KR20160096839A - Nourising cream containing blueberry extract collected in hydrogel - Google Patents
Nourising cream containing blueberry extract collected in hydrogel Download PDFInfo
- Publication number
- KR20160096839A KR20160096839A KR1020150018403A KR20150018403A KR20160096839A KR 20160096839 A KR20160096839 A KR 20160096839A KR 1020150018403 A KR1020150018403 A KR 1020150018403A KR 20150018403 A KR20150018403 A KR 20150018403A KR 20160096839 A KR20160096839 A KR 20160096839A
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- South Korea
- Prior art keywords
- agarose
- blueberry
- extract
- hydrogel
- acetone
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Ceased
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- 235000005493 rutin Nutrition 0.000 description 1
- 229960004555 rutoside Drugs 0.000 description 1
- 235000015067 sauces Nutrition 0.000 description 1
- 230000002000 scavenging effect Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- PUZPDOWCWNUUKD-UHFFFAOYSA-M sodium fluoride Chemical compound [F-].[Na+] PUZPDOWCWNUUKD-UHFFFAOYSA-M 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 235000021012 strawberries Nutrition 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- FDDDEECHVMSUSB-UHFFFAOYSA-N sulfanilamide Chemical compound NC1=CC=C(S(N)(=O)=O)C=C1 FDDDEECHVMSUSB-UHFFFAOYSA-N 0.000 description 1
- 229940124530 sulfonamide Drugs 0.000 description 1
- 229920002994 synthetic fiber Polymers 0.000 description 1
- YORCFVZUJHMQHA-UHFFFAOYSA-N syringetin 3-galactoside Natural products COC1=C(O)C(OC)=CC(C2=C(C(=O)C3=C(O)C=C(O)C=C3O2)C2C(C(O)C(O)C(CO)O2)O)=C1 YORCFVZUJHMQHA-UHFFFAOYSA-N 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 125000003831 tetrazolyl group Chemical group 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 125000000391 vinyl group Chemical group [H]C([*])=C([H])[H] 0.000 description 1
- 229920002554 vinyl polymer Polymers 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 235000019154 vitamin C Nutrition 0.000 description 1
- 239000011718 vitamin C Substances 0.000 description 1
- VHBFFQKBGNRLFZ-UHFFFAOYSA-N vitamin p Natural products O1C2=CC=CC=C2C(=O)C=C1C1=CC=CC=C1 VHBFFQKBGNRLFZ-UHFFFAOYSA-N 0.000 description 1
- 238000003260 vortexing Methods 0.000 description 1
- 238000003809 water extraction Methods 0.000 description 1
- 239000003021 water soluble solvent Substances 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/02—Cosmetics or similar toiletry preparations characterised by special physical form
- A61K8/04—Dispersions; Emulsions
- A61K8/042—Gels
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/92—Oils, fats or waxes; Derivatives thereof, e.g. hydrogenation products thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Epidemiology (AREA)
- Birds (AREA)
- Chemical & Material Sciences (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Botany (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Dispersion Chemistry (AREA)
- Dermatology (AREA)
- Cosmetics (AREA)
Abstract
본 발명은 영양크림 및 그 제조방법에 관한 것으로, 보다 구체적으로는 블루베리 추출물과 아가로즈 하이드로젤을 이용한 하이드로젤에 포집된 블루베리 추출물을 함유한 영양크림 및 그 제조방법에 관한 것이다.
이를 위한 본 발명은, 정제수를 100 중량부로 할 때, 블루베리 0.1 ~ 10 중량부, 아가로즈(agarose) 0.01 ~ 1 중량부, 유상성분 0.01 ~ 1 중량부 , 수상성분 0.01 ~ 1 중량부로 이루어지는 것을 특징으로 한다.More particularly, the present invention relates to a nutritive cream containing a blueberry extract and a blueberry extract collected in a hydrogel using an agarose hydrogel, and a method for producing the same.
To this end, the present invention is characterized by comprising 0.1 to 10 parts by weight of blueberry, 0.01 to 1 part by weight of agarose, 0.01 to 1 part by weight of an oil component, and 0.01 to 1 part by weight of water component, .
Description
본 발명은 영양크림 및 그 제조방법에 관한 것으로, 보다 구체적으로는 블루베리 추출물과 아가로즈 하이드로젤을 이용한 하이드로젤에 포집된 블루베리 추출물을 함유한 영양크림 및 그 제조방법에 관한 것이다.
More particularly, the present invention relates to a nutritive cream containing a blueberry extract and a blueberry extract collected in a hydrogel using an agarose hydrogel, and a method for producing the same.
산업화 및 고령 인구의 증가에 따라 소비자 의식수준이 향상되면서 건강하고 여유로운 삶에 대한 욕구가 증가하고 있다. As industrialization and the aging population increase, consumers' consciousness level is increasing and the desire for healthy and laid-back life is increasing.
이에 따라, “아름답게 나이를 먹는 것(successful aging)”을 실현할 수 있는 많은 과학적인 연구가 다양한 영역에서 활발히 진행되고 있다. As a result, many scientific studies have been actively carried out in various fields to realize "successful aging".
과거 화장품은 단순히 외적인 치장의 도구로 여겨졌으나 최근에는 증가된 여성의 사회진출, 고학력화, 개인의 가치관 존중 및 생활패턴의 변화에 따라 화장품에 대한 욕구도 단순히 아름답게 꾸미는 도구에서 고급화, 다양화되는 추세를 보이며, 점차 기능적 측면이 강조된 기능성 화장품(cosmeceuticals)의 수요가 폭발적으로 증가하고 있다. In the past, cosmetics was regarded as a tool of exteriors, but in recent years, with the advancement of women in society, higher education, respect for individual values, and changes in living patterns, the need for cosmetics has become increasingly sophisticated and diversified And the demand for functional cosmetics (cosmeceuticals), which is increasingly emphasized in functional terms, is increasing explosively.
현재 기존의 화장품에 사용되고 있는 합성물질들은 피부의 알레르기를 일으키거나 사람에 따라 유해한 영향을 미칠 수 있으므로 인체에 무해한 천연물질 소재에 관한 연구가 활발히 진행 중에 있다.Currently, synthetic materials used in cosmetics cause skin allergies or harmful effects to humans, so research on natural materials harmless to the human body is actively under way.
이에 따라 기능성이 이미 알려진 많은 한약재나 식품이 기능성 화장품의 소재로 연구되고 있거나 제품으로 판매되고 있다.Accordingly, many herbal medicines and foods whose functions are already known are being studied as materials for functional cosmetics, or are being sold as products.
예를 들면, 대한민국 등록특허공보 제 10-0659138 호(2006. 12. 19. 공고)에서는 유효성분으로 갈로카테킨 갈레이트를 포함하는 보습용 화장료 조성물이 개시되어 있다.For example, Korean Patent Registration No. 10-0659138 (published on December 19, 2006) discloses a cosmetic composition for moisturizing comprising gallocatechin gallate as an active ingredient.
이러한 보습용 화장료 조성물은 퍼옥시즘 증식제 활성화 수용체를 활성화시키고, 피부보습인자의 발현을 촉진시켜 피부 보습효과를 나타낸다. 그러나, 보습용 화장료 조성물이 표피를 통과하여 피부 깊숙하게 침투하는 기능, 및 각질층에서 수분을 고정하는 기능이 매우 부족하여, 적정한 정도의 피부 수분 함량을 지속적으로 유지하는 것이 곤란하다는 문제점을 지니고 있다.Such a moisturizing cosmetic composition activates the peroxisome proliferator activating receptor and promotes the expression of a skin moisturizing factor, thereby exhibiting a skin moisturizing effect. However, there is a problem that the cosmetic composition for moisturizing penetrates deeply into the skin through the epidermis, and the function of fixing the moisture in the stratum corneum is very poor, and it is difficult to maintain the skin moisture content to an appropriate extent.
또한, 대한민국 공개특허공보 제 2006-0067296 호(2006. 06. 20. 공개)에서는 자일리톨과 베타-글루칸을 동시에 함유한 피부 보습용 화장료 조성물이 개시되어 있다.Korean Patent Laid-Open Publication No. 2006-0067296 (published on June 20, 2006) discloses a cosmetic composition for skin moisturizing which simultaneously contains xylitol and beta-glucan.
이러한 피부 보습용 화장료 조성물은 자일리톨과 베타-글루칸을 조합하여 함유함으로써 피부의 보습 효과를 나타낸다. 그러나, 피부 보습용 화장료 조성물이 표피를 통과하여 피부 깊숙하게 침투하는 기능, 및 각질층에서 수분을 고정하는 기능이 매우 부족하여, 적정한 정도의 피부 수분 함량을 지속적으로 유지하는 것이 곤란하다는 문제점을 지니고 있다.
Such a cosmetic composition for moisturizing the skin contains moisturizing effect of the skin by containing xylitol and beta-glucan in combination. However, there is a problem that the cosmetic composition for skin moisturizing penetrates deeply into the skin through the epidermis, and the function of fixing water in the stratum corneum is very poor, and it is difficult to maintain the skin moisture content to an appropriate extent continuously .
본 발명은 상기와 같은 종래의 문제점을 해결하고자 안출된 발명으로서, 본 발명은 블루베리 추출물의 화장품을 제공하는데 그 목적이 있다.Disclosure of Invention Technical Problem [6] The present invention has been devised in order to solve the above-mentioned conventional problems, and it is an object of the present invention to provide cosmetics of blueberry extract.
또한, 본 발명은 블루베리 추출물 세포독성 및 항염증 효능 평가하여 화장품의 기능성을 확인하여 그 적합도를 파악할 수 있다.In addition, the present invention can evaluate the cytotoxicity and anti-inflammatory efficacy of blueberry extract to ascertain the fitness of the cosmetic product.
또한, 본 발명은 홍조류의 일종인 "꼬시래기"를 이용한 "아가로스 하이드로젤"을 이용하여 크림형의 화장품을 제공할 수 있다.In addition, the present invention can provide a cream-type cosmetic using "agarose hydrogel" using "cod", a kind of red algae.
본 발명의 기타 다른 목적은 이하의 상세한 설명으로부터 명확하게 되고, 본 발명의 바람직한 실시예를 나타내는 상세한 설명 및 실시예는 본 발명의 범주를 제한하는 것이 아니다.
Other objects and advantages of the present invention will become apparent from the following detailed description of the present invention when taken in conjunction with the accompanying drawings.
이를 위한 본 발명은, 물을 100 중량부로 할 때, 블루베리 0.1 ~ 10 중량부, 1,3- 부틸렌글리콜, 1,2-헥산디올 20 ~ 40 중량부, 아가로스 0.01 ~ 1 중량부를 포함하여 이루어지는 것을 특징으로 한다.To this end, the present invention includes 0.1 to 10 parts by weight of blueberry, 20 to 40 parts by weight of 1,3-butylene glycol, 1,2-hexanediol and 0.01 to 1 part by weight of water, .
본 발명에 따른 블루베리를 함유한 영양크림은, 블루베리 100g을 착즙기를 사용하여 착즙 후 12,000rpm에서 7분간 원심분리하고 상등액을 취하여 필터링하여 동결건조하여 1%의 불루베리 추출물을 제조하는 단계;In the nutritional cream containing blueberry according to the present invention, 100 g of blueberry is centrifuged at 12,000 rpm for 7 minutes using a juicer, filtered, and lyophilized to prepare 1% of blueberry extract;
아가로즈(agarose) 파우더 1g 을 100 3차 증류수에 넣고 70℃에서 1시간 동안 교반하여 0.1%의 아가로즈를 수득하는 단계;1 g of agarose powder is added to 100 third distilled water and stirred at 70 캜 for 1 hour to obtain 0.1% agarose;
물, 1,3- 부틸렌글리콜, 1,2-헥산디올의 혼합물 1ℓ을 항온조에 넣고 70℃가 될 때 까지 1시간 정도 가온하여 기본제형을 수득하는 단계;1 liter of a mixture of water, 1,3-butylene glycol and 1,2-hexanediol is placed in a thermostatic chamber and heated to 70 DEG C for about 1 hour to obtain a basic formulation;
상기 블루베리 추출물 과 아가로즈(agarose) 용액을 항온조에서 40℃로 가열하는 단계;Heating the blueberry extract and agarose solution to 40 DEG C in a thermostat;
상기 기존제형과 블루베리 추출물, 아가로즈를 교반하여 블루베리 화장품을 제조하는 단계;Preparing blueberry cosmetics by stirring the conventional formulation, blueberry extract, and agarose;
를 포함하여 이루어지는 것을 특징으로 한다.And a control unit.
또한, 본 발명은 상기 제조된 블루베리 화장품 제형을 진공오븐을 사용하여 -0.05mb 에서 기포를 제거하고 각 용기에 담아 화장품을 완성하는 단계를 더 포함하여 이루어지는 것을 특징으로 한다.The present invention further comprises a step of removing the air bubbles at -0.05 mb using the vacuum oven and filling the cosmetic formulations in each container to complete the cosmetic product.
또한, 본 발명에 적용된 아가로즈(agarose)는, 꼬시래기를 90℃ 이상의 끓는 물에 7~8초 정도 삶아서 찬 물에 냉각 과정을 거친 후 협잡물을 제거하기 위해 흐르는 물에 3회 이상 수세를 반복하고, 탈색과 탈취를 위하여 강한 자외선에 투명비닐을 덮어 2~3일 동안 건조하며, 건조된 원물을 밀링(milling)과정을 통하여 한천을 수득하는 것을 특징으로 한다.The agarose used in the present invention is prepared by boiling the cucumber in boiling water of 90 ° C or higher for 7 to 8 seconds and cooling the cold water, and washing the water for 3 or more times with flowing water to remove the contaminants , A transparent ultraviolet ray is covered with a strong ultraviolet ray for discoloration and deodorization, dried for 2 to 3 days, and the agar is milled to obtain an agar.
또한, 본 발명에 적용된 한천은, 건조된 꼬시래기 원물 15g과 6% NaOH에 85℃ 에서 2시간 30분 동안 전처리하고, 전처리 후 0.5% Acetic acid를 이용하여 상온에서 1시간 동안 반응 후 수돗물을 이용하여 충분히 수세한 다음 증류수를 이용하여 85℃ 에서 2시간 30분 동안 가열추출 후 믹서기로 분쇄 후 필터로 필러링 하여 알코올 탈수법으로 탈수시켜 수득하는 것을 특징으로 한다.The agar used in the present invention was pretreated with 15 g of dried codic material and 6% NaOH at 85 ° C for 2 hours and 30 minutes, pretreated, reacted with 0.5% acetic acid at room temperature for 1 hour, After thoroughly rinsed, the mixture is heated and extracted with distilled water at 85 ° C for 2 hours and 30 minutes, pulverized by a blender, and then pelletized with a filter, and dehydrated by an alcohol dehydration method.
또한, 본 발명에 적용된 한천은, 건조 꼬시래기 15g과 증류수 500mL를 약 1시간 동안 방치 시킨 후 1N NaOH를 이용하여 pH8~10사이로 맞춘 다음 30분 동안 교반기를 사용하여 교반하고, 교반시킨 용액을 오토클레이에서 121℃에서 1시간동안 가압가온 추출하며, 가압가온 추출 후 용액을 0.1N HCl을 이용하여 중화시킨 다음 천으로 여과시키고, 여과시킨 용액을 방치시켜 우무(Agar-Agar)상태에서 -20℃에서 동결시킨 후 해동하여 탈수시켜 믹서기로 갈아 수득하는 것을 특징으로 한다.The agar used in the present invention was prepared by allowing 15 g of dried cod and 500 mL of distilled water to stand for about 1 hour, adjusting the pH to 8 to 10 with 1N NaOH, stirring for 30 minutes using a stirrer, The solution was neutralized with 0.1 N HCl, filtered through a cloth, and the filtrate was allowed to stand. The solution was left to stand in an agar-agar state at -20 ° C Freezing, thawing, dehydration, and grinding with a blender.
또한, 본 발명에 적용된 한천은, 건조된 꼬시래기 원물 약 50g을 증류수 200mL에 약 1시간가량 침지시킨 후 얼음을 원물과 1:1 혼합 후 블랜딩(blending) 시킨다. 블렌딩 후 냉장고 넣어 약 하루간 방치한 후 종이필터로 걸러서 밑으로 빠진 수용성 물질을 동량의 MeOH, EtOH, Isopropanol을 첨가하여 침전시키고, 필터링(filtering) 시켜 필터에 남은 물질을 다시 물에 녹이고 다시 알코올을 부어 2차 침전 시켜 분리정제 시켜 수득하는 것을 특징으로 한다.The agar applied to the present invention is obtained by immersing about 50 g of the dried cod raw material in 200 mL of distilled water for about 1 hour, mixing the ice with the raw material 1: 1 and blending. After blending, put in the refrigerator for about one day, filter with a paper filter, add the same amount of water-soluble material to the bottom, precipitate by adding MeOH, EtOH, Isopropanol, filter and filter the remaining material in water. Followed by pouring, secondary precipitation, separation and purification.
본 발명은, 상기 한천 5g을 100mL DMSO와 함께 실온에서 2시간 교반시킨 다음 원심분리(10,000 X g, 30 min)하여 상층액을 4배 부피의 아세톤에 순차적으로 침전시키고, 침전된 아가로즈(agarose)를 아세톤을 이용하여 3회 세척하며, 이를 회전 증발 농축기를 사용하여 40℃ 이하 온도에서 감압 건조킨 후 아세톤에 침전된 아가로즈(agarose)를 100mL 증류수로 12시간 교반하고 교반 후 약 3회 정도 증류수로 세척 후 이를 다시 아세톤 탈수하여 40℃ 이하에서 감압 건조시켜 아가로즈(agarose)를 수득하는 하는 것을 특징으로 한다.In the present invention, 5 g of the above agar is agitated with 100 mL of DMSO at room temperature for 2 hours and then centrifuged (10,000 X g, 30 min) to sequentially precipitate the supernatant in acetone of 4 times volume and agarose ) Was washed three times with acetone and dried under reduced pressure at a temperature of 40 ° C or lower using a rotary evaporator. The agarose precipitated in acetone was stirred for 12 hours with 100 mL of distilled water. After stirring for about 3 times Washed with distilled water, dehydrated with acetone, and dried under reduced pressure at 40 ° C or lower to obtain agarose.
또한, 본 발명은 상기 한천 5g을 100mL DMSO와 함께 실온에서 2시간 교반시킨 다음 원심분리(10,000 X g, 30 min)하여 상층액을 4배 부피의 아세톤에 순차적으로 침전시키고, 침전된 아가로스를 아세톤을 이용하여 3회 세척하며, 이를 회전 증발 농축기를 사용하여 40℃ 이하 온도에서 감압 건조킨 후 아세톤에 침전된 아가로즈(agarose)를 0.01M의 Na2EDTA용액 100mL로 하룻밤 교반하고 증류수로 3회 세척 후 이를 다시 아세톤으로 탈수하여 40℃ 이하에서 감압 건조시켜 아가로즈(agarose)를 수득하는 하는 것을 특징으로 한다.
In the present invention, 5 g of the agar was agitated with 100 mL of DMSO at room temperature for 2 hours and then centrifuged (10,000 X g, 30 min) to sequentially precipitate the supernatant in acetone of 4 times volume, and the precipitated agarose The mixture was washed three times with acetone and dried under reduced pressure at a temperature of 40 ° C or lower using a rotary evaporator. The agarose precipitated in acetone was stirred with 100 ml of 0.01 M Na 2 EDTA solution overnight, After washing with water, it is dehydrated again with acetone and dried under reduced pressure at 40 ° C or lower to obtain an agarose.
이상에서와 같이, 블루베리 추출물을 이용한 피부과학적 효능효과 검증(항산화 활성, 미백 활성 등)하고, 개발한 물질과 기술에 대하여 기업의 매출증대와 농가소득 증대 및 지역 농산물의 산업화 사례로 활용하고, 블루베리의 효능 성분 분석으로 지역 특화 생물소재 개발 연구에 대한 기술 축적할 수 있는 효과가 발생한다.
As described above, the use of the blueberry extract for the verification of dermatological efficacy (antioxidant activity, whitening activity, and the like) is utilized to increase the sales of the company, increase the farm income and industrialization of the local agricultural products, Analysis of the efficacy components of blueberries has the effect of accumulating the technology for the research on the development of the region-specific biomaterials.
먼저, 본 발명은 블루베리를 이용한 항산화기능 강화 화장품을 개발하기 위한 것으로, 이러한 화장품을 개발하기 위하여 블루베리 착즙액의 항산화 활성을 확인하고, 블루베리 착즙액의 HPLC를 이용한 지표를 확인하며, 하이드로젤 원료 제조법을 확립한 다음 블루베리 화장품 제조하는 과정을 거치게 된다.First, the present invention is to develop an antioxidant-enhancing cosmetic product using blueberry. To develop such a cosmetic product, antioxidant activity of a blueberry juice is confirmed, an index using HPLC of a blueberry juice is confirmed, After establishing the raw material of the gel, it will go through the process of producing blueberry cosmetics.
이를 위해 블루베리 착즙액의 항산화 활성 확인을 위한 실험원리 및 실험방법은,For this purpose, the experimental principle and experimental method for confirming the antioxidant activity of the blueberry juice were as follows:
1. 블루베리 착즙액의 항산화 활성 확인1. Identification of Antioxidant Activity of Blueberry Juice
1) DPPH radical scavenging에 의한 실험방법*1) Experimental method by DPPH radical scavenging *
(1) 실험원리 및 실험방법(1) Principle of experiment and experimental method
*DPPH 반응 원리*
* DPPH Reaction Principle *
활성산소의 생성은 자동산화 반응으로 연쇄적으로 일어나는데 체내에서는 항산화제가 수소원자를 자동산화 반응 중에 생성되는 radical에 공여함으로써, 그 반응을 종결시킨다. 이렇게 전자를 주는 능력을 환원력이라 하고, 이 환원력이 클수록 강력한 항산화제가 되는데 이러한 환원력을 측정할 수 있는 방법으로 DPPH radical 제거법이 폭넓게 사용되고 있다.
The production of active oxygen occurs chainwise by an autoxidation reaction. In the body, the antioxidant terminates the reaction by donating a hydrogen atom to the radical generated during the autoxidation reaction. This ability to give electrons is called the reducing power. The larger the reducing power, the stronger the antioxidant. The DPPH radical removal method is widely used as a method for measuring the reducing power.
*Galic acid standard curve(DPPH)
* Galic acid standard curve (DPPH)
DPPH는 자신이 가지는 홀수 전자 때문에 517 nm에서 특징적인 흡수 spectrum을 가지는데 이것이 수소 원자를 제공해 주는 전자 공여체와 반응하면 hydrogen radical 을 받아 phenoxy radical을 생성하게 된다. DPPH는 radical 상태에서 보라색을 띄고, 환원이 되면 노란색의 2,2-diphenyl-1-picrylhydrazine(DPPH-H)으로 변환된다. 안정한 라디칼인 DPPH는 산화방지물질로부터 전자 혹은 수소를 제공받으면 non-radical 로 전환된다. DPPH법은 천연물의 수용성 혹은 유기용매 추출물의 항산화 활성 측정법으로 널리 사용하는 방법이다. DPPH has a characteristic absorption spectrum at 517 nm due to the odd electrons it possesses. When it reacts with an electron donor which provides hydrogen atom, it gets hydrogen radical to generate phenoxy radical. DPPH is purple in the radical state, and when it is reduced, it is converted to yellow 2,2-diphenyl-1-picrylhydrazine (DPPH-H). DPPH, a stable radical, converts to non-radical when it is supplied with electrons or hydrogen from antioxidants. The DPPH method is widely used as a method for measuring the antioxidative activity of a water-soluble or organic solvent extract of a natural product.
본 실험에서는 항산화 활성 측정방법 중 Blois의 (Blois, 1958) 방법을 변형하여 sample의 radical 소거능을 측정하였다.In this experiment, the antioxidant activity of Blois (Blois, 1958) was modified to measure the radical scavenging ability of the sample.
95% ethanol 에 용해시킨 0.1 mM DPPH (2,2-diphenyl-1-picrylhydrazyl, Sigma, USA) 800 l와 각 실험군 200 l을 E.P tube 에 넣고, 3분간 voltexing 하여 암실에서 30분간 반응 시켰다. 800 l of 0.1 mM DPPH (2,2-diphenyl-1-picrylhydrazyl, Sigma, USA) dissolved in 95% ethanol and 200 l of each experimental group were placed in an E.P tube and allowed to react for 30 minutes in a dark room by vortexing for 3 minutes.
남아 있는 radical의 농도를 UV-Visible spectrophotometer (SCINCO, Seoul, Korea)를 사용하여 517 nm에서 측정하였으며, 양성 대조군으로는 gallic acid (Sigma, USA), quercetin (Sigma, USA), ascorbic acid (Sigma, USA)를 사용하였고, 농도범위는 0.5 mg/ml 0.025 mg/ml 로 측정 하였다. The concentration of residual radicals was measured at 517 nm using a UV-Visible spectrophotometer (SCINCO, Seoul, Korea). The positive controls were gallic acid (Sigma, USA), quercetin (Sigma, USA), ascorbic acid USA) and the concentration range was 0.5 mg / ml 0.025 mg / ml.
추출물의 DPPH에 대한 소거능 (IC50)은 용매만을 사용한 대조군의 흡광도를 50% 감소시키는데 필요한 농도로 나타내었다.
The scavenging ability (IC 50 ) of the extract against DPPH was expressed as the concentration required to reduce the absorbance of the control using only the solvent by 50%.
(2) 실험 결과
(2) Experimental results
*블루베리 착즙액의 DPPH radical scavenging 결과
* DPPH radical scavenging results of blueberry juice
블루베리 착즙액의 DPPH radical 소거능을 확인한 결과 양성 대조군인 quercetin, gallic acid, ascorbic acid에 비해 낮은 항산화 활성이 확인 되었으나, 천연물 기준으로 보았을 때 매우 높은 항산화 활성을 나타내었으며, 블루베리 착즙액의 IC50값은 83.20 mg/ml, quercetin의 IC50값은 0.03 mg/ml, gallic acid의 IC50값은 0.01 mg/ml, ascorbic acid의 IC50값은 0.04 mg/ml 이었다.A positive result confirming the DPPH radical scavenging activity of the blueberry juice liquid control of quercetin, gallic acid, but a low antioxidant activity make than ascorbic acid, which showed very high antioxidant activity as seen with natural product based, blueberry IC 50 of the juice liquid The IC 50 value of quercetin was 0.03 mg / ml, the IC 50 value of gallic acid was 0.01 mg / ml, and the IC 50 value of ascorbic acid was 0.04 mg / ml.
블루베리 착즙액의 항산화능은 flavonoids와 polyphenols 계열 성분에 의해 나타나는 것으로 판단되어 지며, 추후 각 성분의 표준물질을 이용하여 항산화능과 정확한 기전을 규명할 필요가 있다. 항산화능 실험의 결과를 종합해 보았을 때 블루베리 착즙액은 천연 화장품의 소재로서 매우 적합하다고 판단된다.
Antioxidant activity of blueberry juice is judged to be caused by flavonoids and polyphenols, and it is necessary to identify the antioxidant ability and precise mechanism of the antioxidant activity by using standard ingredients of each ingredient. When the results of the antioxidant ability test are summarized, it is considered that the blueberry juice is very suitable as a material of natural cosmetics.
2) ABTS radical scavenging
2) ABTS radical scavenging
(1) 실험원리 및 실험방법
(1) Principle of experiment and experimental method
*ABTS 반응원리
* ABTS reaction principle
본 실험에서는 Jeong 등의 방법 (Jeong et al., 2009)을 변형하여 측정하였다. 증류수에 용해한 7 mM의 ABTS potassium persulfate 2.45 mM을 1:1로 넣어 흡광도 값이 0.70이 되도록 PBS (pH 7.4)로 희석하였다. 12-16시간 동안 암소에 방치하고 Radical stock solution은 희석된 용액 800 l에 농도별로 제조된 sample 200 l씩을 가하여 15분 동안 방치 후 흡광도를 측정하였다. In this experiment, Jeong et al. (2009) was modified and measured. Diluted with PBS (pH 7.4) so as to have an absorbance value of 0.70 by adding 1: 1 of 2.45 mM of ABTS potassium persulfate dissolved in distilled water. The sample was placed in a dark place for 12-16 hours. Radial stock solution was added to 200 liters of diluted solution (200 l each), and the absorbance was measured for 15 minutes.
양성 대조군으로는 gallic acid (Sigma, USA), quercetin (Sigma, USA), ascorbic acid (Sigma, USA)를 사용하였고, 농도범위는 0.5 mg/ml 0.025 mg/ml 로 측정 하였다. 추출물의 ABTS 에 대한 소거능 (IC50)은 용매만을 사용한 대조군의 흡광도를 50% 감소시키는데 필요한 농도로 나타내었다.
As a positive control, gallic acid (Sigma, USA), quercetin (Sigma, USA) and ascorbic acid (Sigma, USA) were used and the concentration range was 0.5 mg / ml 0.025 mg / ml. The abstraction (IC 50 ) of the extracts against ABTS was expressed as the concentration required to reduce the absorbance of the control using only solvent by 50%.
(2) 실험결과
(2) Experimental results
* 블루베리 착즙액의 ABTS radical scavenging activity 및 IC50 ,
* ABTS radical scavenging activity and IC 50 of blueberry juice ,
*블루베리 착즙액의 ABTS radical scavenging 실험 결과
* ABTS radical scavenging test results of blueberry juice
실험결과 블루베리 착즙액의 IC50값은 68.00 mg/ml, quercetin의 IC50값은 0.11 mg/ml, gallic acid의 IC50값은 0.14 mg/ml, ascorbic acid의 IC50값은 0.06 mg/ml 이었다. ABTS radical scavenging 결과는 앞서 실험한 DPPH radical scavenging 보다 약간 높은 항산화 활성을 나타내었는데 이것은 ABTS radical scavenging가 친수성 시료와 소수성 시료의 radical 소거 활성을 모두 측정 할 수 있기 때문에 다른 항산화 실험결과보다 높은 항산화능을 보인 것으로 판단된다.
The IC 50 value of the blueberry juice was 68.00 mg / ml, the IC 50 value of quercetin was 0.11 mg / ml, the IC 50 value of gallic acid was 0.14 mg / ml and the IC 50 value of ascorbic acid was 0.06 mg / ml . The results of ABTS radical scavenging showed a slightly higher antioxidative activity than that of the DPPH radical scavenging, which showed higher antioxidative activity than the other antioxidant tests because ABTS radical scavenging can measure both radical scavenging activities of hydrophilic and hydrophobic samples .
3) Hydroxyl radical scavenging
3) Hydroxyl radical scavenging
(1) 실험원리 및 실험방법
(1) Principle of experiment and experimental method
Hydroxyl radical은 활성산소 라디칼 중에서 화학적으로 가장 반응성이 크며 지질산화를 개시하고 DNA손상을 주거나 돌연변이를 유발하는 물질로 알려져 있고, 생체의 대사 과정에서 생성되는 지질의 과산화물이나 과산화수소가 Fe2+나 Cu2+ 이온의 존재 하에서 생성되며 가장 독성이 강한 free radical이다.
Hydroxyl radicals are the most chemically reactive radicals in the active oxygen radicals. They are known to initiate lipid oxidation and to cause DNA damage or mutagenesis. The lipid peroxides or hydrogen peroxide produced in the metabolism of the body are either Fe 2+ or Cu 2 + Ions and is the most toxic free radical.
* Hydroxyl Radical 반응 원리
* Hydroxyl Radical Reaction Principle
(2) 실험결과(2) Experimental results
*블루베리 착즙액의 Hydroxyl radical scavenging activity 및 IC50 실험 결과*
* Hydroxyl radical scavenging activity and IC 50 test results of blueberry juice *
Hydroxyl radical scavenging 실험결과 블루베리 착즙액의 IC50값은 91.14 mg/ml, quercetin의 IC50값은 0.28 mg/ml, gallic acid의 IC50값은 0.15 mg/ml, ascorbic acid의 IC50값은 0.11 mg/ml 이었다.
In the hydroxyl radical scavenging experiment, the IC 50 value of the juice of blueberry was 91.14 mg / ml, the IC 50 value of quercetin was 0.28 mg / ml, the IC 50 value of gallic acid 0.15 mg / ml and the IC 50 value of ascorbic acid 0.11 mg / ml.
4) 블루베리 착즙액의 총 폴리페놀 함량
4) Total polyphenol content of blueberry juice
(1) 폴리페놀의 효과와 작용원리
(1) Effects of polyphenols and their working principles
폴리페놀은 식물 내에 존재하는 여러 페놀화합물의 총칭이다. 식물이 자라면서 여러가지 유해환경으로부터 자신을 보호하기 위한 보호물질이다. 하나의 식물에는 심지어 수천가지의 폴리페놀이 함유되어 있으며 각기 하는 역할은 조금씩 차이가 있다. 모든 폴리페놀은 공통적으로 항산화와 항암효과를 보이며 다시 말해, 우리 몸 안에서 당뇨병, 동맥경화, 암 등을 유발하는 활성산소를 없애는 기능을 하며 이로 인하여 노화의 속도를 늦추는 기능까지 하는 것으로 알려져 있다. Polyphenols are a generic term for several phenolic compounds present in plants. It is a protective material to protect itself from various harmful environments as plants grow. One plant contains even thousands of polyphenols, each with slightly different roles. All polyphenols have antioxidant and anticancer effects in common, which means that they have the function of eliminating the active oxygen that causes diabetes, arteriosclerosis and cancer in our body and it is known to slow down the aging speed.
폴리페놀 소스로써, 사과껍질 추출물에는 프로시아니딘이라는 성분이 있는데 아토피 질환에 효과적인 것으로 알려져 있다. 특히 사과 유래의 폴리페놀에는 퀘르세틴 등 잘 알려져 있는 성분들 외에 플로리진(phloridzin, phlorizin)이라는 폴리페놀류 물질이 있는데 이 성분은 특히 껍질에 많이 함유되어 있다. 이 물질의 bioavailability에 대한 연구에서는 글리코시드 형태, 혹은 배당체 형태로 투여시 장내로 급속히 흡수돼 대사를 거쳐 소변으로 배출되는 것으로 확인할 수 있었다고 보고되어 있으며, bone metabolism의 개선에 큰 영향을 미치는 것으로 확인되어 학계의 주목을 받았다.As a polyphenol sauce, the apple bark extract has a component called procyanidins, which is known to be effective for atopic disease. Especially apple-derived polyphenols have well-known components such as quercetin, and polyphenol substances called phloridzin (phlorizin), which are especially contained in the shell. The bioavailability of this substance has been reported to be rapidly excreted into the intestines through the glycoside or glycoside form and released into the urine via metabolism and has been shown to have a significant effect on the improvement of bone metabolism I got attention from academia.
폴리페놀은 위에서 언급한 플라보노이드가 대표적이며 타닌, 리그닌 (페닐프로파노이드)으로 분류된다. 노락색을 띠는 안토진틴, 붉은색, 보라색, 청색을 띠는 안토시아닌, 또는 무색의 플라본 색소를 지니고 있다. Polyphenols are classified as tannins and lignins (phenylpropanoids). It has yellowish-colored antagonist, red, purple, blue anthocyanin, or colorless flavone pigment.
안토시아닌은 포도, 딸기, 체리, 적포주에 다량 함유되어 있으며, 프로안토시아닌은 사과와 배에 많다. 히스페리딘과 나린진은 감귤류에, 케르세틴은 양파와 사과 껍질, 녹차에 많은 것으로 알려져 있다. 루틴은 메밀, 이소플라본은 콩류에 함유되어 있으며 알칼리에서 노란색으로 변하는 특징을 보인다.
Anthocyanins are abundant in grapes, strawberries, cherries and red pimples, and proanthocyanins are found in apples and pears. Hissperidin and naringin are known to be found in citrus, quercetin in onions, apple peels and green tea. Rutin is contained in buckwheat, and isoflavone is contained in leguminous beans and shows the characteristic of changing from alkali to yellow.
*플로리진의 2차원 분자 구조*
* 2D molecular structure of floridine *
*과실류의 총 폴리페놀 및 플라보노이드 함량 (농촌진흥청 식품성분표(2009), 제9판)** Total polyphenol and flavonoid content of fruits (RDA, Food Ingredients Table (2009), 9th edition) *
블루베리 속에 함유된 페놀류 화합물들이 간암세포주인 'HepG2'의 성장을 억제하고, 암세포들의 자기괴사(apoptosis)를 유도했음이 보고 되었으며, 각종 페놀산과 탄닌, 플라보놀, 안토시아닌 등의 폴리페놀 성분과 항산화 물질들을 풍부히 함유한 과일로 알려져 있다. 아울러 레스베라트롤과 유사한 성분인 프테로스틸벤(pterostilbene)이 콜레스테롤 수치를 낮추는데 효과적인 작용을 발휘한다는 사실이 최근 밝혀진 바 있다. It has been reported that the phenol compounds contained in the blueberry inhibit the growth of hepatocellular carcinoma cell line HepG2 and induce apoptosis of cancer cells. It has been reported that polyphenol components such as various phenolic acids, tannins, flavonols and anthocyanins, It is known as a fruit rich in substances. It has recently been shown that pterostilbene, a component similar to resveratrol, is effective in lowering cholesterol levels.
또한 블루베리는 비타민C보다 두 배 강력한 항산화 작용을 하는 안토시아닌이 포도의 30배 이상 함유되어 있는 천연 항산화제이다. 이러한 블루베리의 효능은 피부에 활력을 공급하고 탄력을 증진하며 미백효과도 보고된 바 있다. 따라서 본 연구에서는 블루베리의 총 폴리페놀 함량을 분석하여 화장품 소재로서의 가능성을 검토하였다.
Blueberries are a natural antioxidant that contains more than 30 times the amount of grapes of anthocyanin, which has twice as much antioxidant activity as vitamin C. The efficacy of these blueberries has been reported to provide skin vitality, promote elasticity and whitening effects. Therefore, in this study, the total polyphenol contents of blueberries were analyzed to examine the possibilities as a cosmetic material.
(2) 총 폴리페놀 함량 (2) Total polyphenol content
*Galic acid standard curve
* Galic acid standard curve
*블루베리 착즙액의 총 폴리페놀 함량
* Total polyphenol content of blueberry juice
블루베리 착즙액의 폴리페놀 함량을 분석한 결과, 블루베리 착즙액 0.5 mg/ml은 gallic acid 0.098 mg/ml에 해당하였으며, 0.25 mg/ml은 gallic acid 0.085 mg/ml, 0.1 mg/ml은 gallic acid 0.069 mg/ml이었다. 이 같은 결과는 앞서 표5에 제시했던 농촌진흥청 식품성분표(2009), 제9판)보다는 낮은 결과이나 블루베리의 추출 방법 측면에서 보았을때 블루베리 착즙액의 상등액 기준이므로 추후 추출방법을 달리하여 총 폴리페놀 함량을 다시 제시할 필요가 있다.
The polyphenol content of the blueberry juice solution was 0.5 mg / ml of gallic acid, 0.085 mg / ml of gallic acid, 0.085 mg / ml of gallic acid and 0.1 mg / ml of gallic acid acid 0.069 mg / ml. This result is lower than the RDA's Food Ingredient List (2009) and the 9th edition), but it is based on the supernatant of the blueberry juice in terms of the extraction method of blueberry. The total polyphenol content needs to be reexamined.
2. 블루베리 착즙액의 HPLC를 이용한 지표 성분 확인
2. Identification of surface components of blueberry juice by HPLC
1) HPLC 시료 준비
1) Preparation of HPLC sample
블루베리 착즙액을 동결건조한 시료를 준비하여 1g을 취하였다. 취한 시료를 0.1% 염산을 포함한 80% Ethanol 50 ml를 2번 가하여 침지하였고, 80% methanol 50 ml을 가하여 침지하여 추출하였다. 추출된 추출물을 Syringe filter(45 m)를 사용하여 여과하고, 35에서 감압농축을하여 ethanol만 선택적으로 증발하였다. 증발되고 남은 물층을 petroleum ether 25 ml을 가하여 상분리하여 비극성화합물을 제거하고, ethyl acetate 25 ml을 3번 가하여 flavonoid류 화합물을 제거하였다. 제거되고 남은 물층을 감압농축하여 분말형태로 남도록 농축하였고, HCOOH 사용하여 pH 2로 적정한 D.W.를 가하여 농도가 100 mg/ml이 되도록 제조하고, HPLC 분석을 위해 Syringe filter(45 m)로 여과하였고 빛을 차단한 상태에서 4℃에서 보관하였다.
A sample obtained by lyophilizing the blueberry juice was prepared and 1 g was taken. The obtained sample was immersed in 50 ml of 80% ethanol containing 0.1% hydrochloric acid twice and extracted with 50 ml of 80% methanol. The extracted extract was filtered using a syringe filter (45 m) and concentrated under reduced pressure at 35 to selectively evaporate ethanol. The remaining water layer was separated by phase separation with petroleum ether (25 ml), and the nonpolar compound was removed. The flavonoid compounds were removed by adding 25 ml of ethyl acetate three times. The remaining water layer was concentrated under reduced pressure to remain in the form of powder. The pH was adjusted to 2 with DW using HCOOH, and the concentration was adjusted to 100 mg / ml. The resulting solution was filtered with a syringe filter (45 m) And then stored at 4 ° C.
* 블루베리 추출물의 HPLC 분석 시료 준비 diagram
* HPLC analysis of blueberry extract Sample preparation diagram
2) HPLC 분석 방법
2) HPLC analysis method
지표성분은 베리류에서 발견 되는 Cyanidin-3-galactoside, Cyanidin-3-glucoside이고, 블루베리 추출물에서 정량분석을 시작하였다. 정량분석 방법은 각 지표물질의 머무름시간(Retention time, R. Time)과 비교 분석하였다. HPLC는 SIMADZU(Japan)사의 LC-20A Serise를 사용하였고, 이동상 A는 HCOOH:D.W. (10:90, v/v), 이동상 B는 HCOOH:D.W.:ACN (10:60:30, v/v/v)를 사용하였고, C18 column을 사용하였으며 유량은 1 ml/min, 검출 파장은 546 nm에서 검출하였다. 샘플 주입 부피는 2μl이다. Cyanidin-3-galactoside and cyanidin-3-glucoside, which are found in berries, were quantitatively analyzed by blueberry extract. The quantitative analysis method was compared with the retention time (R. Time) of each indicator. LC-20A Serise from SIMADZU (Japan) was used as HPLC, and mobile phase A was HCOOH: D.W. (10:90, v / v), mobile phase B was HCOOH: DW: ACN (10:60:30, v / v / v) and C18 column was used. The flow rate was 1 ml / 546 nm. The sample injection volume is 2 μl.
* 블루베리 HPLC 분석 조건* Blueberry HPLC analysis conditions
3) Standard 물질 HPLC 분석 결과
3) Standard HPLC analysis
*standard 물질의 HPLC 분석 peak.
* HPLC analysis of standard material peak.
* standard 물질의 HPLC 분석 결과
* HPLC analysis of standard material
기준 물질 8종을 HPLC 분석한 결과 13.279, 14.379, 15.085, 15.847, 16.758, 20.083, 24.831, 31.471분에서 각각 Cyanidin-3,5-di-O-glucoside, Cyanidin-3-galactoside, Cyanidin-3-glucoside, Cyanidin-3-O-rutinoside, Delphinidin, Cyanidin, Pelargonidin, Malvidin이 검출되었다. 지표물질의 농도 대비 검출 면적을 이용하여 보정곡선을 그렸고, 보정식은 각각 
3,5-di-O-glucoside, Cyanidin-3-galactoside, and cyanidin-3-glucoside were measured at 13.279, 14.379, 15.085, 15.847, 16.758, 20.083, 24.831 and 31.471 min, , Cyanidin-3-O-rutinoside, Delphinidin, Cyanidin, Pelargonidin and Malvidin were detected. The calibration curve was drawn using the detection area versus the concentration of the indicator material,
4) 블루베리 추출물 HPLC 정량분석 결과
4) HPLC quantitative analysis of blueberry extract
* 블루베리 추출물의 HPLC 분석 peak.
* HPLC analysis of blueberry extract peak.
블루베리 추출물의 농도는 100 mg/ml이었고, 13.191, 14.475, 15.169, 15.892, 16.865, 20.193, 25.234, 31.499분에서 각각 Cyanidin-3,5-di-O-glucoside, Cyanidin-3-O-galactoside, Cyanidin-3-O-glucoside, Cyanidin-3-O-rutinoside, Delphinidin, Cyanidin, Pelargonidin, Malvidin이 검출되었고, 이들의 함량을 측정한 결과 각각 0.135, 0.133, 0.092, 0.079, 0.029, 0.141, 0.152, 0.269 mg/g 이다.
3,5-di-O-glucoside, Cyanidin-3-O-galactoside, and cyanidin were obtained at a concentration of 100 mg / ml of blueberry extract and at 13.191, 14.475, 15.169, 15.892, 16.865, 20.193, 25.234, Cyanidin-3-O-glucoside, Cyanidin-3-O-rutinoside, Delphinidin, Cyanidin, Pelargonidin and Malvidin were detected and their content was measured to be 0.135, 0.133, 0.092, 0.079, 0.029, 0.141, 0.152, 0.269 mg / g.
J. Correa-Betanzo의 조사에 따르면 블루베리에는 페놀성 화합물인 Chlorogenic acid, Quercetin arabinoside, Syringetin-3-galactoside와, 안토시아닌 화합물인 Dephinidin-3-galactoside, Delphinidin-3-glucoside, Delphinidin-3-arabinoside, Cyanidin-3-glucoside, Petunidin-3-galactoside, Peonidin-3-galactoside, Malvidin-3-galactoside, Malvidin-3-glucoside, Malvidin-3-arabinoside, Delphinidin-6-acetyl-3-glucoside, Malvidin-6-acetyl-3-glucoside가 함유되어있는 것으로 확인되었다. 본 실험에서는 Delphinidin, Cyanidin, Pelargonidin, Malvidin 기본형태의 화합물과 당과 결합된 형태인 Cyanidin-3,5-di-O-glucoside, Cyanidin-3-O-galactoside, Cyanidin-3-O-glucoside, Cyanidin-3-O-rutinoside가 있는 것으로 확인 되었다. According to research by J. Correa-Betanzo, the phenolic compounds Chlorogenic acid, Quercetin arabinoside, Syringetin-3-galactoside and the anthocyanin compounds Dephinidin-3-galactoside, Delphinidin-3-glucoside, Delphinidin- 3-galactoside, Malidin-3-galactoside, Malvidin-3-glucoside, Malvidin-3-arabinoside, Delphinidin-6-acetyl-3-glucoside, Malvidin- acetyl-3-glucoside. In this experiment, the basic forms of Delphinidin, Cyanidin, Pelargonidin, and Malvidin and the sugar-bound forms of Cyanidin-3,5-di-O-glucoside, Cyanidin-3-O-galactoside, Cyanidin- -3-O-rutinoside.
*블루베리 추출물 HPLC 정량분석 결과
* HPLC quantitative analysis results of blueberry extract
3. 꼬시래기로 부터 하이드로젤 제조
3. Manufacture of hydrogel from cornucopia
1) 하이드로젤의 정의 및 성질
1) Definition and properties of hydrogels
하이드로젤은 삼차원의 친수성 고분자 망상구조를 가진 물질로 다량의 수분을 함유할 수 있다. 하이드로젤은 적어도 전체 중량의 20% 이상의 수분을 흡수할 수 있으며, 95%이상의 물을 흡수하는 것을 고흡수성 하이드로젤이라고 부른다. 하이드로젤은 공중합체 또는 단일중합체로 형성되는데 외부 이력에 의한 유동성이 거의 없는 구조적으로 안정한 삼차원 구조를 형성하는 요인으로 공유결합, 수소결합, 반 데르 발스 결합, 물리적인 응집 등 여러 요인에 기인된다. 하이드로젤은 액상에서 팽윤된 후 열역학적으로 안정된 상태로 존재하며 액체와 고체의 중간 형태의 물리화학적 특성을 가진다. 이러한 물리화학적 특성 중 팽윤도는 구성성분과 제조방밥에 따라 다양한 형태의 성질을 가진 하이드로젤의 제조가 가능하다.
Hydrogels are materials with a three-dimensional hydrophilic polymer network and may contain a large amount of water. The hydrogel is capable of absorbing at least 20% of the total weight of the water, and absorbing at least 95% of the water is called a highly absorbing hydrogel. The hydrogel is formed of a copolymer or a homopolymer, which is caused by various factors such as covalent bond, hydrogen bond, van der Waals bond, physical agglomeration, and the like as a factor for forming a structurally stable three-dimensional structure with little fluidity due to external history. The hydrogel is thermodynamically stable after swelling in the liquid phase and has an intermediate form of physicochemical properties between liquid and solid. Among these physicochemical properties, the degree of swelling is possible to produce hydrogels having various types of properties depending on the constituent components and the preparation.
*Characterization parameters for hydrogels
* Characterization parameters for hydrogels
약물전달체나 생체조직공학용 지지체로서 하이드로젤의 적용을 위해서는 하이드로젤 내의 네트워크 구조를 이해하고 팽윤상태의 하이드로젤의 구조와 메쉬크기를 적절하게 조절함으로써 적절한 약물방출속도나 원할한 세포의 성장을 이룰 수 있는 하이드로젤의 정확한 설계가 필요하다.
For the application of hydrogel as a carrier for drug delivery or biotissue engineering, understanding the network structure in the hydrogel and appropriately controlling the structure and mesh size of the hydrogel in a swollen state can achieve an appropriate drug release rate and a favorable cell growth The exact design of the hydrogel is needed.
* Schematic of mesh size in hydrogels at swollen or shrunken states.
* Schematic of mesh size in hydrogels at swollen or shrunken states.
2) 꼬시래기로 부터 아가 및 아가로스 제조 방법
2) How to make agar and agarose from the cod
-전처리-
-Pretreatment-
전남 해남에서 채취한 꼬시래기를 90 이상의 끓는 물에 7~8초 정도 삶아서 찬 물에 냉각 과정을 거친 후 협잡물을 제거하기 위해 흐르는 물에 3회 이상 수세를 반복한다. 탈색과 탈취를 위하여 강한 자외선에 투명비닐을 덮어 2~3일 동안 건조시킨다. 건조된 원물을 밀링(milling)과정을 통하여 한천의 수율을 극대화 시킨다.
Boil juice extracted from Haenam, Chonnam Province for more than 90 or more boiling water for 7 ~ 8 seconds, cool it in cold water, and wash it 3 or more times in running water to remove the contaminants. For decolorization and deodorization, cover with strong UV light transparent vinyl and dry for 2 ~ 3 days. Maximizing the yield of the agar by milling the dried raw material.
-한천의 추출-
- Extraction of agar -
한천의 추출의 다양성과 한천의 수율을 극대화시키기 위하여 3가지 방법을 사용하여 추출을 하였다. 첫 번째로 알칼리추출법이다. 건조된 꼬시래기 원물 15g과 6% NaOH에 85에서 2시간 30분 동안 전처리 시킨다. 전처리 후 0.5% Acetic acid를 이용하여 상온에서 1시간 동안 반응 후 수돗물을 이용하여 충분히 수세한다. 증류수를 이용하여 85에서 2시간 30분 동안 가열추출한다. 추출 후 믹서기로 분쇄 후 필터로 필러링 하여 알코올 탈수법으로 탈수 시킨다. 두 번째로 가압추출법이다.To maximize the diversity of agar and the yield of agar, three methods were used for extraction. The first is alkali extraction. Pre-treatment is carried out with 15 g dried cocoa butter and 6% NaOH at 85 for 2 hours and 30 minutes. After pretreatment, react with 0.5% acetic acid at room temperature for 1 hour and wash thoroughly with tap water. Heat with 85% distilled water for 2 hours and 30 minutes. After extraction, it is pulverized with a blender and then pelletized with a filter, and dehydrated by an alcohol dehydration method. The second is pressure extraction.
건조 꼬시래기 15g과 증류수 500mL를 약 1시간 동안 방치 시킨 후 1N NaOH를 이용하여 pH8~10사이로 맞춘 후 30분 동안 교반기를 사용하여 교반시킨다. 교반시킨 용액을 오토클레이에서 121에서 1시간동안 가압가온 추출 한다. 가압가온 추출 후 용액을 0.1N HCl을 이용하여 중화시킨 후 천으로 여과시킨다. 여과시킨 용액을 방치시키면 우무(Agar-Agar)상태가 되는데 영하 20에서 동결시킨 후 해동하여 탈수시켜 믹서기로 갈아준다. 세 번째로 냉수 추출법이다. 냉수추출법(Ice extraction method)은 인도네시아 지역에서 상업적 방법으로 많이 사용되고 있다. 건조된 꼬시래기 원물 약 50g을 증류수 200mL에 약 1시간가량 침지시킨 후 얼음을 원물과 1:1 혼합 후 블랜딩(blending) 시킨다. 블렌딩 후 냉장고 넣어 약 하루간 방치한다. 종이필터로 걸러서 밑으로 빠지는 수용성 물질은 아가로스이고 위에 남는 것은 섬유소와 기타 색소이다. 종이필터로 걸러서 밑으로 빠진 수용성 물질을 동량의 MeOH, EtOH, Isopropanol을 첨가하여 침전 시키고 필터링(filtering) 시켜 필터에 남은 물질을 다시 물에 녹이고 다시 알코올을 부어 2차 침전 시켜 아가로스를 분리정제 시킨다.
15 g of dried cod and 500 ml of distilled water are left for about 1 hour, adjusted to pH 8 to 10 with 1N NaOH, and then stirred for 30 minutes using a stirrer. The stirred solution is heated under pressure at 121 ° C for 1 hour in an autoclave. After heating under pressure, the solution is neutralized with 0.1 N HCl and filtered through a cloth. Agar-Agar state is obtained by letting the filtered solution stand. After freezing at -20 ℃, it is thawed and dehydrated and changed into a blender. The third is cold water extraction. The ice extraction method is widely used in commercial areas in Indonesia. Approximately 50 g of the dried raw material is immersed in 200 mL of distilled water for about 1 hour, and the ice is mixed with the raw material 1: 1 and blended. After blending, leave the refrigerator for about one day. The water-soluble substance that is filtered down by the paper filter is agarose, and the remaining fiber and other pigment are on the top. The water-soluble material that has been filtered by a paper filter is precipitated by addition of the same amount of MeOH, EtOH, and Isopropanol, filtered, and the material remaining in the filter is again dissolved in water. Secondly, alcohol is poured to separate and purify the agarose .
* 가온가압 아가 추출 순서도 *
* Heating pressurized agar extraction flowchart *
-아가로즈(agarose)의 추출-
- Extraction of agarose -
상기 3가지 방법으로 추출된 한천 5g을 100mL DMSO와 함께 실온에서 2시간 교반시킨 다음 원심분리(10,000 g, 30 min)하여 상층액을 4배 부피의 아세톤에 순차적으로 침전시키고, 침전된 아가로스를 아세톤을 이용하여 3회 세척하였다. Five grams of agar extracted by the above three methods were stirred together with 100 mL of DMSO at room temperature for 2 hours, and then centrifuged (10,000 g, 30 min) to sequentially precipitate the supernatant in acetone of 4 times volume and precipitated agarose And washed three times with acetone.
이를 회전 증발 농축기를 사용하여 40 이하 온도에서 감압 건조시킨다. 아세톤에 침전된 아가로스를 100mL 증류수로 12시간 교반하고 교반 후 약 3회 정도 증류수로 세척 후 이를 다시 아세톤 탈수하여 40 이하에서 감압 건조시킨방법과 0.01M의 Na2EDTA용액 100mL로 하룻밤 교반하고 증류수로 3회 세척 후 이를 다시 아세톤으로 탈수하여 40 이하에서 감압 건조하는 방법으로 분류한다.
This is dried under reduced pressure at a temperature of 40 or less using a rotary evaporator. The agarose precipitated in acetone was stirred for 12 hours with 100 mL of distilled water. After stirring, the solution was washed with distilled water three times, dehydrated with acetone, dried under reduced pressure at 40 or less and 100 mL of 0.01 M Na 2 EDTA solution overnight, , Followed by dehydration with acetone, followed by vacuum drying at 40 or less.
* 아가로즈(agarose) 제조 순서도 *
* Agarose manufacturing flow chart *
3) 꼬시래기로 부터 아가 및 아가로즈(agarose) 제조 결과
3) Results of agarose production from fishes and agarose
* 꼬시래기로 부터 제조한 아가로즈 *
* Agarose from crocodile *
앞서 서술한 아가 추출법과 추출된 아가로 부터 아가로즈의 제조법에 따라 꼬시래기로 부터 아가를 추출하여 아가로즈를 제조하였다. The agarose was prepared by extracting agar from the above-described agar extraction method and a method of producing agarose from the extracted agar.
꼬시래기로 부터 제조된 아가로즈는 하이드로겔을 제조하는데 일반적으로 쓰이고 있지만 강도가 약해 취급이 어렵고, 동물 유래 소재인 콜라겐보다 생체 친화성도 높고 강도에서도 큰 차이가 있을 것으로 보인다. 현재 제조된 아가로즈를 이용하여 하이드로겔을 제조하였다.
Although agarose made from cucumber is generally used to produce hydrogels, it is difficult to handle because of its weak strength, and it seems that there is a big difference in strength and biocompatibility compared with animal-derived collagen. The hydrogel was prepared using agarose which is currently produced.
4. 블루베리 착즙액의 세포독성평가 및 항염효능평가4. Evaluation of cytotoxicity and anti-inflammatory activity of blueberry juice
1) 블루베리 착즙액의 세포독성평가
1) Evaluation of cytotoxicity of blueberry juice
(1) 세포배양
(1) Cell culture
세포주는 Dulbecco's Modified Eagle's Medium (DMEM) 배지에 10% Fetal bovine serum (FBS)과 1% streptomycin/penicillin을 첨가하여 37에서 5% CO2 조건 하에서 배양하였다. 기타 세포배양에 관련된 사항은 ATCC animal Cell culture guide를 따라 수행하였다 (http://www.atcc.org/en/Guides/Guides.aspx).
Cell lines were cultured in Dulbecco's Modified Eagle's Medium (DMEM) medium supplemented with 10% Fetal bovine serum (FBS) and 1% streptomycin / penicillin at 37 to 5% CO 2 . Other cell culture related matters were performed according to the ATCC animal cell culture guide (http://www.atcc.org/en/Guides/Guides.aspx).
* 실험에 사용될 세포주* Cell lines to be used for experiments
(2) MTT assay
(2) MTT assay
MTT assay는 Tetrazolium dye 를 이용하여 세포의 NAD(P)H-dependent oxidoreductase enzymes 에 환원되어 색이 변하는 것을 측정하는 방법으로서 세포의 성장 또는 사멸을 측정하는 방법이다. MTT assay is a method for measuring the change in color of NAD (P) H-dependent oxidoreductase enzymes of cells using Tetrazolium dye, which measures the growth or death of cells.
즉, 세포가 살아있으면 염색약 MTT 을 환원시켜 색이 변하게 되지만, 세포가 죽어 있거나 성장을 멈추게 되면 염색약을 환원시키지 않게 된다. 이러한 원리를 이용하여 블루베리 추출물의 독성을 검사할 있으며, 색깔의 변화를 간접적으로 측정하였다.
In other words, if the cells are alive, MTT is reduced to change the color, but if the cells die or stop growing, the dye will not be reduced. Using this principle, we examined the toxicity of blueberry extracts and indirectly measured changes in color.
* 세포독성시험 단계 diagram *
* Cytotoxicity test step diagram *
(3) MTT assay 세포독성실험 결과
(3) MTT assay Cytotoxicity test results
세포주를 96well plate에 각각 1x104 cells/well을 접종하고 24 hr 동안 배양하였다. 농도별로 시료를 처리하고 24 hr 배양 후 배양이 끝난 세포에 MTT stock용액 (5 mg/ml)을 배양배지 부피에 최종적으로 1/10 되게 첨가하여 37에서 4 hr 동안 반응 시켰다. 반응 후 배양 상등액을 제거하고 각각의 well에 DMSO 200 ul를 첨가하여 세포에서 생성된 MTT-formazan 결정체를 용해시킨 후 ELISA reader로 570 nm에서 흡광도를 측정하였다. Cell lines were inoculated into 96-well plates at 1 × 10 4 cells / well and incubated for 24 hrs. The MTT stock solution (5 mg / ml) was added to the culture medium at a final volume of 1/10 and then reacted at 37 for 4 hr. After the reaction, the culture supernatant was removed and 200 μl of DMSO was added to each well to dissolve the MTT-formazan crystals produced in the cells. The absorbance at 570 nm was measured with an ELISA reader.
본 실험에서는 블루베리를 착즙기로 착즙하여 각각 20 , 100 , 250 , 500 1000농도범위에서 세포 독성을 검사하였으며, 실험한 농도 전체에서 세포독성은 나타나지 않았다.
In this experiment, blueberries were juiced and tested for cytotoxicity in the concentration range of 20, 100, 250, and 500, respectively.
* 블루베리 추출물의 MTT assay 결과 *
* MTT assay results of blueberry extract *
* 블루베리 추출물의 MTT assay 세포독성실험 결과 *
* MTT assay cytotoxicity of blueberry extract *
(4) NO assay
(4) NO assay
NO 는 L-arginine 이 L-citrulline 으로 변환하는 과정 중에 형성되는데 여기에는 nitric oxide synthase (이하 NOS)이라는 효소가 관여한다. 현재까지 3종류의 NOS가 밝혀져 있으며, 제 1형 NOS (neu-ronal nitric oxide synthase, nNOS)는 신경세포에서 처음 발견 되었고, 제 2형 NOS (inducible nitric oxide synthase, iNOS)는 큰포식세포에서, 그리고 제 3형인 endothelial nitric oxdide syn-thase (eNOS)는 내피 세포에서 발견 되었다. iNOS 는 정상 성인의 뇌에서는 발현되지 않으나 여러 가지 자극에 의하여 유도될 수 있으며 iNOS가 발현되면 단 시간내에 대량의 NO를 만들어 내는데 이러한 대량의 NO는 자가 산화하여 각종의 질소화합물을 형성하고 유리기의 형태로 조직에 작용하여 강력한 세포독성 작용을 나타낸다. NO is formed during the conversion of L-arginine into L-citrulline, which involves the enzyme nitric oxide synthase (NOS). To date, three types of NOS have been identified. The first type of neuronal nitric oxide synthase (nNOS) has been found in neurons and the second type of inducible nitric oxide synthase (iNOS) And type 3 endothelial nitric oxdide syn-thase (eNOS) was found in endothelial cells. iNOS is not expressed in the normal adult brain but can be induced by various stimuli. When iNOS is expressed, it produces a large amount of NO in a short time. This large amount of NO is autoxidized to form various nitrogen compounds, It acts on tissues and shows strong cytotoxic action.
본 실험에서는 NO 대사과정에서 발생하는 nitrite (NO2 -)를 측정함으로써 NO 의 양을 간접적으로 결정하였다.
In this experiment, the amount of NO was indirectly determined by measuring the nitrite (NO 2 - ) produced in the NO metabolism.
* 피부 염증 발생의 원리 *
* The principle of skin inflammation occurrence *
RAW264.7을 1x104 cells/well을 96well plate에 접종하고 24 hr동안 배양한 시료를 농도별로 처리하고 1 hr 후 LPS 20 ng을 처리하고 18 hr 배양 후 배양이 끝난 세포배양 상등액 50 ul를 취하여 96well plate로 옮긴다. RAW264.7 was inoculated on a 96-well plate at 1 × 10 4 cells / well and cultured for 24 hrs. After 1 hr, 20 ng of LPS was treated and cultured for 18 hr. After culturing, 50 μl of the cell culture supernatant was cultured. plate.
Griess reagent (1% sulfanilamide in 5% phosphoric acid and 0.1% N-(1-Naphthy1) ethylene-diamine dihydrochloride in distilled water) 50 ul를 혼합하고 10 min 동안 반응시킨 후 ELISA reader로 540 nm에서 흡광도를 측정한다. NO의 안정된 최종산물인 nitrite를 NO 생성의 indicator로 사용하여 Green(1982) 등이 제시한 방법에 따라 측정한다.
50 μl of Griess reagent (1% sulfanilamide in 5% phosphoric acid and 0.1% N- (1-Naphthyl) ethylene-diamine dihydrochloride in distilled water) was mixed and reacted for 10 min and absorbance was measured at 540 nm with an ELISA reader . Nitrite, a stable end product of NO, is measured as an indicator of NO production according to the method proposed by Green (1982).
(5) NO assay 항염효능평가 결과
(5) NO assay The anti-inflammatory activity evaluation result
-NO radical assay 측정 결과-
- Results of -NO 2 radical assay -
블루베리 착즙액을 Raw 264. 7 세포주에 처리 하였는데 LPS 200 ng 처리하였을 때, 블루베리 20 g + LPS 200 ng 에서는 Control과 비교하였을 때 NO 생성이 거의 감소하지 않았고 블루베리 500 g + LPS 200 ngsample과 블루베리 1mg + LPS 200 ng에서는 NO 생성이 약간 감소 되었다. When treated with LPS 200 ng, blueberry 20 g + LPS 200 ng showed almost no decrease in NO production compared with Control, and blueberry 500 g + LPS 200 ngsample NO production was slightly reduced at 1 mg of blueberry plus 200 ng of LPS.
* 블루베리 추출물의 NO assay 결과 ** NO assay result of blueberry extract *
* 블루베리 추출물의 NO assay 항염효능평가 결과*
* NO assay of anti-inflammatory activity of blueberry extract *
5. 블루베리 추출물 함유 하이드로겔 보습제 제조5. Preparation of hydrogel moisturizer containing blueberry extract
1) 블루베리 추출물 제조
1) Preparation of blueberry extract
본 실험에 사용한 블루베리는 담양시 소재 블루베리 향토사업단 출하공장에서 구입하였으며, 블루베리 100g을 착즙기(Hurom HE-DBF04)를 사용하여 착즙 후 sample을 12,000 rpm에서 7분간 원심분리하고 상등액을 취하여 filteration(Sartorius 0.45 ul) 하고 동결건조 하였다.
The blueberry used in this experiment was purchased from the shipment factory of Blueberry Domestic Business Unit in Damyang City. 100 g of blueberry was centrifuged at 12,000 rpm for 7 minutes after juicing using a juicer (Hurom HE-DBF04) filteration (Sartorius 0.45 ul) and lyophilized.
2) 아가로즈 제조
2) Manufacture of agarose
Aarose (A6013 sigma USA) 파우더 1g 을 100㎖ 3차 증류수에 넣고 70에서 1시간 동안 교반하여 제조 하였다.
1 g of Aarose (A6013 sigma USA) powder was added to 100 ml of tertiary distilled water and stirred at 70 for 1 hour.
3) 블루베리 화장품 제형제조
3) Manufacture of blueberry cosmetic formulations
화장품 제형은 일반 피부도포용 화장품에 배합되는 보통의 성분을 필요한 만큼 적용 배합하는 것이 가능하다. It is possible to formulate cosmetics by applying the usual ingredients blended with cosmetics for general skin as necessary.
화장품 제형의 유상 성분 함량은 유화 능력의 보강 및 경제성 등을 고려하여 선택되며, 유상 성분으로서 주로 사용될 수 있는 오일로는 식물성 오일, 광물성 오일, 실리콘유 및 합성유 중에서 선택된 하나 이상을 사용할 수 있다. The oil component content of the cosmetic formulation is selected in consideration of the enhancement of emulsifying ability and economical efficiency, and at least one oil selected from vegetable oil, mineral oil, silicone oil and synthetic oil may be used as the oil that can be mainly used as an oil component.
이 외에 유화 능력을 보강하기 위하여 계면활성제, 고급알콜 등을 0.1 내지 5.0 % 첨가할 수 있다. In addition, 0.1 to 5.0% of a surfactant, a higher alcohol and the like may be added to reinforce the emulsifying ability.
이러한 계면 활성제로는 비이온 계면활성제, 인지질 등과 같은 통상적인 계면활성제를 사용할 수 있으며, 고급 알콜으로는 탄소수가 12 내지 20인 알콜을 단독으로 또는 1종 이상 혼합하여 사용할 수 있다. As such surfactants, conventional surfactants such as nonionic surfactants, phospholipids and the like may be used. As the higher alcohols, alcohols having 12 to 20 carbon atoms may be used alone or in admixture of one or more.
또한, 수상 성분은 수상의 점도 또는 경도를 조절하기 위하여 카보머 (carbomer), 잔탄검 (xanthan gum), 벤토나이트 (bentonite) 등과 같은 1종 이상의 점증제를 0.001 내지 5.0 % 더 첨가할 수 있다. The aqueous phase component may further contain 0.001 to 5.0% of at least one thickening agent such as carbomer, xanthan gum, bentonite and the like to control the viscosity or hardness of the aqueous phase.
또한, 필요에 따라 고급 지방산, 비타민 등의 약효 성분과 자외선 차단제, 산화 방지제, 방부제, 향료, 착색제, pH 조절제 등 통상적인 화장품에서 사용되는 성분을 더 첨가할 수 있다. In addition, if necessary, components such as higher fatty acids and vitamins can be further added to components commonly used in cosmetics such as ultraviolet screening agents, antioxidants, preservatives, perfumes, coloring agents and pH adjusting agents.
화장품은 피부를 통해 흡수되는 제품으로, 유연화장수(스킨), 수렴화장수, 영양화장수(로션), 영양크림, 맛사지크림, 에센스, 겔, 팩, 파우더, 피부외용연고, 첩포제, 현탁액, 에멀젼, 스프레이, 또는 미용액 등의 통상의 화장품 형태로 제조될 수 있으며, 이들 각 제형에 적합하고 당업계에 주지된 각종의 통상적인 담체와 첨가제를 포함할 수 있다.
Cosmetics are products that are absorbed through the skin. It is a product that is absorbed through the skin. It is made up of softening longevity (skin), convergent lotion, nutritional lotion (lotion), nutrition cream, massage cream, essence, gel, pack, powder, ointment, Spray, or serum, and may contain various conventional carriers and additives suitable for each of these formulations and known in the art.
위와 같은 기본제형을 항온조에 넣고 70℃가 될 때 까지 1시간 정도 가온한 후 블루베리 추출물과 아가로즈(agarose) 용액을 항온조에서 40℃로 가열하였다.The above basic formulation was placed in a thermostatic chamber and heated for about 1 hour until the temperature reached 70 ° C. The blueberry extract and the agarose solution were heated to 40 ° C. in a thermostatic chamber.
Homogenizer(JSRC-13C)를 가동시켜 3000 rpm 까지 올리고 준비된 기본제형 1 L 와 블루베리 추출물 1%, 아가로즈 0.1%을 넣고 교반하여 블루베리 화장품을 제조하였다. 제조된 블루베리 화장품 제형을 Vaccum Oven(OV-11 Korea)을 사용하여 -0.05 mb에서 화장품 제조 시 발생한 기포를 제거하고 각 용기에 담아 화장품을 완성하였다.
* 화장품 제조의 기본제형 *
* Basic formulation of cosmetics manufacturing *
*블루베리 추출물과 아가로즈를 이용한 화장품 제조*
* Production of cosmetics using blueberry extract and agarose *
4) 블루베리 화장품 시험방법
4) Blueberry cosmetic test method
(1) 피험자
(1) Subjects
피험자들은 조선대학교 화학공학과에 재학중인 20~40 대 사이의 여자 대학원생들로써 일차로 모집된 지원자 16명 중에서 1차로 문진을 하고 본 실험에 적합하다고 판단된 10명을 대상으로 실험군을 최종 선발 하였다.
The subjects were female graduate students between the ages of 20 and 40 attending Chosun University Chemical Engineering Department. Among the 16 candidates who were recruited first, the subjects were interviewed first and the experimental group was finally selected for the 10 students who were judged to be suitable for this experiment.
(2) 평가방법
(2) Evaluation method
피험자 10명을 대상으로 Patch Test를 실시 하였으며, 건선(Psoriasis), 습진(Eczema), 기타 피부병변 보유자나 임신, 수유부 또는 피임제, 항히스차민제 등을 복용하고 있는 사람은 시험에서 제외 하였다. 블루베리 하이드로겔 보습크림을 15 씩 chamber에 적하시킨 뒤, 시험 부위인 상박 부위에 고정하여 첩포하고, 48시간동안 도포하여 관찰하였다. Patch tests were performed on 10 subjects. Psoriasis, eczema, other skin lesions, pregnancy, breastfeeding or contraceptive, and antihistamine were excluded from the study. After 15 drops of blueberry hydrogel moisturizing cream were dripped into the chamber, they were fixed on the upper part of the test area and applied, followed by coating for 48 hours.
판정은 첩포제거 30분, 24시간, 48시간 경과후에 국제접촉피부염연구회(International Contact Dermatitis Research Group:ICDRG)의 판정기준에 따라 자극정도를 분류하였다. 평균 피부반응도는 표? 의 계산공식에 따라 산정하였으며 피부 첩포시험결과 판정표에 따라 자극유무를 판정 하였다. After 30 minutes, 24 hours, and 48 hours after removal of the patch, the test was classified according to the criteria of the International Contact Dermatitis Research Group (ICDRG). The mean skin response was? And the stimulation was judged according to the judgment result of the skin patch test.
* 국제접촉피부염연구회(International Contact Dermatitis Research Group: ICDRG)의 판정기준
* The criteria of the International Contact Dermatitis Research Group (ICDRG)
(3) 피부반응 판정기준
(3) Skin response criteria
Negative(-): 무자극 Negative (-): No stimulation
Doubtful or slight reaction and erythema (±): 미자극 Doubtful or slight reaction and erythema (±):
Erythema+Induration: (+)Erythema + Induration: (+)
Erythema+Induration+Vesicle: (++)Erythema + Induration + Vesicle: (++)
Erythema+Induration+Bullae: (+++)
Erythema + Induration + Bullae: (+++)
(4) 평균 피부반응도 (Mean score) 계산공식
(4) Average skin response (Mean score) calculation formula
* 피부 첩포시험 (Patch test) 결과 판정표 *
* Skin patch test results *
-평가 결과--Evaluation results-
*본 인체시험에 참가한 피험자의 정보와 결과*
* Information and results of the subjects participating in the human body test *
* 피부첩포실험결과 *
* Skin Patch Results *
5) 블루베리 화장품 수분측정시험
5) Blueberry cosmetic moisture test
(1) 피험자
(1) Subjects
피험자들은 조선대학교 화학공학과에 재학 중인 20~40 대 사이의 여자 대학원생들로써 일차로 모집된 지원자 16명 중에서 1차로 문진을 하고 본 실험에 적합하다고 판단된 10명을 대상으로 실험군을 최종 선발 하였다.
The subjects were female graduate students between the ages of 20 and 40 attending Chosun University Chemical Engineering Department. Among the 16 candidates who were recruited first, the subjects were interviewed first and the experimental group was finally selected for the 10 students who were judged to be suitable for this experiment.
(2) 평가방법
(2) Evaluation method
피험자 10명을 대상으로 apply test를 실시하였으며, 건선(Psoriasis), 습진(Eczema), 기타 피부병변 보유자나 임신, 수유부 또는 피임제, 항히스타민제 등을 복용하고 있는 사람은 시험에서 제외 하였다. 블루베리 하이드로겔 보습크림 15 씩을 시험 부위인 얼굴 볼에 바르기 전과 바른 후의 피부를 수분측정기로 촬영하였다.
The test was applied to 10 subjects. Psoriasis, eczema, other skin lesions, pregnant, lactating or contraceptive, and antihistamines were excluded from the study. The skin before and after applying the blueberry hydrogel moisturizing cream 15 to the face ball of the test area was photographed with a moisture meter.
(3) 수분측정시험 결과
(3) Moisture measurement test result
전체적으로 20대, 30대 남성과 20대, 30대, 40대 여성 모두 건조한 피부의 상태가 호전됨을 알 수 있었다. 한편 15번 피험자의 경우 약한 붉은 반점이 나타났고, 약간의 가려움증이 유발되었다. 이것은 블루베리 용매추출액을 사용한 경우 나타난 현상으로서 블루베리 착즙액을 사용한 시료에서는 부작용이 발생하지 않았다. 전반적으로 블루베리 추출물 함유 하이드로겔 보습제는 수분의 흡수를 도울 뿐만 아니라 증발을 막아주는 효과 또한 있는 것으로 판단되어 추후 제품생산을 위한 기초자료로 활용이 가능하다.
As a whole, it was found that dry skin condition improved in both men in their twenties, men in their 30s, and women in their twenties, thirties and forties. On the other hand, 15 subjects showed weak red spots and slight itching. This was a phenomenon that appeared when the blueberry solvent extract was used, and no side effects were observed in the sample using the blueberry juice. Overall, the hydrogel moisturizing agent containing the blueberry extract is thought to be effective not only to absorb moisture but also to prevent evaporation, so that it can be used as basic data for the production of the product in the future.
5) 블루베리 화장품 수분측정시험
5) Blueberry cosmetic moisture test
(1) 피험자
(1) Subjects
피험자들은 조선대학교 화학공학과에 재학 중인 20~40 대 사이의 여자 대학원생들로써 일차로 모집된 지원자 16명 중에서 1차로 문진을 하고 본 실험에 적합하다고 판단된 10명을 대상으로 실험군을 최종 선발 하였다.
The subjects were female graduate students between the ages of 20 and 40 attending Chosun University Chemical Engineering Department. Among the 16 candidates who were recruited first, the subjects were interviewed first and the experimental group was finally selected for the 10 students who were judged to be suitable for this experiment.
(2) 평가방법
(2) Evaluation method
피험자 10명을 대상으로 apply test를 실시하였으며, 건선(Psoriasis), 습진(Eczema), 기타 피부병변 보유자나 임신, 수유부 또는 피임제, 항히스타민제 등을 복용하고 있는 사람은 시험에서 제외 하였다. 블루베리 하이드로겔 보습크림 15 씩을 시험 부위인 얼굴 볼에 바르기 전과 바른 후의 피부를 수분측정기로 촬영하였다.
The test was applied to 10 subjects. Psoriasis, eczema, other skin lesions, pregnant, lactating or contraceptive, and antihistamines were excluded from the study. The skin before and after applying the blueberry hydrogel moisturizing cream 15 to the face ball of the test area was photographed with a moisture meter.
(3) 수분측정시험 결과 (3) Moisture measurement test result
*수분측정시험 test 전과 test 후의 결과
* Moisture measurement test Results before and after test
전체적으로 20대, 30대 남성과 20대, 30대, 40대 여성 모두 건조한 피부의 상태가 호전됨을 알 수 있었다. 한편 15번 피험자의 경우 약한 붉은 반점이 나타났고, 약간의 가려움증이 유발되었다. 이것은 블루베리 용매추출액을 사용한 경우 나타난 현상으로서 블루베리 착즙액을 사용한 시료에서는 부작용이 발생하지 않았다. 전반적으로 블루베리 추출물 함유 하이드로겔 보습제는 수분의 흡수를 도울 뿐만 아니라 증발을 막아주는 효과 또한 있는 것으로 판단되어 추후 제품생산을 위한 기초자료로 활용이 가능하다. As a whole, it was found that dry skin condition improved in both men in their twenties, men in their 30s, and women in their twenties, thirties and forties. On the other hand, 15 subjects showed weak red spots and slight itching. This was a phenomenon that appeared when the blueberry solvent extract was used, and no side effects were observed in the sample using the blueberry juice. Overall, the hydrogel moisturizing agent containing the blueberry extract is thought to be effective not only to absorb moisture but also to prevent evaporation, so that it can be used as basic data for the production of the product in the future.
Claims (9)
상기 유상 성분은 식물성 오일, 광물성 오일, 실리콘유 및 합성유 중에서 선택된 하나 이상을 사용하는 것을 특징으로 하는 하이드로젤에 포집된 블루베리 추출물 함유 화장품.The method according to claim 1,
Wherein the oil component comprises at least one selected from vegetable oil, mineral oil, silicone oil and synthetic oil.
상기 수상 성분은 수상의 점도 또는 경도를 조절하기 위하여 카보머 (carbomer), 잔탄검 (xanthan gum), 벤토나이트 (bentonite) 등과 같은 1종 이상의 점증제를 0.001 ~ 5.0 중량부 더 첨가하는 것을 특징으로 하는 하이드로젤에 포집된 블루베리 추출물 함유 화장품.The method according to claim 1,
The aqueous phase component is further characterized by adding 0.001 to 5.0 parts by weight of at least one thickening agent such as carbomer, xanthan gum, bentonite and the like in order to control the viscosity or hardness of the aqueous phase Containing blueberry extract in a hydrogel.
꼬시래기로부터 얻은 아가로즈(agarose) 파우더 1g 을 100㎖의 3차 증류수에 넣고 70℃에서 1시간 동안 교반하여 0.1%의 아가로즈를 수득하는 단계;
물, 1,3- 부틸렌글리콜, 1,2-헥산디올의 혼합물 1ℓ을 항온조에 넣고 70℃가 될 때 까지 1시간 정도 가온하여 기본제형을 수득하는 단계;
상기 블루베리 추출물과 아가로스 용액을 항온조에서 40℃로 가열하는 단계;
상기 기존제형과 블루베리 추출물, 아가로즈를 교반하여 블루베리 화장품을 제조하는 단계;
를 포함하여 이루어지는 것을 특징으로 하는 하이드로젤에 포집된 블루베리 추출물 함유 화장품의 제조방법.100 g of blueberry was centrifuged at 12,000 rpm for 7 minutes using a juicer, and the supernatant was filtered and lyophilized to prepare 1% of blueberry extract.
1 g of agarose powder obtained from a cicada was added to 100 ml of tertiary distilled water and stirred at 70 ° C for 1 hour to obtain 0.1% of agarose;
1 liter of a mixture of water, 1,3-butylene glycol and 1,2-hexanediol is placed in a thermostatic chamber and heated to 70 DEG C for about 1 hour to obtain a basic formulation;
Heating the blueberry extract and the agarose solution to 40 DEG C in a thermostat;
Preparing blueberry cosmetics by stirring the conventional formulation, blueberry extract, and agarose;
And extracting the extract from the hydrogel.
상기 아가로즈는,
건조된 꼬시래기 원물 15g과 6% NaOH에 85℃에서 2시간 30분 동안 전처리하고, 전처리 후 0.5% Acetic acid를 이용하여 상온에서 1시간 동안 반응 후 수돗물을 이용하여 충분히 수세한 다음 증류수를 이용하여 85℃에서 2시간 30분 동안 가열추출한 후 추출한 다음 믹서기로 분쇄 후 필터로 필러링 하여 알코올 탈수법으로 탈수시켜 수득하는 것을 특징으로 하는 하이드로젤에 포집된 블루베리 추출물 함유 화장품의 제조방법.5. The method of claim 4,
The agarose,
After the pretreatment, the cells were treated with 0.5% acetic acid at room temperature for 1 hour, washed thoroughly with tap water, and then diluted with distilled water to obtain 85 Deg.] C for 2 hours and 30 minutes, extracting the mixture, pulverizing the mixture with a blender, filtering with a filter, and dewatering by an alcohol dehydration method to obtain a cosmetic containing the extract of blueberry.
상기 아가로즈는,
건조 꼬시래기 15g과 증류수 500mL를 약 1시간 동안 방치 시킨 후 1N NaOH를 이용하여 pH8~10사이로 맞춘 다음 30분 동안 교반기를 사용하여 교반하고, 교반시킨 용액을 오토클레이에서 121℃에서 1시간동안 가압가온 추출하며, 가압가온 추출 후 용액을 0.1N HCl을 이용하여 중화시킨 다음 천으로 여과시키고, 여과시킨 용액을 방치시켜 우무(Agar-Agar)상태에서 -20℃에서 동결시킨 후 해동하여 탈수시켜 믹서기로 갈아 수득하는 것을 특징으로 하는 하이드로젤에 포집된 블루베리 추출물 함유 화장품의 제조방법.5. The method of claim 4,
The agarose,
15 g of dried cod and 500 mL of distilled water were left for about 1 hour, adjusted to pH 8 to 10 with 1N NaOH, stirred for 30 minutes using a stirrer, and the stirred solution was heated in autoclave at 121 ° C for 1 hour The solution was neutralized with 0.1 N HCl, filtered through a cloth, left to stand at -20 ° C in an agar-agar state, thawed, and dehydrated by a mixer Wherein the hydrogel-containing cosmetic composition is obtained by grinding.
상기 아가로즈는,
건조된 꼬시래기 원물 약 50g을 증류수 200mL에 약 1시간가량 침지시킨 후 얼음을 원물과 1:1 혼합 후 블랜딩(blending) 시킨다. 블렌딩 후 냉장고 넣어 약 하루간 방치한 후 종이필터로 걸러서 밑으로 빠진 수용성 물질을 동량의 MeOH, EtOH, Isopropanol을 첨가하여 침전시키고 필터링(filtering) 시켜 필터에 남은 물질을 다시 물에 녹이고 다시 알코올을 부어 2차 침전 시켜 분리정제 시켜 수득하는 것을 특징으로 하는 하이드로젤에 포집된 블루베리 추출물 함유 화장품의 제조방법.5. The method of claim 4,
The agarose,
Approximately 50 g of the dried raw material is immersed in 200 mL of distilled water for about 1 hour, and the ice is mixed with the raw material 1: 1 and blended. After blending, put in the refrigerator for about a day, filter with paper filter, add the same amount of MeOH, EtOH and Isopropanol to precipitate the remaining water-soluble material, filter it by filtration, dissolve the remaining material in water and pour alcohol again. And then separating and purifying it by secondary precipitation to obtain a blueberry extract-containing cosmetic product collected on a hydrogel.
상기 아가로즈는 한천 5g을 100mL DMSO와 함께 실온에서 2시간 교반시킨 다음 원심분리(10,000 X g, 30 min)하여 상층액을 4배 부피의 아세톤에 순차적으로 침전시키고, 침전된 아가로즈(agarose)를 아세톤을 이용하여 3회 세척하며, 이를 회전 증발 농축기를 사용하여 40 이하 온도에서 감압 건조킨 후 아세톤에 침전된 아가로스를 100mL 증류수로 12시간 교반하고 교반 후 약 3회 정도 증류수로 세척 후 이를 다시 아세톤 탈수하여 40 이하에서 감압 건조시켜 아가로즈(agarose)를 수득하는 하는 것을 특징으로 하는 하이드로젤에 포집된 블루베리 추출물 함유 화장품의 제조방법.8. The method according to any one of claims 5 to 7,
Agarose was prepared by agarizing 5 g of agar with 100 mL of DMSO at room temperature for 2 hours and then centrifuging (10,000 X g, 30 min) to sequentially precipitate the supernatant in acetone of 4 times volume, Was washed three times with acetone, and then dried under reduced pressure at a temperature of 40 or less using a rotary evaporator. Agarose precipitated in acetone was stirred for 12 hours with 100 mL of distilled water, washed with distilled water about 3 times after stirring, Dehydrating again with acetone and drying under reduced pressure at 40 or less to obtain an agarose. The method for producing a cosmetic comprising the extract of blueberry extract in a hydrogel.
상기 아가로즈는 한천 5g을 100mL DMSO와 함께 실온에서 2시간 교반시킨 다음 원심분리(10,000 X g, 30 min)하여 상층액을 4배 부피의 아세톤에 순차적으로 침전시키고, 침전된 아가로스를 아세톤을 이용하여 3회 세척하며, 이를 회전 증발 농축기를 사용하여 40℃ 이하 온도에서 감압 건조킨 후 아세톤에 침전된 아가로즈(agarose)를 0.01M의 Na2EDTA용액 100mL로 하룻밤 교반하고 증류수로 3회 세척 후 이를 다시 아세톤으로 탈수하여 40℃ 이하에서 감압 건조시켜 아가로즈(agarose)를 수득하는 하는 것을 특징으로 하는 하이드로젤에 포집된 블루베리 추출물 함유 화장품의 제조방법.8. The method according to any one of claims 5 to 7,
Agarose was prepared by stirring 5 g of agar in 100 mL of DMSO at room temperature for 2 hours, and then centrifuging (10,000 X g, 30 min) to sequentially precipitate the supernatant in acetone of 4 times volume. The precipitated agarose was dissolved in acetone , And the precipitate was dried under reduced pressure at a temperature of 40 ° C or lower using a rotary evaporator. The agarose precipitated in acetone was stirred with 100 ml of 0.01 M Na 2 EDTA solution overnight, washed three times with distilled water And then dewatered again with acetone, followed by drying under reduced pressure at 40 ° C or lower to obtain agarose. The method for producing cosmetics containing blueberry extract according to claim 1,
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| KR100659138B1 (en) | 2005-09-16 | 2006-12-19 | (주)아모레퍼시픽 | Moisturizing skin external preparation composition containing gallocatechin gallate as an active ingredient |
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| KR100659138B1 (en) | 2005-09-16 | 2006-12-19 | (주)아모레퍼시픽 | Moisturizing skin external preparation composition containing gallocatechin gallate as an active ingredient |
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