KR101259791B1 - Method for manufacturing health supplement food and health supplement food thereof - Google Patents
Method for manufacturing health supplement food and health supplement food thereof Download PDFInfo
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- KR101259791B1 KR101259791B1 KR1020100041713A KR20100041713A KR101259791B1 KR 101259791 B1 KR101259791 B1 KR 101259791B1 KR 1020100041713 A KR1020100041713 A KR 1020100041713A KR 20100041713 A KR20100041713 A KR 20100041713A KR 101259791 B1 KR101259791 B1 KR 101259791B1
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- South Korea
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- citrus
- vitamin
- concentrate
- mixture
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- 238000000034 method Methods 0.000 title claims abstract description 14
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- A—HUMAN NECESSITIES
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Abstract
본 발명에 의한 감귤류를 함유한 건강보조식품의 제조방법 및 이를 이용한 건강보조식품은 세척된 미성숙 감귤을 분쇄한 후에 슬러지를 분리한 감귤즙을 농축하여 감귤농축액을 준비하는 감귤농축액준비단계; 상기 감귤농축액준비단계의 감귤농축액 4 ~ 8 중량%, 포도당 83 ~ 90중량%, 페닐알긴산을 함유한 아스파탐 0.03 ~ 0.05 중량%, 감귤향분말 1 ~ 2 중량%, 자당지방산에스테르 0.5 ~ 1.5중량%, 비타민C 1.5 ~ 2.5 중량%, 구연산 1.5 ~ 2.5 중량%, 겔라틴 0.01 ~ 0.03 중량%, 비타민B1 0.01 ~ 0.02 중량%, 비타민B2 0.01 ~ 0.02 중량%, 비타민B3 0.01 ~ 0.02 중량%, 판토텐산칼슘(B5) 0.05 ~ 0.07 중량%, 엽산 0.002 ~ 0.004 중량%, 비타민A 0.05 ~ 0.12 중량%, 비오틴(B7) 0.0001 ~ 0.0005 중량%, 나이아신 0.1 ~ 0.2 중량%, 콜라겐 0.05 ~ 0.15 중량%, 밀크칼슘 0.1 ~ 0.15 중량%, 탄산칼슘 0.03 ~ 0.08 중량%를 혼합 반죽하여 혼합물을 형성하는 혼합반죽단계; 상기 조성물혼합단계의 혼합물을 성형 및 건조하는 성형단계; 로 이루어진다.
따라서 본 발명은 감귤류의 미숙과를 이용하여 식품화하여 소비자에게 섭취시킬 수 있어 감귤류에 함유된 시트러스 플라보노이드를 용이하게 섭취할 수 있어 시트러스 플라보노이드의 유익한 성분으로 인체의 건강을 용이하게 유지시킬 수 있으며, 쓰임새가 없는 감귤류 미숙과를 이용하므로 자원 활용도를 용이하게 증대하여 감귤류의 생산을 용이하게 증진시킬 수 있는 등의 효과를 발휘한다.Method for preparing a health supplement food containing citrus fruits and health supplement food using the same according to the present invention is a citrus concentrate preparation step of preparing a citrus concentrate by concentrating the sewage separated citrus juice after crushing the washed immature citrus; Citrus concentrate 4 ~ 8% by weight of the preparation of the citrus concentrate, glucose 83 ~ 90% by weight, 0.03 ~ 0.05% by weight aspartame containing phenyl alginic acid, 1 ~ 2% by weight citrus flavor powder, sucrose fatty acid ester 0.5 ~ 1.5% by weight , Vitamin C 1.5 to 2.5% by weight, Citric acid 1.5 to 2.5% by weight, Gelatin 0.01 to 0.03% by weight, Vitamin B 1 0.01 to 0.02% by weight, Vitamin B 2 0.01 to 0.02% by weight, Vitamin B 3 0.01 to 0.02% by weight , Calcium pantothenate (B5) 0.05 to 0.07 wt%, folic acid 0.002 to 0.004 wt%, vitamin A 0.05 to 0.12 wt%, biotin (B7) 0.0001 to 0.0005 wt%, niacin 0.1 to 0.2 wt%, collagen 0.05 to 0.15 wt% Mixing dough step of mixing and kneading milk calcium 0.1 ~ 0.15% by weight, calcium carbonate 0.03 ~ 0.08% by weight; A molding step of molding and drying the mixture of the composition mixing step; .
Therefore, the present invention can be ingested to consumers by using the citrus fruits inexperienced and can be easily ingested citrus flavonoids contained in citrus fruits can be easily maintained as a beneficial component of citrus flavonoids, the health of the human body, The use of unsweetened citrus fruits makes it easier to increase the utilization of resources, thus facilitating the production of citrus fruits.
Description
본 발명은 감귤류를 함유한 건강보조식품의 제조방법 및 이를 이용한 건강보조식품에 관한 것으로서, 더욱 상세하게는 감귤류 미숙과를 이용하여 식품화함으로써 감귤류에 함유된 시트러스 플라보노이드(Citrus Flavonoid)를 용이하게 섭취할 수 있도록 하는 감귤류를 함유한 건강보조식품의 제조방법 및 이를 이용한 건강보조식품에 관한 것이다.The present invention relates to a method for preparing a health supplement containing citrus fruits and a health supplement using the same, more specifically, by easily citrus citrus flavonoids (Citrus Flavonoid) contained in the citrus by food It relates to a method of manufacturing a health supplement containing citrus fruits and a health supplement using the same.
남성과 여성의 사회 활동의 증가와 야간활동의 인구증가, 대중소비시대, 소비패턴의 다양화, 편리성 추구경향 등으로 인하여 사회적 경제적 환경이 변화됨과 아울러 식생활 구조가 변화되며, 식생활의 변화에 따라 고칼로리의 인스턴트 식품으로 인하여 비만증, 고지혈증, 고혈압을 비롯하여 각종 성인병이 급증하고 있다.Due to the increase in social activities of men and women, the increase in the number of night activities, the age of mass consumption, the diversification of consumption patterns, and the trend of convenience, the social and economic environment is changed and the dietary structure is changed. Due to the high-calorie instant food, various adult diseases, such as obesity, hyperlipidemia and hypertension, are rapidly increasing.
즉 잘못된 식생활은 영양부족, 영양 과잉 및 잘못된 영양 정보에 의한 영양불량을 초래하기 쉬우며, 영양부족으로 인한 영양불량은 빈혈 등 여러 질병을 일으키게 되고, 영양소의 과잉 섭취는 체중 과다 및 비만을 유발하여 고지혈증, 동맥경화증, 당뇨병, 고혈압 등과 같은 만성 퇴행성 질환의 발병을 더욱 가중시키게 된다.In other words, poor eating habits are likely to cause malnutrition due to malnutrition, malnutrition, and incorrect nutritional information. Malnutrition due to malnutrition causes various diseases such as anemia, and excessive intake of nutrients causes overweight and obesity. It further increases the onset of chronic degenerative diseases such as hyperlipidemia, arteriosclerosis, diabetes, hypertension and the like.
그래서 종래에는 홍삼, 함초, 키토산, 솔잎, 신선초, 삼백초, 쑥 등과 같은 약초, 해산물 등을 함유한 건강보조식품이 많이 개발되었으나, 쓰임새가 없는 감귤류 미숙과를 이용하여 건강보조식품이 개발되지 않았다.Therefore, in the past, many health supplements containing herbs, seafood, etc., such as red ginseng, hamcho, chitosan, pine needles, fresh vinegar, triticale, wormwood, etc., have been developed, but health supplements have not been developed by using unripe citrus fruits.
참고로 감귤류는 감귤, 당유자, 진귤, 지각, 편귤 등이 있으며, 감귤류의 과피에는 테르페노이드(terpenoid), 플라보노이드(flavonoid), 리모노이드(limonoid), 카로테노이드(carotenoid) 등 생리활성 2차대사물이 포함됨과 아울러 헤스페리딘(hesperidin), 나린진(naringin) 등 바이오플라보노이드(bioflavonoid)가 많이 존재하며, 감귤류의 과실은 대표적으로 비타민C 및 다양한 바이오플라보노이드(bioflavonoid)를 함유하고 있다.Citrus fruits include citrus fruits, sugary citrus fruits, tangerines, crust, and cinnamon.The citrus skins contain terpenoids, flavonoids, limonoids, and carotenoids. In addition to water, hesperidin, naringin, and other bioflavonoids are present, and citrus fruits typically contain vitamin C and various bioflavonoids.
바이오플라보노이드(Bioflavonoid)는 필수적인 영양요소가 아니지만 건강에 도움을 주는 역할을 하고 있는 물질군으로서, 비타민C의 생성을 도와주는 영양소이고, 비타민C와 건강한 모세혈관을 형성하여 콜라겐의 형성을 도와주고, 상처가 아무는 것을 도우고 건강한 면역계를 유지시키며, 감귤류에서는 헤스페리딘(hesperidin), 나린진(naringin) 외에도 루틴(rutin), 나리루틴(narirutin), 네오헤스페리딘(neohesperidin), 시넨세틴(sinensetin), 폰시린(poncirin), 다이디민(didymin) 등이다.Bioflavonoid is not an essential nutrient but is a group of substances that play a role in health. It is a nutrient that helps produce vitamin C. It helps to form collagen by forming vitamin C and healthy capillaries. Helps to heal wounds and maintain a healthy immune system.In citrus fruits, in addition to hesperidin and naringin, rutin, narirutin, neohesperidin, sinencetin, sinensetin and poncirine (poncirin), didymin, and the like.
상기와 같은 유익한 성분이 감귤류에 많이 함유하고 있으며, 종래에는 쥬스로 많이 사용하기는 하나, 쓰임새가 없는 감귤류 미숙과를 이용하여 건강보조식품으로 개발하지 못했다는 단점이 있다.The beneficial ingredients as described above contain a lot of citrus fruits, but conventionally used a lot as a juice, there is a disadvantage that did not develop as a dietary supplement using citrus unripe fruit without any use.
본 발명은 상기와 같은 종래 기술의 문제점을 해결하기 위하여 발명된 것으로서, 본 발명의 목적은 감귤류의 미숙과를 이용하여 식품화함으로써, 감귤류에 함유된 시트러스 플라보노이드(Citrus Flavonoid)를 용이하게 섭취할 수 있고, 시트러스 플라보노이드의 유익한 성분으로 인체의 건강을 용이하게 유지시킬 수 있는 감귤류를 함유한 건강보조식품의 제조방법 및 이를 이용한 건강보조식품을 제공하는 데 있다.The present invention has been invented to solve the problems of the prior art as described above, the object of the present invention is to food intake using citrus fruits immature, can easily ingest citrus flavonoid (Citrus Flavonoid) contained in citrus fruits The present invention provides a method of preparing a health supplement containing citrus fruits and a health supplement using the same as an advantageous component of the citrus flavonoid.
상기와 같은 목적을 달성하기 위하여, 본 발명은 세척된 미성숙 감귤류를 분쇄한 후에 슬러지를 분리한 감귤즙을 농축하여 감귤농축액을 준비하는 감귤농축액준비단계; 상기 감귤농축액준비단계의 감귤농축액 4 ~ 8 중량%, 포도당 83 ~ 90중량%, 페닐알긴산을 함유한 아스파탐 0.03 ~ 0.05 중량%, 감귤향분말 1 ~ 2 중량%, 자당지방산에스테르 0.5 ~ 1.5중량%, 비타민C 1.5 ~ 2.5 중량%, 구연산 1.5 ~ 2.5 중량%, 겔라틴 0.01 ~ 0.03 중량%, 비타민B1 0.01 ~ 0.02 중량%, 비타민B2 0.01 ~ 0.02 중량%, 비타민B3 0.01 ~ 0.02 중량%, 판토텐산칼슘(B5) 0.05 ~ 0.07 중량%, 엽산 0.002 ~ 0.004 중량%, 비타민A 0.05 ~ 0.12 중량%, 비오틴(B7) 0.0001 ~ 0.0005 중량%, 나이아신 0.1 ~ 0.2 중량%, 콜라겐 0.05 ~ 0.15 중량%, 밀크칼슘 0.1 ~ 0.15 중량%, 탄산칼슘 0.03 ~ 0.08 중량%를 혼합 반죽하여 혼합물을 형성하는 혼합반죽단계; 상기 조성물혼합단계의 혼합물을 성형 및 건조하는 성형단계; 로 이루어지고, 상기 감귤농축액준비단계는 감귤농축액의 100중량부를 기준으로 감귤류 미숙과로부터 분획된 시트러스 플라보노이드 3 ~ 9중량부를 혼합하고, 상기 감귤농축액준비단계에서는 세척된 미성숙 감귤류을 습식분쇄기로 1차 분쇄한 후에 습식마이크로분쇄기로 2차 분쇄하고, 추출기에 효소와 함께 투입 교반하여 효소 분해하고, 원심분리기에 의해 분리된 감귤즙을 농축기로 50 ~ 100브릭스(Brix) 농도로 농축하여 감귤농축액을 형성하고, 상기 혼합반죽단계의 혼합물을 0.3mm ~ 3mm 크기로 과립화한 후에 과립화한 혼합물을 성형하고, 상기 혼합반죽단계의 혼합물을 32 ~ 38 ℃에서 3 ~ 12시간 숙성한 후에 상기 성형단계에서 성형하고, 상기 혼합반죽단계에서 천연색소인 치자황색소 0.05 ~ 0.13 중량%를 혼합하는 것을 포함한다.In order to achieve the above object, the present invention is a citrus concentrate preparation step of preparing a citrus concentrate by concentrating the citrus juice separated from the sludge after grinding the washed immature citrus; Citrus concentrate 4 ~ 8% by weight of the preparation of the citrus concentrate, glucose 83 ~ 90% by weight, 0.03 ~ 0.05% by weight aspartame containing phenyl alginic acid, 1 ~ 2% by weight citrus flavor powder, sucrose fatty acid ester 0.5 ~ 1.5% by weight , Vitamin C 1.5 to 2.5% by weight, Citric acid 1.5 to 2.5% by weight, Gelatin 0.01 to 0.03% by weight, Vitamin B 1 0.01 to 0.02% by weight, Vitamin B 2 0.01 to 0.02% by weight, Vitamin B 3 0.01 to 0.02% by weight , Calcium pantothenate (B5) 0.05 to 0.07 wt%, folic acid 0.002 to 0.004 wt%, vitamin A 0.05 to 0.12 wt%, biotin (B7) 0.0001 to 0.0005 wt%, niacin 0.1 to 0.2 wt%, collagen 0.05 to 0.15 wt% Mixing dough step of mixing and kneading milk calcium 0.1 ~ 0.15% by weight, calcium carbonate 0.03 ~ 0.08% by weight; A molding step of molding and drying the mixture of the composition mixing step; The citrus concentrate preparation step consists of 3 to 9 parts by weight of citrus flavonoids fractionated from citrus fruits immature based on 100 parts by weight of citrus concentrates, and in the preparation step of citrus concentrates, the immature citrus fruits are washed with a wet mill. After the secondary grinding with a wet micro crusher, and enzymatic decomposition by stirring with the enzyme in the extractor, the concentrated citrus juice by a centrifuge to a concentration of 50 ~ 100 Brix (Brix) concentration to form a citrus concentrate After granulating the mixture of the kneading step in a size of 0.3 mm to 3 mm, the granulated mixture is molded, and the mixture of the kneading step is aged at 32 to 38 ° C. for 3 to 12 hours and then molded in the forming step. And, in the mixing and kneading step, it comprises mixing 0.05 ~ 0.13% by weight of the natural pigments of gardenia yellow.
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이와 같이 이루어지는 본 발명에 의한 감귤류를 함유한 건강보조식품은 감귤류의 미숙과를 이용하여 식품화하여 소비자에게 섭취시킬 수 있으므로, 감귤류에 함유된 시트러스 플라보노이드(Citrus Flavonoid)를 용이하게 섭취할 수 있어 시트러스 플라보노이드의 유익한 성분으로 인체의 건강을 용이하게 유지시킬 수 있다는 이점이 있다.The health supplement containing the citrus fruits according to the present invention can be ingested to consumers by using the citrus fruits immature fruit, can be easily ingested citrus flavonoid (Citrus Flavonoid) contained in citrus citrus The beneficial component of the flavonoids has the advantage that it is easy to maintain the health of the human body.
또한 본 발명은 쓰임새가 없는 감귤류 미숙과를 이용하기 때문에, 자원 활용도를 용이하게 증대하여 농가의 감귤류 생산을 용이하게 증진시킬 수 있다는 이점이 있다.In addition, since the present invention uses a citrus unripe fruit, there is an advantage that it is possible to easily increase the utilization of resources to facilitate the production of citrus fruits of farms.
도 1은 보고된 당유자의 시기별 플라보노이드(flavonoid) 함량 변화 추이를 나타내는 도표이다.
도 2는 MPLC를 이용한 neohesperidin의 분리 그래프이다.
도 3은 분리된 플라보노이드 분획 및 분리과정중의 HPLC chromatograph이다.
도 4는 분리된 neohesperidin의 1H NMR spectrum (100MHz in DMSO-d 6)이다.
도 5는 분리된 neohesperidin의 13C NMR spectrum (100MHz in DMSO-d 6)이다.
도 6은 sigma사의 표준품 HPLC chromatogram(1mg/mL)이다.
도 7은 분리된 neohesperidin의 HPLC chromatogram(1mg/mL)이다.
도 8은 DPPH 라디칼 소거활성 결과를 나타내는 도표이다.
도 9는 NO 자유기 소거활성 결과를 나타내는 도표이다.
도 10은 수퍼옥사이드(Superoxidase) 억제 활성결과를 나타내는 도표들이다.
도 11은 젠신 옥시데이즈(Xanthine oxidase) 억제 활성결과를 나타내는 도표들이다.
도 12는 RAW 264.7 cell을 이용한 세포독성과 항염활성 결과를 나타내는 도표들이다.
도 13은 세포내 타이로시나아제 활성 결과를 나타내는 도표이다.Figure 1 is a chart showing the change in the flavonoid content (flavonoid) content of the sugar sugars reported time.
2 is a graph of separation of neohesperidin using MPLC.
3 is an isolated flavonoid fraction and HPLC chromatograph during the separation process.
4 is a 1 H NMR spectrum (100 MHz in DMSO- d 6 ) of neohesperidin isolated.
5 is a 13 C NMR spectrum (100 MHz in DMSO- d 6 ) of neohesperidin isolated.
6 is a standard HPLC chromatogram (1 mg / mL) from sigma.
7 is an HPLC chromatogram (1 mg / mL) of neohesperidin isolated.
8 is a graph showing the results of DPPH radical scavenging activity.
9 is a chart showing the result of NO free radical scavenging activity.
10 is a chart showing the results of superoxide inhibitory activity.
11 is a table showing the results of Xanthine oxidase inhibitory activity.
12 is a chart showing the results of cytotoxicity and anti-inflammatory activity using RAW 264.7 cells.
13 is a chart showing the results of intracellular tyrosinase activity.
이하 본 발명에 의한 감귤류를 함유한 건강보조식품의 제조방법 및 이를 이용한 건강보조식품의 실시예를 더욱 상세하게 설명한다.Hereinafter, a method of preparing a health supplement containing citrus fruits and an embodiment of a health supplement using the same will be described in more detail.
본 발명에 의한 제조방법은 감귤농축액을 준비하는 감귤농축액준비단계, 상기 감귤농축액과 포도당 등을 혼합한 혼합물을 형성하는 혼합반죽단계, 상기 조성물혼합단계의 혼합물을 성형 및 건조하는 성형단계로 이루어진다.The production method according to the present invention comprises a preparation step of preparing a citrus concentrate, a mixing kneading step of forming a mixture of the citrus concentrate and glucose, and forming and drying the mixture of the composition mixing step.
여기서 상기 감귤농축액준비단계는 세척된 미성숙 감귤인 감귤, 당유자, 진귤, 지각, 편귤 등을 분쇄한 후, 슬러지를 분리한 감귤즙을 농축하여 감귤농축액을 준비하게 된다.Here, the step of preparing the citrus concentrate is to prepare a citrus concentrate by crushing the immature citrus, citrus, sugar citrus, cinnamon, crust, cinnamon, etc. washed immature citrus, concentrated citrus juice separated from the sludge.
또한 상기 감귤농축액준비단계에서는 세척된 미성숙 감귤류를 습식분쇄기로 1차 분쇄한 후에 습식마이크로분쇄기로 2차 분쇄하고, 원심분리기로 슬러지를 분리한 감귤즙을 농축기로 50 ~ 100브릭스(Brix) 농도로 농축하여 감귤농축액을 형성하게 된다.In addition, in the citrus concentrate preparation step, the washed immature citrus fruits are first crushed by a wet mill, and then secondly crushed by a wet micro mill, and the citrus juice separated from the sludge by a centrifuge is concentrated to 50 to 100 Brix concentrations. Concentrate to form citrus concentrate.
다시 말하면 제주 지방에서 7월 내지 8월에 생산되는 감귤 미숙과 또는 6월과 10월 중순 경의 당유자 미숙과 등의 껍질에 묻은 농약이나 왁스 등을 세척한 후, 가정용 녹즙기 등과 같은 습식분쇄기로 1차 분쇄하고, 습식마아크로분쇄기를 이용하여 800 ~ 1100 메쉬로 2차 분쇄한다.In other words, after washing the pesticides and waxes on the skin of the citrus unripe fruit produced in July-August or the sugar unripe fruit in June and mid-October in Jeju Province, and then using a wet grinder such as
그리고 상기 원심분리기로 슬러지와 감귤즙을 분리한 후, 원심분리기에 의해 분리된 감귤즙을 농축기에 넣어서 50 ~ 100 브릭스(Brix) 농도로 농축하여 감귤농축액을 형성하게 되며, 감귤농축액을 제조한 뒤에 바로 사용하는 것이 아니므로 장기간 보관하기 위하여 영하 70 ℃로 24시간 냉동시킨 후, 영하 20 ℃로 냉동 보관하며, 필요한 양을 해동하여 사용하게 된다.And after separating the sludge and citrus juice by the centrifuge, and put the citrus juice separated by the centrifuge in a concentrator to concentrate to 50 ~ 100 Brix (Brix) concentration to form a citrus concentrate, after preparing the citrus concentrate Since it is not used immediately, it is frozen for 24 hours at minus 70 ℃ for long-term storage, and then frozen at minus 20 ℃ and thawed as necessary.
본 발명에서는 후술하겠지만 상기 감귤농축액준비단계는 감귤농축액의 100중량부를 기준으로 감귤류 미숙과로부터 분획된 시트러스 플라보노이드 3 ~ 9중량부를 혼합함으로써, 소비자가 최종 제품인 건강보조식품을 섭취하였을 때 감귤농축액에 함유된 시트러스 플라보노이드(Citrus Flavonoid)가 인체에 그대로 섭취되도록 할 수 있다.Although the present invention will be described later, the step of preparing the citrus concentrate by mixing 3 to 9 parts by weight of citrus flavonoids fractionated from citrus fruits immature based on 100 parts by weight of citrus concentrates, when the consumer ingested the final dietary supplements contained in citrus concentrates Citrus Flavonoids can be ingested into the human body.
더우기 감귤과피에 포함된 플라보노이드 성분 추출량을 높이기 위하여, 본 발명에서는 분쇄된 감귤류를 효소 분해하는 것을 포함한다.Furthermore, in order to increase the extraction amount of flavonoid components contained in the citrus peel, the present invention includes enzymatically digesting the pulverized citrus fruits.
즉 분쇄된 감귤류 전체 중량의 0.15 ~ 0.25중량%의 분해 효소 펙티네이즈(pectinase)와 함께 추출기에 투입하여 50 ~ 60℃로 5 ~ 8시간동안 지속 교반한 후, 원심분리기로 슬러지와 감귤즙을 분리하고, 원심분리기로 분리된 감귤즙을 농축기로 농축하는 것이다.In other words, 0.15 ~ 0.25% by weight of the digested enzyme pectinase (pectinase) of the total weight of the pulverized citrus fruit was added to the extractor and stirred continuously for 5 to 8 hours at 50 ~ 60 ℃, the sludge and citrus juice is separated by a centrifuge Then, the citrus juice separated by a centrifuge is concentrated by a concentrator.
예컨대 분쇄된 감귤류 전체 중량의 0.2중량%의 분해 효소 펙티네이즈(pectinase)와 함께 추출기에 투입하여 57℃로 7시간동안 지속 교반한 후, 원심분리기로 슬러지와 감귤즙을 분리할 경우, 상술한 바와 같이 분쇄한 감귤류를 그대로 원심분리기로 분리된 감귤즙보다 효소 분해 과정을 거친 감귤즙에 포함된 플라보노이드 성분 추출량이 48% 이상 높아질 수 있다.For example, when the sludge and citrus juice are separated by a centrifuge after being added to the extractor with 0.2% by weight of digestive enzyme pectinase of the total weight of the pulverized citrus fruits and continuously stirred at 57 ° C. for 7 hours. Extracted flavonoids contained in the citrus juice after the enzymatic decomposition process can be more than 48% higher than the citrus juice as it is separated by the centrifuge.
아무튼 본 발명에 적용되는 혼합반죽단계는 상기 감귤농축액준비단계의 감귤농축액 4 ~ 8 중량%, 포도당 83 ~ 90중량%, 페닐알긴산을 함유한 아스파탐 0.03 ~ 0.05 중량%, 감귤향분말 1 ~ 2 중량%, 자당지방산에스테르 0.5 ~ 1.5중량%, 비타민C 1.5 ~ 2.5 중량%, 구연산 1.5 ~ 2.5 중량%, 겔라틴 0.01 ~ 0.03 중량%, 비타민B1 0.01 ~ 0.02 중량%, 비타민B2 0.01 ~ 0.02 중량%, 비타민B3 0.01 ~ 0.02 중량%, 판토텐산칼슘(B5) 0.05 ~ 0.07 중량%, 엽산 0.002 ~ 0.004 중량%, 비타민A 0.05 ~ 0.12 중량%, 비오틴(B7) 0.0001 ~ 0.0005 중량%, 나이아신 0.1 ~ 0.2 중량%, 콜라겐 0.05 ~ 0.15 중량%, 밀크칼슘 0.1 ~ 0.15 중량%, 탄산칼슘 0.03 ~ 0.08 중량%를 혼합 반죽하여 혼합물을 형성하게 된다.In any case, the mixed dough step applied to the present invention is 4 to 8% by weight of the citrus concentrate preparation step, 83 to 90% by weight of glucose, 0.03 to 0.05% by weight of phenyl alginic acid, 1 to 2 weight of citrus flavor powder %, Sucrose fatty acid ester 0.5 ~ 1.5%, vitamin C 1.5 ~ 2.5%, citric acid 1.5 ~ 2.5%, gelatin 0.01 ~ 0.03%, vitamin B 1 0.01 ~ 0.02% by weight, vitamin B 2 0.01 ~ 0.02% %, Vitamin B 3 0.01 to 0.02 weight%, calcium pantothenate (B5) 0.05 to 0.07 weight%, folic acid 0.002 to 0.004 weight%, vitamin A 0.05 to 0.12 weight%, biotin (B7) 0.0001 to 0.0005 weight%, niacin 0.1 to 0.2% by weight, 0.05% to 0.15% by weight of collagen, 0.1% to 0.15% by weight of milk calcium, and 0.03% to 0.08% by weight of calcium carbonate are mixed and kneaded to form a mixture.
그리고 상기 성형단계는 상기 조성물혼합단계의 혼합물을 성형기로써 성형한 후에 건조하여 건강보조식품을 제조하게 되며, 상기 건강보조식품을 일정한 개수로 포장하여 시중에 유통할 수 있는 것이다.In the molding step, the mixture of the composition mixing step is molded by a molding machine and then dried to produce a health supplement food, and the health supplement food may be packaged in a predetermined number and distributed on the market.
그래서 본 발명에 의한 건강보조식품은 감귤 맛과 향, 단맛, 신맛, 영양 성분 등을 고려하여 감귤농축액, 포도당, 아스파탐, 감귤향분말, 자당지방산에스테르, 비타민C, 구연산, 겔라틴, 비타민B1, 비타민B2, 비타민B3, 판토텐산칼슘(B5), 엽산, 비타민A, 비오틴(B7), 나이아신, 콜라겐, 밀크칼슘, 탄산칼슘를 상술한 바와 같은 비율로 혼합 반죽하고, 이러한 혼합물을 성형 및 건조한 것이다.Therefore, the health supplement food according to the present invention in consideration of citrus flavor and aroma, sweetness, sour taste, nutritional ingredients, citrus concentrate, glucose, aspartame, citrus flavor powder, sucrose fatty acid ester, vitamin C, citric acid, gelatin, vitamin B 1 , Mixes vitamin B 2 , vitamin B 3 , calcium pantothenate (B5), folic acid, vitamin A, biotin (B7), niacin, collagen, milk calcium, calcium carbonate in the proportions described above, and forms the mixture and will be.
상기 성형단계에서 성형기로 성형할 때, 반죽물 형태의 혼합물을 그대로 성형기에 투입하여 성형하면 성형기에 묻거나 최종 제품의 형태가 흐트러질 수 있어 혼합물을 과립 상태로 형성한 뒤에 이를 성형함이 바람직하다.When forming the molding machine in the molding step, when the mixture in the form of the dough is put into the molding machine as it is molded, it may be buried in the molding machine or the shape of the final product may be disturbed to form the mixture in a granular state and then molding it. .
따라서 본 발명에서는 상기 혼합반죽단계의 혼합물을 0.3mm ~ 3mm 크기로 과립화한 후에 과립화한 혼합물을 성형하는 것을 포함한다.Therefore, the present invention includes molding the granulated mixture after granulating the mixture of the mixing and kneading step to a size of 0.3mm ~ 3mm.
더우기 성형한 후에 건조하는 과정에서 다소 숙성될 수 있으나, 최종 제품에서 숙성되어 많은 조성물이 상호 작용시킬 필요가 있어 상기 혼합반죽단계의 혼합물을 숙성할 필요가 있다.Furthermore, it may be somewhat aged in the drying process after molding, but it is necessary to mature the mixture of the mixing and kneading step as it needs to be aged in the final product to interact with many compositions.
그래서 본 발명에서는 상기 혼합반죽단계의 혼합물을 32 ~ 38 ℃에서 3 ~ 12시간 숙성한 후에 상기 성형단계에서 성형하는 것을 포함한다.So, in the present invention, after the mixture of the mixing and kneading step is aged for 3 to 12 hours at 32 ~ 38 ℃ comprises molding in the forming step.
또한 소비자가 취향에 맞추어서 제품화하기 위해서는 최종 제품의 색상을 가미할 필요가 있으므로, 본 발명에서는 상기 혼합반죽단계에서 천연색소인 치자황색소 0.05 ~ 0.13 중량%를 혼합하는 것을 포함한다.In addition, since the consumer needs to add the color of the final product in order to commercialize the product, the present invention includes mixing 0.05 to 0.13% by weight of the natural pigments of gardenia yellow in the mixing and kneading step.
물론 최종 제품의 크기를 소비자가 쉽게 먹을 수 있도록 상기 혼합물을 입경 10 ~ 25mm 두께 3 ~ 8mm 크기로 성형 및 건조하는 것이 바람직하다.Of course, it is desirable to mold and dry the mixture to a particle size of 10 to 25 mm thick and 3 to 8 mm in size so that consumers can easily eat the size of the final product.
이렇게 본 발명에서는 감귤류를 이용하여 소비자에게 감귤류에 함유된 유익한 성분을 편리하게 섭취하게 하고, 제주도 등과 같은 지역에서 많이 생산되는 감귤류 중에 미성숙되어 쓰임새가 없는 미숙과를 이용할 수 있어 이러한 감귤류를 생산하는 농가의 생산 증대를 촉진할 수 있는 것이다.Thus, in the present invention, consumers can conveniently consume the beneficial ingredients contained in the citrus fruits using citrus fruits, and can use the immature fruit that is immature among citrus fruits produced in a lot of regions such as Jeju-do, and thus produces these citrus fruits. Will increase production.
한편 본 발명에서는 상기 감귤농축액준비단계에서 감귤농축액에 감귤류 미숙과로부터 분획된 시트러스 플라보노이드(Citrus Flavonoid)를 혼합하게 되는 바, 이러한 시트러스 플라보노이드를 감귤류 미숙과로부터 분획하는 실시예를 아래에서 상세하게 설명한다.Meanwhile, in the present invention, the citrus concentrate is prepared by mixing citrus flavonoids (Citrus Flavonoids) fractionated from citrus unripe fruits in the citrus concentrate preparation step. An embodiment of dividing citrus flavonoids from citrus unripe fruits will be described in detail below. .
<실시예><Examples>
본 실시예에서는 열매솎기로 해마다 많은 양의 미숙과가 노지에서 버려지고 있지만, 이러한 감귤류 미숙과를 이용하여 건강보조식품을 제조하는 바, 건강보조식품에 함유되는 시트러스 플라보노이드를 분획하게 된다.In the present embodiment, a large amount of immature fruit is thrown away in the field each year as a fruiting machine. However, citrus flavonoids contained in the health supplement are fractionated when the health supplement is prepared using the citrus immature fruit.
참고로 감귤류의 과피에는 테르페노이드(terpenoid), 플라보노이드(flavonoid), 리모노이드(limonoid), 카로테노이드(carotenoid) 등 생리활성 2차대사물이 포함되어 있다. 감귤 테르페노이드의 주성분은 리모넨(limonene)으로서 감귤 과피 특유의 방향성분이다.For reference, citrus peel contains bioactive secondary metabolites such as terpenoid, flavonoid, limonoid, carotenoid, and the like. The main component of citrus terpenoids is limonene (limonene), which is a characteristic ingredient of citrus peels.
그리고 감귤류의 과피에는 헤스페리딘(hesperidin), 나린진(naringin) 등 바이오플라보노이드(bioflavonoid)가 많이 존재한다. 오렌지, 귤, 유자 등에는 헤스페리딘이 더 많으며, 자몽, 레몬 등에는 나린진이 더 많은 것으로 알려져 있다.In the citrus peel, there are many bioflavonoids such as hesperidin and naringin. Oranges, tangerines, citrons, etc. are more hesperidin, grapefruit, lemons, etc. are known to have more naringin.
아래의 표 1은 감귤의 과피에서 일반적으로 얻어지는 성분의 일 예를 나타낸 것이다.Table 1 below shows an example of the components generally obtained from the tangerine peel.
유리당
Glass sugar
여기에 제시되어 있는 감귤과피의 성분에서 헤스페리딘은 분자식 C28H34O15의 물질로서, 헤스페리딘은 플라보노이드(flavonoid)인 헤스페레틴(hesperetin)에 두 개의 당이 결합되어 있는 배당체이며, 항산화활성, 항염활성 및 항알러지활성이 알려져 있다.Hesperidin is a substance of the molecular formula C 28 H 34 O 15 in the citrus peels presented here, and hesperidin is a glycoside with two sugars attached to hesperetin, a flavonoid, Anti-inflammatory and anti-allergic activities are known.
감귤류의 과실은 대표적으로 비타민C 및 다양한 바이오플라보노이드(bioflavonoid)를 함유하고 있다. 바이오플라보노이드(Bioflavonoid)란 비타민P로 알려져 있으며 필수적인 영양요소는 아니지만, 건강에 도움을 주는 역할을 하고 있는 물질군이다. 바이오플라보노이드(Bioflavonoid)의 유익한 효과가 발견되기 시작한 것은 비교적 최근의 일이며, 여러 가지 과실이나 채소에서 800종류 이상의 바이오플라보노이드(bioflavonoid)가 보고되어 있다. 이 중에서 감귤류에서 주로 발견되는 주성분은 헤스페리딘(hesperidin), 나린진(naringin) 외에 루틴(rutin), 나리루틴(narirutin), 네오헤스페리딘(neohesperidin), 시넨세틴(sinensetin), 폰시린(poncirin), 다이디민(didymin) 등이다. 이들은 대부분 감귤류에서 나타나는 노란색소의 물질로서, 화학구조적으로 플라보노이드 골격을 포함하고 있다.Citrus fruits typically contain vitamin C and various bioflavonoids. Bioflavonoids are known as vitamin P and are not essential nutrients, but are a group of substances that contribute to health. It has been relatively recent that the beneficial effects of bioflavonoids have begun to be discovered, and more than 800 bioflavonoids have been reported in various fruits and vegetables. The main components found in citrus fruits are hesperidin and naringin, in addition to rutin, narirutin, neoohsperidin, sinensetin, poncirin, and didimin. (didymin) and the like. These are mostly yellow pigments found in citrus fruits and contain a flavonoid backbone chemically.
시트러스 플라보노이드(Citrus flavonoid)가 건강에 도움을 준다고 알려져 있으며, 바이오플라보노이드는 비타민C의 생성을 도와주는 영양소이다. 바이오플라보노이드는 비타민C와 상승적으로 건강한 모세혈관을 형성하여 커넥티브 티슈(connective tissue)에서 콜라겐의 형성을 도와주며, 상처가 아무는 것을 도우며, 건강한 면역계가 유지되도록 한다고 알려져 있다.Citrus flavonoids are known to help health, and bioflavonoids are nutrients that help produce vitamin C. Bioflavonoids are known to synergistically form healthy capillaries with vitamin C to help collagen formation in connective tissue, help to heal wounds, and maintain a healthy immune system.
국내의 감귤류 27종에 대하여 네오헤스페리딘 함량을 조사한 결과, 당유자, 지각, 편귤에서 함량이 많음을 확인 한 바 있다. 특히 당유자 미숙과(반지름 7.7 mm 내외)의 네오헤스페리딘 함량은 33%(건조 중량 기준)에 이르러, 지금까지 보고된 어떤 품종 보다도 높은 것으로 나타났다. 그러나 당유자의 숙성 정도에 따라, 네오헤스페리딘 함량이 급격히 감소함을 확인하였다.As a result of examining the neohesperidin content of 27 kinds of citrus fruits in Korea, it was confirmed that the content is high in sugary citron, crust, and citrus. In particular, the neohesperidin content of sugary immature fruit (about 7.7 mm in radius) reached 33% (by dry weight), which was higher than any of the varieties reported so far. However, it was confirmed that the neohesperidine content rapidly decreases according to the degree of ripening of the glucose.
따라서 이른 시기의 미숙과에는 상당량의 네오헤스페리딘(neohesperidin)이 기대되며, 6월 22일경 수확된 당유자의 경우에는 건조중량 기준으로 33%의 네오헤스페리딘(neohesperidin)이 함유되어 있는 것으로 보고되어 있다.Therefore, it is expected that a considerable amount of neohesperidin is expected in early immature fruit, and 33% of neohesperidin by dry weight is reported in sugary milk harvested around June 22.
아무튼 당유자 미숙과는 6월 중순 경 및 10월 중순 경 농가로부터 직접 구입하여 건조하지 않고 신선품 그대로 이용하였다. 장시간 보존이 필요한 경우는 영하75℃의 냉동고 안에 보관하였다. 가정용 녹즙기 또는 제주대학교의 TIC설비인 습식 분쇄기를 이용 분쇄하여 이용하였다.In any case, the sugar-dried immatures were purchased directly from farmers in mid-June and mid-October and used as fresh products without drying. If long time storage is required, it was stored in freezer at minus 75 ℃. It was pulverized using a household green juicer or a wet mill, a TIC facility of Cheju National University.
[시료 및 기기][Samples and Instruments]
추출에 사용된 메탄올(methanol)과 에탄올(ethanol)은 동양제철화학주식회사의 Extra pure급을 사용하였고, 고분자량의 물질을 제거하기 위해서 사용된 Ultrafiltration Kit은 Millipore 사의 Prep/Scale TFF Cartridge를 사용하였다. 플라보노이드(Flavonoid)만을 선택적으로 획득하기 위해 충진제로서 Amberlite XAD-16을 Sigma에서 구매하였다. HPLC 검출의 Standard로 사용된 naringin, neohesperidin의 표품은 각각 sigma 사에서 구입하여 사용하였다. HPLC는 Waters 사의 Delta Prep 4000을 이용하였고, 컬럼은 prep novapack HR C18 7.8X300mm을 이용하였다. 검출은 Waters의 2487 UV detecter를 이용하여 254nm에서 하였고, MPLC는 Yamazen 사의 Pump 540과 Prep·UV-10V Set을 이용하였다. MPLC의 컬럼은 Yamazne 사의 ULTRA PACK SI-40B, 26X300mm 컬럼을 사용하였다.Methanol and ethanol used for the extraction were used extra pure grade of Dongyang Steel Chemical Co., Ltd., and the Ultrafiltration Kit used to remove high molecular weight material was Prep / Scale TFF Cartridge of Millipore. Amberlite XAD-16 was purchased from Sigma as a filler to selectively acquire only flavonoids. Naringin and neohesperidin used as standard for HPLC detection were purchased from sigma, respectively. HPLC was used for Delta Prep 4000 of Waters, column was prep novapack HR C18 7.8X300mm. Detection was performed at 254nm using Waters 2487 UV detecter and MPLC using Yamazen Pump 540 and PrepUV-10V Set. MPLC's column was a ULTRA PACK SI-40B, 26X300mm column from Yamazne.
정량에 사용한 HPLC는 PDA detector Waters 2996을 갖춘 ailiance 2695 Separation Module 시스템을 사용하였으며, 컬럼은 Waters 사의 Sunfire 4.6mmX250mm(5um)를 이용하였다.HPLC was used for quantification using an ailiance 2695 Separation Module system equipped with a PDA detector Waters 2996, and the column used Waterfire's Sunfire 4.6mm × 250mm (5um).
[플라보노이드(Flavonoid) 분획의 제조][Preparation of Flavonoid Fractions]
추출은 80%(v/v) 메탄올 수용액을 시료 무게의 1.5배를 가하고, 중탕 가온하여 1시간 동안 끓게 하고 상온으로 식힌 후 착즙하여 고형분을 제거한후 필터 하는 것을 2회 반복하였다.Extraction was performed twice by adding 80% (v / v) methanol aqueous solution 1.5 times the weight of the sample, warming in a bath and boiling for 1 hour, cooling to room temperature, juiced to remove solids and then filter.
감압 농축 과정 도중 용액이 시럽상이 되면 약간의 메탄올을 첨가하여 펙틴으로 추정되는 노란색의 고체를 원심분리(9,000 rpm supra 22k)하는 방법으로 제거하였다. 시제품의 생산에 있어서는 제주대 TIC내의 장비인 습식 분쇄기와 추출기, 농축기와 탈수기 및 고속 원심분리기를 이용하였다.When the solution became syrup during the concentration under reduced pressure, a little methanol was added to remove the yellow solid, which is assumed to be pectin, by centrifugation (9,000 rpm supra 22k). In the production of prototypes, wet grinders and extractors, concentrators and dehydrators, and high-speed centrifuges were used.
Calvarano 등(1996)이 Bergamot peels의 산업적 이용을 위하여 naringin을 추출하는 방법을 보고하였는데, hot water로 추출 후 ultrafiltration 하고, macroporous cross-linked polystyrene인 Amberlite XAD-16 resin에 흡착시켜서 얻는 방법이 있다. 이에 XAD-16에 의한 flavonoid의 흡착 및 분리를 시도하였다.Calvarano et al. (1996) reported a method of extracting naringin for industrial use of Bergamot peels, which is obtained by ultrafiltration after extraction with hot water and adsorption on Amberlite XAD-16 resin, a macroporous cross-linked polystyrene. The adsorption and separation of flavonoid by XAD-16 was attempted.
원심분리한 시료는 메탄올을 제거하기 위하여 완전히 농축 시키고 동결건조를 거친 다음 다시 무게의 25배량에 해당하는 증류수에 녹였다. 다시 증류수에 녹은 시료는 Adventec No 2. 필터로 Ultrafiltration에 알맞도록 여과하였다. Ultrafiltration Kit(Cut Off 10K와 3K)은 Millipore사의 Prep/ Scale TFF 2.5ft2 Cartridge 와 1ft2 Cartridge, Wilo 사의 화학용 펌프 PM-015NM을 사용하였고, 순환되는 용액의 양을 측정한 결과 사용 권장 범위 내인 3-4L/min의 용액이 순환되었으며, 여과액은 약 1L/hour 정도의 속도로 용출되었다.The centrifuged sample was completely concentrated to remove methanol, lyophilized and then dissolved in distilled water equal to 25 times its weight. The sample dissolved in distilled water was filtered with Adventec No. 2 filter to suit Ultrafiltration. Ultrafiltration Kits (Cut Off 10K and 3K) are available from Millipore's Prep / Scale TFF 2.5ft 2 Cartridge and 1ft 2 Cartridge, Wilo's chemical pump PM-015NM was used. As a result of measuring the amount of circulating solution, 3-4 L / min of the solution was circulated and the filtrate was eluted at a rate of about 1 L / hour. It became.
여과되지 않고 순환되는 용액은 여과된 용액만큼을 보충하여 농도가 점점 옅어짐에도 불구하고 필터를 통과하여 빠져나가는 용액의 양이 많아지면 많아질수록 고형분이 생겨나므로 수시로 용액을 다시 필터하여 주었다.The solution circulated without filtration was supplemented as much as the filtered solution, and although the concentration gradually became thinner, the more the amount of the solution passed through the filter, the more solids were formed, so the solution was filtered again from time to time.
분자량에 따른 여과물이 계속해서 나오는지를 확인해 보기 위해서 Yamazen사의 MPLC system에 연결하여 UV 흡광도를 관찰하였다. 이렇게 얻어진 여과액은 XAD-16 resin column에 농축 없이 바로 통과시켰다.UV absorbance was observed by connecting to Yamazen's MPLC system to check if the filtrate continued to yield molecular weight. The filtrate thus obtained was passed directly through the XAD-16 resin column without concentration.
Ultrafiltration 후의 용액에서 flavonoid 분획을 획득하기 위하여 nonionic polymeric absorbent인Amberlite XAD-16을 이용하였다.Amberlite XAD-16, a nonionic polymeric absorbent, was used to obtain the flavonoid fraction from the solution after ultrafiltration.
600g의 XAD-16 resin을 판매시에 충진되어 있는 이온성 물질을 24시간 동안 충분히 물로 씻어준 후 앞에서 얻어진 여과액 4L를 흘리며 column은 감압하거나, MPLC에 장착하는 방식으로 가압하여 속도를 높였다. 일단 흡착된 물질 중에서 당류 등 극성 물질을 제거하기 위해 4L의 증류수를 다시 통과 시켰으며, 그 이후 60% ethanol 용액 2L를 통과시켜 flavonoid 분획을 얻고 나서, methanol 2L를 통과시켜 흡착된 다른 비극성 물질들을 회수한 다음, 원래의 부피만큼으로 수축할 때가지 물을 흘려 알콜을 제거하여 다시 반복 사용하였다.After washing 600g of XAD-16 resin with ionic substance filled with water for 24 hours, 4L of the filtrate obtained above was poured and the column was decompressed or pressurized by mounting on MPLC to increase the speed. 4L of distilled water was passed again to remove polar substances such as sugars from the adsorbed material, and then 2L of 60% ethanol solution was obtained to obtain a flavonoid fraction, and then 2L of methanol was recovered to adsorb other nonpolar substances. Then, the water was removed by pouring water until it contracted to the original volume and used again.
상기와 같이 ultrafiltration과정이 있는 경우, 역산하여 볼때 약 100~120g의 시료가 600g의 resin에 대해 통과 될 수 있고, 흡착되어 나오는 flavonoid 분획은 25~30g/1회 정도였다. Ultrafiltration을 적용하지 않은 경우와 비교하여, 추출물 용액을 바로 XAD-16 resin 컬럼을 시도하는 경우 600g에 대해서 10g내외만 통과 시켜도 압력이 생겨 전개용액의 흐름이 차단되며, 2~3회 사용후 컬럼을 세척해야 할 정도로 오염이 심하였다.In the case of the ultrafiltration process as described above, about 100-120 g of the sample can pass through about 600 g of resin, and the adsorbed flavonoid fraction was about 25-30 g / 1 times. Compared to the case where no ultrafiltration is applied, when the extract solution is directly tried for the XAD-16 resin column, pressure is generated even if only 10g is passed for 600g, and the flow of the developing solution is blocked. The contamination was severe enough to be cleaned.
[네오헤스페리딘(Neohesperidin)의 정제][Purification of Neohesperidin]
본 발명에서는 일반 용매를 사용할 수 있는 중압크로마토그래피(MPLC)를 이용하고자 하였다. MPLC를 이용한 컬럼에서도 대다수의 2용매계에서는 효과적인 분리가 이루어지지 않았으며 최종적으로 CHCl3 : MeOH : H2O = 26 : 14 : 3이라는 용매 조성으로 효과적인 분리를 이룰 수 있었다.In the present invention, it was intended to use a medium pressure chromatography (MPLC) that can use a general solvent. In the column using MPLC, most of the two-solvent systems were not effectively separated. Finally, the separation was achieved by a solvent composition of CHCl 3 : MeOH: H 2 O = 26: 14: 3.
즉 도 2는 MPLC를 이용한 neohesperidin의 분리 그래프로서, neohesperidin과 naringin 사이는 약 4분이며 각 injection간의 간격은 약 16분 내외이다.That is, Figure 2 is a graph of the separation of neohesperidin using MPLC, neohesperidin and naringin is about 4 minutes and the interval between each injection is about 16 minutes.
[플라보노이드(Flavonoid) 분획의 활성 검색] [Activity Screening of Flavonoid Fractions]
자유 유리기 소거 활성 검색을 살펴본다.We look at the free free radical scavenging activity search.
DPPH 자유유리기 소거활성은 시료의 여러 농도에 0.4mM DPPH용액을 동량 첨가하여 실온에서 10분간 방치한 후 517 nm에서 흡광도를 측정하였다.DPPH free free radical scavenging activity was measured by adding the same amount of 0.4mM DPPH solution to various concentrations of the sample, and leaving it at room temperature for 10 minutes to measure absorbance at 517 nm.
NO 자유기 소거활성은 생리적 조건에서 자연적으로 NO를 생성하는 물질인 sodium nitroprusside(SNP)를 사용하여 550nm에서 흡광도를 측정하여 검색하였다.NO free radical scavenging activity was determined by measuring absorbance at 550 nm using sodium nitroprusside (SNP), a naturally occurring NO substance under physiological conditions.
Superoxide 자유기 소거활성은 PMS/NADH system을 이용하여 생성된 superoxide anion의 양을 NBT 환원방법에 의해 517nm에서 측정하여 검색하였다. 실험의 정확도는 superoxide dismutase(SOD)를 가지고 확인하였다.Superoxide free radical scavenging activity was detected by measuring the amount of superoxide anion produced by PMS / NADH system at 517 nm by NBT reduction method. The accuracy of the experiment was confirmed with superoxide dismutase (SOD).
Xanthine oxidase 억제활성은 Xanthine을 기질로 하여 xanthine oxidase에 의해 생성된 uric acid의 양을 290nm에서 흡광도를 측정하여 검색하였고 생성된 superoxide anion의 양은 NBT 환원방법에 의해 517nm에서 측정하여 검색하였다. 실험의 정확도는 xanthine oxidase 억제제인 allopurinol을 가지고 확인하였다.Xanthine oxidase inhibitory activity was determined by measuring the absorbance at 290 nm for the amount of uric acid produced by xanthine oxidase using Xanthine as a substrate, and the amount of superoxide anion was measured at 517 nm by NBT reduction. The accuracy of the experiment was confirmed with allopurinol, an xanthine oxidase inhibitor.
항염활성 검색을 살펴본다.Look for anti-inflammatory activity screening.
세포 독성 검색은 RAW 264.7 세포에 여러 가지 농도의 시료를 첨가한 후 24시간 동안 배양하였다. 배지를 제거한 후 0.5mg/ml의 MTT용액을 첨가하고 1시간 동안 배양하였다. MTT 용액을 제거하고 DMSO를 첨가하여 MTT의 환원에 의해 생성된 formazan 침전물을 용해시킨 후 540nm에서 흡광도를 측정하였다. 대조군과 비교하여 RAW 264.7 세포에 대한 시료의 세포독성을 평가하였다.Cytotoxicity screening was performed for 24 hours after adding various concentrations of samples to RAW 264.7 cells. After removing the medium, 0.5mg / ml MTT solution was added and incubated for 1 hour. The MTT solution was removed and DMSO was added to dissolve the formazan precipitate produced by the reduction of MTT and the absorbance was measured at 540 nm. The cytotoxicity of the sample against RAW 264.7 cells was evaluated in comparison to the control.
in vitro 항염활성 검색은 RAW 264.7 세포에 LPS(100ng/ml)와 여러 가지 농도의 시료를 동시에 첨가한 후 24시간 동안 배양하였다. 생성된 NO의 양은 Griess 시약을 이용하여 세포배양액 중에 존재하는 nitrite의 형태로 측정하였다. in In vitro anti-inflammatory activity was screened for 24 hours after adding LPS (100ng / ml) and various concentrations of samples to RAW 264.7 cells. The amount of NO produced was measured in the form of nitrite present in the cell culture using Griess reagent.
타이로시나아제 활성 검색은 세포내 타이로시나제 활성분석(Cell tyrosinase activity assay in B16/F10) 쥐(Mus musculus)의 melanoma cell line 중 B16/F10 cell을 6well plate에 ml당 5X104이 되게 준비한 후, 24시간동안 37℃ CO2 항온기에서 배양하고, 멜라닌 생합성 유도물질인 alpha-MSH 50nM을 처리하고 동시에 시료분획(EtOH:PBS=1:1)을 31, 63, 125, 250, 500ug/ml로 각각 처리하였다. 72시간 동안 37℃항온기에서 배양하였으며 36시간이 지난 후, 배지교체와 동시에 시료를 다시 처리하였다.Tyrosinase activity screening was performed by preparing B16 / F10 cells in a 6-well plate at 5X10 4 per ml in a cell tyrosinase activity assay in B16 / F10 melanoma cell line of rat (Mus musculus). After incubation for 24 hours at 37 ℃ CO 2 incubator, and treated with melanin biosynthesis induction alpha-MSH 50nM and at the same time sample fractions (EtOH: PBS = 1: 1) 31, 63, 125, 250, 500ug / ml Respectively. The cells were incubated at 37 ° C. for 72 hours and after 36 hours, the samples were treated again at the same time as the medium was replaced.
세포를 trypsin-EDTA를 이용하여 수확했다. 이렇게 수확된 cell에 0.2mM phenylmethylsulfonyl fluoride(PMSF)와 1% Triton X-100을 함유한 67mM sodium phosphate buffer(pH 6.8)를 500ul 처리하고 sonication 하였으며, 이 상태로 1시간동안 얼음에서 보관하였다. 1시간 후 4℃ 원심분리기에서 15,000 rpm으로 15분간 원심분리하였으며 상층액을 취하여 tyrosinase 활성분석에 사용하였다.Cells were harvested using trypsin-EDTA. The harvested cells were treated with 500ul of 67mM sodium phosphate buffer (pH 6.8) containing 0.2mM phenylmethylsulfonyl fluoride (PMSF) and 1% Triton X-100 and sonicated, and stored on ice for 1 hour. After 1 hour, the mixture was centrifuged at 15,000 rpm for 15 minutes at 4 ° C centrifuge, and the supernatant was taken for tyrosinase activity analysis.
세포내 tyrosinsae 활성분석은 세포내의 존재하는 tyrosinase의 작용결과 생성되는 DOPA chrome을 미색법에 의핵 측정했다. 120ul의 67mM sodium phosphate buffer(pH 6.8)에 기질로서 5uL의 1.5mM L-tyrosine과 40ul의 25mM L-DOPA를 혼합한 후 위에서 얻은 상층액 100ul를 처리하여 37℃ 항온기에서 2시간 반응시켰다.Intracellular tyrosinsae activity assay was performed by whitening of DOPA chrome produced by the action of tyrosinase in cells. After mixing 5ul of 1.5mM L-tyrosine and 40ul of 25mM L-DOPA as a substrate in 120ul of 67mM sodium phosphate buffer (pH 6.8), 100ul of the supernatant obtained above was treated and reacted at 37 ° C for 2 hours.
반응액 중에 생성된 DOPA chrome을 475nm에서 측정하여 Mushroom tyrosinase의 활성을 기준으로 하여 단백질 1ug당 unit으로 효소 활성을 나타내었다.DOPA chrome produced in the reaction solution was measured at 475 nm and showed enzymatic activity per unit per protein based on the activity of Mushroom tyrosinase.
[실험 결과][Experiment result]
플라보노이드(Flavonoid) 분획의 제조Preparation of Flavonoid Fractions
과실에서 플라보노이드류를 대량 분획하기 위해 자주 사용되는 XAD-16 흡착법을 이용하였으며, 도 3과 같이 나린진(naringin)과 네오헤스페리딘(neohesperidin)이 주성분인 플라보노이드(flavonoid) 분획을 분취하였다.XAD-16 adsorption method, which is frequently used for mass fractionation of flavonoids in fruits, was used. As shown in FIG.
네오헤스페리딘(Neohesperidin)의 정제Purification of Neohesperidin
시료로 사용한 당유자 미숙과피에는 나린진(naringin)과 네오헤스페리딘(neohesperidin)이 같이 존재하여, 당유자 알칼리 추출액의 산성화 과정에서의 나린진 하이드로레이트(naringin hydrolate) 석출에 의한 네오헤스페리딘(neohesperidin)과의 분리를 시도하였으나, 3개월 이상 지난 시점까지도 결정이 생기지 않았다.Naringin and neohesperidin are present in the immature skin of the sugar-oil used as a sample, and separation from neohesperidin by the precipitation of naringin hydrolate during acidification of the sugar-oil alkali extract. At least three months later, no decision was made.
80% 메탄올 추출법에 따라 네오헤스페리딘(neohesperidin)의 석출을 시도하였으나. 역시 3개월 이상 방치해도 결정은 생기지 않았다. 다만 클로로폼(chloroform)과 물의 액면에 회백색의 고체를 수차례 관찰하였으나 이를 얻기 위한 조작에서 위의 물층과 닿으면 바로 녹아들어가므로 인해 얻어내기가 불가능하였다.The precipitation of neohesperidin was attempted by 80% methanol extraction. After three months or more, no decision was made. However, gray white solids were observed several times on the surface of chloroform and water, but it was impossible to obtain them due to melting as soon as they were in contact with the water layer.
이 분획의 성분 분석을 TLC로 시도하였으나, 여러 문헌을 참고하여 여러 용매조성을 적용하더라도 쉽게 Rf 값이 차이나지 않았으며, 두 성분이 공존하더라도 한개의 spot으로 나타나는 현상이 잦아 TLC 또는 일반적인 순상 컬럼으로는 분리 여부가 거의 확인할 수가 없었다.Although TLC was attempted to analyze the components of this fraction, Rf was not easily different even when various solvent compositions were applied with reference to various literatures, and even if two components coexisted, they often appear as one spot. Almost no separation was confirmed.
이러한 현상으로 인하여 XAD-16 column 통과 시 60% ethanol 용액의 앞 분획은 naringin과 뒷 분획은 neohesperidin과 TLC Rf 값이 일치하는 경향을 보이나, HPLC로 분석해 보면(mobile phase : Water : MeOH : acetic acid = 57 : 43 : 0.5, Flow rate:1mL/min) 두 분획의 비율만 다를 뿐 서로 상당량 섞여 있음을 확인할 수 있었다.Due to this phenomenon, naringin and neohesperidin and TLC R f values of the 60% ethanol solution showed a tendency to coincide with the XAD-16 column, but the HPLC analysis (mobile phase: Water: MeOH: acetic acid = 57: 43: 0.5, Flow rate: 1mL / min) It was confirmed that only the ratio of the two fractions are mixed with each other.
일반적인 flash column chromatography로는 효과적인 분리는 이룰 수 없었고, HPLC의 방법으로는 경제성을 얻을 수 없다고 판단한 결과, MPLC를 이용하여 효율적인 분리를 이룰 수 있었으며, 사용한 용매는 CHCl3 : MeOH : H2O = 26 : 14 : 3 있었다. 26mmX300mm 컬럼에 20mL/min의 유속으로 용매를 흘렸으며, injection 양은 앞서 기술한 전개용매에 0.2g/mL의 농도로 녹인 플라보노이드 분획을 4mL씩 주입하였다.The effective separation was not achieved by general flash column chromatography, and it was determined that economic efficiency could not be obtained by HPLC method. As a result, efficient separation was achieved by MPLC. The solvent used was CHCl 3 : MeOH: H 2 O = 26: 14: 3 were. The solvent was flowed into a 26mmX300mm column at a flow rate of 20mL / min, and the injection amount was injected into 4 mL of flavonoid fractions dissolved in a concentration of 0.2g / mL in the above-described developing solvent.
1회 총 0.8g의 플라보노이드 분획을 주입하여, Detect range(A) 2.56에서 30%를 상회할 때 수집하도록 프로그램하였다. 이러한 프로그램에 의하여 수집된 네오헤스페리딘(neohesperidin) 분획은 소량의 물에 부유 시킨 후 클로로포름을 가한 후 약 12시간 냉장고에 방치한다. 클로로포름 층에 있는 네오헤스페리딘 침전물을 glass filter를 이용하여 filtration하고 소량의 메탄올로 세척 후 회수하였다.A total of 0.8 g of flavonoid fractions were injected once and programmed to collect above 30% in Detect range (A) 2.56. The neohesperidin fraction collected by this program is suspended in a small amount of water and left in the refrigerator for about 12 hours after adding chloroform. The neohesperidine precipitate in the chloroform layer was filtered through a glass filter, washed with a small amount of methanol, and recovered.
이 과정에서 얻어지는 네오헤스페리딘(neohesperidin)은 평균 214mg/1g 플라보노이드 분획 이었다. neohesperidin의 구조 확인을 위한 NMR 스펙트럼은 도 4와 도 5에 나타나 있다. 도 6과 같이 순도는 HPLC를 이용하여 정량하였으며, 도 7과 같이 98%로 제시된 sigma 사의 제품과 비교 95%로 정량되었다.Neohesperidin obtained in this process was an average 214mg / 1g flavonoid fraction. NMR spectra for confirming the structure of neohesperidin are shown in FIGS. 4 and 5. Purity was quantified using HPLC as shown in FIG. 6, and was quantified at 95% compared with the product of sigma shown as 98% as shown in FIG. 7.
자유 유리기 소거 활성 검색을 살펴본다Examine free free radical scavenging activity detection
DPPH 자유유리기 소거활성 결과는 도 8과 표 2와 같으며, 시료의 여러 농도에 0.4mM DPPH용액을 동량 첨가하여 실온에서 10분간 방치한 후 517 nm에서 흡광도를 측정하였다.The results of DPPH free free radical scavenging activity are shown in FIG. 8 and Table 2. The same amount of 0.4mM DPPH solution was added to various concentrations of the sample, and the absorbance was measured at 517 nm after 10 minutes at room temperature.
DPPH 자유기 소거 활성에 대한 시료의 IC50 값은 492.2ug/ml 이었으며, 이는 양성대조군으로 사용한 시료들보다는 낮은 활성이지만 제주대학교 TIC가 보유하고 있는 30% 플라보노이드를 함유한 감귤과피 분획물 TIC B(IC50=591.4ug/ml)보다 좋은 활성이었으며, 불용성의 물질을 필터한 것에 따른 활성 차이는 없는 것으로 나타났다.The IC 50 value of the sample for DPPH free radical scavenging activity was 492.2 ug / ml, which was lower than that of the positive control group, but the citrus peel fraction TIC B (IC) containing 30% flavonoids possessed by TIC of Jeju National University. 50 = 591.4 ug / ml), and there was no difference in activity by filtering insoluble material.
필터후 시료분획
TIC B
BHA
Trolox
TroloxL-Ascorbic acid
QueretinSample fraction
Sample fraction after filter
TIC B
BHA
Trolox
TroloxL-Ascorbic acid
Queretin
504.3±9.7
591.4±11.0
11.1±0.9
11.4±0.1
12.2±0.1
6.1±0.03492.2 ± 14.8
504.3 ± 9.7
591.4 ± 11.0
11.1 ± 0.9
11.4 ± 0.1
12.2 ± 0.1
6.1 ± 0.03
NO 자유기 소거활성 결과는 도 9과 표 3와 같으며, 생리적 조건에서 자연적으로 NO를 생성하는 물질인 sodium nitroprusside(SNP)를 사용하여 550nm에서 흡광도를 측정하여 검색하였다.The results of NO free radical scavenging activity are shown in FIGS. 9 and 3, and the absorbance was measured at 550 nm using sodium nitroprusside (SNP), a material that naturally generates NO under physiological conditions.
이 분획과 분획의 불용성물질을 필터한 뒤의 nitric oxide 자유기 소거활성은 500ug/ml 농도에서 각각 30.8과 40.7%로 나타났으며 필터를 한 시료에서 다소 높은 활성을 보였으며 전체적으로는 감귤과피 에탄올 추출물인 TIC A와 비슷한 활성을 나타내었다.The nitric oxide free radical scavenging activity after filtering this fraction and its insoluble substance was 30.8 and 40.7% at 500 ug / ml concentration, respectively, and showed slightly higher activity in the filtered sample. It showed similar activity with TIC A.
시료 분획
필터후 시료분획
TIC A
TIC B
TroloxQuercetin
Curcumin
Sample fraction
Sample fraction after filter
TIC A
TIC B
TroloxQuercetin
Curcumin
30.8 ±0.5
40.7±3.0
38.5±0.2
51.5±0.1
At 100ug/ml
12.1±2.2
20.6±0.07
47.2±0.9At 500ug / ml (%)
30.8 ± 0.5
40.7 ± 3.0
38.5 ± 0.2
51.5 ± 0.1
At 100ug / ml
12.1 ± 2.2
20.6 ± 0.07
47.2 ± 0.9
*
*
*
367.7±29.7
*
*
121.1±8.4IC 50 (ug / ml)
*
*
*
367.7 ± 29.7
*
*
121.1 ± 8.4
* Can't calculate the value of IC50 * Can't calculate the value of IC 50
수퍼옥사이드(Superoxide) 자유기 소거활성 결과는 도 10과 표 4와 같으며, PMS/NADH system을 이용하여 생성된 superoxide anion의 양을 NBT 환원방법에 의해 517nm에서 측정하여 검색하였다. 실험의 정확도는 superoxide dismutase(SOD)를 가지고 확인하였다.The results of the superoxide free radical scavenging activity are shown in FIGS. 10 and 4, and the amount of superoxide anion generated using the PMS / NADH system was measured and measured at 517 nm by the NBT reduction method. The accuracy of the experiment was confirmed with superoxide dismutase (SOD).
Superoxide 자유기 소거활성에 대한 IC50 값은 140.6ug/ml이었다. 이는 양성대조군으로 사용한 Trolox보다 약간 낮은 활성이었으나 제주대학교 TIC에서 보유한 감귤과피 에탄올 추출물인 TIC A(IC50=329.0ug/ml) 및 30% 플라보노이드를 함유한 분획 TIC B(IC50 > 1000ug/ml)보다 좋은 활성이, 불용성의 물질을 필터한 것에 따른 활성 차이는 없는 것으로 나타났다.The IC 50 value for the superoxide free radical scavenging activity was 140.6 ug / ml. This activity was slightly lower than Trolox used as a positive control, but the fraction TIC B (IC 50 > 1000 ug / ml) containing TIC A (IC 50 = 329.0 ug / ml) and 30% flavonoids, a citrus peel ethanol extract of Cheju National University TIC. It was shown that better activity was not the difference in activity by filtering insoluble material.
필터후 시료분획
TIC A
Trolox
TroloxL-Ascorbic acid
SODSample fraction
Sample fraction after filter
TIC A
Trolox
TroloxL-Ascorbic acid
SOD
135.1±0.1
329.0±4.3
101.8±6.2
12.2±0.1
6.1±0.03(U/mL)140.6 ± 5.4
135.1 ± 0.1
329.0 ± 4.3
101.8 ± 6.2
12.2 ± 0.1
6.1 ± 0.03 (U / mL)
젠신 옥시데이즈(Xanthine oxidase) 억제활성 결과는 도 11과 표 5와 같으며, 젠신(Xanthine)을 기질로 하여 젠신 옥시데이즈(xanthine oxidase)에 의해 생성된 uric acid의 양을 290nm에서 흡광도를 측정하여 검색하였고, 생성된 superoxide anion의 양은 NBT 환원방법에 의해 517nm에서 측정하여 검색하였다. 실험의 정확도는 젠신 옥시데이즈(Xanthine oxidase) 억제제인 allopurinol을 가지고 확인하였다.Xanthine oxidase inhibitory activity results are shown in Figure 11 and Table 5, by measuring the absorbance at 290 nm the amount of uric acid produced by the genine (Xanthine oxidase) produced by xanthine oxidase (Xanthine oxidase) as a substrate The amount of superoxide anion produced was measured at 517 nm by NBT reduction. The accuracy of the experiment was confirmed with allopurinol, a Xanthine oxidase inhibitor.
분획은 젠신 옥시데이즈(Xanthine oxidase) 억제 활성을 보였으며 IC50 값은 132.7ug/ml으로 TIC A 및 TIC B보다 높은 활성이었다.The fraction showed Xanthine oxidase inhibitory activity and the IC 50 value was 132.7 ug / ml, which was higher than TIC A and TIC B.
분획은 젠신 옥시데이즈(Xanthine oxidase) 억제 활성과 더불어 수퍼옥사이드(Superoxide) 생성억제 활성을 보여주었으며 이는 PMS/NADH법에 의한 superoxide 소거 활성과 일치하는 결과이다.Fractions showed Xanthine oxidase inhibitory activity as well as superoxide inhibitory activity, which is consistent with superoxide scavenging activity by PMS / NADH.
시료
sample
필터 후 시료분획
TIC A
TIC B
Allopurinol
Curcumin
Quercetin
SODSample fraction
Sample fraction after filter
TIC A
TIC B
Allopurinol
Curcumin
Quercetin
SOD
49.5±1.4
*
39.3±3.3
2.9±0.1
87.7±0.4
5.6±0.4
0.55±0.003(U/ml)41.9 ± 0.07
49.5 ± 1.4
*
39.3 ± 3.3
2.9 ± 0.1
87.7 ± 0.4
5.6 ± 0.4
0.55 ± 0.003 (U / ml)
117.0±10.7
*
*
1.5±0.1
*
*
**132.7 ± 15.4
117.0 ± 10.7
*
*
1.5 ± 0.1
*
*
**
* Can't calculate the value of IC50 * Can't calculate the value of IC 50
** <5% radical scavenging activity at maximum concentration used for assay** <5% radical scavenging activity at maximum concentration used for assay
항염활성 검색을 살펴본다.Look for anti-inflammatory activity screening.
세포 독성 검색 결과는 도 12과 표 6와 같으며, RAW 264.7 세포에 여러 가지 농도의 시료를 첨가한 후 24시간 동안 배양하였다. 배지를 제거한 후 0.5mg/ml의 MTT용액을 첨가하고 1시간 동안 배양하였다. MTT 용액을 제거하고 DMSO를 첨가하여 MTT의 환원에 의해 생성된 formazan 침전물을 용해시킨 후 540nm에서 흡광도를 측정하였다.The results of the cytotoxicity search are shown in FIGS. 12 and 6, and various samples were added to RAW 264.7 cells and cultured for 24 hours. After removing the medium, 0.5mg / ml MTT solution was added and incubated for 1 hour. The MTT solution was removed and DMSO was added to dissolve the formazan precipitate produced by the reduction of MTT and the absorbance was measured at 540 nm.
대조군과 비교하여 RAW 264.7 세포에 대한 시료의 세포독성을 평가하였다.The cytotoxicity of the sample against RAW 264.7 cells was evaluated in comparison to the control.
in vitro 항염활성 검색을 살펴보면, RAW 264.7 세포에 LPS(100ng/ml)와 여러 가지 농도의 시료를 동시에 첨가한 후 24시간 동안 배양하였다. 생성된 NO의 양은 Griess 시약을 이용하여 세포배양액 중에 존재하는 nitrite의 형태로 측정하였다. in In vitro anti-inflammatory activity screening, LPS (100ng / ml) and various concentrations of the sample was added to RAW 264.7 cells and incubated for 24 hours. The amount of NO produced was measured in the form of nitrite present in the cell culture using Griess reagent.
50%의 세포독성을 나타내는 농도인 TC50 값을 LPS로 유도된 NO의 생성을 50% 억제하는 IC50값으로 나누어 선택지수를 구하였다.The TC 50 value, a concentration showing 50% cytotoxicity, was divided by the IC 50 value that inhibits the production of LPS-induced NO by 50%.
분획의 불용성 물질을 여과한 뒤는 비교군으로 사용한 TIC B보다 NO 생성에 대한 억제활성은 낮지만 독성이 약하여 선택지수가 3.08 이상으로 TIC B와 유사하게 나타냈다. 또한, 감귤과피 에탄올 추출물인 TIC A보다 좋은 활성을 나타내었다.After filtering the fraction of insoluble substance, the inhibitory activity on NO production was lower than that of TIC B used as a comparative group, but the toxicity was weak. In addition, it showed better activity than TIC A, a citrus peel ethanol extract.
(A)TC 50 (ug / ml)
(A)
(B)IC 50 (ug / ml)
(B)
A/BChoice index
A / B
TIC A
TIC BSample fraction after filter
TIC A
TIC B
> 2000
495.5±4.8> 2000
> 2000
495.5 ± 4.8
1035.7±34.2
132.6±8.0649.3 ± 21.6
1035.7 ± 34.2
132.6 ± 8.0
>1.93
3.7> 3.08
> 1.93
3.7
세포내 타이로시나제 활성분석을 살펴본다.Examine the intracellular tyrosinase activity assay.
세포내 타이로시나제 활성분석(Cell tyrosinase activity assay in B16/F10)결과는 도 13과 같으며, 쥐(Mus musculus)의 melanoma cell line 중 B16/F10 cell을 6well plate에 ml당 5X104이 되게 준비한 후, 24시간동안 37℃ CO2 항온기에서 배양하고, 멜라닌 생합성 유도물질인 alpha-MSH 50nM을 처리하고 동시에 시료분획(EtOH:PBS=1:1)을 31, 63, 125, 250, 500ug/ml로 각각 처리하였다. 72시간 동안 37℃항온기에서 배양하였으며 36시간이 지난 후, 배지교체와 동시에 시료를 다시 처리하였다.Cell tyrosinase activity assay in B16 / F10 results are shown in FIG. 13. B16 / F10 cells in the melanoma cell line of musculus were 5X10 4 / ml on 6well plate. After the preparation, the cells were incubated in a 37 ° C. CO 2 incubator for 24 hours, treated with melanin biosynthetic inducer alpha-MSH 50nM, and sample fractions (EtOH: PBS = 1: 1) were simultaneously 31, 63, 125, 250, 500ug / Each was treated with ml. The cells were incubated at 37 ° C. for 72 hours and after 36 hours, the samples were treated again at the same time as the medium was replaced.
세포를 trypsin-EDTA를 이용하여 수확했다. 이렇게 수확된 cell에 0.2mM phenylmethylsulfonyl fluoride(PMSF)와 1% Triton X-100을 함유한 67mM sodium phosphate buffer(pH 6.8)를 500ul 처리하고 sonication 하였으며, 이 상태로 1시간동안 얼음에서 보관하였다. 1시간 후 4℃ 원심분리기에서 15,000 rpm으로 15분간 원심분리하였으며 상층액을 취하여 tyrosinase 활성분석에 사용하였다.Cells were harvested using trypsin-EDTA. The harvested cells were treated with 500ul of 67mM sodium phosphate buffer (pH 6.8) containing 0.2mM phenylmethylsulfonyl fluoride (PMSF) and 1% Triton X-100 and sonicated, and stored on ice for 1 hour. After 1 hour, the mixture was centrifuged at 15,000 rpm for 15 minutes at 4 ° C centrifuge, and the supernatant was taken for tyrosinase activity analysis.
세포내 tyrosinsae 활성분석은 세포내의 존재하는 tyrosinase의 작용결과 생성되는 DOPA chrome을 미색법에 의핵 측정했다. 120ul의 67mM sodium phosphate buffer(pH 6.8)에 기질로서 5uL의 1.5mM L-tyrosine과 40ul의 25mM L-DOPA를 혼합한 후 위에서 얻은 상층액 100ul를 처리하여 37℃ 항온기에서 2시간 반응시켰다.Intracellular tyrosinsae activity assay was performed by whitening of DOPA chrome produced by the action of tyrosinase in cells. After mixing 5ul of 1.5mM L-tyrosine and 40ul of 25mM L-DOPA as a substrate in 120ul of 67mM sodium phosphate buffer (pH 6.8), 100ul of the supernatant obtained above was treated and reacted at 37 ° C for 2 hours.
반응액 중에 생성된 DOPA chrome을 475nm에서 측정하여 Mushroom tyrosinase의 활성을 기준으로 하여 단백질 1ug당 unit으로 효소 활성을 나타내였다.DOPA chrome produced in the reaction solution was measured at 475 nm and showed enzyme activity in units per ug of protein based on Mushroom tyrosinase activity.
Reference로 사용한 Arbutin의 저해활성이 50ug/ml에서 약 50% 정도 나오는 조건에서 실험하였다. 이 조건에서 시료 분획은 농도의존적으로 melanogenesis를 유도하고 있었으며, 100mg/ml 이하에서는 두드러진 활성을 나타내지 않았다.The inhibitory activity of Arbutin used as reference was about 50% at 50ug / ml. Under these conditions, the sample fraction induced melanogenesis in a concentration-dependent manner, and showed no significant activity below 100 mg / ml.
시료분획 병행투여군의 melanogenesis 유도활성은 500mg/ml에서 MSHaks 처리된 대조군보다 6배정도 높게 나타나고 있음을 알 수 있다. 따라서 분획 A는 MSH 병행 투여조건에서 melanogenesis를 유도하고 있다.The melanogenesis-inducing activity of the sample fraction group was shown to be 6 times higher than that of the MSHaks group at 500 mg / ml. Therefore, fraction A induces melanogenesis under MSH concurrent administration.
본 발명에서는 국내에서 생산된 당유자의 미숙과를 이용하여 neohesperidine의 함량이 많은 플라보노이드(flavonoid) 분획을 얻고자 한 것으로서, 당유자 미숙과를 6월 21일경 및 10월 중순 수확하여 신선품 그대로를 2회 80% 메탄올로 추출하여 ultrafiltration, XAD-16 레진을 사용한 MPLC 등 일련의 정제 과정을 거쳐 flavonoid 분획을 획득하였으며, 이 과정의 수율은 신선품 기준 9.2%, 건조중량 기준으로 15.3%였다. 이 분획의 성분은 neohesperidin이 약 64%, naringin이 약 29%를 차지하였다.In the present invention, to obtain a flavonoid fraction with a high content of neohesperidine by using the immature fruit of sugar-milk produced in Korea, the sugar-milk fruit is harvested around June 21 and mid-October Flavonoid fractions were obtained through a series of purification procedures, such as ultrafiltration and MPLC using XAD-16 resin, extracted twice with 80% methanol. The yield was 9.2% for fresh products and 15.3% for dry weight. The components of this fraction accounted for about 64% of neohesperidin and about 29% of naringin.
그 결과, 상기 분획은 높은 DPPH 및 NO, superoxide 소거활성과 xanthine oxidase 억제활성을 가지고 있으며, 또한 우수한 in vitro 항염 활성을 보여주었다. 이 분획은 항산화 및 항염 활성이 우수한 성분이 함유되어 있는 것으로 판단되며, 이들의 자유기 소거활성과 in vitro 항염활성은 두 활성의 시너지 효과로 인해 다양하고 유익한 생리활성을 보여줄 것이다.As a result, the fraction has high DPPH and NO, superoxide scavenging activity and xanthine oxidase inhibitory activity, and also excellent in In vitro anti-inflammatory activity was shown. These fractions are believed to contain components with excellent antioxidant and anti-inflammatory activity, and their free phase scavenging activity and in vitro anti-inflammatory activity will show various and beneficial physiological activities due to the synergistic effect of the two activities.
상기 플라보노이드 분획으로부터 고순도(95%이상)의 네오헤스페리딘(neohesperidin)을 얻었으며, 이 과정의 수율은 플라보노이드 분획에 대하여 21.4%였다.High purity (> 95%) neohesperidin (neohesperidin) was obtained from the flavonoid fraction, and the yield of this process was 21.4% relative to the flavonoid fraction.
Claims (10)
상기 감귤농축액준비단계의 감귤농축액 4 ~ 8 중량%, 포도당 83 ~ 90중량%, 페닐알긴산을 함유한 아스파탐 0.03 ~ 0.05 중량%, 감귤향분말 1 ~ 2 중량%, 자당지방산에스테르 0.5 ~ 1.5중량%, 비타민C 1.5 ~ 2.5 중량%, 구연산 1.5 ~ 2.5 중량%, 겔라틴 0.01 ~ 0.03 중량%, 비타민B1 0.01 ~ 0.02 중량%, 비타민B2 0.01 ~ 0.02 중량%, 비타민B3 0.01 ~ 0.02 중량%, 판토텐산칼슘(B5) 0.05 ~ 0.07 중량%, 엽산 0.002 ~ 0.004 중량%, 비타민A 0.05 ~ 0.12 중량%, 비오틴(B7) 0.0001 ~ 0.0005 중량%, 나이아신 0.1 ~ 0.2 중량%, 콜라겐 0.05 ~ 0.15 중량%, 밀크칼슘 0.1 ~ 0.15 중량%, 탄산칼슘 0.03 ~ 0.08 중량%를 혼합 반죽하여 혼합물을 형성하는 혼합반죽단계;
상기 조성물혼합단계의 혼합물을 성형 및 건조하는 성형단계;
로 이루어지고,
상기 감귤농축액준비단계는 감귤농축액의 100중량부를 기준으로 감귤류 미숙과로부터 분획된 시트러스 플라보노이드 3 ~ 9중량부를 혼합하고,
상기 감귤농축액준비단계에서는 세척된 미성숙 감귤류를 습식분쇄기로 1차 분쇄함과 아울러 습식마이크로분쇄기로 2차 분쇄하고, 분쇄된 감귤류를 분해 효소와 함께 추출기에 투입 교반한 후, 원심분리기로 슬러지와 감귤즙을 분리하고, 원심분리기로 분리된 감귤즙을 농축기로 50 ~ 100브릭스(Brix) 농도로 농축하여 감귤농축액을 형성하고, 분쇄된 감귤류 전체 중량의 0.15 ~ 0.25중량%의 분해 효소 펙티네이즈(pectinase)와 함께 추출기에 투입하여 50 ~ 60℃로 5 ~ 8시간 동안 지속 교반하고,
상기 혼합반죽단계의 혼합물을 0.3mm ~ 3mm 크기로 과립화한 후에 과립화한 혼합물을 성형하고,
상기 혼합반죽단계의 혼합물을 32 ~ 38 ℃에서 3 ~ 12시간 숙성한 후에 상기 성형단계에서 성형하고,
상기 혼합반죽단계에서 천연색소인 치자황색소 0.05 ~ 0.13 중량%를 혼합하는 것을 포함하여 이루어지는 감귤류를 함유한 건강보조식품의 제조방법.Preparing a citrus concentrate by crushing the washed immature citrus fruits and then preparing a citrus concentrate by concentrating the separated citrus juice;
Citrus concentrate 4 ~ 8% by weight of the preparation of the citrus concentrate, glucose 83 ~ 90% by weight, 0.03 ~ 0.05% by weight aspartame containing phenyl alginic acid, 1 ~ 2% by weight citrus flavor powder, sucrose fatty acid ester 0.5 ~ 1.5% by weight , Vitamin C 1.5 to 2.5% by weight, Citric acid 1.5 to 2.5% by weight, Gelatin 0.01 to 0.03% by weight, Vitamin B 1 0.01 to 0.02% by weight, Vitamin B 2 0.01 to 0.02% by weight, Vitamin B 3 0.01 to 0.02% by weight , Calcium pantothenate (B5) 0.05 to 0.07 wt%, folic acid 0.002 to 0.004 wt%, vitamin A 0.05 to 0.12 wt%, biotin (B7) 0.0001 to 0.0005 wt%, niacin 0.1 to 0.2 wt%, collagen 0.05 to 0.15 wt% Mixing dough step of mixing and kneading milk calcium 0.1 ~ 0.15% by weight, calcium carbonate 0.03 ~ 0.08% by weight;
A molding step of molding and drying the mixture of the composition mixing step;
Lt; / RTI >
The step of preparing the citrus concentrate mixes 3 to 9 parts by weight of citrus flavonoids fractionated from citrus fruits immature based on 100 parts by weight of citrus concentrate,
In the citrus concentrate preparation step, the washed immature citrus fruits are firstly pulverized with a wet mill, followed by a second pulverization with a wet microcrusher, and the pulverized citrus fruits are added to the extractor with a degrading enzyme and stirred, followed by sludge and citrus with a centrifuge. The juice was separated, and the citrus juice separated by a centrifuge was concentrated to a concentration of 50 to 100 Brix with a concentrator to form a citrus concentrate, and 0.15 to 0.25% by weight of digestive enzyme pectinase of the total weight of the pulverized citrus fruits. ) Into the extractor and continuously stirred at 50 to 60 ° C. for 5 to 8 hours,
After granulating the mixture of the kneading step to a size of 0.3mm ~ 3mm, to form a granulated mixture,
After the mixture of the mixing and kneading step is aged for 3 to 12 hours at 32 to 38 ℃, it is molded in the molding step,
Method of producing a health supplement containing citrus fruits comprising mixing 0.05 ~ 0.13% by weight of the natural pigments in gardenia yellow pigment in the mixing dough step.
상기 감귤농축액의 100중량부를 기준으로 감귤류 미숙과로부터 분획된 시트러스 플라보노이드 3 ~ 9중량부를 혼합하고,
상기 혼합물을 입경 10 ~ 25mm 두께 3 ~ 8mm 크기로 성형 및 건조하는 것을 특징으로 하는 건강보조식품.4 to 8% by weight citrus concentrate with a concentration of 50 to 100 Brix, 83 to 90% by weight glucose, 0.03 to 0.05% by weight aspartame containing phenylalginate, 1 to 2% by weight citrus fruit powder, 0.5 to 0.5% sucrose fatty acid ester 1.5% by weight, vitamin C 1.5 to 2.5% by weight, citric acid 1.5 to 2.5% by weight of gelatin 0.01 ~ 0.03 wt% of vitamin B 1 0.01 ~ 0.02 wt% of vitamin B 2 0.01 ~ 0.02% by weight, of vitamin B 3 0.01 ~ 0.02% by weight, calcium pantothenate (B5) 0.05-0.07%, folic acid 0.002-0.004%, vitamin A 0.05-0.12%, biotin (B7) 0.0001-0.0005%, niacin 0.1-0.2%, collagen 0.05- 0.15% by weight, milk calcium 0.1-0.15% by weight, calcium carbonate 0.03-0.08% by weight of the mixture is kneaded to form a mixture, the mixture is molded and dried,
3 to 9 parts by weight of citrus flavonoids fractionated from citrus fruits immature fruit based on 100 parts by weight of the citrus concentrate,
Health supplement, characterized in that for molding and drying the mixture to a particle size of 10 ~ 25mm thickness 3 ~ 8mm.
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| WO2015002393A1 (en) | 2013-07-03 | 2015-01-08 | 바이오스펙트럼(주) | Composition for treating or preventing inflammatory skin disease, comprising, as active ingredient, immature citrus fruit extract, or synephrine or salt thereof |
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| KR102062160B1 (en) * | 2017-12-29 | 2020-01-03 | 제주대학교 산학협력단 | A chewable tablet including colostrum and citrus concentrate |
| KR102622250B1 (en) * | 2021-07-15 | 2024-01-08 | 유영민 | Method for producing collagen |
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| KR100852258B1 (en) | 2006-05-18 | 2008-08-14 | 전현철 | Spicy and Sweety Collagen Orange Vita Green Tea Composition and Process for Producing of the same |
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| JP2008137985A (en) | 2006-11-10 | 2008-06-19 | Univ Kinki | A drug that enhances the antiallergic effect without increasing the side effects of a corticosteroid, and a method of enhancing the antiallergic effect without increasing the side effects of a corticosteroid. |
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