KR100401472B1 - Functional beverage containing extract of Artermisia capillaris THUNB and process for preperation thereof - Google Patents
Functional beverage containing extract of Artermisia capillaris THUNB and process for preperation thereof Download PDFInfo
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- KR100401472B1 KR100401472B1 KR10-2000-0086199A KR20000086199A KR100401472B1 KR 100401472 B1 KR100401472 B1 KR 100401472B1 KR 20000086199 A KR20000086199 A KR 20000086199A KR 100401472 B1 KR100401472 B1 KR 100401472B1
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- South Korea
- Prior art keywords
- extract
- weight
- phosphorus
- mugwort
- injin
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- A—HUMAN NECESSITIES
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- A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
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- A—HUMAN NECESSITIES
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- A23L21/00—Marmalades, jams, jellies or the like; Products from apiculture; Preparation or treatment thereof
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- A—HUMAN NECESSITIES
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- A—HUMAN NECESSITIES
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- A—HUMAN NECESSITIES
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Abstract
본 발명은 인진쑥 추출물 함유 기능성 음료 및 그 제조방법에 관한 것으로, 인진 추출물 제조시 밀폐하지 않은 용기에서 90∼110℃의 저온으로 인진과 정제수를 넣어 가열한 후 환류냉각장치를 이용하여 냉각시켜 인진쑥 추출물을 획득하고 여기에 단백질 분해효소를 첨가하여 부유물을 제거한 후 감압농축함으로써 향취미가 뛰어나고 맛이 부드러우며 침전물의 양이 적어 관능이 뛰어나며, 상대적으로 많은 양의 인진 추출물을 음료에 첨가할 수 있어 기호성 및 관능이 개선된 인진 추출물을 획득하였으며 이러한 인진쑥 추출물 10∼50중량%, 산미제 0.01∼0.50중량%, 감미제 5.0∼10.0중량%, 과일농축액 1.0∼5.0중량% 및 벌꿀 0∼10중량% 등을 혼합하여 제조한 본 발명 인진쑥 추출물 함유 기능성 음료는 혈관이완효과, 뇌졸중 예방효과 및 항돌연변원성에 의한 항암효과를 높일 수 있는 뛰어난 효과가 있다.The present invention relates to a functional beverage containing jinjin mugwort extract and a method for preparing the same, jinjin extract extracted by heating phosphorus and purified water at a low temperature of 90 ~ 110 ℃ in an unsealed container when the ginjin extract is prepared by cooling using a reflux cooling device After removing the suspended solids by adding proteolytic enzymes to it, and concentrating under reduced pressure, it has excellent taste, soft taste, and small amount of sediment, which makes it excellent in sensory taste. And the phosphorus extract with improved organoleptic extract, such as 10 to 50% by weight of the extract of Injin mugwort, 0.01 to 0.50% by weight of acidulant, 5.0 to 10.0% by weight of sweetener, 1.0 to 5.0% by weight of fruit concentrate, and 0 to 10% by weight of honey. Functional drink containing the present invention Injin mugwort extract prepared by mixing the vascular relaxation effect, stroke prevention effect and anti-mutagenicity There are excellent effects that can increase cancer effect.
Description
본 발명은 인진쑥 추출물 함유 기능성 음료 및 그 제조방법에 관한 것이다. 더욱 상세하게는, 본 발명은 인진쑥의 유효성분 및 기능성을 조사한 결과 혈관 이완 효과, 국소뇌혈류량의 증가로 인한 뇌졸중 예방 효과 및 항돌연변이원성에 의한 항암 효과가 있는 것으로 판명된 인진쑥을 다양한 조건 및 방법으로 물 추출하여 인진쑥 추출물을 획득하고, 상기 인진쑥 추출물을 첨가함으로써 혈관 이완 효과, 뇌졸중 예방 효과 및 항암효과가 탁월하고 관능면에서도 우수한 인진쑥 추출물 함유 기능성 음료 및 그 제조방법에 관한 것이다.The present invention relates to a functional beverage containing jinjin wormwood extract and a method for producing the same. More specifically, the present invention is a study on the active ingredient and functionality of Injin mugwort as a result of vascular relaxation effect, prevention of stroke due to the increase of local cerebral blood flow and anti-mutagenic anti-cancer effect that turned out to be various conditions and methods By extracting the water to obtain Injin mugwort extract, and the addition of the Injin mugwort extract relates to a functional drink containing the extract of Injin mugwort extract excellent in the vascular relaxation effect, stroke prevention effect and anticancer effect and excellent in terms of sensory function and its preparation method.
쑥은 우리나라 전역에 자생하는 식용식물로 단백질, 지방질, 섬유질, 당질 칼슘, 인, 비타민 A, B, C가 풍부한 식품자원으로 예로부터 나물, 쑥국, 죽 및 쑥떡 등의 음식으로 널리 섭취되어 왔다. 이 중 인진 (茵蔯)은 국화과 쑥속에 속하는 다년생 초본형 낙엽관목으로, 생당쑥, 애당쑥, 사철쑥, 인진초, 더위지기 (Artemisia iwayomogi), 흰산쑥 (Artemisa sacrorum subsp.Kitamura), 털산쑥 (Artemisa sacrorum subsp. manshuria var.vestita Kitamura) 등으로 불리워지며 특히, 어린잎을 인진 또는 더위지기라 부르는데 그 특유의 향기와 약효로 인하여 전통적으로 민간에서 식용, 약용, 단방약으로 애용하여 왔다. 동의보감과 본초강목에서는 손상된 간 회복, 지방간, 간암, 간염, 황달 등의 간 질환의 치료 및 예방, 식욕의 증진, 소화불량, 위장병 등의 위장질환의 치료, 중풍, 혈액순환, 청혈작용 등의 순환기계 기능의 개선 및 당뇨병의 치료 등에 그 효능이 있다고 기록하고 있다. 최근에는 더위지기 쑥이 흰쥐의 혈청지질, 간지질 저하효과 및 발암억제효과가 있음이 보고되었다 [함승시, 한국식품영양학과학회지 27, 338. 157 (1998)]. 또한 인진쑥의 지표성분으로 쿠마린 화합물인 메틸에스쿠레틴과 스코폴린 성분이 분리동정되었다 [서울대학교 천연물과학연구소 연구보고서 (1993)]. 인진에 함유되어 있는 주성분은 배당체인 스코폴레틴 (scopoletin)을 비롯한 정유성분 방향족 oxycarbonic acid, 사포닌 (saponin), 탄닌 (tannin), sitosterin resin 등으로, 해독, 항균, 면역 등의 효과가 예상되며 혈청지질의 감소효과로 고혈압, 당뇨, 비만, 뇌졸중 등의 치료와 예방에 효과가 보고되어 있다. 이러한 인진을 이용한 가공품으로는 생당쑥엿, 편, 환 또는 추출차, 과립차 등의 제품이 개발되어 있는 실정이다.Mugwort is an edible plant native to Korea, and is a food resource rich in protein, fat, fiber, saccharide calcium, phosphorus, vitamins A, B, and C. It has been widely consumed as foods such as herbs, mugwort, porridge, and mugwort rice cakes since ancient times. Among them, Injin is a perennial herbaceous deciduous shrub belonging to the genus Asteraceae, raw mugwort, Asteraceae, Evergreen mugwort, Injincho, heather ( Artemisia iwayomogi ), Artemisa sacrorum subsp.Kitamura, and Tulsan mugwort ( Artemisa sacrorum subsp.manshuria var.vestita Kitamura), and young leaves are called jinjin or heather, and they have traditionally been used as edible, medicinal, and herbal medicines in the private sector. In Dongbobom and Herbal Medicine, the circulation of damaged liver recovery, fatty liver, liver cancer, hepatitis, jaundice and other liver diseases, improvement of appetite, indigestion, gastrointestinal diseases such as gastrointestinal diseases, stroke, blood circulation, blood circulation It has been reported to be effective in improving mechanical function and treating diabetes. Recently, it has been reported that heat wormwood mugwort has the effect of lowering serum lipids, liver lipids and carcinogenesis in rats [Ham Seung-si, Journal of the Korean Society of Food Science and Nutrition 27, 338. 157 (1998)]. In addition, coumarin compounds, methyl escurin and scopoline, were identified as indicators of Injin mugwort [Seoul National University Research Report (1993)]. The main ingredients in phosphorus are essential oils such as glycoside scopoletin, aromatic oxycarbonic acid, saponin, tannin, and sitosterin resin, which are expected to have detoxification, antibacterial and immunity effects. It has been reported to be effective in the treatment and prevention of hypertension, diabetes, obesity, stroke, etc. as a reduction of lipids. As processed products using such phosphorus, products such as raw mugwort syrup, flakes, pill or extract tea, and granular tea have been developed.
현대인은 각종 환경오염 및 불가피한 위해 요소의 접촉으로 인한 과도한 정신적, 신체적 스트레스가 지속되어 생활에 위협을 받고 있다. 이에 따라 건강 지향적인 식문화가 형성되어 가고 있으며 건강을 위한 기능성 식품을 선호하는 추세이다. 현재 인진에 대한 문헌상의 효과, 효능만을 부각시킬 뿐 인진의 기능식품으로의 상품가치를 나타내는 주요성분에 대한 약리 효과와 기능성을 검증하고, 손쉽게 음용할 수 있는 기술적인 연구와 검증을 거친 제품은 아직 개발되지 않았다. 간암, 간경변, 황달 등 간 질환에 그 효능을 보이는 인진쑥 (Artemisia capillarisTHUNB)에 관한 연구는 활발히 이루어져 왔으나, 위암 및 폐암을 유발시키는 벤조피렌 (benzo(a)pyrene, B(a)P)에 대한 인진쑥의 연구는 거의 찾아볼 수 없었다. 이에 본 발명자들은 세균돌연변이 분석법 (bacterial mutation assay)을 이용하여 인진쑥 추출물이 벤조피렌의 변이원성에 대한 돌연변이원성 억제효과가 뛰어남을 확인하였다.Modern people are threatened with excessive mental and physical stress caused by contact with various environmental pollution and inevitable hazards. As a result, health-oriented food cultures are being formed, and functional foods for health are preferred. Currently, only the literature's effects and efficacy on Injin are highlighted, and the pharmacological effects and functionalities of the main ingredients that represent the product value of Injin have been verified. Not developed. Artemisia capillaris THUNB has been actively studied for its liver diseases such as liver cancer, cirrhosis and jaundice, but it has been actively studied. However, jinpyurem against benzo (a) pyrene (B (a) P), which causes stomach and lung cancer, has been studied. Has hardly been found. Therefore, the present inventors confirmed that the extract of Injin mugwort has an excellent mutagenicity inhibitory effect on the mutagenicity of benzopyrene using a bacterial mutation assay.
본 발명자들은 인진의 산지별, 시기별 유효성분의 검색을 통하여 인진 원료의 선택 및 채취 조건의 설정과 인진의 기호성 및 관능을 개선하는 추출방법을 개발하고 인진 추출물의 유효성분인 스코폴레틴 (scopoletin)이 혈관에 미치는 영향, 인진의 추출물이 뇌혈류량에 미치는 영향 및 항산화 항돌연변이원성을 조사하여 인진 추출물의 효능을 규명하였다. 본 발명에서는 인진쑥으로부터 인진 추출물의 제조시 통상적으로 이용되는 고온고압의 추출조건을 배제하고 90∼110℃의 낮은 온도에서 추출함으로써 추출액 중의 부유물 침전량을 최소화하였으며 이러한 저온추출 결과 생성된 인진 추출물은 그 향취미가 뛰어나고 부드러워 많은 양의 인진 추출물을 음료에 첨가할 수 있어 인진 추출물의 효능을 높일 수 있었고, 환류냉각장치를 이용하여 추출할 경우 증발 수증기에 함유된 방향성 물질의 일부가 배출될 수 있도록 밀폐되지 않은 용기를 이용하여 추출한 결과 관능을 향상시킬 수 있었다. 또한 한방 식물체 추출액에서 다량으로 생성되는 부유물의 제거를 위하여 글루코아밀라제 또는 프로테아제와 같은 단백질 분해효소를 처리함으로써 침전물의 양이 현저히 감소된 관능적으로 우수한 인진 추출액을 획득할 수 있었다. 특허등록 0155231호의 인진쑥 단백 다당체 및 그의 추출정제법에 관한 특허에서는 물 추출물을 감압농축하고 에탄올을 가하여 부유물을 침전시킨 후 인진쑥 단백 다당체를 획득하는 방법이 공지된 바 있다. 또한 특허공개 2000-0024959에서는 밀폐용기에서 제조한 인진 추출물에서 발생된 수증기를 냉각시켜 인진쑥 증류수를 제조하는 방법이 공지된 바 있다. 그러나 이들 방법들은 인진쑥으로부터 단순히 단백다당체를 얻거나 인진의 방향성 물질이 녹아있는 증류수만을 취득하는 것에 불과하였다. 이는 본 발명의 단백질 효소를 사용한 부유물 제거 및 저온추출법에 의한 추출물 제조공정과는 서로 상이한 것이다.The present inventors have developed an extraction method for selecting the raw materials and setting the harvesting conditions and improving the palatability and sensory properties of human extracts by searching for active ingredients by place and time of phosphorus and scopoletin as an active ingredient of the extracts. ), The effect of jinjin extract on the blood vessels, the effects of jinjin extract on cerebral blood flow, and the antioxidant antimutagenicity were investigated. In the present invention, by extracting at a low temperature of 90 ~ 110 ℃ to remove the extraction conditions of the high temperature and high pressure commonly used in the preparation of the phosphorus extract from Injin mugwort to minimize the amount of suspended solids in the extract and the extract of the resulting phosphorus extract Excellent hobby and tenderness, a large amount of phosphorus extract can be added to beverages to enhance the efficacy of extracts, and when extracted using a reflux condenser, some of the aromatic substances contained in evaporated water vapor are not sealed. Extraction using a non-container was able to improve the sensory. In addition, by treating proteolytic enzymes such as glucoamylase or protease to remove a large amount of suspended solids from the herbal plant extract, it was possible to obtain a sensory good human extract extract with a significantly reduced amount of precipitate. Patent registration of 0155231 Injin mugwort protein polysaccharide and extract purification method thereof has been known to obtain a phosphorus mugwort protein polysaccharide after the water extract is concentrated under reduced pressure and ethanol is added to precipitate the suspended matter. In addition, Japanese Patent Application Laid-Open No. 2000-0024959 discloses a method of preparing distilled water from jinjin jinjin by cooling water vapor generated from a jinjin extract prepared in a closed container. However, these methods simply obtained protein polysaccharides from Injin mugwort or only distilled water in which aromatic substances of Injin were dissolved. This is different from the extract manufacturing process by the suspension removal and low temperature extraction method using the protein enzyme of the present invention.
따라서 본 발명의 목적은 상기 인용발명들과는 달리 밀폐되지 않은 용기에 인진쑥을 넣어 저온 및 환류냉각장치 처리하고 단백질 분해효소를 처리한 결과 관능면에서 탁월하고 향취미가 향상된 인진쑥 추출물 함유 기능성 음료용 조성물의 제조방법을 제공하는데 있다. 본 발명의 다른 목적은 상기 음료용 조성물이 혈관 이완 효과, 국소뇌혈류량의 증가로 인한 뇌졸중 예방 효과 및 항돌연변이원성에 의한 항암 효과가 있음을 규명하는데 있다.Therefore, the object of the present invention, unlike the above-mentioned inventions, put the jinjinmugi in an unsealed container and treated with low-temperature and reflux cooling system and the proteolytic enzyme treatment resulted in a functional beverage composition containing the ginjinguru extract excellent in functionality and taste. It is to provide a manufacturing method. Another object of the present invention is to determine that the beverage composition has a vascular relaxation effect, a stroke prevention effect due to an increase in local cerebral blood flow, and an anticancer effect due to antimutagenicity.
본 발명의 상기 목적은 시기별, 산지별, 부위별로 인진을 채취한 후 수분, 회분, 산불용성 회분, 고형분의 함량을 측정하고 TLC 및 HPLC 분석하여 인진 시료에 함유된 유효성분을 확인한 후 상기 인진 유효성분이 혈관이완효과, 국소뇌혈류량의 증가에 따른 뇌졸중 예방효과 및 항돌연변이원성에 의한 항암효과가 있음을 확인하고 기존의 고온고압 추출법으로 농도별로 인진을 추출하거나, 저온추출법 및환류냉각장치를 이용하여 개폐용기에서 인진을 추출하거나 또는 고온고압으로 추출한 인진추출물의 부유물을 제거하기 위하여 효소처리한 인진추출물의 특성을 조사하고 관능평가를 실시하여 인진 추출물의 최적 및 최대 처리농도 및 조건을 규명하고 인진 추출물 혹은 농축액에 각종의 감미제, 향신료, 산미제 등을 첨가하여 본 발명 인진 추출물 함유 기능성 음료를 제조함으로써 달성하였다.The object of the present invention is to collect the phosphorus by time, place and region, by measuring the content of moisture, ash, acid insoluble ash, solids and TLC and HPLC analysis to determine the active ingredient contained in the phosphorus sample after the phosphorus Confirmed that the active ingredient has vascular relaxation effect, stroke prevention effect due to increase of local cerebral blood flow, and anti-cancer effect by antimutagenicity. Extraction of phosphorus by concentration using existing high temperature and high pressure extraction method, or using low temperature extraction method and reflux cooling device In order to extract the phosphorus from the opening or closing container or to remove the suspended solids extracted from the high temperature and high pressure, the characteristics of enzymatically treated phosphorus extracts were investigated and sensory evaluation was performed to identify the optimum and maximum treatment concentrations and conditions of the extracts, Contains the present invention phosphorus extract by adding various sweeteners, spices, acidulants, etc. to the extract or concentrate Achieved by preparing a functional beverage.
이하, 본 발명의 구성을 상세히 설명한다.Hereinafter, the configuration of the present invention will be described in detail.
도 1은 본 발명 인진쑥의 유효성분인 스코폴레틴, 에스쿠레틴 및 에스쿠린의 확인을 위하여 박층크로마토그래피에 대해 자외선을 조사한 결과를 나타낸다.Figure 1 shows the results of irradiating ultraviolet light on the thin layer chromatography for the confirmation of scopoletin, escurin and escurin as the active ingredients of the present invention Injin mugwort.
도 2는 대장균 PQ37에 대한 벤조피렌 [B(a)P]의 돌연변이원성에 대한 본 발명 인진쑥 (Artemisia capillarisTHUNB)의 물 추출물로부터 분리한 수용성 분획의 억제효과를 나타낸다.Figure 2 shows the inhibitory effect of the water-soluble fraction isolated from the water extract of Artemisia capillaris THUNB of the present invention on the mutagenicity of benzopyrene [B (a) P] against E. coli PQ37.
도 3은 대장균 PQ37에 대한 벤조피렌 [B(a)P]의 돌연변이원성에 대한 본 발명 인진쑥 (Artemisia capillarisTHUNB)의 물 추출물로부터 분리한 수용성 분획의 세포내,외 항돌연변이원성을 비교한 결과이다. 이 때 시료는 50㎍/assay의 농도로 처리하였다.Figure 3 is a result of comparing the intracellular and external antimutagenicity of the water-soluble fraction isolated from the water extract of Artemisia capillaris THUNB of the present invention for the mutagenicity of benzopyrene [B (a) P] against E. coli PQ37. At this time, the sample was treated at a concentration of 50 µg / assay.
도 4는 2,3,7,8-tetrachlorodibenzo-P-dioxin으로 유도된 쥐간 마이크로좀에 의해 형성된 AFB1의 산화물을 HPLC 법으로 측정한 결과를 나타낸다. A는 대조군의 결과이고, B는 본 발명 인진쑥 물 추출물의 에탄올 불용성 분획을 첨가하여 반응시킨 결과이다.Figure 4 shows the result of measuring the oxide of AFB 1 formed by the rat liver microsomes induced with 2,3,7,8-tetrachlorodibenzo-P-dioxin by HPLC method. A is the result of the control group, B is the result of the reaction by adding the ethanol insoluble fraction of the water extract of the present invention.
본 발명은 강화 및 진안 지역으로부터 채취시기 및 부위를 달리하여 채취한 인진의 수분, 회분 및 산불용성 회분의 함량을 측정하고 에탄올, 물 및 에테르 추출물의 고형분 함량을 측정하는 단계; 상기 인진 시료에 에탄올을 가하고 환류추출한 후 여과한 시료를 TLC 및 HPLC하여 인진 시료에 함유된 유효성분인 스코폴레틴, 에스쿠레틴 및 에스쿠린의 함량을 측정하는 단계; 사육한 Rabbit 및 Rat으로부터 적출한 기관의 각 혈관에 혈관수축물질인 노레피네프린 ED50및 진안 9월산 인진 추출물 시료를 투입하여 인진쑥 추출물이 혈관에 미치는 효과를 확인하는 단계; 사육한 sprague-dawley계 흰쥐의 두정골을 노출시키고 개두술을 시술한 후 농도별로 인진 추출물을 투여하여 인진쑥 추출물이 국소뇌혈류량에 미치는 효과를 확인하는 단계; 돌연변이원성인 벤조피렌 [B(a)P] 및 진안 6월산 인진 물 추출물의 에탄올 가용성 및 수용성 분획을 각각 농도별로 대장균 PQ37, 살모넬라 TA98 및 살모넬라TA100에 반응시켜 인진쑥 추출물의 항돌연변이원성을 확인하고 에탄올 가용성 및 수용성 분획의 항돌연변이원성을 확인하는 단계; 시토크롬 P-450 1A1에 의해 산화되어 돌연변이원으로 작용하는 벤조피렌 [B(a)P]에 대한 인진 추출물의 시토크롬 P-450 1A1 효소계 저해효과를 확인하기 위하여 간접적으로 인진 추출물의 시토크롬 P-450 1A1에 의해 산화되는 AFM1형성능을 HPLC법으로 확인하는 단계; 기존의 고온고압의 추출방법 (120℃, 1.5kg/㎠)으로 인진의 추출비율을 5%, 10%, 15% 농도로 하여 추출한 후 가용성 고형분 농도 및 pH를 측정하는 단계; 경제적인 인진 추출물을 획득하기 위하여 90, 97, 100℃의 온도조건으로 환류냉각장치를 이용하여 밀폐용기에서 추출한 인진 추출물의 당도, 투광도 및 pH를 측정하는 단계; 밀폐 및 밀페시키지 않은 조건 및 온도조건에 따른 인진 추출물의 관능평가를 실시하는 단계; 상기 고온고압으로 추출한 인진 추출물의 부유물을 제거하기 위하여 글루코아밀라제 및 프로테아제를 처리하여 적정 처리농도를 규명하는 단계; 인진 추출물의 음료화를 위하여 인진 추출액 또는 농축액에 사과 농축액, 시트르산, 수크로스, 사이클로 덱스트린, 실리콘 오일, 향료, 꿀 및 산미료의 첨가비를 달리하여 첨가한 후 pH와 당도 및 향미를 테스트하여 인진 추출액의 최적 및 최대 첨가량 및 추출조건을 획득하여 본 발명 인진 추출물 함유 항암 및 혈액순환 기능성 음료를 제조하는 단계로 구성된다.The present invention comprises the steps of measuring the content of moisture, ash and acid insoluble ash of phosphorus collected by varying the time and location of the collection from the Ganghwa and Jinan region and the solid content of ethanol, water and ether extracts; Adding ethanol to the phosphorus sample, refluxing and filtering the filtered sample by TLC and HPLC to determine the contents of scopoline, escurin and escurin, which are active ingredients contained in the phosphorus sample; Checking the effect of the jinsugugui extract on blood vessels by injecting the vascular contracting material norepinephrine ED 50 and Jingan September jinjin extract samples to the blood vessels of the organs extracted from the rabbit and rats; Exposing the parietal bone of the bred sprague-dawley rats and performing a craniotomy procedure to confirm the effect of Injin mugwort extract on local cerebral blood flow by administering Injin extract for each concentration; Ethanol Soluble and Water Soluble Fractions of Mutagenic Benzopyrene [B (a) P] and Jinan Jun Phosphate Water Extracts were reacted with Escherichia coli PQ37, Salmonella TA98 and Salmonella TA100 at different concentrations to confirm antimutagenicity and And identifying the antimutagenicity of the water soluble fraction; In order to confirm the cytochrome P-450 1A1 enzymatic inhibitory effect of jinjin extract on benzopyrene [B (a) P], which is oxidized by cytochrome P-450 1A1 and acts as a mutagen, it was indirectly applied to cytochrome P-450 1A1 of jinjin extract. Confirming AFM 1 forming ability to be oxidized by HPLC method; Extracting the extraction ratio of phosphorus in a conventional high temperature and high pressure extraction method (120 ° C., 1.5 kg / cm 2) at a concentration of 5%, 10%, and 15%, and then measuring soluble solids concentration and pH; Measuring sugar, light transmittance and pH of the phosphorus extract extracted from the hermetic container using a reflux condenser at a temperature of 90, 97, 100 ° C. to obtain an economic phosphorus extract; Performing sensory evaluation of the phosphorus extract according to sealed and unsealed conditions and temperature conditions; Identifying a proper treatment concentration by treating glucoamylase and protease to remove suspended solids of the phosphorus extract extracted at high temperature and high pressure; For the saponification of phosphorus extracts, apple extracts, citric acid, sucrose, cyclodextrin, silicone oil, flavors, honey and acidulants were added to the extracts or concentrates at different concentrations, and then the pH, sugar and flavor were tested. It is composed of the steps of preparing the anti-cancer and blood circulation functional beverage containing the present invention extract by obtaining the optimum and maximum addition amount and extraction conditions of.
본 발명의 과일농축액은 사과, 포도, 레몬, 오렌지, 배, 살구, 딸기, 복숭아, 멜론 등 기타 과일의 농축액을 단독 또는 혼합하여 선택적으로 사용할 수 있다.The fruit concentrate of the present invention can be selectively used alone or mixed with a concentrate of other fruits such as apples, grapes, lemons, oranges, pears, apricots, strawberries, peaches, melons.
본 발명의 당류 또는 감미제로는 정백당, 과당, 포도당, 올리고당, 아스파탐, 솔비톨, 만니톨 등의 기타 당류를 단독 또는 혼합하여 사용할 수 있다.As the sugar or sweetener of the present invention, other sugars such as white sugar, fructose, glucose, oligosaccharide, aspartame, sorbitol, and mannitol may be used alone or in combination.
본 발명의 산미제로는 구연산, DL-사과산, 호박산을 단독 또는 혼합하여 사용할 수 있다.As the acidulant of the present invention, citric acid, DL-apple acid, succinic acid may be used alone or in combination.
본 발명에 있어 통계처리는 student's paired and/or unpaired t-test에 의하였으며, p-value가 최소한 0.05의 값을 보이는 경우 유의한 차이의 한계로 보았다.In the present invention, statistical processing was performed by student's paired and / or unpaired t-test, and the p-value was considered to be the limit of significant difference when showing a value of at least 0.05.
이하, 본 발명의 구체적인 방법을 실시예를 들어 상세히 설명하고자 하지만 본 발명의 권리범위는 이들에만 한정하는 것은 아니다.Hereinafter, specific examples of the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited thereto.
실시예 1 : 시기별, 산지별, 부위별로 채취한 인진쑥의 유효성분에 대한 이화학적인 성분분석Example 1 Physicochemical Analysis of Active Ingredients of Injin mugwort Extracted by Season, Mountain, and Site
제1단계. 인진쑥의 수분, 회분, 산불용분 및 고형분 함량의 조사First step. Investigation of Moisture, Ash, Acid Insoluble and Solid Contents
강화와 진안 지역으로부터 시기별, 산지별, 부위별로 채취한 인진의 유효성분에 대한 이화학적인 성분을 분석하였다. 이 때 진안산 인진은 6∼10월 중 재배된 것을 채취하여 이용하였다.The physicochemical components of the active ingredients of Injin collected from Ganghwa and Jinan by time, mountain and site were analyzed. At this time, Jinansan Injin was used to harvest the cultivated in June-October.
각 인진 시료를 일정량 취하여 확인실험, 건조감량, 회분, 산불용성 회분, 엑스 (묽은 에탄올, 수성, 에테르) 함량을 대한약전에 준하여 행하였다. 산불용성 회분은 회화된 회분에 10% HCl 25mL을 가하여 5분간 끓인 후 정량용 여과지로 여과하여 회분 분석과 동일한 방법으로 산불용성 회분을 구하였다.A certain amount of each phosphorus sample was taken and carried out in accordance with the Korean Pharmacopoeia of the confirmatory test, drying loss, ash, acid insoluble ash, and X (dilute ethanol, aqueous, ether) content. Acid insoluble ash was added to 25% of 10% HCl in the ashed ash and boiled for 5 minutes, and filtered with a quantitative filter paper to obtain an acid insoluble ash in the same manner as the ash analysis.
국내산 인진의 수분, 회분 및 산불용성 회분 함량을 표 1에 나타내었다. 각 지역에서 생산된 인진의 수분함량은 4.2∼9.4%, 회분함량은 5.5∼8.8%, 산불용분은 1.20∼1.79% 수준으로 회분의 경우 시료들간에 함량 차이가 큰 편이나 한약생약규격집의 인진호 규격기준에 적합하였다. 수용성 추출물의 엑스분의 함량은 4.47∼26.21% 범위로 강화쑥 줄기 및 진안쑥 중 10월에 채취한 쑥을 제외하고는 대한생약규격집의 묽은 에탄올 추출물 함량 기준에 적합하였다.Table 1 shows the moisture, ash and acid insoluble ash content of domestic phosphorus. The moisture content of phosphorus produced in each region ranges from 4.2 to 9.4%, ash content from 5.5 to 8.8%, and acid insolubles from 1.20 to 1.79%. The criteria were met. The extract content of water-soluble extracts ranged from 4.47 to 26.21%, which was in compliance with the dilute ethanol extract content of the Korean Herbal Standards except for the mugwort collected in October.
에탄올 추출물의 엑스분의 함량은 8.37∼28.94% 범위로 강화쑥, 강화쑥 줄기, 진안 6, 7월 산을 제외하고는 대한생약규격집의 묽은 에탄올 추출물 함량 기준에 적합하였다. 에테르 추출물의 엑스분의 함량은 한약생약규격집에는 규격이 기재되지 않았으나 2.92∼8.60% 범위로 함량이 적정한 것으로 판단되었다.The extract content of ethanol extract ranged from 8.37 to 28.94%, and was suitable for the dilute ethanol extract content of the Korean Herbal Standards except for Ganghwa mugwort, Ganghwa mugwort, Jinan Jun, and July acid. The content of the extract of the ether extract was not specified in the herbal medicine standard collection, but was determined to be appropriate in the range of 2.92 to 8.60%.
제2단계. 인진 추출물에 함유된 유효성분의 분석Second step. Analysis of Active Ingredients in Injin Extract
인진에 함유된 스코폴레틴 (scopoletin), 에스쿠레틴 (esculetin), 에스쿠린 (esculin)의 확인을 위하여 상기 1단계에서 사용한 각 산지별, 시기별, 부위별 인진 시료를 이용하여 TLC (Thin Layer Chromatography) 및 HPLC (High Pressure Liquide Chromatography) 분석을 실시하였다.In order to identify scopoletin, esculetin, and esculin contained in the phosphorus, TLC (Thin Layer) was used by using the phosphorus samples of each region, time, and region used in step 1 above to identify scopoletin, esculetin, and esculin. Chromatography) and HPLC (High Pressure Liquide Chromatography) analyzes were performed.
UV lamp로는 Spectroline (Model FBPDS150, Spectronis Corpo., U.S.A.), 자외-가시 분광 스펙트럼은 UV/VIS spectrophotometer (Model DMS-200, Varian Co., Australia), HPLC는 (Water Co., U.S.A.)을 실험 기기로 사용하였다. 시약으로는 스코플레틴 (esculetin 6-dimethylether), 에스쿠레틴 (esculetin), 에스쿠린 (esculin), 아세토니트릴 (acetonitril), 아세트산 (acetic acid), 에탄올 (ethanol), 메탄올 (methanol), n-부탄올 (n-butanol), PIC Reagent B6(HPLC용 특급시약, Sigma Co.), 3rd D.W., 실리카겔 플레이트 (silica gel plate, Kieselgel 60F254) 외 30종의 시약을 사용하였다.For UV lamp, Spectroline (Model FBPDS150, Spectronis Corpo., USA), UV-VIS spectrophotometer (UV / VIS spectrophotometer (Model DMS-200, Varian Co., Australia), HPLC (Water Co., USA) Used as. Reagents include esculetin 6-dimethylether, esculetin, escuin, acetonitrile, acetic acid, ethanol, methanol, n- Butanol (n-butanol), PIC Reagent B 6 (HPLC, Sigma Co.), 3rd DW, silica gel plate (Kieselgel 60F 254 ) and 30 kinds of reagents were used.
인진 시료 5g에 에탄올 30mL을 가하여 80℃로 30분간 환류추출하여 얻은 용액을 여과한 다음 분석시료로 사용하였다. 표준물질은 2% 메탄올 용액으로 만들고, TLC 전개는 흡착제 (silica gel 60F254,Merck Co.)를 사용하였으며, 인진 추출물은 20㎕, 표준용액은 10㎕씩 선적하고 전개용매로 톨루엔-에테르 (toluene -ether) (1:1, saturated with 10% acetic acid)을 이용하여 전개한 후 365nm에서 투광도를 측정하였다. 또한 HPLC 분석은 컬럼을 μ BondapakTMC18로, 검출 (detector)은 UV 254nm 0.02AUFS로, 이동상 (mobile phase)은 0.2M 인산 (phosphoric acid, pH 2.1):메탄올=58:42로, flow rat는 1.5 mL/min로, 시료의 양 (sample size) 10㎕로, chart speed는 0.25cm/min로 하여 분석하였다. 표준액 및 검액은 각각 10, 20㎕를 취하여 박층크로마토그래프용 실리카겔 (Kieselgel 60F254) 판에 선적한 후 톨루엔:에테르 (1:1, v/v; 10% 초산포화액) 전개용액으로 전개시킨 다음 박층판을 꺼내고 바람에 말린 후 분리된 스코폴레틴 (F.W. 192.17), 에스쿠레틴 (F.W. 178.15), 에스쿠린 (F.W. 340.3)의 확인을 위하여 365nm을 자외선을 조사한 결과 도 1과 같이 밴드가 분리되었다.30 mL of ethanol was added to 5 g of the phosphorus sample, and the solution obtained by reflux extraction at 80 ° C. for 30 minutes was filtered and used as an analytical sample. The standard material was made of 2% methanol solution, and TLC development was performed using an adsorbent (silica gel 60F 254, Merck Co.), 20 μl phosphorus extract and 10 μl standard solution were loaded and toluene ether was developed as a developing solvent. -ether) (1: 1, saturated with 10% acetic acid) was used to determine the light transmittance at 365nm. HPLC analysis also showed that the column was μ Bondapak TM C 18 , the detector was UV 254nm 0.02AUFS, the mobile phase 0.2M phosphoric acid (pH 2.1): methanol = 58: 42, flow rat Was 1.5 mL / min, the sample size (10 μl) and the chart speed was 0.25 cm / min. The standard solution and the sample solution are each 10, 20 μl and loaded onto silica gel (Kieselgel 60F 254 ) plate for thin layer chromatography, and then developed with toluene: ether (1: 1, v / v; 10% acetic acid saturated solution) development solution. After removing the thin plate and drying in the wind, the band was separated as shown in FIG. 1 by irradiation of UV at 365 nm for identification of the separated scopollet (FW 192.17), escurin (FW 178.15), and escurin (FW 340.3). .
인진의 유효물질로 알려진 스코폴레틴의 Rf값은 0.55, 에스쿠레틴의 Rf값은 0.375이었으나 에스쿠린의 경우 시료의 선적선으로부터 거의 이동이 되지 않아 Rf값이 0부근이었다. 인진의 산지 및 채취시기별 시료를 분석한 결과, 스코폴레틴과 에스쿠레틴의 표준품의 Rf값의 위치에서 존재를 확인할 수 있었으나, 에스쿠린은 미약하여 확인할 수 없었다. 각 시료 스코폴레틴의 형광강도를 비교한 결과, 산지별로 강화산줄기(S4)<강화산전체(S2)<진안산전체(S1)<강화산잎(S3)순이었고, 채취시기별로는진안9월산(S8)≤진안10월산(S9)<진안8월산(S7)<진안6월산(S5)<진안7월산(S6)순이었으며 분석한 인진을 모두 비교하면 진안9월산(S8)≤진안10월 산(S9)<강화산줄기(S4)<진안8월(S7)<강화산전체(S2)<진안산전체(S1)<진안7월산(S5)<강화산잎(S3)<진안7월산(S6)순이었다.The Rf value of scopoline, known as the active substance of phosphorus, was 0.55, and the Rf value of escurin was 0.375. However, in the case of escurin, the Rf value was near 0 because it was hardly moved from the sample ship. As a result of analyzing the samples of each plant and sampling time, it was possible to confirm the presence at the position of Rf value of the standard products of scopoline and escuretin, but the escurin was weak. As a result of comparing the fluorescence intensity of each sample of scopoline, it was found that in order of acidity, Ganghwasan Stem (S4) <Ganghwasan (S2) <Jinansan (S1) <Ganghwasan (S3) was in order. S8) ≤Jinan October Mountain (S9) <Jinan August Mountain (S7) <Jinan June Mountain (S5) <Jinan July Mountain (S6) The total Jinan September Mountain (S8) ≤Jinan October Mountain (S9) <Granite volcano stem (S4) <Jin'an August (S7) <Ganghwa volcano (S2) <Jin'an whole mountain (S1) <Jinan July mountain (S5) <Ganghwasan leaf (S3) <Jinan July mountain (S6) It was net.
HPLC에 의한 유효물질을 분석한 결과, 표 2와 같이 지역별 인진쑥의 스코폴레틴 함량은 강화산 인진, 강화산 줄기, 강화산 잎, 진안산 전체가 각각 126.15, 392,16, 331.53, 326.13 ppm였으며, 에스쿠린은 진안산에서만 1,905.2 ppm함유되어 있었다. 시기별 함량은 6, 7, 8, 9, 10월 산 인진이 각각 스코폴레틴은 747.04, 1206.53, 791.07, 349.57, 54.50 ppm, 에스쿠린은 396.72, 491.05, 699.94, 1535.57, 262.88 ppm이었으나 에스쿠레틴은 8월의 인진에만 함유되어 있었다. 따라서, 인진의 채취 적기는 실시예 1의 결과를 종합하여 볼 때 7월과 9월 사이이며 특히 8월에 채취하는 것이 바람직할 것으로 판단된다As a result of analyzing the active substance by HPLC, the scopoline content of Injin mugwort in each region was 126.15, 392, 16, 331.53, and 326.13 ppm, respectively. , Escurin contained 1,905.2 ppm only in Mt. The content of each period was 6, 7, 8, 9, and October, respectively. Was contained only in August's humanity. Therefore, it is judged that the timely harvesting time is between July and September, and especially in August.
실시예 2 : 인진 추출물이 혈관에 미치는 영향 조사Example 2 Investigation of the Effect of Injin Extract on Blood Vessels
인진은 비, 위, 간, 담경에 들어가 청리습열, 황달 치료 효능이 있고 주된 약리 작용으로는 해열, 이담, 항균, 항바이러스, 항진균, 지질저하 등이 있다고 알려져 왔다. 이와 같은 효능이 있는 인진이 각종 평활근 및 혈관에 미치는 효과를 관찰하고자, 노레피네프린 ED50(norepinephrine ED50)으로 수축을 유발한 복부대동맥과 대퇴부동맥을 이용하여 실험을 수행하였다.Injin has been known to enter rain, stomach, liver, and bile ducts, and it is effective in treating wet fever and jaundice, and its main pharmacological actions are antipyretic, edema, antibacterial, antiviral, antifungal and lipid lowering. As to the injin with the same efficacy observed the effect on various smooth muscle and blood vessel, an experiment was conducted using the abdominal aorta and femoral artery caused by the shrinkage nore Pinero printer ED 50 (norepinephrine ED 50).
본 실험에서는 노레피네프린 ED50(Sigma, U.S.A.)을 사용하였으며 완충액을 위한 시약으로는 특급시약을 사용하였다. 인진은 상기 실시예 1에서 사용한 시료 중 진안 9월산의 줄기와 잎을 혼합하고 음건한 후 100g을 증류수 900㎖와 혼합하고 고온고압의 추출조건인 120℃에서 5시간동안 1.5㎏/㎠의 상태로 추출한 후 이 추출액을 여과지로 압착 여과하였다. 추출 후 여과액은 가수량에 대하여 68%의 수율을 나타내었으며, 그 여과액을 0.08% 아밀라아제, 프로테아제로 효소처리한 후 사용하였다.In this experiment, norepinephrine ED 50 (Sigma, USA) was used and the reagent was used as a reagent for the buffer. Injin is mixed with the stem and leaves of Jinan September acid in the sample used in Example 1 and dried, and then mixed with 100g of 900ml of distilled water and 1.5kg / ㎠ for 5 hours at 120 ℃ high temperature and extraction conditions After extraction, the extract was compressed by filtration with filter paper. The filtrate after extraction showed a yield of 68% with respect to the amount of hydrolyzate. The filtrate was used after enzymatic treatment with 0.08% amylase and protease.
인진쑥이 혈관에 미치는 영향을 알아보기 위하여 Rabbit 혈관 및 Rat의 기관지평활근에 대한 실험을 수행하였다. 체중 2kg 내외의 웅성 Rabbit (Newzealand White)과 300g 내외의 웅성 백서 (白鼠)를 실험실 환경에서 2주일 이상 사료를 충분히 공급하면서 적응시킨 후 이산화탄소 가스를 주입 (吸入)시켜 질식사시킨 다음 기관을 적출하여 기관지 평활근과 혈관에 손상이 가지 않도록 절취한 후 기관지 및 혈관의 크기가 4∼5mm가 되게 하여 Magnus 법에 따라 Kreb-Henseleit bicarbonate buffer solution (조성:118mM NaCl, 27.2mM NaHCO3, 4.8mM KCl, 1.0mM KH2PO4, 1.8mM CaCl2, 12.1mM MgSO4및 11.1mM glucose)이 들어있는 organ bath에 현수하였다. 혈관과 기관지의 수축력을 측정하기 위하여 각 혈관과 기관지의 일단을 isometic transducer에 연결하여 혈관은 1.5g, 기관지는 0.5g의 resting tension을 각각 가하였고, 근수축역은 physiograph (Grass, U.S.A.)상에 묘기하였다.In order to investigate the effect of jinseng worms on blood vessels, experiments were performed on rabbit blood vessels and bronchial smooth muscles of rats. A male rabbit (Newzealand White) weighing about 2 kg and a male white paper weighing about 300 g were adapted while feeding enough for 2 weeks or more in a laboratory environment, injected with carbon dioxide gas, suffocated, and the organs were extracted and bronchialized. Cut to smooth muscles and blood vessels to ensure that the size of the bronchus and blood vessels to 4 ~ 5mm, Kreb-Henseleit bicarbonate buffer solution (Composition: 118mM NaCl, 27.2mM NaHCO 3 , 4.8mM KCl, 1.0mM) according to Magnus method KH 2 PO 4 , 1.8 mM CaCl 2 , 12.1 mM MgSO 4 and 11.1 mM glucose) were suspended in an organ bath. In order to measure the contractile force of blood vessels and bronchus, one end of each blood vessel and bronchus was connected to an isometic transducer, and the resting vessels of 1.5g and bronchus were applied with a resting tension of 0.5g, respectively. Stunted.
노레피네프린 ED50에 의한 혈관수축에 인진이 미치는 효과를 알아보기 위하여 상기 각 혈관에 노레피네프린 ED50을 투여하고 organ bath내의 인진 농도가 5, 10, 35, 50㎕/㎖가 되게 투여하였다. 그 결과, 노레피네프린만을 투여한 경우의 혈관 수축력을 100% 수축력으로 한 경우 복부동맥에서는 99.88, 100.38, 96.00, 68.75%의 수축력을, 대퇴동맥에서는 100.00, 96.00, 82.17, 0.33% 수축력으로 유의한 혈관의 이완작용을 관찰할 수 있었다 (표 3). 따라서, 본 발명 인진 추출물은 혈관의 이완작용을 촉진한다는 것을 알 수 있다.In order to study the effect on the injin vasoconstriction by nore Pinero printer ED 50 dose of nore Pinero printer ED 50 in each of the vessels and the injin concentration in the organ bath it was administered to be a 5, 10, 35, 50㎕ / ㎖ . As a result, when the norepinephrine-only vascular contraction force was 100% contraction force, the contractile force of 99.88, 100.38, 96.00, 68.75% in the abdominal artery, and 100.00, 96.00, 82.17, 0.33% in the femoral artery were significant. Vascular relaxation could be observed (Table 3). Therefore, it can be seen that the extract of the present invention promotes relaxation of blood vessels.
실시예 3 : 인진 추출물이 국소뇌혈류량에 미치는 효과Example 3 Effect of Injin Extract on Local Cerebral Blood Flow
인진에 대한 약리학적 기전을 찾기 위해 국소뇌혈류량에 미치는 인진 추출물의 효과를 측정하였다.To find out the pharmacological mechanisms for phosphorus, we measured the effect of phosphorus extract on focal cerebral blood flow.
상기 실시예 1에서 사용한 진안산 인진을 수확기별로 채취하여 충분히 세척한 다음 줄기와 잎을 혼합하고 음건한 후 각각 1cm이하로 절단하고 10∼20배수 정도의 비율로 정제수를 가하여 4∼5시간 동안 100∼120℃정도의 온도를 가하여 추출하였다. 추출액은 각 실험의 조건에 맞게 전처리하고, 음료화를 위해서는 투명한 상태로 만들어야 하므로 침전물을 제거하기 위해 0.08% 아밀라아제, 프로테아제로 효소처리한 후 여과 및 정제하였다.Collect the Jinan-san injin used in Example 1 for each harvester, wash it sufficiently, mix the stem and leaves, dry it, cut it to 1cm or less, and add purified water at a ratio of 10 to 20 times 100 for 4 to 5 hours. It extracted by adding a temperature of about -120 degreeC. The extract was pretreated according to the conditions of each experiment, and in order to be cleared for the sake to be purified, and then filtered and purified after enzymatic treatment with 0.08% amylase and protease to remove the precipitate.
국소뇌혈류량에 대한 실험을 실시하기 위하여 체중 300g내외의 웅성 스프라그-다우리 (Sprague-Dawley)계 흰쥐를 항온 항습 장치가 부착된 사육장에서 고형사료와 야채를 충분히 공급하면서 2주일 이상 실험실 환경에 적응시킨 후, 상기 백서를 stereotactic frame에 고정시키고 정중선을 따라 두피를 절개하여 두정골을 노출시킨 후 brema의 4∼6mm 측방, 2∼1mm 전방에 직경 5∼6mm의 개두술 (craniotomy)을 시술하였다. 이때 두개골의 두께를 최대한 얇게 남겨 경막외 출혈을 방지토록 한다. stereotactic micromanipulator를 사용하여 Laser-Doppler flowmeter (Transonic Instrument, U.S.A.)용 needle probe (직경 0.8mm)를 대뇌 (두개엽) 피질표면에 수직이 되도록 하여 뇌연막동맥에 조심스럽게 근접시켰다. 일정시간 안정시킨 후 실험 프로토콜에 따라 국소뇌혈류량 (regional cerebral blood flow, rCBF)을 측정하였다.Local cerebral blood flow In order to conduct the experiment, the male Sprague-Dawley rats having a body weight of about 300 g were adapted to a laboratory environment for two weeks or longer while supplying sufficient solid feed and vegetables in a kennel equipped with a constant temperature and humidity device. The white paper was fixed to the stereotactic frame, and the scalp was cut along the midline to expose the parietal bone, and then a craniotomy with a diameter of 5 to 6 mm at the 4 to 6 mm lateral and 2 to 1 mm front of the brema was performed. At this time, leave the thickness of the skull as thin as possible to prevent epidural bleeding. Using a stereotactic micromanipulator, the needle probe (0.8 mm in diameter) for the Laser-Doppler flowmeter (Transonic Instrument, U.S.A.) was placed perpendicular to the cerebral (cranial) cortical surface and carefully approached to the periosteum artery. After stabilizing for a while, regional cerebral blood flow (rCBF) was measured according to the experimental protocol.
국소뇌혈류량에 대한 인진의 효과를 살펴보기 위하여 농도별로 100%의 인진호 추출물을 투여하여 국소뇌혈류량의 변동을 Laser-Doppler flowmeter로 측정하여 그 결과를 표 4에 나타내었다. 대조군의 국소뇌혈류량은 100.00±0.02%(AU)이었으며, 인진호를 농도별로 0.01, 0.1, 1.0 및 10.0mg/kg을 정맥주사한 결과, 105.26±0.05, 111.74±0.06, 129.14±0.06, 131.86±0.04%(AU)로 유의한 증가를 나타내었다. 이와 같은 결과를 통하여 볼 때, 인진 추출물은 국소뇌혈류량을 증가시키므로 뇌졸중 예방 효과가 있음을 알 수 있다.In order to examine the effect of phosphorus on local cerebral blood flow, 100% of the phosphorus extract was administered by concentration, and the change in the cerebral blood flow was measured by Laser-Doppler flowmeter and the results are shown in Table 4. Local cerebral blood flow in the control group was 100.00 ± 0.02% (AU), and intravenous injection of 0.01, 0.1, 1.0, and 10.0 mg / kg of Injinho showed 105.26 ± 0.05, 111.74 ± 0.06, 129.14 ± 0.06, and 131.86 ± 0.04. A significant increase in% (AU) was shown. From these results, it can be seen that the phosphorus extract increases the local cerebral blood flow, thereby preventing stroke.
실시예 4 : 인진쑥 추출물의 항돌연변이원성 검색Example 4 Antimutagenic Screening of Injin mugwort Extract
벤조피렌 [B(a)P]에 대한 인진쑥의 항돌연변이원성을 검색하고, 활성이 인정된 분획물에 대한 세포내ㆍ외의 항 돌연변이원성과 폐암발생에 중요한 역할을 하는 시토크롬 P-450 1A1 (cytocrome P-450 1A1)의 억제능을 실험하였다.Cytochrome P-450 1A1 (Cytochrome P-450 1A1), which plays an important role in the development of antimutagenicity and intracellular and external antimutagenicity of lungs of jinpyri worms against benzopyrene [B (a) P]. 450 1A1) inhibitory ability was tested.
제1단계. 시료 및 재료의 준비First step. Preparation of Samples and Materials
변이원인 벤조피렌과 시토크롬 P-450 1A1 유도물질인 2, 3, 7, 8-테트라클로로디벤조-P-디옥신 (tetrachlorodibenzo-P-dioxin, TCDD)은 Sigma (U.S.A.)사 제품을, S9 분획은 Aroclor 1254로 유도된 제품 (ICN pharmaceutical, Inc.)을 구입하여 사용하였다. 시험균주인 대장균 PQ37 (Escherichia coliPQ37)은 Quillardet 박사 (Pasteur 연구소, France)로부터, 살모넬라 TA98 (Salmonella typhimuriumTA98, 100) 및 TA100은 원광대학교 약학대학으로부터 기증받아 사용하였다. HPLC용 컬럼은 ultrasphere ODSC-18 reversed-phase column (Beckman, 5 ㎛, 4.6×250 mm)을 사용하였다.Mutants benzopyrene and cytochrome P-450 1A1 inducers 2, 3, 7, 8-tetrachlorodibenzo-P-dioxin (TCDD) are from Sigma (USA) and S9 fraction Aroclor 1254 derived product (ICN pharmaceutical, Inc.) was purchased and used. The test strain E. coli PQ37 (Esherichia coli PQ37) was used by Dr. Quillardet (Pasteur Institute, France) and Salmonella TA98 ( Salmonella typhimurium TA98, 100) and TA100 from Wonkwang University College of Pharmacy. HPLC column was used ultrasphere ODSC-18 reversed-phase column (Beckman, 5 ㎛, 4.6 × 250 mm).
진안 6월산 인진쑥을 121℃로 1시간 증자한 후 건조물을 파쇄하였다. 여기에 6배 (w/v)의 증류수를 가한 후 83±5℃에서 4시간동안 약탕기에서 추출하여 물 추출물을 준비하였다. 상기 시료에 6배 (w/v)의 에탄올을 가하고 45℃에서 12시간 정치하여 2회 추출한 후 여과, 감압농축 및 동결건조하여 에탄올 추출물을 준비하였다. 물 추출물을 100% 에탄올로 가용성 및 불용성 분획물 (침전물)로 분리한 후 감압농축 및 동결건조하였다.Jinan Junjin Injin mugwort was added to 121 ℃ for 1 hour and then the dried product was crushed. 6 times (w / v) of distilled water was added thereto, followed by extraction in a water bath at 83 ± 5 ° C. for 4 hours to prepare a water extract. 6 times (w / v) of ethanol was added to the sample, and the mixture was extracted twice for 12 hours at 45 ° C., followed by filtration, concentration under reduced pressure, and lyophilization to prepare an ethanol extract. The water extract was separated into soluble and insoluble fractions (precipitates) with 100% ethanol, concentrated under reduced pressure and lyophilized.
제2단계. 벤조피렌에 대한 인진쑥의 항돌연변이성 검색을 위한 SOS chromotestSecond step. SOS chromotest for the screening of antimutagenicity of ginseng worm against benzopyrene
SOS Chromotest는 Quillardet와 Hofnung의 방법에 준하여 수행하였다. 즉 L 배지에서 5×108CFU/mL 농도로 배양된 종배양액을 2% (v/v)가 되도록 접종하고 37℃에서 약 2시간 진탕배양 (5×106CFU/mL)하였다. 3% S9 mixer에 0.1배 (v/v)량의 균을 첨가하여 0.6mL의 균 희석액을 분취하고 여기에 10㎕의 변이원과 10 ㎕의 시료를 혼합한 다음, 37℃에서 210rpm으로 2시간 배양하였다. 이 때 첨가된 양성 대조 물질 (돌연변이원)의 사용량은 변이원의 용량반응을 실시하여 최적의 농도 (2500ng/assay)를 설정한 후 사용하였다. 시료 색소로 인한 흡수 스펙트럼에 대한 영향을 차단하기 위하여 배양액을 4℃에서 3,500rpm으로 2분간 원심분리하여 상징액을 제거한 후 잔사에 0.6mL의 L 배지를 현탁시켰다. 이 현탁액 0.2mL을 취하여 Quillardet와 Hofnung의 방법으로 각각의 효소 활성을 측정하였다. 활성 단위는 흡광도×1,000/반응시간(분)으로 나타내었으며, R (Ratio)값은 β-galactosidaseunit/alkaline phosphatase unit로 계산하였고 R(0)값을 1로 정하였다. 유도지수 (induction factor, IF)는 SOS 유전자의 유도정도를 나타내며, R(C)/R(0)로 계산하였고 R(C)은 변이원 및 변이원과 시료를 첨가한 시험구의 ratio 값이다. R(0)은 변이원을 첨가하지 않는 농도의 ratio 값이며, 음성 대조구 IF 값은 1로 정하였다. 시료의 돌연변이 억제효과는 [(IFo-IFs/IFo×100]으로 산출하였고 여기서 IFo는 양성대조구의 IF 값이며, IFs는 시험물질의 IF 값이다.SOS Chromotest was performed according to the method of Quillardet and Hofnung. That is, inoculated to 2% (v / v) of the culture medium incubated at a concentration of 5 × 10 8 CFU / mL in L medium and shaking culture (5 × 10 6 CFU / mL) at 37 ℃ for about 2 hours. Add 0.1 fold (v / v) of bacteria to the 3% S9 mixer, collect 0.6 mL of bacterial dilutions, mix 10 μL of the mutagen and 10 μL of the sample, and then mix for 2 hours at 37 rpm at 210 rpm. Incubated. At this time, the amount of the positive control substance (mutant) added was used after setting the optimum concentration (2500ng / assay) by performing the dose response of the mutagen. In order to block the influence on the absorption spectrum caused by the sample pigment, the culture solution was centrifuged at 3,500 rpm for 2 minutes at 4 ° C. to remove the supernatant, and 0.6 mL of L medium was suspended in the residue. 0.2 mL of this suspension was taken and their respective enzyme activities were measured by the method of Quillardet and Hofnung. The activity unit was expressed as absorbance × 1,000 / reaction time (minutes). The R (Ratio) value was calculated as β-galactosidase unit / alkaline phosphatase unit and the R (0) value was set to 1. The induction factor (IF) represents the degree of induction of the SOS gene, which is calculated as R (C) / R (0), and R (C) is the ratio of the mutant, the mutant and the sample added. R (0) is the ratio value of the concentration without adding the mutagen, and the negative control IF value was set to 1. Mutation inhibitory effect of the sample was calculated as [(IF o -IF s / IF o × 100], where IF o is the IF value of the positive control, IF s is the IF value of the test substance.
Ames test는 전배양 (preincubation)법을 이용하였다. 돌연변이 억제효과는 [(M-S1/M-S0)×100]으로 계산하였고 돌연변이 물질만 존재한 경우의 복귀 돌연변이 수는 M, 자연복귀 돌연변이 수는 S0,돌연변이원과 시료를 첨가하였을 경우의 복귀 돌연변이수는 S1로 나타내었다.Ames test was performed by preincubation. Mutation inhibitory effect was calculated as [(MS 1 / MS 0 ) × 100] and the number of return mutations in the presence of mutant material was M, the number of natural return mutations was S 0 , and the return mutations when mutations and samples were added. The number is represented by S 1 .
B(a)P에 대한 대장균 PQ37의 돌연변이에 대한 인진 물 추출물의 에탄올 가용성 분획 및 수용성 분획의 돌연변이원성 억제효과를 조사한 결과, 표 5와 같이 25㎍/assay의 농도에서 수용성 분획은 18.5%의 억제활성을 나타낸 반면, 에탄올 가용성 분획은 5.50%의 억제활성을 나타내어 수용성 분획물에서 돌연변이원성 억제효과가 강하게 나타났음을 알 수 있다. 약한 검정색을 띠는 수용성 분획은 쓴맛을 전혀 느낄 수 없었으나 노란색을 나타내는 에탄올 가용성 분획은 쓴맛이 매우 강하였다. 또한 추출물 제조과정 및 스팀과정에 있어서 가을에 수확한 시료의 경우 쓴맛이 매우 강한 반면, 여름에 수확한 시료는 쓴맛이 약하였다. 쓴맛이 약한 봄의 시료와쓴맛이 없는 수용성 분획에서 모두 강한 돌연변이원성 억제효과가 나타난 것으로 보아, 쓴맛과 돌연변이원성 억제효과는 무관하다는 것을 알 수 있으며, 인진쑥 물 추출물의 쓴맛 성분은 에탄올에 완전히 용해된다는 것을 알 수 있다. 지방간의 치료로 음용되는 인진쑥 추출물은 쓴맛으로 인하여 기호성에 적합하지 않은 단점을 지니고 있는데 이러한 단점의 개선 방안으로 인진의 추출물을 농축한 후 에탄올을 첨가하여 쓴맛을 제거하고 불용된 침전물을 사용할 수 있을 것이다.As a result of investigating the mutagenic effects of the ethanol soluble fraction and the water soluble fraction of human extracts against the mutation of E. coli PQ37 against B (a) P, the water soluble fraction was inhibited by 18.5% at the concentration of 25 µg / assay as shown in Table 5. On the other hand, the ethanol soluble fraction showed an inhibitory activity of 5.50%, indicating that the mutagenicity effect was strong in the water-soluble fraction. The slightly black water-soluble fraction could not feel bitter taste, but the yellow ethanol soluble fraction was very bitter. In addition, the bitter taste was very strong in the autumn harvest during the extract preparation and steam process, while the bitter taste was weak in the summer harvest. The strong mutagenicity inhibitory effect was found in both the spring samples with weak bitterness and the water-soluble fraction without bitterness, indicating that the bitterness and mutagenicity inhibitory effects are irrelevant. It can be seen that. Injin mugwort extract, which is used for the treatment of fatty liver, has disadvantages that are not suitable for palatability due to bitter taste.In order to improve these disadvantages, it is possible to concentrate the extract of Injin and remove the bitter taste by adding ethanol and use the insoluble precipitate. .
간 보호 성분으로 알려진 인진 다당체를 에탄올로 침전시켜 분리하였다. 인진 물 추출물의 농축액에 2∼3배량의 에탄올을 첨가하고 냉장보관하여 여과하는 방법으로는 활성성분이 뚜렷하게 분리되지 않으므로 100% 에탄올을 사용하여 분리하였다. 인진의 활성성분은 물에 잘 용해되는 성분 중에서도 에탄올에 침전되는 극성이 강한 당단백질, 다당체 및 그 이외의 성분으로 예측되므로, 적합한 추출온도를 100℃이하로 설정하였다. 가을에 수확한 인진 시료를 고온 (121℃)으로 4시간 동안 추출한 추출물을 1/3용량으로 농축하여 냉장보관한 결과, 레진으로 추측되는 불용물이 다량 침전됨과 동시에 에탄올 가용분획의 수율도 21.7%로 증가하였다.Phosphorus polysaccharide, known as a hepatoprotective component, was isolated by precipitation with ethanol. Addition of two to three times the amount of ethanol to the concentrate of the phosphorus water extract and the method of cold storage to filter the active ingredient is not clearly separated and separated using 100% ethanol. Since the active ingredient of phosphorus is predicted to be a polar polar glycoprotein, polysaccharide and other components precipitated in ethanol among the components that are well dissolved in water, a suitable extraction temperature was set below 100 ℃. After extracting the extracted human extract in high temperature (121 ℃) for 4 hours and refrigerated by 1/3 volume, the insoluble matter that is supposed to be resin was precipitated and the yield of ethanol soluble fraction was 21.7%. Increased to.
돌연변이 억제효과가 확인된 수용성 분획의 용량반응 (dose-response) 활성도 측정 결과를 도 2에 나타내었다. 수용성 분획의 농도를 assay당 12.5, 25, 50, 100, 200㎍으로 증가시켰을 때 각각 12.5%, 18.5%, 38%, 46%, 51.5%의 억제효과를 나타내었다. 100㎍/assay 농도까지는 그 농도가 증가함에 따라 B(a)P로 유도된 돌연변이원성에 대한 억제효과가 뚜렷하게 증가되었으나, 그 이상의 농도에서는 증가효과가 두드러지게 나타나지 않았으며 50%의 저해농도 (IC50)를 나타낸 200㎍ 이상의 농도에서는 억제효과가 거의 증가되지 않았다. 돌연변이원성 억제효과와 더불어 알카라인 포스파테이즈 (alkaline phosphatase) 활성도가 일정한 것으로 보아, 수용성 물질에 의한 균수의 증가 결과 나타날 위양성 가능성을 배제함과 동시에 세포독성이 없음을 알 수 있다.The dose-response activity of the water-soluble fraction in which the mutation inhibitory effect was confirmed is shown in FIG. 2. Increasing the concentration of the water-soluble fraction to 12.5, 25, 50, 100, 200 ㎍ per assay showed inhibitory effects of 12.5%, 18.5%, 38%, 46%, and 51.5%, respectively. The inhibitory effect on B (a) P-induced mutagenicity was significantly increased as the concentration increased up to 100 ㎍ / assay, but at higher concentrations, the inhibitory effect was not remarkable and the inhibitory concentration of 50% (IC The inhibitory effect was hardly increased at the concentration of 200 µg or more. In addition to mutagenicity, the alkaline phosphatase activity was constant, indicating that there is no cytotoxicity while excluding the possibility of false positives that would result from an increase in the number of bacteria by water-soluble substances.
SOS Chromotest는 히스티딘의 영향을 받지 않으므로 생약재 시료의 추출과정에서 혼재할 수 있는 히스티딘을 제거할 필요는 없으나, 선택적으로 β-갈락토시다제를 억제하거나 ONPG을 분해, 또는 RecA 효소를 불활성화하여 위양성 효과를 나타낼 수도 있다. 또한 돌연변이 활성은 시험법, 변이원 및 균 농도, 균 종에 따라 억제율의 차이는 물론 활성의 유, 무까지도 다르게 나타날 수 있으므로, 서로 다른시험계에서 돌연변이 억제활성을 일관되게 측정하여야 결과를 인정하고 해석할 수 있을 것이다. 이에 따라 대장균 PQ37을 이용한 상기 SOS Chromotest 1차 검색 결과를 살모넬라 TA98, TA100 (Salmonella. typhimuriumTA98, TA100) reversion 시험계에서 확인하였다. 용량에 따른 억제효과는 상기 SOS chromotest 결과와 동일하게 나타났으며 (표 6,7), 50%의 저해농도 (IC50)는 살모넬라 TA98, 100에서 800, 600㎍/plate로 나타났다. 단, IC50의 차이는 base substitution 돌연변이가 일어나는 살모넬라 TA100 균주가 frame shift 돌연변이가 일어나는 살모넬라 TA98보다 B(a)P에 의한 돌연변이율이 높기 때문에 상대적으로 억제율이 낮은 것으로 나타났다.Since SOS Chromotest is not affected by histidine, it is not necessary to remove the mixed histidine during the extraction of herbal samples, but it is possible to selectively inhibit β-galactosidase, decompose ONPG, or inactivate RecA enzyme to false-positive It may work. In addition, mutation activity may vary depending on the test method, mutagen and bacteria concentration, and species, as well as the presence or absence of activity. Therefore, it is necessary to consistently measure the mutation inhibitory activity in different test systems to recognize and interpret the results. You can do it. Accordingly, the SOS Chromotest primary search results using E. coli PQ37 were confirmed in Salmonella TA98, TA100 ( Salmonella. Typhimurium TA98, TA100) reversion test system. The inhibitory effect according to the dose was the same as the SOS chromotest results (Table 6,7), 50% inhibition concentration (IC 50 ) was found in Salmonella TA98, 100, 800, 600 ㎍ / plate. However, the difference of IC 50 showed that Salmonella TA100 strain having a base substitution mutation had a relatively low inhibition rate due to B (a) P mutation rate than Salmonella TA98 having a frame shift mutation.
제3단계. 인진 추출물의 항돌연변이원성 실험Third step. Antimutagenic Test of Injin Extract
세포내 항돌연변이원성 실험은 Sato등의 방법으로 세포내 항돌연변이 효과를 조사하였다. 멸균된 cap tube에 L 배지로 10배 (v/v) 희석된 본 배양액 0.6mL와 10㎕의 변이원을 혼합하여, 37℃에서 210rpm으로 20분 동안 전배양하여 DNA 손상을 유도한 후, 5mL의 PBS (phosphate buffer saline, pH 7.2) 용액으로 4℃에서 4,500×g로 20분간 원심분리하여 세척하였다. 그 후 50 ㎍/assay의 시료와 0.6mL의 L배지를 첨가하여 동일한 조건에서 100분간 배양하였다. 배양종료 후 효소활성 측정과 세포내 항돌연변이원성 산출은 돌연변이원성 억제시험법과 동일하게 실시하였다.Intracellular antimutagenicity was investigated by Sato et al. After mixing 10 mL of the culture medium diluted with 10 times (v / v) L culture medium and 10 μl of the mutant in a sterile cap tube, preincubation at 210 rpm for 20 minutes to induce DNA damage, and then 5 mL. PBS (phosphate buffer saline, pH 7.2) solution was washed by centrifugation at 4,500 × g for 20 minutes at 4 ℃. Thereafter, 50 μg / assay sample and 0.6 mL L medium were added and incubated for 100 minutes under the same conditions. Enzyme activity and intracellular antimutagenicity were determined in the same manner as the mutagenicity assay after the end of the culture.
AFM1형성능은 AFB1대사 활성을 측정한 Oh의 방법과 동일하게 HPLC 법을 이용하여 측정하였으며 시토크롬 P-450 1A1의 유도를 위해 2,3,7,8-테트라클로로디벤조-P-디옥신 (TCDD)으로 유도된 쥐의 마이크로좀을 이용하였다. 용출 용매는 20mM 암모늄 아세테이트 (pH4.0)용매 A와 아세토니트릴:메탄올:물을 4.5:4.5:1.0의 비율로 혼합된 용매 B로 이루어졌으며 용매의 용출속도는 1.5mL/min이었다. 또한 AFB1의대사물질은 360nm에서 측정하였다.AFM 1 formation ability was measured by HPLC method in the same manner as Oh's method for measuring AFB 1 metabolic activity, and 2,3,7,8-tetrachlorodibenzo-P-dioxin for the induction of cytochrome P-450 1A1. Rat microsomes induced with (TCDD) were used. The elution solvent consisted of solvent B mixed with 20 mM ammonium acetate (pH 4.0) solvent A and acetonitrile: methanol: water in a ratio of 4.5: 4.5: 1.0 and the elution rate of the solvent was 1.5 mL / min. In addition, the metabolite of AFB 1 was measured at 360nm.
돌연변이 억제물질은 작용방식에 따라 세포외 항돌연변이원 (desmutagen)과 세포내 항돌연변이원 (bio-antimutagen)으로 구분되는데, 이러한 세포내 및 세포외 항돌연변이원성은 변이원에 대한 억제작용 시스템을 규명할 목적으로 이용된다. 간접변이원은 시토크롬 P-450 (cytocrome P-450)에 의한 대사활성화로 산화되어져 변이원으로 작용하므로 세포외 항돌연변이원성의 작용기구는 매우 다양하다. 즉 대사활성 효소를 저해하여 변이원을 억제하거나, 대사활성화된 최종 돌연변이원 (ultimate mutagen)에 직접 작용해서 불활성화시키는 인자도 포함된다. 따라서 간접변이원에 대한 항돌연변이원성을 검색할 때에는 세포내 및 세포외에서 변이원의 작용을 직접적으로 불활성화시키는 작용 이외에도 시토크롬 P-450에 의한 변이원의 산화 억제효과 또한 고려해야 한다.Mutant inhibitors are classified into extracellular and mutant bio-antimutagens, depending on the mode of action. These intracellular and extracellular antimutagenicities identify inhibitory systems for mutants. It is used for the purpose. Indirect mutagens are oxidized by metabolic activation by cytochrome P-450 and act as mutagens, so the mechanism of extracellular mutagenicity is very diverse. In other words, factors that inhibit the mutagen by inhibiting the metabolic enzyme or directly inactivate the final mutagen metabolized (ultimate mutagen) is also included. Therefore, in the search for antimutagenicity against indirect mutagens, in addition to the direct inactivation of the mutagenesis in cells and extracellularly, the inhibitory effect of cytochrome P-450 mutagenization should be considered.
상기 세포내 항돌연변이 효과 실험 중 SOS Chromotest 용량 반응결과 적정농도로 규명된 50㎍/assay 농도에서 변이원성 억제시험을 수행한 결과, 세포외 및 세포내 항돌연변이 효과가 각각 37.0%, 2.0%로 나타났으며 세포외 항돌연변이성 효과 (desmutagenic effect)임을 알 수 있었다 (도 3). 변이원과 S9 mixer를 전배양 (20min)시킨 후 원심 세척하여 인진 시료를 첨가하는 세포내 항돌연변이 실험결과, 억제활성을 나타내지 못하였으며 그 결과는 세포 분열의 과정에서 돌연변이가 고정화되는 것 또는 이미 DNA에 손상이 일어난 경우에 세포의 DNA 수복 및 복제과정에서 변이원의 발생 빈도를 억제하지 못한다는 것을 의미한다.As a result of SOS Chromotest dose response in the intracellular antimutagenic effect test, mutagenicity inhibition test was performed at the concentration of 50㎍ / assay, and the extracellular and intracellular antimutagenic effects were 37.0% and 2.0%, respectively. And it can be seen that the extracellular antimutagenic effect (desmutagenic effect) (Fig. 3). Intracellular antimutation experiments in which the mutagen and the S9 mixer were preincubated (20 min) and then centrifuged to add a human sample showed no inhibitory activity. The result was that the mutations were immobilized or DNA was already present during cell division. In case of damage to cells, it means that the incidence of mutagen is not suppressed during DNA repair and replication of cells.
시토크롬 P-450계에 의해 대사되는 B(a)P의 산화물은 대부분 소수성이나, 시토크롬 P-450 1A1 효소에 의해 대사되어진 B(a)P diol-epoxide 산화체는 가장 강한 친전자성으로 DNA 결합물을 강하게 형성시킨다고 알려져 있다. 또한 사람의 간세포 효소 시토크롬 P-450 1A1에 의해 B(a)P은 7,8-diol, 9,10-epoxide 및 3-hydroxy-B(a)P 산화물로 대사됨으로 이들의 생성을 억제하는 물질은 B(a)P과 같이 오염된 대기나, 특히 담배연기에 많은 polycyclic aromatic hydrocarbon들에 의해 발생되는 발암성을 억제할 것이다. 이러한 B(a)P의 대사물을 측정하려고 시도하였으나 시토크롬 P-450 1A1 효소에 의해 B(a)P로부터 대사되는 산화물을 정확히 분리, 측정하기가 어려우므로 간접적으로 (side effect) 시토크롬 P-450 1A1에 의해 산화되는 AFM1의 형성능을 측정하였다.Most of the oxides of B (a) P metabolized by cytochrome P-450 are hydrophobic, but the B (a) P diol-epoxide oxidized by cytochrome P-450 1A1 enzyme is the strongest electrophilic DNA binding. It is known to form water strongly. In addition, B (a) P is metabolized to 7,8-diol, 9,10-epoxide and 3-hydroxy-B (a) P oxides by human hepatocellular enzyme cytochrome P-450 1A1, which inhibits their production. Will suppress the carcinogenicity caused by many polycyclic aromatic hydrocarbons in contaminated air, especially tobacco smoke, such as B (a) P. Attempted to measure the metabolites of B (a) P, but it is difficult to accurately isolate and measure the oxides metabolized from B (a) P by cytochrome P-450 1A1 enzyme. The formation ability of AFM 1 oxidized by 1A1 was measured.
도 4에서 볼 수 있듯이 AFB1산화산물 중 AFM1의 피크 면적이 많은 결과는 시토크롬 P-450 효소 중 P-450 1A1 효소가 2,3,7,8-테트라클로로디벤조-P-디옥신 (TCDD)으로 유도시킨 쥐의 간세포에서 절대적으로 다량 생성되었음을 확인해 주는 것이며, 시료를 첨가하여 배양한 마이크로좀에서는 AFM1이 거의 생성되지 않았다. 따라서 P450 1A1 효소의 대사에 인진쑥 물 추출물이 활성을 나타냄을 확인할 수 있었다. 이러한 결과로부터 인진의 B(a)P에 대한 항돌연변이 효과(desmutagenic effect)는 인진쑥 물 추출물이 시토크롬 P-450 1A1에 의한 최종 돌연변이 대사 (ultimate mutagenic metabolites)로 활성화됨을 차단하는 것으로 예측된다.As can be seen in FIG. 4, the result of having a large peak area of AFM 1 in the AFB 1 oxidation product was that 2,3,7,8-tetrachlorodibenzo-P-dioxine (P-450 1A1 enzyme in cytochrome P-450 enzyme). TCDD) induced a large amount of hepatocytes induced by rats, and almost no AFM 1 was produced in the microsomes cultured with the sample. Therefore, it was confirmed that the extract of Insugyeong water showed activity in the metabolism of P450 1A1 enzyme. From these results, the antimutagenic effect of human genes on B (a) P is predicted to block the activation of phosphorus mugwort water extracts by the final mutagenic metabolites by cytochrome P-450 1A1.
결론적으로 인진쑥 추출물의 벤조피렌 [B(a)P] 변이원성에 대한 억제효과를 SOS Chromotest로 시험한 결과, 에탄올 가용성 분획과 수용성 분획 중 수용성 분획에서 돌연변이 억제효과가 더 높게 나타났다. 수용성 분획은 SOS Chromotest 및 Ames test에서 정확한 용량반응의 억제효과를 나타내었으며, 50% 돌연변이 억제농도 (IC50)는 대장균 PQ37에 대하여는 200㎍/assay,S.typhimuriumTA98에 대하여는 800㎍/plate,S.typhimuriumTA100에서는 600㎍/plate였다. 세포내·외 항돌연변이 효과를 비교한 결과 세포내 항돌연변이 효과는 나타내지 않았다.In conclusion, SOS Chromotest tested the inhibitory effect of jinpyrui extract on benzopyrene [B (a) P] mutagenicity. The water-soluble fraction showed the correct inhibitory effect on the SOS Chromotest and Ames test, and the 50% mutation inhibition concentration (IC 50 ) was 200 µg / assay for E. coli PQ37 and 800 µg / plate for S. typhimurium TA98. In typhimurium TA100, 600 μg / plate. Comparing intracellular and extracellular mutagenic effects did not show intracellular antimutagenic effects.
HPLC를 사용하여 B(a)P의 변이원성의 주효소인 시토크롬 P-450 1A1에 의해 대사되어지는 아플라톡신 M1(AFM1, aflatoxin M1) 농도를 측정한 결과 AFM1의 형성이 두드러지게 감소되었다. B(a)P의 변이원성에 대한 인진쑥 물추출물의 항돌연변이 효과는 아마도 B(a)P를 최종돌연변이원 (ultimate mutagen)으로 대사시키는 시토크롬 P-450 1A1 효소계를 저해함으로써 나타나는 결과로 해석된다.B (a) Aflatoxin being metabolized by primary enzyme cytochrome P-450 1A1 in the mutagenicity of the P M 1 (AFM 1, aflatoxin M 1) As a result of measuring the concentration of the form of AFM 1 was reduced remarkably by using the HPLC . The antimutagenic effect of the water extract of jinseng wormwood on the mutagenicity of B (a) P is probably interpreted as the result of inhibiting the cytochrome P-450 1A1 enzymatic system that metabolizes B (a) P to the ultimate mutagen.
실시예 5 : 인진 추출물의 최적의 추출조건 탐색Example 5 Search for Optimal Extraction Conditions of Injin Extract
제1단계. 인진 추출비율에 따른 가용성 고형분 농도 및 pH 변화First step. Soluble Solids Concentration and pH Changes with Extraction Rate
수확기 구분 없이 잎 및 줄기를 포함하여 세절한 인진으로부터 인진 추출물을 분리하였다.Phosphorus extracts were isolated from fine phosphorus, including leaves and stems, without harvesting season.
최근 한방탕재의 조재시 보편적으로 행해지고 있는 고온고압의 추출조건인 120℃, 1.5kg/cm2의 조건하에서 잎과 줄기를 구분하고 10%의 함량으로 4∼5시간동안 추출한 후 여과포로 압착 여과한 여과액에 효소를 처리하고 잎과 줄기를 혼합한 인진의 추출비율을 5%, 10%, 15%의 농도로 하여 추출하였다.The leaves and stems were separated under the conditions of 120 ℃ and 1.5kg / cm 2 , which are commonly used in the preparation of herbal medicines, under the condition of 120 ℃ and 1.5kg / cm 2 . Enzyme was treated to the filtrate, and the extraction ratio of phosphorus mixed with leaves and stems was extracted at a concentration of 5%, 10%, and 15%.
인진을 고온고압의 추출방법에 따라 추출한 추출액의 브릭스 (Brix)와 pH는 표 8에 나타낸 바와 같이 인진의 추출 비율이 증가함에 따라 가용성 고형분(Brix)의 농도도 증가하였으며 10% 전후의 추출조건이 추출에 가장 적합한 농도를 나타냈다.As the extraction rate of phosphorus increased, the concentration of soluble solids increased and the concentration of soluble solids increased as the extraction rate of phosphorus was extracted according to the extraction method of high temperature and high pressure. The concentration best suited for extraction was shown.
제2단계. 온도조건 및 개폐 조건에 따른 인진 추출물의 특성 및 관능평가Second step. Characteristics and Sensory Evaluation of Injin Extract According to Temperature and Opening Conditions
기존의 고온고압 추출방식을 배제하고 경제적인 추출물을 얻기 위하여 90℃, 97℃, 100℃의 상태에서 각각 2시간 동안 추출용기에 10%에 해당하는 시료를 넣고 밀폐 추출할 때 인진 특유의 고미 (쓴맛)를 줄여 관능 평가시 향상된 결과를 얻기 위하여 환류 냉각장치를 이용하여 추출하였다. 한편, 추출온도에 따른 추출효율의 비교를 위하여 밀폐용기에 시료 10%와 정제수를 넣고 90, 97, 100, 110℃ 온도조건으로 4시간 동안 추출하였으며 110℃의 경우에는 0.8㎏/㎠의 조건으로 유지하였다.In order to exclude the existing high-temperature and high-pressure extraction method and obtain economical extracts, injecting 10% of the sample into the extraction container for 2 hours at 90 ℃, 97 ℃, and 100 ℃ respectively, it is unique to the human taste Bitter taste) was extracted using a reflux condenser to obtain improved results in sensory evaluation. On the other hand, in order to compare the extraction efficiency according to the extraction temperature, put 10% sample and purified water in a sealed container and extracted for 4 hours at 90, 97, 100, 110 ℃ temperature conditions, in the case of 110 ℃ 0.8 ℃ / ㎠ Maintained.
추출온도에 따른 밀폐조건 하에서의 추출액의 상태변화는 표 9와 같이 추출온도가 높을수록 당도는 상승하였으나 상대적으로 추출액의 투광도는 떨어졌다. 본 발명에서는 추출물의 효능 및 효과의 차이가 크게 없는 한 추출온도를 90∼110℃범위에서 낮추어 추출함으로써 효소처리를 하지 않고 추출액의 침전물을 최소화 할 수 있었다. 본 발명에서는 투광도 및 추출당도로 보아 97℃가 가장 바람직한 추출온도임을 알 수 있었다.As the extraction temperature increased, the sugar content increased as the extraction temperature increased, but the light transmittance of the extract decreased. In the present invention, the extraction temperature was lowered in the range of 90 to 110 ° C. as long as there is no significant difference in the efficacy and effect of the extract, thereby minimizing the precipitate of the extract without enzyme treatment. In the present invention, it was found that 97 ° C is the most preferable extraction temperature from the light transmittance and extraction sugar.
환류냉각 추출시 증발하는 수증기에 함유된 방향성 물질 일부가 배출될 수 있도록 프리드리히 냉각기를 이용하여 환류냉각시켜 추출한 추출액에 대한 상태를 비교하기 위하여 표 10의 처방으로 30% 추출액을 기준으로 하여 동일 처방으로 배합하여 관능 실험을 실시하였다. 관능평가는 pennel 요원을 남녀구분 없이 시험군 별로 시험일 오전 중 음용하여 제품의 전체적인 기호성을 5점법으로 기록하고 평균 점수를 구하여 표 11과 같이 나타내었다. 제품의 향과 고미에 있어 추출액 별로 큰 차이를 보이지 않았지만 100℃ 추출액은 다소 강한 향취와 고미로 가장 낮은 점수를 보여 추출온도가 낮을수록 전체적으로 부드러운 향취미를 나타낸 것으로 보인다. 또한 밀폐조건의 추출액과의 상태를 비교한 결과 밀폐 조건에서 추출한 추출액의 경우 밀폐시키지 않은 조건보다 10∼20%가량 낮은 관능의 점수를 보였다.In order to compare the state of the extract extracted by reflux cooling using Friedrich Cooler so that some aromatic substances contained in the vapor evaporated during reflux cooling can be discharged. The compounding was conducted for sensory experiments. In sensory evaluation, pennel agents were drunk in the morning of each test group without gender distinction, and the overall palatability of the product was recorded by the 5-point method, and the average score was obtained as shown in Table 11 below. There was no significant difference in the flavor and taste of the product, but 100 ℃ extract showed the lowest score with rather strong flavor and bitter taste. In addition, as a result of comparing the state with the extract in the sealed condition, the extract extracted in the sealed condition showed a sensory score of about 10 to 20% lower than the unsealed condition.
추출조건별 추출액에 대한 제품화 실험은 환류냉각에 의한 추출액의 함량을 높여가며 고미의 정도를 기준으로 하여 적절한 추출액의 배합량을 결정하였다. 표 10에서와 같이 추출온도가 낮은 추출액일수록 향취미가 부드러워 추출온도가 90℃일 때 최고 42%까지 많은 량의 인진 추출액을 함유시키는 것이 가능해져 인진에 의한 효과를 기대할 수 있을 것이다.In the experiments on the extraction of extracts for each extraction condition, the content of extracts by reflux cooling was increased, and the appropriate amount of extracts was determined based on the degree of bitterness. As shown in Table 10, the lower the extraction temperature, the softer the flavor, so that it is possible to contain a large amount of human extract up to 42% when the extraction temperature is 90 ℃ can be expected by the effect of phosphorus.
제3단계. 인진 추출물의 부유물을 제거하기 위한 단백질 분해효소반응Third step. Proteolytic Enzyme Reaction to Remove Suspensions of Human Gene Extracts
추출액에 대한 효소처리 [G-ZYME G990ZR (glucoamylase, Enzyme Bio-Systems Ltd.)] 방법은 120℃의 고온추출의 경우에만 행하였고 추출액의 농도별로 55±5℃에서 120분 동안 교반하여 반응시켰다. 효소는 글루코아밀라제 (glucoamylase), 프로테아제 (protase)를 1차 시료와 같은 조건으로 반응시킨 후 90℃ 이상으로 가열하여 효소를 실활시킨 후 냉각하고 NO.2 여과지와 규조토 500으로 여과하였다. 효소반응에 의한 상징정도를 비교하기 위하여 여과액을 10배 희석하여 spectrophotometer로 465nm에서 투광도를 측정하였다.Enzyme treatment for the extract [G-ZYME G990ZR (glucoamylase, Enzyme Bio-Systems Ltd.)] method was performed only for the high temperature extraction of 120 ℃ and reacted by stirring for 120 minutes at 55 ± 5 ℃ by the concentration of the extract. The enzyme was reacted with glucoamylase and protease under the same conditions as the first sample, and then heated to 90 ° C. or higher to inactivate the enzyme, cooled, and filtered with NO.2 filter paper and diatomaceous earth 500. To compare the degree of symbol by enzyme reaction, the filtrate was diluted 10-fold and the light transmittance was measured at 465 nm with a spectrophotometer.
한방 식물체 추출액의 부유물은 완전제거가 어렵기 때문에 추출액을 정치하였을 때 부유물이 분리되어 침전되는 현상을 최소화하기 위하여 고온고압 추출액을 효소처리하였다. 그 결과를 표 12에 나타내었다. 추출액은 인진 10% 추출액을 사용하였으며, 글루코아밀라제 0.05중량%, 프로테아제 0.03중량%를 혼합처리하여 가장 적합한 처리 농도를 획득하였으며 투광도로 볼 때 가장 적절한 효소처리 농도는 0.08%-0.04%의 수준이었다. 이로써 침전물의 형성을 감소시킬 수 있었다.Since the suspended solids of the herbal plant extracts are difficult to remove completely, the high-temperature and high-pressure extracts were enzymatically treated to minimize the phenomenon that the suspended solids were separated and precipitated when the extract was left standing. The results are shown in Table 12. As the extract, 10% phosphorus extract was used, and 0.05% by weight of glucoamylase and 0.03% by weight of protease were mixed to obtain the most suitable treatment concentration. This could reduce the formation of precipitates.
시료에 대한 전처리 결과, 인진의 잎, 줄기 및 혼합 추출액의 침전물 발생시 효소처리하여 정제한 상기의 결과에서와 같이 글루코아밀라제 0.08%의 농도, 프로테아제 0.04%와 같은 단백질분해효소로 처리한 후 여과 정제하여 획득한 추출액을 이용하여 음료화하는 것이 관능적으로 매우 우수할 것으로 판단된다.As a result of pretreatment of the sample, the enzyme was purified when the precipitates of the leaves, stems and mixed extracts of the phosphorus were purified by treatment with proteolytic enzymes such as glucoamylase 0.08% and protease 0.04% It is judged to be very sensory to drink using the obtained extract.
실시예 6 : 인진 추출물의 음료화를 위한 최적의 추출조건 및 음료의 제조Example 6 Preparation of Optimal Extraction Conditions and Beverages for Drinking of Injin Extract
상기의 실시예 5에 따른 여러 가지 전처리 과정과 결과에 따라 얻어진 인진의 추출액을 표 13과 같이 음료로 제조하였다. 표 13의 인진 추출물 1 (extract 1)은 상기 실시예 5의 표 8에서의 잎과 줄기를 별도로 10% 추출한 것을 혼합하여 사용한 것이고, 표 14의 인진 추출물 2 (3.3브릭스, 4.3브릭스)는 상기 실시예 5의 표 8에서의 잎, 줄기 혼합 10% 추출물을 사용한 것이다.Extracts of phosphorus obtained according to various pretreatment procedures and results according to Example 5 were prepared as beverages as shown in Table 13. The extract 1 of Table 13 (extract 1) is a mixture of 10% extracted separately from the leaves and stems in Table 8 of Example 5 was used, the extract of Table 2 (3.3 Brix, 4.3 Brix) of Table 14 In Example 8, the leaf and stem mixture of Table 8 was used for 10% extract.
인진 추출액 1을 10-20%을 음료 배합실험에 적용하여 보았으나 전체적으로 쓴맛이 강해서 감미를 높여야 하는 것과 향료를 첨가하여야 하는 문제로 인진 고유의 이미지를 살리는데는 적합하지 않았다. 한편 2번 인진 추출액에 대한 음료화 실험은 표 13에 나타낸 바와 같이, 인진 혼합 추출액이 비교적 추출액의 조성이 균일하여 다양한 실험을 할 수 있었다. 표 14에서 보는 바와 같이 인진 추출액 15%에 각 원료별 배합비를 달리하여 제조한 시료 2071∼2076을 만들어 맛의 변화를 조사하였다. 그 결과, 인진 추출액만을 이용한 것보다 감미제나 산미제, 사이클로덱스트린 등을 첨가한 경우 고미의 감소효과가 한정되었으며, 이 중 사이클로덱스트린에 의한 효과가 가장 컸다.Inginol extract 1 was applied to 10-20% of the beverage blending experiments. However, the bitter taste was so strong that the sweetness and flavoring were added. On the other hand, as shown in Table 13, the drinking experiment for the phosphorus extract No. 2, the phosphorus mixture extract was relatively uniform composition of the extract was able to perform a variety of experiments. As shown in Table 14, samples 2071 to 2076 prepared by varying the compounding ratio of each raw material in 15% phosphorus extract were prepared to investigate the change in taste. As a result, when the sweetener, acidulant, cyclodextrin, etc. were added to the extract rather than using only the human extract, the effect of delicacy was limited, and the cyclodextrin had the greatest effect.
본 발명 개발음료 중 인진 추출액의 함량을 높이기 위한 실험으로2211∼2223에서는 추출액을 20%에서 30%가 되도록 구성비율을 늘렸다. 그 결과, 역시 고미가 가장 문제가 되어 감미제를 첨가하였으나 수크로스의 함량을 증가시키는 것은 제품당도를 높이므로 그 한계가 있어 대신 글리신을 첨가하였으나 제품의 맛에는 영향을 미치지 못하였다.In the experiment for increasing the content of the phosphorus extract in the beverage of the present invention, the composition ratio of the extract was increased from 20% to 30% in 2211 to 2223. As a result, the sweetener was the most problematic, but the addition of sweeteners, but increasing the content of sucrose increases the sugar content of the product because there is a limit, but glycine was added instead, but it did not affect the taste of the product.
시료 3011∼3012는 천연 감미제인 스테비오시드 (stevioside)를 첨가하여 당의 함량을 감소시키고 고미를 개선하려 하였으나, 인진의 향미를 반감시키는 결과를 가져왔다. 3131∼3134 시료는 인공 감미제를 배재하고 인진 음료수에 어울릴 수 있는 적절한 감미를 제공하기 위하여 꿀과 사이클로덱스트린를 첨가하고 각 농도별 실험을 수행한 결과, 사이클로덱스트린의 첨가 시료에서 고미를 감소시키는 결과를 보여 인진 추출액의 농도를 30%로 증가시켜 만든 3133, 3134 시료의 음용시 맛이 부드러워 제품화에 큰 문제가 없는 것으로 판단되었다.Samples 3011-3012 added stevioside, a natural sweetener, to reduce sugar content and improve taste, but resulted in half the flavor of injin. Samples 3131 to 3134 were tested for each concentration of honey and cyclodextrins to eliminate artificial sweeteners and provide an appropriate sweetness for human beverages. 3133, 3134 samples made by increasing the concentration of phosphorus extract to 30% of the soft taste when drinking it was judged that there is no big problem in commercialization.
인진을 이용한 음료를 개발하기 위하여 여러 가지 추출방법과 전처리 방법을 실시한 결과, 제품화에 적합한 인진 추출액의 전처리 방법과 제품화 방법을 획득하였다. 즉, 인진 고유의 향미를 순화시켜 음료화하기 위해서는 밀폐하지 않은 용기에서 90∼110℃의 저온 조건 및 환류 추출조건을 이용할 때 최적의 상태를 획득할 수 있으며, 이것은 추출조건의 완화로 기존의 고온고압에서의 추출시 따르는 추출액의 순화뿐만 아니라 제품화하였을 때의 품질안정성과 전처리 비용이 저렴해지므로 경제적인 이점이 있다. 또한 고압 또는 밀폐조건으로 얻은 인진 추출액을 사용한 경우 관능 평가값이 저하되므로 인진 추출액의 함량을 높이기 어려우나, 저온의 환류 추출조건에서 얻은 추출액의 경우 음료제품 중 인진 추출물이 40% 이상 함유되어도 관능평가치가 크게 떨어지지 않아 40%의 인진 추출물을 함유시킨 효과적인 제품의 개발이 가능하여 인진 고유의 기능성을 높일 수 있는 음료를 개발할 수 있을 것으로 기대된다. 인진의 상품성을 높이기 위한 제품화 과정에서 감미료로 최소량의 벌꿀을 사용하였으며 산미제 및 기타 첨가물 사용의 최소화로 인공적인 풍미를 배제한 결과, 벌꿀 9.8%이하, 구연산 0.035%, 사이클로덱스트린 (Cydex-S) 1.47%의 배합비로 구성된 음료를 개발하였다.As a result of various extraction and pretreatment methods to develop drinks using phosphorus, we obtained the pretreatment method and commercialization method of phosphorus extract suitable for commercialization. In other words, in order to purify the intrinsic flavor of Injin, it is possible to obtain an optimal state when using low temperature and reflux extraction conditions of 90 ~ 110 ℃ in an unsealed container. There is an economical advantage because the quality stability and pretreatment cost of the extract as well as the purification of the extraction solution at high pressure is reduced. In addition, it is difficult to increase the content of the phosphorus extract when the phosphorus extract obtained under high pressure or closed conditions is lowered. However, the extract obtained under reflux extraction conditions at low temperature may contain more than 40% of phosphorus extract in the beverage product. It is expected to be able to develop an effective product containing 40% phosphorus extract, so that it can develop a drink that can enhance the intrinsic functionality of phosphorus. As a sweetener in the process of commercialization of Injin, the minimum amount of honey was used, and artificial flavors were excluded by minimizing the use of acidifiers and other additives. Honey 9.8% or less, citric acid 0.035%, cyclodextrin (Cydex-S) 1.47 A beverage consisting of% blend was developed.
실시예 7 : 인진 추출물의 음료화를 위한 농축액의 이용 및 음료의 제조Example 7 Use of Concentrates for the Drinking of Human Extracts and Preparation of Beverages
인진 추출물 함유 음료 제품 중 높은 농도의 인진 추출액을 포함하며 기호성이 뛰어난 음료를 개발하기 위하여 1차 관능적으로 거부감이 없는 추출액으로 음료 조성물을 개발하였다.In order to develop a beverage having a high concentration of phosphorous extract and containing a high concentration of phosphorus extract in the phosphorus extract-containing beverage product, the beverage composition was developed as an extract that is not primarily sensory.
인진을 기호성이 높은 추출액으로 추출할 수 있는 조건은 실시예 6에 의한 결과로 90℃의 환류 추출조건을 2차적인 처리로 거치는 방법을 부가하였다.The conditions under which phosphorous extract can be extracted with a highly palatable extract were added as a result of Example 6 through a reflux extraction condition of 90 ° C. as a secondary treatment.
실험 방법은 5% 인진을 90℃의 환류 추출조건에서 추출한 추출액을 감압 농축기를 이용하여 20 브릭스 (brix)로 농축하여 만든 농축액을 이용하였다. 농축효과를 비교하기 위하여 농축하지 않은 추출액을 대조구로 하고 농축액을 추출액과 동일한 농도로 희석하여 음료의 배합에 이용하였다. 여기에서 추출액의 당도 기준은 1.2브릭스를 기준으로 하여 10∼25%의 농도로 배합하였다. 배합한 음료에 대한 관능실험은 실시예 6에서 실시한 방법으로 실시하여 그 결과를 표 15에 나타내었다. 쓴맛에 대한 결과로 추출액의 농도가 같은 경우 농축액을 사용시 25% 이상의 높은 추출액의 함량을 투여했지만 쓴맛의 정도는 추출액을 10%사용한 제품보다 관능점수가 우수하여 추출액을 농축하여 사용할 경우 관능적으로도 우수하고 1.5배 높은 함량의 인진 추출액을 함유시켜 높은 기능성을 기대할 수 있는 방법으로 사료된다.In the experimental method, the extract obtained by concentrating the extract extracted from 5% phosphorus at 90 ° C. under reflux extraction conditions using a vacuum concentrator to 20 brix was used. In order to compare the concentration effect, the non-concentrated extract was used as a control, and the concentrate was diluted to the same concentration as the extract and used for blending the beverage. Here, the sugar content of the extract was formulated at a concentration of 10 to 25% based on 1.2 brix. Sensory experiment on the blended beverage was carried out by the method described in Example 6 and the results are shown in Table 15. As a result of the bitterness, the concentration of the extract was the same when the concentration of the extract was higher than 25%, but the bitter taste was higher than the product using 10% of the extract. It is considered to be a method that can expect high functionality by containing 1.5 times higher phosphorus extract.
본 실시예에서 인진을 제외한 첨가물의 배합비는 사과 농축액 2.0%, 설탕 8.0%, 구연산 0.14%, 베타사이클로덱스트린 0.3%, 벌꿀 3.0%를 기준으로 만들었다.In the present embodiment, the compounding ratio of the additive except phosphorus was based on apple concentrate 2.0%, sugar 8.0%, citric acid 0.14%, betacyclodextrin 0.3%, and honey 3.0%.
이상의 실시예에서 설명한 바와 같이, 본 발명은 인진 추출물의 유효성분인 스코폴레틴 (scopoletin)이 혈관에 미치는 영향, 인진의 추출물이 뇌혈류량에 미치는 영향 및 항산화 항돌연변이원성을 조사하여 효능이 입증된 인진 추출물의 기호성 및 관능을 개선할 수 있는 추출방법으로, 인진 추출물의 제조시 밀폐하지 않은용기에서 90∼110℃의 저온추출법으로 추출하거나, 단백질 분해효소를 첨가하거나 또는 감압농축한 농축액의 형태로 추출함으로써 향취미가 뛰어나고 맛이 부드러우며 침전물의 양이 적어 관능이 뛰어나며, 상대적으로 많은 양의 인진 추출물을 음료에 첨가할 수 있어 인진 추출물의 혈관이완, 뇌졸중 예방효과 및 항암효과를 높일 수 있는 인진쑥 추출물 함유 기능성 음료 및 그 제조방법을 제공하는 뛰어난 효과가 있으므로 국민건강증진 및 식품가공산업상 매우 유용한 발명인 것이다.As described in the above embodiment, the present invention has been proved efficacy by investigating the effect of scopoletin (scopoletin), an active ingredient of the jinjin extract on blood vessels, the effect of the extract of jinjin on the blood flow of the brain and antioxidant antimutagenicity Extraction method to improve palatability and sensory properties of jinjin extract, extracted by cold extraction method of 90 ~ 110 ℃ in an unsealed container in the production of jinjin extract, or added in the form of a concentrated solution concentrated under reduced pressure By extracting, it has excellent flavour, soft taste and small amount of sediment, so it has excellent sensory ability, and it is possible to add a relatively large amount of human extract to beverages, which can enhance vascular relaxation, stroke prevention effect and anticancer effect of human extract. Functional health beverage containing extracts It is a very useful invention in the promotion and food processing industries.
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KR100429141B1 (en) * | 2001-05-17 | 2004-04-28 | 남궁순자 | health food beverage use mugwort and it's manufacture method |
KR100420767B1 (en) * | 2002-01-31 | 2004-03-02 | 현 용 이 | The preparing method of the ingin-mugwort extract in which the components of bitter taste were selectively removed, and the functional liquid food of using it |
KR20040034897A (en) * | 2002-10-17 | 2004-04-29 | 내츄럴네오팜(주) | Antioxidant composition comprising herbal extracts |
KR100601265B1 (en) * | 2002-10-24 | 2006-07-14 | 알앤엘생명과학주식회사 | A composition for promoting growth or secretion of growth hormone comprising an extract of artemisia capillaris thunberg. and functional cereals coated by same |
RU2007126833A (en) * | 2004-12-16 | 2009-01-27 | Хилл`С Пет Ньютришн, Инк. (Us) | METHODS FOR IMPROVING THE TASTE OF FOOD COMPOSITIONS |
KR102269260B1 (en) * | 2019-12-11 | 2021-06-25 | 대한민국 | Method for preparing high cynarin extract from Artemisia capillaris by high temperature and high pressure treatment |
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JPH08107779A (en) * | 1994-10-11 | 1996-04-30 | Chojiro Noguchi | Mugwort juice for healthy beverage and its production |
KR980000186A (en) * | 1997-10-07 | 1998-03-30 | 용태웅 | Manufacturing method of mugwort drink |
KR0164840B1 (en) * | 1996-04-24 | 1998-12-01 | 손경식 | Process of wormwood beverage and its products |
KR20000024959A (en) * | 1998-10-07 | 2000-05-06 | 김부권 | Method for preparing artemisia capillaris thunb distilled water |
KR101223099B1 (en) * | 2010-12-31 | 2013-01-17 | 주식회사 한샘 | Method for embossing on sheet |
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KR860006947A (en) * | 1985-03-13 | 1986-10-06 | 2) 구자연(외 | Mugwort Beverage Manufacturing Method |
JPH08107779A (en) * | 1994-10-11 | 1996-04-30 | Chojiro Noguchi | Mugwort juice for healthy beverage and its production |
KR0164840B1 (en) * | 1996-04-24 | 1998-12-01 | 손경식 | Process of wormwood beverage and its products |
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KR101267509B1 (en) * | 2012-12-03 | 2013-05-27 | (주)맥스파워이앤디 | Method for manufacturing multi-functional energy drink and multi-functional energy drink manufactured thereby |
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