 발명에서 사용한 헛개나무, 운지버섯, 인진호 및 미나리는 영석영농조합법인으로부터 공급받아 사용하였으며, 황칠나무는 동부생약영농조합에서 보관되어있는 것을 사용하였다. 상기 황칠나무, 헛개나무, 운지버섯, 인진호 및 미나리를 세척한 후 물기를 제거하여 시료로 사용하였다.
The hinoki, the fingi mushroom, the hinjinho, and the buttermilk used in the present invention were supplied from the Yeongseok farming association corporation. The Hwigae-jinja, Houttuynia japonica, Fenugumi mushroom, Injin-jin and Ranunculus were washed and then used as a sample.

2. 최적 추출조건 설정을 위한 실험모델 계획2. Experimental model planning for optimum extraction condition setting

추출조건 설정을 위한 추출물의 추출방법은 1:1:1:1:1의 중량비로 혼합한 황칠나무, 헛개나무, 운지버섯, 인진호 및 미나리 분말 혼합물 1.5kg에 추출 조건별로 물의 첨가량, 추출온도 및 추출시간을 달리하면서 추출한 후, 감압여과하여 황칠나무, 헛개나무, 운지버섯, 인진호 및 미나리 혼합 추출물(하기 '시료'라 함)을 제조하였으며, 각 조건별로 3회 반복 추출하였다.The extracting method for the extraction condition was 1.5kg of mixture of Hwangwoo tree, Houttuynia japonica, Echinho mushroom, Injinhwa and Minnaris powder mixed at a weight ratio of 1: 1: 1: 1: 1, After extracting with different extraction times, the mixture was filtered under reduced pressure to prepare a mixed extract of Hwangchil, Hwanggi, Yunji mushroom, Injinhwa and Minari (hereinafter referred to as "sample").

추출특성의 모니터링과 추출조건의 최적화를 위하여 반응표면분석법(repose surface methodology, RSM)을 이용하였으며, 상기 황칠나무, 헛개나무, 운지버섯, 인진호 및 미나리 혼합 추출물의 최적 추출조건을 확립하기 위해 중심합성계획법(central composite design)을 실시하였다. 중심합성법에 의한 요인(factor; 독립변수)(Xi)의 실험계획은 하기 표 1에 개시한 바와 같이 추출온도(X1), 추출시간(X2) 및 추출용매:시료 비율(X3)를 5단계의 코드값(coded value)(-2, -1, 0, 1, 2)로 부호화하였으며, 중심합성계획에 따라 16구로 설정하여 추출 실험을 실시하였다. 또한, 이들 독립변수에 영향을 받을 종속변수(Yn)로는 추출수율(Y1), DPPH 라디칼 소거능(DPPH radical scavenging activity)(Y2), 총 폴리페놀 함량(TPC; total polyphenol content)(Y3) 및 ROS(reactive oxygen species) 생성 억제활성(Y4)으로 하였으며, 3회 반복 측정하여 평균값을 계산한 후, 회귀분석에 사용하였다. 회귀분석에 의한 최적 조건의 예측은 SAS(STATISTICAL Analysis System) 통계패키지를 이용하였으며, 회귀분석 결과 임계점이 최대점이거나 최소점이 아니고 안장점(saddle point)일 경우에는 능선분석을 하여 최적점을 구하였다. 반응표면분석에서 독립변수의 종속변수에 대한 2차 회귀식은 하기와 같다. In order to monitor the extraction characteristics and to optimize the extraction conditions, the repose surface methodology (RSM) was used. In order to establish optimal extraction conditions for the above extracts of Hwangchu, Houttuynia japonica, A central composite design was implemented. The experimental design of the factor (X i ) by the central synthesis method is shown in Table 1 below as the extraction temperature (X 1 ), extraction time (X 2 ) and extraction solvent: sample ratio (X 3 ) (-1, 0, 1, 2) and coded values were coded in 5 steps. In addition, the dependent variable (Y n ) to be affected by these independent variables are extraction yield (Y 1 ), DPPH radical scavenging activity (Y 2 ), total polyphenol content (TPC) 3 ) and reactive oxygen species (ROS) production inhibitory activity (Y 4 ). The mean value was calculated by repeating the measurement three times and then used for regression analysis. The statistical analysis system (SAS) statistical package was used to predict the optimal conditions by regression analysis. Regression analysis showed that the optimum point was obtained by ridge analysis when the critical point was not the maximum point or the minimum point and the saddle point . The second-order regression equation for dependent variables of independent variables in response surface analysis is as follows.

Y = b0 + b1X1 + b2X2 + b3X3 + b11X2 2 + b21X2X1 + b22X2 2 + b31X3X1 + b32X3X2 + b33X3 2 Y = b 0 + b 1 X 1 + b 2 X 2 + b 3 X 3 + b 11 X 2 2 + b 21 X 2 X 1 + b 22 X 2 2 + b 31 X 3 X 1 + b 32 X 3 X 2 + b 33 X 3 2

(Y: 종속변수, X1, X2 및 X3: 독립변수, b0: 절편, bn: 회귀계수)(Y: dependent variable, X 1 , X 2 and X 3 : independent variable, b 0 : intercept, b n : regression coefficient)

최적 추출조건을 위한 실험 디자인Experimental design for optimal extraction conditions 실험 조건의 개수1 ) Number of experimental conditions 1 ) 추출 조건Extraction condition 추출 온도(X1)The extraction temperature (X 1 ) 추출 시간(X2)Extraction time (X 2 ) 시료에 대한 추출용매비(X3)The extraction solvent ratio (X 3 ) 1One 90 (-1)90 (-1) 4 (-1)4 (-1) 7.5 (-1)7.5 (-1) 22 90 (-1)90 (-1) 4 (-1)4 (-1) 12.5 (+1)12.5 (+1) 33 90 (-1)90 (-1) 8 (+1)8 (+1) 7.5 (-1)7.5 (-1) 44 90 (-1)90 (-1) 8 (+1)8 (+1) 12.5 (+1)12.5 (+1) 55 110 (+1)110 (+1) 4 (-1)4 (-1) 7.5 (-1)7.5 (-1) 66 110 (+1)110 (+1) 4 (-1)4 (-1) 12.5 (+1)12.5 (+1) 77 110 (+1)110 (+1) 8 (+1)8 (+1) 7.5 (-1)7.5 (-1) 88 110 (+1)110 (+1) 8 (+1)8 (+1) 12.5 (+1)12.5 (+1) 99 100 (0)100 (0) 6 (0)6 (0) 10 (0)10 (0) 1010 100 (0)100 (0) 6 (0)6 (0) 10 (0)10 (0) 1111 80 (-2)80 (-2) 6 (0)6 (0) 10 (0)10 (0) 1212 120 (+2)120 (+2) 6 (0)6 (0) 10 (0)10 (0) 1313 100 (0)100 (0) 2 (-2)2 (-2) 10 (0)10 (0) 1414 100 (0)100 (0) 10 (+2)10 (+2) 10 (0)10 (0) 1515 100 (0)100 (0) 6 (0)6 (0) 5 (-2)5 (-2) 1616 100 (0)100 (0) 6 (0)6 (0) 15 (+2)15 (+2)

1) 중심합성계획에 의한 실험 조건의 개수
1) Number of experimental conditions by central synthesis plan

3. 추출 수율 측정3. Measurement of extraction yield

시료의 수율은 항량을 구한 수기에 20mL의 황칠나무, 헛개나무, 운지버섯, 인진호 및 미나리 혼합 추출물을 취하여 105℃에서 증발 건고시킨 후 그 무게를 측정하였으며, 추출물 제조에 사용된 원료량(건물량)에 대한 백분율로서 추출수율(%, total extraction yield)을 나타내었다.
The yield of the sample was determined by weighing 20 mL of Hwangchujang, Houttuynia chinensis, Echinho, and Mulberry mixed extracts at 20 ℃, evaporating at 105 ℃ and weighing them. The amount of raw material (%, Total extraction yield).

4. 4. DPPHDPPH 라디칼  Radical 소거능Scatters (( DPPHDPPH radical scavenging activity) 측정 radical scavenging activity

DPPH 라디칼 소거능 측정은 블로이스의 방법(Blois MS., 1958, Nature, 181:1199-1200)에 따라 조건별 황칠나무, 헛개나무, 운지버섯, 인진호 및 미나리 혼합 추출물의 DPPH(1,1-diphenyl-2-picrylhydrazyl)에 대한 수소공여 효과로 측정하였다. 즉, 일정 농도의 시료 2mL에 2×10-4M의 DPPH(dissolved in 99% ethanol) 용액을 1mL를 가하고, 혼합하여 37℃에서 30분간 반응시킨 후, 흡수분광광도계를 사용하여 517nm에서 흡광도를 측정하였다. DPPH 라디칼 소거능(DPPH radical scavenging activity, %)은 하기 식으로 계산하였으며, 3회 반복 실험하여 얻은 결과를 평균과 표준편차로 나타내었다. The DPPH radical scavenging activity was measured by DPPH (1,1-diphenyl (diphenylphosphino) phenyl) diphenylphosphate (DPPH) -2-picrylhydrazyl). ≪ / RTI > That is, 1 mL of a solution of 2 × 10 -4 M of dissolved in 99% ethanol was added to 2 mL of a certain concentration of the sample, and the mixture was reacted at 37 ° C. for 30 minutes. Then, absorbance at 517 nm was measured using an absorption spectrophotometer Respectively. The DPPH radical scavenging activity (%) was calculated by the following formula, and the results obtained by three repeated experiments were expressed as mean and standard deviation.

DPPH(%)= (대조군(control)의 흡광도-시료의 흡광도)/대조군(control)의 흡광도×100
DPPH (%) = (absorbance of control - absorbance of sample) / absorbance of control × 100

5. 총 폴리페놀 함량(5. Total polyphenol content ( TPCTPC ; total ; total polyphenolpolyphenol content) 측정 content measurement

총 폴리페놀 함량 측정은 Folin-Denis법(Folin O. and Denis W., 1912, J Biol chem. 12:239-249)에 따라 실시하였다. 메탄올에 희석한 25㎕의 시료에 75㎕의 메탄올과 25㎕의 폴린-시오칼투 페놀 시약(Folin-Ciocalteu phenol reagent)을 첨가하여 6분간 실온에서 반응시킨 후, 포화용액 Na2Co3 100㎕를 첨가하여 혼합하고, 90분간 실온에서 방치한 후 765nm에서 흡광도를 측정하였다. 표준물질인 갈릭산(gallic acid)을 사용하여 표준 검량선을 작성하였으며, 추출물의 총 폴리페놀 함량을 mg GAE(gallic acid equivalent)/g로 나타내었다. 표준물질은 갈릭산(gallic acid)을 각각 0.5, 1.0, 5.0, 10, 50 및 100mg/mL로 제조하여 처리한 후 흡광도와 농도와의 관계를 나타내는 표준곡선을 만들어 총 폴리페놀 함량(total polyphenol content)을 나타내었다.
Total polyphenol content was determined according to the Folin-Denis method (Folin O. and Denis W., 1912, J Biol chem. 12: 239-249). 75 μl of methanol and 25 μl of Folin-Ciocalteu phenol reagent were added to 25 μl of the sample diluted in methanol, reacted at room temperature for 6 minutes, and then 100 μl of a saturated solution of Na 2 Co 3 The mixture was allowed to stand at room temperature for 90 minutes, and the absorbance was measured at 765 nm. A standard calibration curve was prepared using gallic acid as a standard substance, and the total polyphenol content of the extract was expressed as mg GAE (gallic acid equivalent) / g. The standard material was prepared by preparing gallic acid at 0.5, 1.0, 5.0, 10, 50 and 100 mg / mL, respectively, and then a standard curve showing the relationship between absorbance and concentration was prepared to obtain total polyphenol content ).

6. 6. ROSROS (reactive oxygen species) 생성 억제활성 측정(reactive oxygen species) production inhibitory activity measurement

세포 내에서 산화 스트레스에 의해 발생하는 ROS 생성에 대한 본 발명의 혼합 추출물의 억제활성을 알아보기 위해, HepG2 세포에 t-BHP로 산화적 스트레스를 유도한 뒤 DCFH-DA(2',7'-dichlorodihydrofluorescein diacetate)를 이용하여 ROS의 양에 따른 형광의 발생 정도를 측정하였다. 구체적으로, HepG2 세포(1×104cells/well)를 CO2 배양기 내에서 24시간 배양한 후, 본 발명의 혼합 추출물을 전처리하여 12시간 후 간독성 유발물질인 t-BHP을 50μM의 농도로 처리하고 12시간 동안 CO2 배양기에서 배양하였으며, 배양된 세포를 PBS로 세척한 후, 10μM의 DCFH-DA를 포함하는 HBSS(Hank's balanced salt solution)에서 30분 동안 반응시킨 후 세포의 형광광도(SpectramaxGemini XS, Molecular Devices, Sunnyvale, CA, U.S.A.)를 측정하였다.
In order to investigate the inhibitory activity of the mixed extract of the present invention against the production of ROS caused by oxidative stress in the cells, oxidative stress was induced in HepG2 cells by t-BHP, and DCFH-DA (2 ', 7'- dichlorodihydrofluorescein diacetate) was used to measure the amount of fluorescence produced by the amount of ROS. Specifically, HepG2 cells (1 × 10 4 cells / well) were cultured in a CO 2 incubator for 24 hours. Pretreatment of the mixed extract of the present invention was conducted to treat t -BHP, a hepatotoxicity inducing substance, at a concentration of 50 μM And incubated for 12 hours in a CO 2 incubator. The cultured cells were washed with PBS and reacted in HBSS (Hank's balanced salt solution) containing 10 μM of DCFH-DA for 30 minutes. Then, the fluorescence intensity of the cells (Spectramax Gemini XS , Molecular Devices, Sunnyvale, CA, USA).

실시예Example 1. 모델 구축 및 통계분석 1. Model building and statistical analysis

본 발명의 황칠나무, 헛개나무, 운지버섯, 인진호 및 미나리 혼합 추출물의 최적 추출조건을 확립하기 위해, 중심합성계획법(central composite design)을 실시하여 독립변수(Xi)인 추출온도(X1), 추출시간(X2) 및 추출용매:시료 비율(X3)에 따른 추출수율, DPPH 라디칼 소거능, 총 폴리페놀 함량 및 ROS 생성 억제활성 결과를 얻을 수 있었다(표 2). To establish hwangchil wood heotgae wood fingering mushrooms, injinho and optimum extraction of parsley mixed extract conditions of the present invention, by implementing the central composite design (central composite design) the independent variable (X i) the extraction temperature (X 1) , DPPH radical scavenging ability, total polyphenol content and ROS production inhibition activity were obtained according to the extraction time (X 2 ) and the extraction solvent: sample ratio (X 3 ) (Table 2).

추출온도, 추출시간 및 추출용매:시료 비율에 따른 추출수율, DPPH 라디칼 소거능, 총 폴리페놀 함량 및 ROS 생성 억제활성 측정 결과Extraction temperature, extraction time and extraction solvent: extraction yield according to sample ratio, DPPH radical scavenging ability, total polyphenol content and ROS production inhibitory activity 실험 조건의 개수1) Number of experimental conditions 1) 추출조건Extraction condition 추출
수율(%)
(Y1)extraction
yield(%)
(Y 1 ) DPPH
소거능(%)
(Y2)DPPH
Scavenging ability (%)
(Y 2 ) TPC
(mg/g)
(Y3)TPC
(mg / g)
(Y 3) ROS
억제활성(%)
(Y4)ROS
Inhibitory activity (%)
(Y 4 ) 온도
(X1)Temperature
(X 1 ) 시간
(X2)time
(X 2 ) 추출용매비
(X3)Extraction solvent ratio
(X 3) 1One 9090 44 7.57.5 8.738.73 77.1077.10 32.3132.31 190.32190.32 22 9090 44 12.512.5 11.6811.68 81.3581.35 42.7042.70 196.31196.31 33 9090 88 7.57.5 7.437.43 80.1480.14 41.7441.74 187.86187.86 44 9090 88 12.512.5 10.3810.38 71.4171.41 40.1940.19 190.44190.44 55 110110 44 7.57.5 11.4911.49 69.3869.38 34.3434.34 172.35172.35 66 110110 44 12.512.5 12.4012.40 80.5380.53 43.5143.51 183.86183.86 77 110110 88 7.57.5 18.5718.57 71.6471.64 36.7536.75 171.33171.33 88 110110 88 12.512.5 23.4623.46 68.4868.48 40.0940.09 170.86170.86 99 100100 66 1010 16.3316.33 74.7174.71 38.7438.74 176.56176.56 1010 100100 66 1010 15.2215.22 76.3976.39 38.1438.14 175.36175.36 1111 8080 66 1010 12.1612.16 64.8164.81 37.1437.14 190.31190.31 1212 120120 66 1010 24.0524.05 72.2372.23 36.0136.01 173.63173.63 1313 100100 22 1010 7.227.22 80.7280.72 47.6047.60 180.32180.32 1414 100100 1010 1010 18.0618.06 66.1866.18 34.7534.75 172.86172.86 1515 100100 66 55 8.508.50 65.2065.20 37.5537.55 162.65162.65 1616 100100 66 1515 18.0918.09 77.4577.45 44.5044.50 185.87185.87

1) 중심합성계획에 의한 실험 조건의 개수
1) Number of experimental conditions by central synthesis plan

이러한 결과를 바탕으로 반응표면 회귀분석을 실시하였으며, 각 종속변수(Yn)인 추출수율(Y1), DPPH 라디칼 소거능(DPPH radical scavenging activity)(Y2), 총 폴리페놀 함량(TPC; total polyphenol content)(Y3) 및 ROS 생성 억제활성(Y4)에 대한 회귀식을 얻을 수 있었다.
Was performed and the reaction surface regression analysis on the basis of this result, each of the dependent variable (Y n) the extraction yield (Y 1), DPPH radical scavenging activity (DPPH radical scavenging activity) (Y 2), polyphenol content (TPC; total regression equations for polyphenol content (Y 3 ) and ROS production inhibitory activity (Y 4 ) were obtained.

(1) 추출 조건에 따른 수율 변화 확인(1) Confirmation of yield change according to extraction conditions

각 추출 조건(독립변수; Xi)에 따른 황칠나무, 헛개나무, 운지버섯, 인진호 및 미나리 혼합 추출물의 추출수율(Y1)을 측정한 결과, 7.22~24.05%의 범위로 측정되었으며(표 2), 이에 대한 반응표면 회귀식은 하기와 같다. 회귀식의 R2의 값은 0.9432로 높은 상관관계를 보였으며, 5% 이내의 범위에서 유의성이 인정되는 것으로 확인되었다(표 3). 또한, 도 1 및 표 4에 개시한 바와 같이 각 추출조건에 따른 수율 변화의 반응표면 분석 결과, 추출온도에 큰 영향을 받는 것으로 나타났으며, 예측된 정상점에 대해 안장점(saddle point)으로 능선분석(ridge analysis)을 실시한 결과, 하기 표 3에 개시한 바와 같이 최대값은 28.68%였으며, 이때의 추출조건은 116.43℃의 추출온도, 8.13시간의 추출시간 및 11:1의 추출용매:시료 비율로 확인되었다.The extraction yields (Y 1 ) of the extracts of Hwangchujigyeo, Houttuynia japonica, Yunji mushroom, Injinhwa and Minari were measured in the range of 7.22 ~ 24.05% according to each extraction condition (independent variable; X i ) ), And the reaction surface regression equation is as follows. The R 2 value of the regression equation was 0.9432, which was highly correlated with significance within the range of 5% (Table 3). Also, as shown in FIG. 1 and Table 4, the reaction surface analysis of the yield change according to each extraction condition was found to be strongly influenced by the extraction temperature, and the saddle point As a result of ridge analysis, the maximum value was 28.68% as shown in Table 3. The extraction conditions were 116.43 ° C extraction time, 8.13 hours extraction time, and 11: 1 extraction solvent: sample Respectively.

Y1 = 92.45 - 1.62X1 + 10.44X2 + 2.21X3 + 0.01X1 2 + 0.13X1X2 - 0.19X2 2 - 0.001X1X3 + 0.1X2X3 - 0.1X3 2
Y 1 = 92.45 - 1.62X 1 + 10.44X 2 + 2.21X 3 + 0.01X 1 2 + 0.13X 1 X 2 - 0.19X 2 2 - 0.001 X 1 X 3 + 0.1 X 2 X 3 - 0.1X 3 2

반응표면분석 범위에 따른 최대 및 최소 반응의 최적 추출조건의 예상값Estimation of Optimum Extraction Condition of Maximum and Minimum Response according to Response Surface Analysis Range 반응reaction R2 R 2 유효성effectiveness 추출조건Extraction condition 예상값Expected value 형태
(Morphology)shape
(Morphology) 온도(X1)Temperature (X 1 ) 시간(X2)Time (X 2 ) 추출용매비(X3)The extraction solvent ratio (X 3 ) 수율(%)yield(%) 0.94320.9432 0.00420.0042 87.11887687.118876 7.9026517.902651 7.0044347.004434 6.166568
(MIN)6.166568
(MIN) Saddle pointSaddle point 116.434424116.434424 8.1316648.131664 11.00963911.009639 28.682258
(MAX)28.682258
(MAX) DPPH 라디칼
소거능(%)DPPH radical
Scavenging ability (%) 0.70570.7057 0.29240.2924 95.00214295.002142 9.0207459.020745 13.03004613.030046 64.803263
(MIN)64.803263
(MIN) Saddle pointSaddle point 103.030195103.030195 2.9610412.961041 13.16164613.161646 87.665673
(MAX)87.665673
(MAX) TPC(mg/g)TPC (mg / g) 0.71840.7184 0.26670.2667 118.138603118.138603 7.1216967.121696 8.4280598.428059 33.863028
(MIN)33.863028
(MIN) Saddle pointSaddle point 102.920407102.920407 3.3257463.325746 13.64589113.645891 50.520560
(MAX)50.520560
(MAX) ROS
억제활성(%)ROS
Inhibitory activity (%) 0.80770.8077 0.11140.1114 110.320794110.320794 5.8798055.879805 5.7198135.719813 165.105426
(MIN)165.105426
(MIN) Saddle pointSaddle point 81.34717181.347171 5.2451245.245124 11.53757111.537571 198.564855
(MAX)198.564855
(MAX)

추출조건에 따른 회귀모델에 대한 변수분석Analysis of Variables for Regression Model Based on Extraction Condition 추출조건Extraction condition F-valueF-value 수율(%)yield(%) DPPH 라디칼
소거능(%)DPPH radical
Scavenging ability (%) TPC(mg/g)TPC (mg / g) ROS 생성 억제활성(%)ROS production inhibitory activity (%) 온도(X1)Temperature (X 1 ) 13.82** 13.82 ** 0.740.74 0.300.30 3.863.86 시간(X2)Time (X 2 ) 9.35** 9.35 ** 2.362.36 1.671.67 0.700.70 시료에 대한 추출용매비(X3)The extraction solvent ratio (X 3 ) 4.164.16 1.851.85 2.642.64 1.781.78

**은 5% 레벨에서의 유의성(significant)
** is significant at the 5% level,

(2) 추출 조건에 따른 (2) Depending on extraction conditions DPPHDPPH 라디칼  Radical 소거능Scatters 변화 확인 Confirm change

각 추출 조건(독립변수)에 따른 황칠나무, 헛개나무, 운지버섯, 인진호 및 미나리 혼합 추출물의 DPPH 라디칼 소거능(Y2)을 측정한 결과, 64.81~81.35%의 범위로 측정되었으며(표 2), 이에 대한 반응표면 회귀식은 하기와 같다. 회귀식의 R2의 값은 0.7057으로 나타났으며(표 3), 도 2 및 표 4에 개시한 바와 같이 각 추출조건에 따른 DPPH 라디칼 소거능 변화의 반응표면 분석 결과, 추출시간에 큰 영향을 받는 것으로 나타났으며, 예측된 정상점에 대해 안장점(saddle point)으로 능선분석(ridge analysis)을 실시한 결과, 표 3에 개시한 바와 같이 최대값은 87.66%였으며, 이때의 추출조건은 103.03℃의 추출온도, 2.96시간의 추출시간 및 13.16:1의 추출용매:시료 비율로 확인되었다. The DPPH radical scavenging ability (Y 2 ) of the mixed extracts of Hwangchujang, Hovenia dulcis, Echinho and Minari was measured in the range of 64.81 ~ 81.35% according to each extraction condition (independent variable) (Table 2) The reaction surface regression equation is as follows. The R 2 value of the regression equation was 0.7057 (Table 3). As shown in FIG. 2 and Table 4, the response surface analysis of the DPPH radical scavenging effect according to each extraction condition revealed that the As shown in Table 3, the maximum value was 87.66%, and the extraction condition was 103.03 ° C. As shown in Table 3, ridge analysis was performed at a saddle point with respect to the predicted normal point. Extraction temperature, extraction time of 2.96 hours and extraction solvent: sample ratio of 13.16: 1.

Y2 = -105.07 + 2.97X1 + 8.77X2 + 1.94X3 - 0.02X1 2 - 0.02X1X2 - 0.13X2 2 + 0.06X1X3 - 0.68X2X3 - 0.17X3 2
Y 2 = -105.07 + 2.97X 1 + 8.77X 2 + 1.94X 3 - 0.02X 1 2 0.02 X 1 X 2 - 0.13X 2 2 + 0.06 X 1 X 3 - 0.68 X 2 X 3 - 0.17 X 3 2

(3) 추출 조건에 따른 총 폴리페놀 함량((3) Total polyphenol content by extraction condition ( TPCTPC )의 변화 확인) Change confirmation

각 추출 조건에 따른 황칠나무, 헛개나무, 운지버섯, 인진호 및 미나리 혼합 추출물의 총 폴리페놀 함량(Y2)의 결과, 32.31mg/g~47.60mg/g의 범위로 측정되었으며, 이에 대한 반응표면 회귀식은 하기와 같다. 회귀식의 R2의 값은 0.7184로 나타났으며(표 3), 도 3 및 표 4에 개시한 바와 같이 각 추출조건에 따른 총 폴리페놀 함량 변화의 반응표면 분석 결과, 시료에 대한 추출용매비가 큰 영향을 받는 것으로 나타났으며, 예측된 정상점에 대해 안장점(saddle point)으로 능선분석(ridge analysis)을 실시한 결과, 표 3에 개시한 바와 같이 최대값은 50.52%였으며, 이때의 추출조건은 102.92℃의 추출온도, 3.32시간의 추출시간 및 13.64:1의 추출용매:시료 비율로 확인되었다. The total polyphenol contents (Y 2 ) of the mixed extracts of Hwangchujigyeo, Hovenia dulcis, Mulberry Mushroom, Injinhwa and Minari were measured in the range of 32.31mg / g to 47.60mg / g, The regression equation is as follows. The R 2 value of the regression equation was 0.7184 (Table 3). As shown in FIGS. 3 and 4, the reaction surface analysis of the total polyphenol content changes according to each extraction condition revealed that the extraction solvent ratio As shown in Table 3, the maximum value was 50.52%. As a result of the ridge analysis using the saddle point for the predicted normal point, the extraction condition Was determined at an extraction temperature of 102.92 占 폚, an extraction time of 3.32 hours, and an extraction solvent: sample ratio of 13.64: 1.

Y3 = -32.19 + 1.02X1 + 6.73X2 - 0.36X3 - 0.01X1 2 - 0.05X1X2 + 0.17X2 2 + 0.02X1X3 - 0.44X2X3 + 0.10X3 2
Y3= -32.19 + 1.02XOne + 6.73X2 - 0.36X3 - 0.01XOne 2 - 0.05XOneX2 + 0.17X2 2 + 0.02XOneX3- 0.44X2X3+ 0.10X3 2

(4) 추출 조건에 따른 ROS 생성 억제활성 변화 확인(4) Determination of ROS production inhibitory activity according to extraction conditions

세포 내에서 산화 스트레스에 의해 발생하는 ROS(reactive oxygen species) 생성 억제 활성을 황칠나무, 헛개나무, 운지버섯, 인진호 및 미나리 혼합 추출물의 각 추출 조건에 따라 제조하여 확인한 결과, 162.65~196.31%의 범위로 측정되었으며, 이에 대한 반응표면 회귀식은 하기와 같다. 회귀식의 R2의 값은 0.8077로 나타났으며(표 3), 도 4 및 표 4에 개시한 바와 같이 각 추출조건에 따른 ROS 생성 억제 활성 변화의 반응표면 분석 결과, 추출온도에 큰 영향을 받는 것으로 확인되었으며, 예측된 정상점에 대해 안장점(saddle point)으로 능선분석(ridge analysis)을 실시한 결과, 표 3에 개시한 바와 같이 최대값은 198.56%로 이때의 추출조건은 81.35℃의 추출온도, 5.25시간의 추출시간 및 11.54:1의 추출용매:시료 비율로 확인되었다.The inhibitory activity of ROS (reactive oxygen species) formation induced by oxidative stress in the cells was investigated according to the extraction conditions of Hwangchil, Hovenia dulcis, Echinho, And the reaction surface regression equation is as follows. The R 2 value of the regression equation was 0.8077 (Table 3). As shown in FIG. 4 and Table 4, the reaction surface analysis of the ROS production inhibitory activity according to each extraction condition showed that the extraction temperature had a large effect on the extraction temperature As shown in Table 3, the maximum value was 198.56%, and the extraction condition at this time was 81.35 ° C. extraction, as shown in Table 3. As a result of ridge analysis, Temperature, an extraction time of 5.25 hours and an extraction solvent: sample ratio of 11.54: 1.

Y4 = 344.52 - 3.54X1 + 5.77X2 + 4.09X3 + 0.02X1 2 - 0.04X1X2 + 0.04X2 2 + 0.01X1X3 - 0.38X2X3 - 0.07X3 2 Y 4 = 344.52 - 3.54X 1 + 5.77X 2 + 4.09X 3 + 0.02X 1 2 - 0.04X 1 X 2 + 0.04X 2 2 + 0.01X 1 X 3 - 0.38X 2 X 3 - 0.07X 3 2

Claims (5)

1) 물, C1~C4의 저급알코올 또는 이들의 혼합 용매를 추출용매로 사용하고, 80~120℃의 추출온도, 2~10시간의 추출시간 및 5~15:1의 추출용매(㎖):시료(g)의 비율의 추출 조건으로, 황칠나무, 헛개나무, 운지버섯, 인진호 및 미나리 혼합물을 추출하는 단계;
2) 상기 단계 1)에서 추출한 황칠나무, 헛개나무, 운지버섯, 인진호 및 미나리 혼합 추출물을 여과하는 단계; 및
3) 상기 단계 2)의 여과물을 감압농축하는 단계;를 포함하며, 추출수율, DPPH 라디칼 소거능, 총 폴리페놀 함량 및 ROS(reactive oxygen species) 생성 억제활성이 증진된 황칠나무, 헛개나무, 운지버섯, 인진호 및 미나리 혼합 추출물의 제조 방법.1) water, a C 1 -C 4 lower alcohol or a mixed solvent thereof is used as an extraction solvent, and an extraction temperature of 80 to 120 ° C, an extraction time of 2 to 10 hours and an extraction solvent of 5 to 15: 1 ): Extracting a mixture of Hwangchujang, Hovenia dulcis, Fungus mushroom, Injinhwa and Muller mixture as the extraction condition of the ratio of the sample (g);
2) filtering out the mixed extract of Hwangchil, Hovenia dulcis, Echinacea, Injinho and Minnaris extracted in step 1); And
3) The step of concentrating the filtrate of step 2) under reduced pressure, wherein the extraction yield, the DPPH radical scavenging activity, the total polyphenol content and the reactive oxygen species (ROS) Mushroom, hinjinho and parsley mixed extract. 제1항에 있어서, 상기 황칠나무, 헛개나무, 운지버섯, 인진호 및 미나리 혼합 추출물은 황칠나무, 헛개나무, 운지버섯, 인진호 및 미나리를 1~10:1~10:1~10:1~10:1~10의 중량비로 혼합하여 추출한 것을 특징으로 하는 추출수율, DPPH 라디칼 소거능, 총 폴리페놀 함량 및 ROS(reactive oxygen species) 생성 억제활성이 증진된 황칠나무, 헛개나무, 운지버섯, 인진호 및 미나리 혼합 추출물의 제조 방법.The method according to claim 1, wherein the mixed extract of Hwangbok, Hwanggi, Yunji mushroom, Injinbyeo and Minari is used at a ratio of 1 to 10: 1 to 10: 1 to 10: 1 to 10 : 1 to 10, wherein the extract yield, DPPH radical scavenging activity, total polyphenol content, and reactive oxygen species (ROS) production inhibitory activity are enhanced. A method for producing a mixed extract. 제1항에 있어서, 상기 단계 1)의 추출 조건은,
(a) 박스 벤켄 계획법(BBD, Box-Behnken design)으로 추출온도(X1), 추출시간(X2), 추출용매:시료의 비율(X3)에 대하여, 5단계의 -2, -1, 0, 1 및 2로 코드화하여 실험범위를 설계하는 단계;
(b) 상기 단계 (a)의 설계된 실험범위로, 상기 황칠나무, 헛개나무, 운지버섯, 인진호 및 미나리 혼합물을 추출하여 추출수율, DPPH 라디칼 소거능, 총 폴리페놀 함량 또는 ROS 생성 억제활성에 대한 실험값을 획득하는 단계;
(c) 상기 단계 (b)의 실험값을 이용하여 하기 수학식 1~4로 표시되는 이차 회귀식 모델을 도출하는 단계; 및
(d) 상기 단계 (c)에서 도출된 수학식 1~4로 표시되는 이차 회귀식 모델을 변랑분석(ANOVA)하여 신뢰도를 입증하는 단계;를 포함하는 것을 특징으로 하는 추출수율, DPPH 라디칼 소거능, 총 폴리페놀 함량 및 ROS(reactive oxygen species) 생성 억제활성이 증진된 황칠나무, 헛개나무, 운지버섯, 인진호 및 미나리 혼합 추출물의 제조 방법:
[수학식 1]
Y1 = 92.45 - 1.62X1 + 10.44X2 + 2.21X3 + 0.01X1 2 + 0.13X1X2 - 0.19X2 2 - 0.001X1X3 + 0.1X2X3 - 0.1X3 2
[수학식 2]
Y2 = -105.07 + 2.97X1 + 8.77X2 + 1.94X3 - 0.02X1 2 - 0.02X1X2 - 0.13X2 2 + 0.06X1X3 - 0.68X2X3 - 0.17X3 2
[수학식 3]
Y3 = -32.19 + 1.02X1 + 6.73X2 - 0.36X3 - 0.01X1 2 - 0.05X1X2 + 0.17X2 2 + 0.02X1X3 - 0.44X2X3 + 0.10X3 2
[수학식 4]
Y4 = 344.52 - 3.54X1 + 5.77X2 + 4.09X3 + 0.02X1 2 - 0.04X1X2 + 0.04X2 2 + 0.01X1X3 - 0.38X2X3 - 0.07X3 2
(상기 수학식 1 내지 4에 있어서, Y1는 추출수율의 예측값(%), Y2는 DPPH 라디칼 소거능의 예측값(%), Y3는 총 폴리페놀 함량의 예측값(mg/g), Y4는 ROS 생성 억제활성의 예측값(%), X1은 추출온도(코드 단위), X2는 추출시간(코드단위) 및 X3는 추출용매:시료 비율(코드단위)를 의미한다).The method according to claim 1, wherein the extraction condition of step (1)
(a) For the extraction temperature (X 1 ), extraction time (X 2 ), and extraction solvent: sample ratio (X 3 ) in the Box-Bencken design method (BBD) , 0, 1, and 2 to design the experimental range;
(b) extracting the Hwangbokgol, Hwanggi mushroom, Yinjinho and Mung bean mixture from the experimental range of the step (a) to determine the extract yield, DPPH radical scavenging ability, total polyphenol content or ROS production inhibitory activity ;
(c) deriving a secondary regression model expressed by the following equations (1) to (4) using the experimental value of the step (b); And
and (d) proving the reliability by performing a diagonal analysis (ANOVA) on the secondary regression model expressed by Equations (1) to (4) derived in the step (c). The DPPH radical scavenging ability, Production of mixed extracts of Hwangchil, Hovenia dulcis, Fengji mushroom, Injinhwa and Minari, with increased total polyphenol content and reactive oxygen species (ROS)
[Equation 1]
Y 1 = 92.45 - 1.62X 1 + 10.44X 2 + 2.21X 3 + 0.01X 1 2 + 0.13X 1 X 2 - 0.19X 2 2 - 0.001 X 1 X 3 + 0.1 X 2 X 3 - 0.1 X 3 2
&Quot; (2) "
Y 2 = -105.07 + 2.97X 1 + 8.77X 2 + 1.94X 3 - 0.02X 1 2 0.02 X 1 X 2 - 0.13X 2 2 + 0.06 X 1 X 3 - 0.68 X 2 X 3 - 0.17 X 3 2
&Quot; (3) "
Y 3 = -32.19 + 1.02X 1 + 6.73X 2 - 0.36X 3 - 0.01X 1 2 - 0.05X 1 X 2 + 0.17X 2 2 + 0.02X 1 X 3 - 0.44X 2 X 3 + 0.10X 3 2
&Quot; (4) "
Y 4 = 344.52 - 3.54X 1 + 5.77X 2 + 4.09X 3 + 0.02X 1 2 - 0.04X 1 X 2 + 0.04X 2 2 + 0.01X 1 X 3 - 0.38X 2 X 3 - 0.07X 3 2
(In the equation 1 to 4, Y 1 is the predicted value (%) of the extraction yield, Y 2 are predicted values (%) of DPPH radical scavenging activity, Y 3 is the predicted value (mg / g) of the total polyphenol content, Y 4 X 1 is the extraction temperature (code unit), X 2 is the extraction time (code unit), and X 3 is the extraction solvent: sample ratio (code unit). 제1항 내지 제3항 중 어느 한 항의 제조 방법으로 제조된 추출수율, DPPH 라디칼 소거능, 총 폴리페놀 함량 및 ROS(reactive oxygen species) 생성 억제활성이 증진된 황칠나무, 헛개나무, 운지버섯, 인진호 및 미나리 혼합 추출물.A method for producing the extracts of DPPH radical scavenging activity, total polyphenol content, and reactive oxygen species (ROS) production inhibitory activity by the production method according to any one of claims 1 to 3, And parsley mixed extract. 제4항의 추출수율, DPPH 라디칼 소거능, 총 폴리페놀 함량 및 ROS(reactive oxygen species) 생성 억제활성이 증진된 황칠나무, 헛개나무, 운지버섯, 인진호 및 미나리 혼합 추출물을 포함하는 음료 조성물.A beverage composition comprising the extract of Hwanghaek, Hinoki, Fenghi mushroom, Injinhwa, and Minari mixed with the extract of claim 4, wherein DPPH radical scavenging activity, total polyphenol content and reactive oxygen species (ROS) production inhibitory activity are enhanced.
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