KR102393417B1 - Antidiabetic composition comprising Hemp stem extract - Google Patents
Antidiabetic composition comprising Hemp stem extract Download PDFInfo
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- KR102393417B1 KR102393417B1 KR1020200067572A KR20200067572A KR102393417B1 KR 102393417 B1 KR102393417 B1 KR 102393417B1 KR 1020200067572 A KR1020200067572 A KR 1020200067572A KR 20200067572 A KR20200067572 A KR 20200067572A KR 102393417 B1 KR102393417 B1 KR 102393417B1
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- South Korea
- Prior art keywords
- extract
- hemp
- hemp stem
- stem extract
- composition
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Abstract
본 발명은 대마 줄기 추출물로 이루어지거나 대마 줄기 추출물을 유효성분으로 포함하는 조성물을 제공한다. 본 발명에 따른 조성물의 유효성분인 대마 줄기 추출물은 대마 종자 추출물 또는 상업적인 경구용 혈당강하제인 아카보스(Acarbose)에 비해 현저하게 우수한 α-glucosidase 효소 저해활성을 나타내므로 항당뇨 또는 혈당강화 소재로 사용될 수 있다. 따라서, 본 발명에 따른 조성물은 당뇨병, 특히 제2형 당뇨병을 예방, 개선 또는 치료하기 위한 용도의 의약품, 건강기능식품으로 구현될 수 있다.The present invention provides a composition comprising a hemp stem extract or comprising a hemp stem extract as an active ingredient. The hemp stem extract, which is an active ingredient of the composition according to the present invention, exhibits significantly superior α-glucosidase enzyme inhibitory activity compared to hemp seed extract or a commercial oral hypoglycemic agent, Acarbose, so it can be used as an antidiabetic or blood sugar enhancing material. there is. Therefore, the composition according to the present invention can be implemented as a pharmaceutical or health functional food for preventing, improving or treating diabetes, particularly type 2 diabetes.
Description
본 발명은 항당뇨용 조성물 등에 관한 것으로서, 더 상세하게는 α-글루코시다제(α-glucosidase) 효소 활성을 저해할 수 있는 천연물 기반의 유효성분을 포함하는 항당뇨용 조성물 및 상기 유효성분이 적용된 의약품, 건강기능식품 등에 관한 것이다.The present invention relates to an antidiabetic composition and the like, and more particularly, to an antidiabetic composition comprising a natural product-based active ingredient capable of inhibiting α-glucosidase enzyme activity, and a drug to which the active ingredient is applied. , health functional food, etc.
대마(Cannabis sativa L.)는 삼과(Cannabinaceae)에 속하는 한해살이 식물로 중앙아시아가 원산지이고, 온화하고 습한 기후에서 잘 자라며, 국내에는 기원전 1세기 무렵부터 재배되었다고 알려져 있다. 대마의 섬유질은 옷감, 실 등을 만드는데 이용되며, 대마씨는 오일의 원료로 사용된다. 대마의 환각 성분은 주로 잎과 줄기에 함유되어 있으며, 씨에는 환각 성분이 소량 함유되어 있으나 껍질을 제거하면 건강식품으로 활용이 가능하다. 이러한 대마씨의 효능으로는 항산화, 면역력 강화, 동맥경화 예방, 정력 향상, 고혈압 개선, 변비 예방, 다이어트, 피부 미용, 고지혈증 예방, 그리고 항염 및 항암작용 등이 있다고 보고되고 있다[Smeriglio, A., Galati, E. M., Monforte, M. T., Lanuzza, F., D'Angelo, V. and Circosta, C. 2016. Polyphenolic compounds and antioxidant activity of cold-pressed seed oil from finola cultivar of Cannabis sativa L. Phytother. Res. 30, 1298-307.]. 또한, 대마씨에는 칼슘, 감마리놀렌산(Gamma Linolenic acid)이 함유되어 있어 슈퍼 푸드(Superfood)로 인식되고 있다. 잣과 비슷한 형태를 가지는 대마씨(Cannabis sativa L. seed; HS)는 다량의 필수 아미노산과 불포화 지방산과 탄수화물 함량이 적고 체내에서 생성되지 않는 비타민 B1, 2, 6, 그리고 식이 섬유가 다량 함유되어 있다고 알려져 있다. 대마씨의 함유 물질은 테트라하이드로칸나비놀(Tetrahydrocanabinol; THC)과 칸나비디올(Cannabidiol;CBD)이 대표적이다. THC는 환각 성분으로 알려져 있으며, CBD는 THC의 환각 효과를 억제한다고 보고 되고 있으며, 대마씨에는 THC 뿐만 아니라, 이를 억제하는 CBD 성분도 다량 함유되어 있다. 최근 보고에 따르면 THC의 경우 신경 보호 활성, 뇌 성장인자 유전자 발현 조절 등이 있으며, CBD는 항경련 효과, 알츠하이머, 항간질 등이 알려져 있으며 이에 대한 다양한 연구가 진행되고 있다.Hemp ( Cannabis sativa L.) is an annual plant belonging to the Cannabinaceae family and is native to Central Asia, grows well in a mild and humid climate, and is known to have been cultivated in Korea since the 1st century BC. The fibers of hemp are used to make fabrics and threads, and hemp seeds are used as raw materials for oil. The hallucinogenic components of hemp are mainly contained in leaves and stems, and the seeds contain a small amount of hallucinogenic components, but if the skin is removed, it can be used as a health food. It has been reported that the effects of hemp seeds include antioxidant, immunity enhancement, arteriosclerosis prevention, stamina improvement, hypertension improvement, constipation prevention, diet, skin beauty, hyperlipidemia prevention, and anti-inflammatory and anticancer action [Smeriglio, A., Galati, EM, Monforte, MT, Lanuzza, F., D'Angelo, V. and Circosta, C. 2016. Polyphenolic compounds and antioxidant activity of cold-pressed seed oil from finola cultivar of Cannabis sativa L. Phytother. Res. 30, 1298-307.]. In addition, hemp seeds contain calcium and gamma linolenic acid, which is recognized as a superfood. Cannabis sativa L. seed (HS), which has a shape similar to that of pine nuts, contains a large amount of essential amino acids, unsaturated fatty acids and carbohydrates, and contains a large amount of vitamins B1, 2, 6, and dietary fiber that are not produced by the body. is known Hemp seeds contain tetrahydrocannabinol (THC) and cannabidiol (CBD). THC is known as a hallucinogenic component, and CBD is reported to inhibit the hallucinogenic effect of THC. According to a recent report, THC has neuroprotective activity and regulation of brain growth factor gene expression.
예를 들어, 김동희 등[김동희, 성재신, 정태을, 이규재. 2015. 대마씨 추출물이 당뇨 및 지질개선에 미치는 영향. 한국물학회, 4권, 2호, 1-6.]에는 대마씨 추출물의 혈당 감소 효과가 개시되어 있다. 또한, 윤여총 등[윤여초, 김병혁, 김중규, 이준형, 박예은, 권기석, 황학수, 이중복. 2018. 대마씨 발효 추출물의 생리 활성 및 미백 활성 검증. Journal of Life Science, 28권, 6호, 688-696.]에는 유산균으로 발효한 대마씨 추출물의 항산화, α-glucosidase 저해 활성 및 tyrosinase 저해 활성이 발효하지 않은 대마씨 추출물에 비해 현저히 증가된다는 내용이 개시되어 있다.For example, Kim Dong-hee et al. [Kim Dong-hee, Sung Jae-shin, Jeong Tae-eul, Lee Kyu-jae. 2015. Effect of Hemp Seed Extract on Diabetes and Lipid Improvement. The Korean Water Society, Vol. 4, No. 2, 1-6.] discloses the blood sugar reducing effect of hemp seed extract. In addition, Yeo Chong Yoon, et al. [Yeocho Yoon, Byeong-hyeok Kim, Jung-gyu Kim, Jun-hyung Lee, Ye-eun Park, Ki-seok Kwon, Hak-su Hwang, and Lee Jung-bok. 2018. Verification of physiological and whitening activity of fermented hemp seed extract. Journal of Life Science, Vol. 28, No. 6, 688-696.] reported that the antioxidant, α-glucosidase inhibitory activity and tyrosinase inhibitory activity of hemp seed extract fermented with lactic acid bacteria were significantly increased compared to that of unfermented hemp seed extract. has been disclosed.
그러나 대마씨를 이용한 다양한 연구와 제품들이 개발되어 있지만, 현재 대마 뿌리나 대마 줄기의 생리활성 및 효능 증대에 관한 기술적 연구는 거의 이루어지지 않고 있으며, 기능성 식품에 관한 제품화 개발 또한 미미한 실정이다. 또한, 해외에서는 점차 대마의 뛰어난 약리작용에 대한 중요성이 인식되고 대마의 치료적 목적의 사용을 허용하자는 움직임이 늘고 있는 반면, 국내에서는 대마에 대한 규제가 엄격하여 대마에 대한 연구가 부족한 실정이고, 특히 의약품용 또는 건강기능식품용 대마에 대한 효능 검정 등에 관한 연구가 매우 미비한 실정이다.However, although various researches and products using hemp seeds have been developed, technical research on increasing the physiological activity and efficacy of hemp roots or hemp stems has not been conducted at present, and development of commercialization of functional foods is also insignificant. In addition, while overseas, the importance of the excellent pharmacological action of cannabis is gradually recognized, and the movement to allow the use of cannabis for therapeutic purposes is increasing, whereas in Korea, there is a lack of research on cannabis due to strict regulations on cannabis, In particular, research on efficacy testing of cannabis for pharmaceuticals or health functional foods is very scarce.
본 발명은 종래의 기술적 배경하에서 도출된 것으로서, 본 발명의 목적은 대마 줄기 추출물의 다양한 생리활성에 기초한 의약품 용도 또는 건강기능식품 용도를 제공하는데에 있다.The present invention has been derived from the prior technical background, and an object of the present invention is to provide a pharmaceutical use or a health functional food use based on various physiological activities of a hemp stem extract.
본 발명의 발명자들은 합성 화학물질에 비해 안전성이 확보된 수많은 천연물을 대상으로 생리활성이 우수한 추출물을 개발하기 위하여 연구를 수행하였다. 그 결과, 대마 줄기 추출물이 대마 종자 추출물 또는 상업적인 경구용 혈당강하제인 아카보스(Acarbose)에 비해 현저하게 우수한 α-glucosidase 효소 저해활성을 가지는 점을 확인하고 본 발명을 완성하였다.The inventors of the present invention conducted research to develop extracts with excellent physiological activity for numerous natural products that have secured safety compared to synthetic chemicals. As a result, it was confirmed that the hemp stem extract had significantly superior α-glucosidase enzyme inhibitory activity compared to the hemp seed extract or a commercial oral hypoglycemic agent, Acarbose, and completed the present invention.
상기 목적을 달성하기 위하여, 본 발명의 일 예는 대마 줄기 추출물로 이루어지거나 대마 줄기 추출물을 유효성분으로 포함하는 항당뇨 또는 혈당강하용 조성물을 제공한다.In order to achieve the above object, an example of the present invention provides a composition for antidiabetic or blood sugar lowering consisting of or comprising a hemp stem extract as an active ingredient.
상기 목적을 달성하기 위하여, 본 발명의 일 예는 대마 줄기 추출물로 이루어지거나 대마 줄기 추출물을 유효성분으로 포함하는 당뇨병 예방, 개선 또는 치료용 조성물을 제공한다.In order to achieve the above object, an example of the present invention provides a composition for preventing, improving or treating diabetes comprising a hemp stem extract or comprising a hemp stem extract as an active ingredient.
상기 조성물은 바람직하게는 약학 조성물 또는 식품 조성물의 형태를 가진다.The composition is preferably in the form of a pharmaceutical composition or a food composition.
본 발명에 따른 조성물의 유효성분인 대마 줄기 추출물은 대마 종자 추출물 또는 상업적인 경구용 혈당강하제인 아카보스(Acarbose)에 비해 현저하게 우수한 α-glucosidase 효소 저해활성을 나타내므로 항당뇨 또는 혈당강화 소재로 사용될 수 있다. 따라서, 본 발명에 따른 조성물은 당뇨병, 특히 제2형 당뇨병을 예방, 개선 또는 치료하기 위한 용도의 의약품, 건강기능식품으로 구현될 수 있다.The hemp stem extract, which is an active ingredient of the composition according to the present invention, exhibits significantly superior α-glucosidase enzyme inhibitory activity compared to hemp seed extract or a commercial oral hypoglycemic agent, Acarbose, so it can be used as an antidiabetic or blood sugar enhancing material. there is. Therefore, the composition according to the present invention can be implemented as a pharmaceutical or health functional food for preventing, improving or treating diabetes, particularly type 2 diabetes.
도 1은 대마 추출물의 DPPH 라디칼 소거능 측정 결과를 나타낸 그래프이다.
도 2는 대마 추출물의 총 폴리페놀 함량 측정 결과를 나타낸 그래프이다.
도 3은 대마 추출물의 총 플라보노이드 함량 측정 결과를 나타낸 그래프이다.
도 4는 대마 추출물의 SOD 유사활성 측정 결과를 나타낸 그래프이다.
도 5는 대마 추출물의 ABTS+ 라디칼 소거능 측정 결과를 나타낸 그래프이다.
도 6은 대마 추출물의 3T3-L1 Cell에 대한 세포 독성 평가(MTT assay) 결과를 나타낸 그래프이다.
도 7은 대마 추출물의 3T3-L1 Cell에 대한 지방세포로의 분화 억제능 측정 결과를 나타낸 그래프이다.
도 8은 대마 추출물의 Tyrosinase 효소 저해활성 측정 결과를 나타낸 그래프이다.
도 9는 대마 추출물의 B16F10 Cell에 대한 세포 독성 평가(MTT assay) 결과를 나타낸 그래프이다.
도 10은 대마 추출물의 B16F10 Cell에 대한 멜라닌 생성 저해활성 평가 결과를 나타낸 그래프이다.
도 11은 대마 추출물의 RAW 264.7 Cell에 대한 NO 생성 저해활성 평가(NO assay) 결과를 나타낸 그래프이다.
도 12는 대마 추출물의 α-glucosidase 효소 저해활성(α-glucosidase enzyme inhibition activity) 측정 결과를 나타낸 그래프이다.1 is a graph showing the measurement results of DPPH radical scavenging ability of cannabis extract.
Figure 2 is a graph showing the measurement result of the total polyphenol content of the hemp extract.
3 is a graph showing the measurement result of the total flavonoid content of the hemp extract.
4 is a graph showing the measurement results of SOD-like activity of the hemp extract.
5 is a graph showing the measurement result of ABTS + radical scavenging ability of cannabis extract.
6 is a graph showing the results of cytotoxicity evaluation (MTT assay) for 3T3-L1 cells of cannabis extract.
7 is a graph showing the measurement results of the inhibition of differentiation into adipocytes for 3T3-L1 cells of cannabis extract.
8 is a graph showing the results of measuring the tyrosinase enzyme inhibitory activity of the hemp extract.
9 is a graph showing the results of cytotoxicity evaluation (MTT assay) for B16F10 cells of cannabis extract.
10 is a graph showing the evaluation results of melanin production inhibitory activity against B16F10 Cells of cannabis extract.
11 is a graph showing the results of evaluation of NO production inhibitory activity (NO assay) for RAW 264.7 Cells of cannabis extract.
12 is a graph showing the measurement results of α-glucosidase enzyme inhibition activity of the cannabis extract.
이하, 본 발명에서 사용한 용어를 설명한다.Hereinafter, the terms used in the present invention will be described.
본 발명에서 사용되는 용어 "조성물"은 2가지 이상의 성분이 균일하게 혼합되어 있는 상태의 물질을 의미하며, 완제품뿐만 아니라 완제품 제조를 위한 중간 소재를 포함하는 개념이다.The term "composition" used in the present invention refers to a material in which two or more components are uniformly mixed, and is a concept including intermediate materials for manufacturing finished products as well as finished products.
본 발명에서 사용되는 용어 "약학적으로 허용가능한" 및 "식품학적으로 허용가능한"이란 생물체를 상당히 자극하지 않고 투여 활성 물질의 생물학적 활성 및 특성을 저해하지 않는 것을 의미한다.As used herein, the terms "pharmaceutically acceptable" and "food acceptable" mean not significantly stimulating the organism and not inhibiting the biological activity and properties of the administered active substance.
본 발명에서 사용되는 용어 "예방"은 본 발명의 조성물의 투여로 특정 질환의 증상을 억제하거나 진행을 지연시키는 모든 행위를 의미한다.As used herein, the term “prevention” refers to any action that suppresses the symptoms of a specific disease or delays the progression by administration of the composition of the present invention.
본 발명에서 사용되는 용어 "치료"는 본 발명의 조성물의 투여로 특정 질환의 증상을 호전 또는 이롭게 변경시키는 모든 행위를 의미한다.As used herein, the term “treatment” refers to any action that improves or beneficially changes the symptoms of a specific disease by administration of the composition of the present invention.
본 발명에서 사용되는 용어 "개선"은 치료되는 상태와 관련된 파라미터, 예를 들면 증상의 정도를 적어도 감소 또는 완화시키는 모든 행위를 의미한다.As used herein, the term “improvement” refers to any action that at least reduces or alleviates a parameter related to the condition being treated, for example, the severity of symptoms.
본 발명에서 사용되는 용어 "투여"는 임의의 적절한 방법으로 개체에 소정의 본 발명의 조성물을 제공하는 것을 의미한다. 이때, 개체는 본 발명의 조성물을 투여하여 특정 질환의 증상이 호전될 수 있는 질환을 가진 인간, 원숭이, 개, 염소, 돼지 또는 쥐 등 모든 동물을 의미한다.As used herein, the term “administration” means providing a given composition of the present invention to a subject by any suitable method. In this case, the subject refers to all animals, such as humans, monkeys, dogs, goats, pigs, or mice, having a disease in which the symptoms of a specific disease can be improved by administering the composition of the present invention.
본 발명에서 용어 "약학적으로 유효한 양"은 의학적 치료에 적용 가능한 합리적인 수혜 또는 위험 비율로 질환을 치료하기에 충분한 양을 의미하며, 이는 개체의 질환의 종류, 중증도, 약물의 활성, 약물에 대한 민감도, 투여 시간, 투여 경로 및 배출비율, 치료기간, 동시에 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다.In the present invention, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit or risk ratio applicable to medical treatment, which is the type, severity, activity of the drug, and the drug. Sensitivity, administration time, administration route and excretion rate, duration of treatment, factors including concurrent drugs and other factors well known in the medical field may be determined.
이하, 본 발명을 상세하게 설명한다.Hereinafter, the present invention will be described in detail.
본 발명은 대마 줄기 추출물로 이루어지거나 대마 줄기 추출물을 유효성분으로 포함하는 조성물을 제공한다. 본 발명의 조성물은 항당뇨; 혈당강하; 당뇨병 예방, 개선 또는 치료 등과 같이 다양한 의약품 용도 또는 건강기능식품 용도로 사용될 수 있다.The present invention provides a composition comprising a hemp stem extract or comprising a hemp stem extract as an active ingredient. The composition of the present invention is antidiabetic; lowering blood sugar; It can be used for various pharmaceutical uses or health functional foods, such as preventing, improving or treating diabetes.
본 발명에 따른 조성물의 유효성분인 대마 줄기 추출물은 다양한 방법으로 제조될 수 있다. 구체적으로, 상기 대마 줄기 추출물은 당업계에 공지된 통상의 추출 방법, 예를 들어 용매 추출법을 사용하여 제조될 수 있다. 용매 추출법에 이용되는 추출 용매는 물, 탄소 수가 1 내지 4인 저급 알코올(예를 들면, 메탄올, 에탄올, 프로판올 및 부탄올) 또는 이들의 혼합물인 함수 저급 알코올, 프로필렌글리콜, 1,3-부틸렌글리콜, 글리세린, 아세톤, 디에틸에테르, 에틸 아세테이트, 부틸아세테이트, 디클로로메탄, 클로로포름, 헥산 및 이들의 혼합물로 구성된 군으로부터 선택될 수 있고, 이중 물, 알코올 또는 이들 중 2종 이상의 혼합 용매에서 선택되는 것이 바람직하고, 함수 알코올인 것이 더 바람직하다. 추출 용매로 물을 사용하는 경우 물은 열수인 것이 바람직하다. 또한, 추출 용매로 알코올을 사용하는 경우 알코올은 탄소 수가 1 내지 4인 저급 알코올인 것이 바람직하고, 저급 알코올은 메탄올 또는 에탄올에서 선택되는 것이 더 바람직하다. 또한, 추출 용매로 함수 알코올을 사용하는 경우 알코올 함량은 55~95%(w/w)인 것이 바람직하고, 60~85%(w/w)인 것이 더 바람직하다. 추출 용매로 함수 에탄올을 사용하는 경우 대마 줄기 추출물은 1) 대마 줄기를 에탄올 농도가 55~95%(w/w)인 함수 에탄올로 추출하여 추출액을 얻는 단계; 2) 상기 추출액을 여과하여 잔사를 제거하고 여과된 추출액을 얻는 단계; 및 3) 상기 여과된 추출액을 농축하고 건조하여 고상의 추출물을 얻는 단계를 통해 얻어질 수 있다. 또한, 상기 1) 추출액을 얻는 단계에서 추출 용매인 함수 에탄올의 사용량은 대마 뿌리 건조 중량 100g을 기준으로 0.25~1 L인 것이 바람직하고, 함수 에탄올의 에탄올 농도는 60~85%(w/w)인 것이 바람직하고, 추출 온도는 45~75℃인 것이 바람직하고, 추출 시간은 12~48 hr인 것이 바람직하다. 한편, 상기한 추출 용매뿐만 아니라, 다른 추출 용매를 이용하여도 실질적으로 동일한 효과를 나타내는 대마 줄기 추출물이 얻어질 수 있다는 것은 당업자에게 자명한 것이다. 또한, 대마 줄기 추출물에는 상술한 추출 용매에 의한 추출물뿐만 아니라, 통상적인 다른 추출 방법을 통해 얻어진 추출물, 분획물, 정제물 및 발효 과정을 거친 추출물도 포함될 수 있다. 예를 들어, 이산화탄소를 이용한 초임계 유체 추출법(supercritical fluid extraction)에 의한 추출, 초음파를 이용한 추출법에 의한 추출, 일정한 분자량 컷-오프 값을 갖는 한외 여과막을 이용한 분리 또는 다양한 크로마토그래피(크기, 전하, 소수성 또는 친화성에 따른 분리를 위해 제작된 것)에 의한 분리, 각종 미생물의 발효 산물에 의한 추출 등, 추가적으로 실시된 다양한 정제 및 추출방법을 통해 얻어진 활성 분획도 본 발명의 추출물에 포함된다. 일반적으로 초임계 유체는 기체가 고온 고압 조건에서 임계점에 도달하였을 때 갖는 액체 및 기체의 성질을 지니고 있으며, 화학적으로 비극성 용매와 유사한 극성을 지니고 있으며 이러한 특성으로 인해 초임계 유체는 지용성 물질의 추출에 사용되고 있다(J. Chromatogr. A. 1998;479:200-205). 이산화탄소는 초임계 유체기기의 작동으로 압력 및 온도가 임계점까지 이르는 과정을 거치면서 액체 및 기체 성질을 동시에 지닌 초임계 유체가 되고 그 결과 지용성 용질에 대한 용해도가 증가한다. 초임계 이산화탄소가 일정량의 시료를 함유한 추출 용기를 통과하게 되면 시료에 함유된 지용성 물질은 초임계 이산화탄소에 추출되어 나온다. 지용성 물질을 추출한 후 추출 용기에 남아있는 시료에 다시 소량의 공용매가 함유된 초임계 이산화탄소를 흘려 통과시키면 순수한 초임계 이산화탄소만으로는 추출되지 않았던 성분들이 추출되어 나오게 할 수 있다. 본 발명의 초임계 추출법에 사용되는 초임계 유체는 초임계 이산화탄소 또는 이산화탄소에 추가적으로 공용매를 혼합한 혼합유체를 사용함으로써 효과적으로 유효 성분을 추출할 수 있다. 이러한 공용매로 클로로포름, 에탄올, 메탄올, 물, 에틸아세테이트, 헥산 및 디에틸에테르로 이루어진 군에서 선택되는 1종 또는 2종 이상의 혼합물을 사용할 수 있다. 추출된 시료는 대부분 이산화탄소를 함유하고 있는데 이산화탄소는 실온에서 공기 중으로 휘발되고, 공용매는 감압증발기로 제거할 수 있다. 또한, 상기 초음파 추출법은 초음파 진동에 의해 발생되는 에너지를 이용하는 추출방법으로, 초음파가 수용성 용매 속에서 시료에 포함된 불용성인 용매를 파괴시킬 수 있으며, 이때 발생되는 높은 국부온도로 인하여 주위에 위치하는 반응물 입자들의 운동에너지를 크게 하기 때문에 반응에 필요한 충분한 에너지를 얻게 되고, 초음파 에너지의 충격효과로 높은 압력을 유도하여 시료에 함유된 물질과 용매의 혼합 효과를 높여주어 추출 효율을 증가시키게 된다. 초음파 추출법에 사용할 수 있는 추출용매는 클로로포름, 에탄올, 메탄올, 물, 에틸아세테이트, 헥산 및 디에틸 에테르로 이루어진 군에서 선택되는 1종 또는 2종 이상의 혼합물을 사용할 수 있다. 추출된 시료는 진공 여과하여 여과액을 회수한 후 감압증발기로 제거하고, 동결 건조하는 통상의 추출물 제조방법을 통해 추출물을 얻을 수 있다. 또한, 대마 줄기 추출물에는 발효과정을 거친 추출물도 포함하는데, 예를 들어 대마 줄기 발효 추출물은 다음과 같이 제조할 수 있다. 대마 줄기를 100~500 메쉬 정도로 미세하게 파쇄한 다음 통상적인 미생물용 배양액에 대마 줄기 농도가 1~50g/L가 되도록 첨가하고 여기에 효모 균주 또는 유산균 등의 미생물을 10,000~100,000 cfu/L의 양으로 첨가한다. 이후, 30~37℃의 배양 온도, 5~7의 pH와 같은 통상적인 미생물 배양조건으로 로 호기적 또는 혐기(anaerobic)적인 상태에서 약 5일 내지 10일간 배양한다. 이후, 숙성 및 여과를 통해 대마 줄기 발효 추출물을 얻을 수 있다.The hemp stem extract, which is an active ingredient of the composition according to the present invention, can be prepared by various methods. Specifically, the cannabis stem extract may be prepared using a conventional extraction method known in the art, for example, a solvent extraction method. The extraction solvent used in the solvent extraction method is water, a lower alcohol having 1 to 4 carbon atoms (eg, methanol, ethanol, propanol, and butanol) or a mixture thereof, which is a hydrous lower alcohol, propylene glycol, 1,3-butylene glycol , glycerin, acetone, diethyl ether, ethyl acetate, butyl acetate, dichloromethane, chloroform, hexane and mixtures thereof may be selected from the group consisting of, among them, water, alcohol, or a mixed solvent of two or more thereof It is preferable, and it is more preferable that it is a hydrous alcohol. When water is used as the extraction solvent, the water is preferably hot water. In addition, when alcohol is used as the extraction solvent, the alcohol is preferably a lower alcohol having 1 to 4 carbon atoms, and the lower alcohol is more preferably selected from methanol or ethanol. In addition, when an aqueous alcohol is used as the extraction solvent, the alcohol content is preferably 55 to 95% (w/w), more preferably 60 to 85% (w/w). When hydrous ethanol is used as the extraction solvent, the hemp stem extract is prepared by 1) extracting the hemp stem with hydrous ethanol having an ethanol concentration of 55 to 95% (w/w) to obtain an extract; 2) filtering the extract to remove residues and obtaining a filtered extract; and 3) concentrating and drying the filtered extract to obtain a solid extract. In addition, in the step of 1) obtaining the extract, the amount of hydrous ethanol as the extraction solvent is preferably 0.25 to 1 L based on 100 g of dry weight of hemp root, and the ethanol concentration of the hydrous ethanol is 60 to 85% (w/w) is preferably, the extraction temperature is preferably 45 to 75 ℃, the extraction time is preferably 12 to 48 hr. On the other hand, it is apparent to those skilled in the art that a cannabis stem extract exhibiting substantially the same effect can be obtained using not only the above extraction solvent but also other extraction solvents. In addition, the cannabis stem extract may include not only the extract using the above-described extraction solvent, but also extracts, fractions, purified products and extracts obtained through other conventional extraction methods. For example, extraction by supercritical fluid extraction using carbon dioxide, extraction by extraction using ultrasonic waves, separation using an ultrafiltration membrane having a constant molecular weight cut-off value, or various chromatography (size, charge, Active fractions obtained through various additional purification and extraction methods, such as separation by hydrophobicity or affinity), extraction by fermentation products of various microorganisms, etc. are also included in the extract of the present invention. In general, supercritical fluids have the properties of liquids and gases when the gas reaches a critical point under high temperature and high pressure conditions, and chemically has a polarity similar to that of a non-polar solvent. (J. Chromatogr. A. 1998;479:200-205). Carbon dioxide becomes a supercritical fluid with both liquid and gas properties as the pressure and temperature reach the critical point through the operation of the supercritical fluid device, and as a result, the solubility in fat-soluble solutes increases. When the supercritical carbon dioxide passes through an extraction vessel containing a certain amount of the sample, the fat-soluble substance contained in the sample is extracted into the supercritical carbon dioxide. After extracting the fat-soluble substance, if supercritical carbon dioxide containing a small amount of co-solvent is again flowed through the sample remaining in the extraction container, components that were not extracted with pure supercritical carbon dioxide alone can be extracted. The supercritical fluid used in the supercritical extraction method of the present invention can effectively extract the active ingredient by using supercritical carbon dioxide or a mixed fluid in which carbon dioxide is additionally mixed with a cosolvent. As such a cosolvent, one or a mixture of two or more selected from the group consisting of chloroform, ethanol, methanol, water, ethyl acetate, hexane and diethyl ether may be used. Most of the extracted samples contain carbon dioxide, which is volatilized into the air at room temperature, and the cosolvent can be removed by a vacuum evaporator. In addition, the ultrasonic extraction method is an extraction method using energy generated by ultrasonic vibration, and ultrasonic waves can destroy an insoluble solvent contained in a sample in an aqueous solvent. Because the kinetic energy of the reactant particles is increased, sufficient energy required for the reaction is obtained, and the high pressure is induced by the impact effect of ultrasonic energy to increase the mixing effect of the material and the solvent contained in the sample, thereby increasing the extraction efficiency. The extraction solvent that can be used for the ultrasonic extraction method may be one or a mixture of two or more selected from the group consisting of chloroform, ethanol, methanol, water, ethyl acetate, hexane and diethyl ether. The extracted sample is vacuum filtered to recover the filtrate, then removed by a vacuum evaporator, and an extract can be obtained through a conventional extract preparation method of freeze-drying. In addition, the hemp stem extract includes an extract that has undergone a fermentation process, for example, the fermented hemp stem extract can be prepared as follows. Hemp stems are finely crushed to about 100-500 mesh, and then added to a normal culture medium for microorganisms so that the concentration of hemp stems is 1-50 g/L, and microorganisms such as yeast strains or lactic acid bacteria are added thereto in an amount of 10,000-100,000 cfu/L add with Thereafter, culture is carried out for about 5 to 10 days in an aerobic or anaerobic state with conventional microbial culture conditions such as a culture temperature of 30 to 37° C. and a pH of 5 to 7. Thereafter, it is possible to obtain a hemp stem fermented extract through aging and filtration.
본 발명의 조성물에서 유효성분으로 사용되는 대마 줄기 추출물은 매우 우수한 α-글루코시다제(α-glucosidase) 효소 저해활성을 가진다. 따라서, 본 발명에 따른 조성물은 항당뇨 용도; 혈당강하 용도; 당뇨병 예방, 개선 또는 치료 용도의 의약품, 건강기능식품으로 구현될 수 있다. 또한, 본 발명의 조성물은 특히 당뇨병 중 α-글루코시다제(α-glucosidase) 효소 저해활성과 높은 관련성을 가진 높은 제2형 당뇨병을 예방, 개선 또는 치료하는 용도의 의약품, 건강기능식품으로 구현될 수 있다.The cannabis stem extract used as an active ingredient in the composition of the present invention has very excellent α-glucosidase enzyme inhibitory activity. Accordingly, the composition according to the present invention may be used for antidiabetic use; blood sugar lowering use; It can be implemented as pharmaceuticals and health functional foods for preventing, improving or treating diabetes. In addition, the composition of the present invention may be implemented as a pharmaceutical or health functional food for preventing, improving or treating high type 2 diabetes, which has a high correlation with α-glucosidase enzyme inhibitory activity in diabetes mellitus. can
본 발명의 조성물은 사용 목적 내지 양상에 따라 약학 조성물, 식품 첨가제, 식품 조성물(특히 건강기능식품), 또는 사료 첨가제 등으로 구체화될 수 있고, 조성물 내에서의 유효성분의 함량도 조성물의 구체적인 형태, 사용 목적 내지 양상에 따라 다양한 범위에서 조정될 수 있다.The composition of the present invention may be embodied as a pharmaceutical composition, a food additive, a food composition (especially a health functional food), or a feed additive, etc., depending on the purpose or aspect of use, and the content of the active ingredient in the composition is also in the specific form of the composition, It may be adjusted in various ranges depending on the purpose or aspect of use.
본 발명의 약학 조성물에서 유효성분인 대마 줄기 추출물의 함량은 크게 제한되지 않으며, 예를 들어 조성물 총 중량을 기준으로 0.1~99 중량%, 바람직하게는 0.5~50 중량%, 더 바람직하게는 1~30 중량%일 수 있다. 또한, 본 발명의 약학 조성물은 유효성분 외에 약학적으로 허용가능한 담체, 부형제 또는 희석제와 같은 첨가제를 더 포함할 수 있다. 본 발명의 약학 조성물에 포함될 수 있는 담체, 부형제 및 희석제로는 락토즈, 덱스트로즈, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 들 수 있다. 또한, 본 발명의 약학 조성물은 대마 줄기 추출물 외에 당뇨병 치료 효과를 갖는 다양한 유효성분을 1종 이상 더 함유할 수 있다. 예를 들어, 본 발명의 약학 조성물은 유효성분으로 대마 줄기 추출물 외에 대마 뿌리 추출물을 더 포함할 수 있다. 본 발명의 약학 조성물은 통상의 방법에 의해 경구 투여를 위한 제형 또는 비경구 투여를 위한 제형으로 제제화될 수 있고, 제제화할 경우 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제될 수 있다. 경구 투여를 위한 고형 제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형 제제는 유효성분인 복합 추출물에 적어도 하나 이상의 부형제 예를 들면, 전분, 칼슘카보네이트(Calcium carbonate), 수크로스(Sucrose), 락토오스(Lactose) 또는 젤라틴 등을 섞어 조제될 수 있다. 또한, 단순한 부형제 이외에 마그네슘 스티레이트 탈크 같은 윤활제들도 사용될 수 있다. 경구 투여를 위한 액상 제제로는 현탁제, 내용액제, 유제 및 시럽제 등이 해당되는데 흔히 사용되는 단순희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조제제, 좌제가 포함될 수 있다. 비수성용제, 현탁용제로는 프로필렌글리콜(Propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔(witepsol), 마크로골, 트윈(tween) 61, 카카오지, 라우린지, 글리세로제라틴 등이 사용될 수 있다. 더 나아가 당 분야의 적정한 방법으로 또는 Remington's Pharmaceutical Science(최근판), Mack Publishing Company, Easton PA에 개시되어 있는 방법을 이용하여 각 질환에 따라 또는 성분에 따라 바람직하게 제제화할 수 있다. 본 발명의 약학 조성물은 목적하는 방법에 따라 인간을 포함한 포유류에 경구 투여되거나 비경구 투여될 수 있으며, 비경구 투여 방식으로는 피부 외용, 복강내주사, 직장내주사, 피하주사, 정맥주사, 근육내 주사 또는 흉부내 주사 주입방식 등이 있다. 본 발명의 약학 조성물의 투여량은 약학적으로 유효한 양이라면 크게 제한되지 않으며, 환자의 체중, 연령, 성별, 건강상태, 식이, 투여시간, 투여방법, 배설율 및 질환의 중증도에 따라 그 범위가 다양하다. 본 발명의 약학 조성물의 통상적인 1일 투여량은 크게 제한되지 않으며, 예를 들어 유효성분을 기준으로 할 때 0.1 내지 1000 ㎎/㎏(body weight)에서 선택될 수 있고, 1 내지 500 ㎎/㎏(body weight)에서 선택되는 것이 바람직하고, 10 내지 250 ㎎/㎏(body weight)에서 선택되는 것이 더 바람직하다. 본 발명의 약학 조성물의 투여 횟수도 크게 제한되지 않으며, 예를 들어 하루 1회 또는 수회로 나누어 투여될 수 있다.The content of the hemp stem extract as an active ingredient in the pharmaceutical composition of the present invention is not particularly limited, for example, 0.1 to 99% by weight, preferably 0.5 to 50% by weight, more preferably 1 to based on the total weight of the composition. 30% by weight. In addition, the pharmaceutical composition of the present invention may further include additives such as a pharmaceutically acceptable carrier, excipient or diluent in addition to the active ingredient. Carriers, excipients and diluents that may be included in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate , cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In addition, the pharmaceutical composition of the present invention may further contain one or more kinds of various active ingredients having a diabetes treatment effect in addition to the hemp stem extract. For example, the pharmaceutical composition of the present invention may further include a hemp root extract in addition to the hemp stem extract as an active ingredient. The pharmaceutical composition of the present invention may be formulated as a formulation for oral administration or a formulation for parenteral administration by a conventional method, and when formulated, commonly used fillers, extenders, binders, wetting agents, disintegrants, surfactants, etc. It may be formulated using diluents or excipients. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient, for example, starch, calcium carbonate, water in the complex extract as an active ingredient. It can be prepared by mixing cross (Sucrose), lactose (Lactose) or gelatin. In addition to simple excipients, lubricants such as magnesium stearate talc may also be used. Liquid formulations for oral administration include suspensions, internal solutions, emulsions and syrups, and various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. there is. Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like can be used. Furthermore, it can be preferably formulated according to each disease or component using an appropriate method in the art or a method disclosed in Remington's Pharmaceutical Science (latest edition), Mack Publishing Company, Easton PA. The pharmaceutical composition of the present invention may be administered orally or parenterally to mammals including humans according to a desired method, and parenteral administration methods include external application to the skin, intraperitoneal injection, intrarectal injection, subcutaneous injection, intravenous injection, and intramuscular administration. There are intra-injection or intra-thoracic injection injection methods. The dosage of the pharmaceutical composition of the present invention is not particularly limited as long as it is a pharmaceutically effective amount, and the range may vary depending on the patient's weight, age, sex, health status, diet, administration time, administration method, excretion rate, and severity of the disease. Varies. A typical daily dose of the pharmaceutical composition of the present invention is not particularly limited, and for example, may be selected from 0.1 to 1000 mg/kg (body weight) based on the active ingredient, and 1 to 500 mg/kg It is preferably selected from (body weight), and more preferably selected from 10 to 250 mg/kg (body weight). The number of times of administration of the pharmaceutical composition of the present invention is not significantly limited, and for example, it may be administered once a day or divided into several times.
또한, 본 발명의 식품 조성물에서 유효성분인 대마 줄기 추출물의 함량은 조성물 총 중량을 기준으로 0.01~50 중량%, 바람직하게는 0.1~25 중량%, 더 바람직하게는 0.5~10 중량%이나, 이에 한정되는 것은 아니다. 본 발명의 식품 조성물은 환제, 분말, 과립, 침제, 정제, 캡슐, 또는 액제 등의 형태를 포함하며, 구체적인 식품의 예로는 육류, 소시지, 빵, 초콜릿, 캔디류, 스넥류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 차, 기능수, 드링크제, 알코올음료 및 비타민 복합제 등이 있으며, 통상적인 의미에서의 건강기능식품(health functional food), 식이보충제(dietary supplement) 및 식품보충제(food supplement)를 모두 포함한다. 본 발명의 식품 조성물은 유효성분 외에 식품학적으로 허용 가능한 담체, 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다. 또한, 본 발명의 식품 조성물은 여러 가지 영양제, 비타민, 전해질, 풍미제, 착색제, 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올, 탄산음료에 사용되는 탄산화제 등을 함유할 수 있다. 그 밖에 본 발명의 식품 조성물은 천연 과일주스, 과일주스 음료 및 야채 음료의 제조를 위한 과육을 함유할 수 있다. 이러한 성분들은 독립적으로 또는 혼합하여 사용할 수 있다. 상술한 천연 탄수화물은 포도당, 과당과 같은 모노사카라이드, 말토스, 슈크로스와 같은 디사카라이드, 및 덱스트린, 사이클로덱스트린과 같은 폴리사카라이드, 자일리톨, 소르비톨, 에리트리톨 등의 당알코올이다. 향미제로는 타우마틴, 스테비아 추출물과 같은 천연 향미제나 사카린, 아스파르탐과 같은 합성 향미제 등을 사용할 수 있다.In addition, the content of the hemp stem extract as an active ingredient in the food composition of the present invention is 0.01 to 50% by weight, preferably 0.1 to 25% by weight, more preferably 0.5 to 10% by weight, based on the total weight of the composition, but It is not limited. The food composition of the present invention includes the form of pills, powders, granules, needles, tablets, capsules, or liquids, and specific examples of food include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, Other dairy products including noodles, gums, and ice cream, various soups, beverages, tea, functional water, drinks, alcoholic beverages and vitamin complexes, etc. Includes both dietary supplement and food supplement. In addition to the active ingredient, the food composition of the present invention may contain, as an additional ingredient, a pharmaceutically acceptable carrier, various flavoring agents, or natural carbohydrates. In addition, the food composition of the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol , a carbonation agent used in carbonated beverages, and the like. In addition, the food composition of the present invention may contain fruit for the production of natural fruit juice, fruit juice beverage, and vegetable beverage. These components may be used independently or in combination. The above-mentioned natural carbohydrates include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As the flavoring agent, a natural flavoring agent such as thaumatin or stevia extract or a synthetic flavoring agent such as saccharin or aspartame may be used.
이하, 본 발명을 실시예를 통하여 보다 구체적으로 설명한다. 다만, 하기 실시예는 본 발명의 기술적 특징을 명확하게 예시하기 위한 것 일뿐, 본 발명의 보호범위를 한정하는 것은 아니다.Hereinafter, the present invention will be described in more detail through examples. However, the following examples are only for clearly illustrating the technical features of the present invention, and do not limit the protection scope of the present invention.
1. 대마 추출물의 제조1. Preparation of Hemp Extract
제조예 1 : 대마 줄기 추출물의 제조Preparation Example 1: Preparation of hemp stem extract
대마 줄기(Hemp stem)를 자연 건조시킨 후 분쇄기로 분말화하여 대마 줄기 건조 분말을 수득하였다. 이후, 대마 줄기 건조 분말 200에 에탄올 농도가 70%(w/w)인 함수 에탄올 1L를 넣고 교반한 후 60℃ 항온 수조상에서 중탕하여 24 hr 동안 추출하였다. 이후, 추출액을 필터페이퍼를 이용하여 여과하고, 여과된 추출액을 감압농축기를 이용하여 농축한 후, 동결건조하여 고상의 대마 줄기 추출물을 수득하였다.Hemp stems were naturally dried and then pulverized with a grinder to obtain a dried hemp stem powder. Thereafter, 1 L of aqueous ethanol having an ethanol concentration of 70% (w/w) was added to 200 hemp stem dry powder, stirred, and then extracted for 24 hr by bathing in a water bath at 60 ° C. Thereafter, the extract was filtered using filter paper, and the filtered extract was concentrated using a vacuum concentrator, and then freeze-dried to obtain a solid hemp stem extract.
제조예 2 : 대마 뿌리 추출물의 제조Preparation Example 2: Preparation of hemp root extract
대마 뿌리(Hemp root)를 자연 건조시킨 후 분쇄기로 분말화하여 대마 뿌리 건조 분말을 수득하였다. 이후, 대마 뿌리 건조 분말 200에 에탄올 농도가 70%(w/w)인 함수 에탄올 1L를 넣고 교반한 후 60℃ 항온 수조상에서 중탕하여 24 hr 동안 추출하였다. 이후, 추출액을 필터페이퍼를 이용하여 여과하고, 여과된 추출액을 감압농축기를 이용하여 농축한 후, 동결건조하여 고상의 대마 뿌리 추출물을 수득하였다.Hemp root was dried naturally and then pulverized with a grinder to obtain a dried hemp root powder. Thereafter, 1 L of aqueous ethanol having an ethanol concentration of 70% (w/w) was added to 200 dried hemp root powder, stirred, and then extracted for 24 hr by bathing in a water bath at 60° C. Thereafter, the extract was filtered using filter paper, and the filtered extract was concentrated using a vacuum concentrator, and then freeze-dried to obtain a solid hemp root extract.
제조예 3 : 대마 종자(또는 씨앗) 추출물의 제조Preparation Example 3: Preparation of hemp seed (or seed) extract
대마 종자(Hemp seed)를 자연 건조시킨 후 분쇄기로 분말화하여 대마 뿌리 건조 분말을 수득하였다. 이후, 대마 뿌리 건조 분말 200에 에탄올 농도가 70%(w/w)인 함수 에탄올 1L를 넣고 교반한 후 60℃ 항온 수조상에서 중탕하여 24 hr 동안 추출하였다. 이후, 추출액을 필터페이퍼를 이용하여 여과하고, 여과된 추출액을 감압농축기를 이용하여 농축한 후, 동결건조하여 고상의 대마 뿌리 추출물을 수득하였다.Hemp seeds were naturally dried and then pulverized with a grinder to obtain a dried hemp root powder. Thereafter, 1 L of aqueous ethanol having an ethanol concentration of 70% (w/w) was added to 200 dried hemp root powder, stirred, and then extracted for 24 hr by bathing in a water bath at 60° C. Thereafter, the extract was filtered using filter paper, and the filtered extract was concentrated using a vacuum concentrator, and then freeze-dried to obtain a solid hemp root extract.
2. 대마 추출물의 항산화 활성 평가2. Evaluation of Antioxidant Activity of Hemp Extract
대마 추출물의 항산화 활성은 DPPH 라디칼 소거능(DPPH radical scavenging activity), 총 폴리페놀 함량(Total polyphenol contents), 총 플라보노이드 함량(Total flavonoid contents), SOD 유사활성(Superoxide dismutase like activity), ABTS+ 라디칼 소거능 등과 같은 다양한 지표를 이용하여 평가하였다.Antioxidant activity of hemp extract is DPPH radical scavenging activity, total polyphenol contents, total flavonoid contents, SOD-like activity, ABTS + radical scavenging activity, etc. It was evaluated using the same various indicators.
(1) DPPH 라디칼 소거능(DPPH radical scavenging activity)(1) DPPH radical scavenging activity
200 μM DPPH(1,1-diphenyl-2-picrylhydrazyl) 180 ㎕와 시료 20 ㎕를 혼합고 37℃의 온도 및 암소 조건에서 30분 동안 반응시킨 후, 517nm에서 흡광도를 측정하였다. 또한, 대조군으로 Vitamin C를 이용하였다. DPPH 라디칼 소거능은 다음과 같은 식으로 계산하였다.180 μl of 200 μM DPPH (1,1-diphenyl-2-picrylhydrazyl) and 20 μl of the sample were mixed, reacted at 37° C. and in the dark for 30 minutes, and then absorbance was measured at 517 nm. In addition, Vitamin C was used as a control. The DPPH radical scavenging ability was calculated as follows.
도 1은 대마 추출물의 DPPH 라디칼 소거능 측정 결과를 나타낸 그래프이다. 자유라디칼 소거 작용은 활성라디칼에 전자를 공여하여 항산화 효과나 인체에서 노화를 억제하는 척도로 이용되고 있다. 천연물이 가지는 자유라디칼 소거활성을 측정하기 위해 대표적으로 사용되는 방법이 DPPH 라디칼 소거능 실험이다. 도 1에서 보이는 바와 같이 대마 줄기 추출물 및 대마 뿌리 추출물은 5,000㎍/㎖ 농도에서 각각 23% 및 18%의 DPPH 라디칼 소거능을 나타내었으나, 표준품으로 사용한 100 ㎍/㎖의 Vitamin C 와 비교하였을 때 50배 높은 농도에서도 Vitamin C 보다 현저히 낮은 값을 보였는바, 대마 줄기 추출물 및 대마 뿌리 추출물은 모두 항산화 효과가 미비한 것으로 사료된다.1 is a graph showing the measurement results of DPPH radical scavenging ability of cannabis extract. Free radical scavenging action is used as a measure of antioxidant effect or aging in the human body by donating electrons to active radicals. A typical method used to measure the free radical scavenging activity of natural products is the DPPH radical scavenging activity. As shown in FIG. 1 , the hemp stem extract and hemp root extract showed DPPH radical scavenging activity of 23% and 18%, respectively, at a concentration of 5,000 µg/ml, but 50 times compared to 100 µg/ml of Vitamin C used as a standard product. Even at high concentrations, the values were significantly lower than those of Vitamin C, so both hemp stem extract and hemp root extract are considered to have insufficient antioxidant effect.
(2) 총 폴리페놀 함량(Total polyphenol contents)(2) Total polyphenol contents
총 폴리페놀 함량 측정은 Folin-Denis법을 일부 변형하여 측정하였다. 시료 10 ㎕에 2% Na2CO3 용액 200 ㎕를 첨가하고 Folin & Ciocalteu’s phenol reagents 10 ㎕를 혼합한 다음 실온에서 30분간 반응시킨 후 760nm에서 흡광도를 측정하였다. 표준물질로는 Galic acid(Sigma co., USA)를 사용하고 시료와 동일한 방법으로 분석하여 작성한 검량선으로부터 총 폴리페놀 함량을 계산하였다.Total polyphenol content was measured by partially modifying the Folin-Denis method. 200 μl of 2% Na 2 CO 3 solution was added to 10 μl of sample, 10 μl of Folin &Ciocalteu's phenol reagents were mixed, and then reacted at room temperature for 30 minutes, and absorbance was measured at 760 nm. Galic acid (Sigma co., USA) was used as a standard material, and the total polyphenol content was calculated from the calibration curve prepared by analyzing in the same way as the sample.
도 2는 대마 추출물의 총 폴리페놀 함량 측정 결과를 나타낸 그래프이다. 폴리페놀은 분자 내에 다수의 수산화기가 있는 페놀을 지칭하며, 페놀 링 구조의 수에 따라 분류된다. 폴리페놀에 존재하는 phenolic hydroxyl(-OH)기는 여러 화합물과 쉽게 결합하는 특성을 가지고 있어 폴리페놀은 항산화 효과 및 항암, 항염증 효과가 뛰어난 것으로 알려져 있다. 일반적으로 총 폴리페놀 함량이 증가할수록 항산화 등의 생리활성이 증가하는 경향을 가지는 것으로 보고되였다. 도 2에서 보이는 바와 같이, 대마 줄기 추출물의 총 폴리페놀 함량은 5381.78 ㎍/g이고 대마 뿌리 추출물의 총 폴리페놀 함량은 4654.67 ㎍/g으로서, 대마 줄기 추출물이 대마 뿌리 추출물보다 높은 총 폴리페놀 함량 값을 나타내었지만 두 결과 값의 차이는 크지 않았다.Figure 2 is a graph showing the measurement result of the total polyphenol content of the hemp extract. Polyphenols refer to phenols with multiple hydroxyl groups in the molecule, and are classified according to the number of phenol ring structures. The phenolic hydroxyl (-OH) group present in polyphenols has the property of easily bonding with various compounds, so polyphenols are known to have excellent antioxidant, anticancer, and anti-inflammatory effects. In general, it has been reported that as the total polyphenol content increases, physiological activities such as antioxidants tend to increase. As shown in FIG. 2 , the total polyphenol content of the hemp stem extract was 5381.78 μg/g and the total polyphenol content of the hemp root extract was 4654.67 μg/g, and the total polyphenol content value of the hemp stem extract was higher than that of the hemp root extract. , but the difference between the two results was not significant.
(3) 총 플라보노이드 함량(Total flavonoid contents)(3) Total flavonoid contents
총 플라보노이드 함량 측정은 Jia 등[1. Jia, Z., Tang, M. and Wu, J. 1999. The determination of flavonoid contents in mulberry and they scavenging effects on superoxide radicals. Food Chem 64: 555-559.]의 방법을 일부 변형하여 측정하였다. 시료 20 ㎕에 증류수 80 ㎕와 5% NaNO2 6 ㎕를 첨가하고 상온에서 5분간 반응시킨 후 10% AlCl3·6H2O 12 ㎕를 첨가하고 6분간 반응시킨 후 1N NaOH 40 ㎕를 첨가하여 반응을 정지 시킨 다음 510 nm에서 흡광도를 측정하였다. 표준물질로는 Rutin(Sigma co., USA)을 사용하고 시료와 동일한 방법으로 분석하여 작성한 검량선으로부터 총 플라보노이드 함량을 계산하였다.Determination of total flavonoid content was performed by Jia et al. [1. Jia, Z., Tang, M. and Wu, J. 1999. The determination of flavonoid contents in mulberry and they scavenging effects on superoxide radicals. Food Chem 64: 555-559.] was measured with some modifications. 80 μl of distilled water and 6 μl of 5% NaNO 2 were added to 20 μl of the sample, and after reacting at room temperature for 5 minutes, 12 μl of 10% AlCl 3 .6H 2 O was added and reacted for 6 minutes, followed by reaction by adding 40 μl of 1N NaOH After stopping, absorbance was measured at 510 nm. Rutin (Sigma co., USA) was used as a standard material, and the total flavonoid content was calculated from the calibration curve prepared by analyzing in the same way as the sample.
도 3은 대마 추출물의 총 플라보노이드 함량 측정 결과를 나타낸 그래프이다. 플라보노이드는 폴리페놀에 속하는 성분으로 자연계에 널리 분포하고 있고 활성산소종을 효과적으로 제거하여 항산화능이 높다고 알려져 있으며 폴리페놀과 동일하게 항염증, 항암 효과가 있는 것으로 알려져 있다. 도 3에서 보이는 바와 같이, 대마 줄기 추출물의 총 폴리페놀 함량은 3461.33 ㎍/g이고 대마 뿌리 추출물의 총 폴리페놀 함량은 2175.33 ㎍/g으로서, 총 폴리페놀 함량 결과와 동일하게 대마 줄기 추출물이 대마 뿌리 추출물보다 높은 값을 나타내었다.3 is a graph showing the measurement result of the total flavonoid content of the hemp extract. Flavonoids are components that belong to polyphenols and are widely distributed in nature and are known to have high antioxidant activity by effectively removing reactive oxygen species. As shown in FIG. 3 , the total polyphenol content of the hemp stem extract was 3461.33 μg/g and the total polyphenol content of the hemp root extract was 2175.33 μg/g, and the hemp stem extract was It showed a higher value than the extract.
(4) SOD 유사활성(Superoxide dismutase like activity)(4) SOD-like activity (Superoxide dismutase like activity)
SOD 유사활성은 Marklund의 방법을 일부 변형하여 측정하였다. 시료 10 ㎕에 pH 8.5로 보정한 tris-HCl buffer[50 mM tris(hydroxymethyl)amino-methane+10 mM EDTA] 130 ㎕와 7.2 mM pyrogallol 10 ㎕를 첨가하고 25℃에서 10분간 반응시킨 후 1N HCl 10 ㎕를 첨가하여 반응을 정지시킨 다음 420 nm에서 흡광도를 측정하였다. 또한, 대조군으로 Vitmin C를 이용하였다. SOD 유사활성은 다음과 같은 식으로 계산하였다.SOD-like activity was measured with some modifications of Marklund's method. 130 μl of tris-HCl buffer [50 mM tris(hydroxymethyl)amino-methane+10 mM EDTA] corrected to pH 8.5 and 10 μl of 7.2 mM pyrrogallol were added to 10 μl of the sample and reacted at 25° C. for 10 minutes, followed by
도 4는 대마 추출물의 SOD 유사활성 측정 결과를 나타낸 그래프이다. SOD(Superoxide dismutase)는 생체 내에서 superoxide의 소거에 관여하는 효소이며 인체 내의 superoxide를 제거함으로써 노화 억제와 더불어 산화적 장해를 방어하는 작용을 한다. SOD 유사활성 물질은 효소는 아니지만 SOD와 유사한 역할을 하여 superoxide로부터 생체를 보호한다고 보고되었다. 도 4에서 보이는 바와 같이 대마 줄기 추출물 및 대마 뿌리 추출물은 5,000 ㎍/㎖ 농도에서 각각 16% 및 28%의 SOD 유사활성을 나타내었고, 같은 농도에서 대마 뿌리 추출물이 대마 줄기 추출물보다 약 2배 정도 높은 값을 나타냈다. 다만, 표준품으로 사용한 500 ㎍/㎖의 Vitamin C 와 비교하였을 때 10배 높은 농도에서도 Vitamin C 보다 낮은 값을 보였는바, 대마 줄기 추출물 및 대마 뿌리 추출물은 모두 항산화 효과가 크지 않은 것으로 사료된다.4 is a graph showing the measurement results of SOD-like activity of the hemp extract. SOD (superoxide dismutase) is an enzyme that is involved in the elimination of superoxide in the body, and by removing superoxide in the body, it inhibits aging and protects against oxidative damage. Although SOD-like active substances are not enzymes, it has been reported that they protect the living body from superoxide by acting similar to SOD. As shown in FIG. 4 , the hemp stem extract and hemp root extract exhibited SOD-like activity of 16% and 28%, respectively, at a concentration of 5,000 μg/ml, and at the same concentration, the hemp root extract was about twice as high as the hemp stem extract. value was indicated. However, compared with the 500 μg/ml Vitamin C used as a standard product, the values were lower than those of Vitamin C even at 10-fold higher concentrations.
(5) ABTS+ 라디칼 소거능(5) ABTS + radical scavenging ability
ABTS+ 라디칼 소거능은 Re 등의 방법[1. Re, R., Pellegrini, N., Proteggente, A., Pannala, A. and Yang, M, Rice-Evans C. 1999. Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radical Bio. Med. 26: 1231-1237.]에 준하여 측정하였다. ABTS를 7.4 mM의 농도로 증류수에 용해한 다음 여기에 2.6 mM potassium persulfate를 1:1의 부피비로 혼합하고 실온의 암소 조건에서 24 hr 동안 반응시켰다. 혼합한 ABTS용액을 PBS용액으로 희석하여 732 nm에서 0.7(±0.02)가 되도록 조정하였다. 이후, ABTS 용액 180 ㎕와 시료 20㎕를 혼합하고 5분간 반응시킨 후 732 nm에서 흡광도를 측정하였다. 또한, 대조군으로 Vitmin C를 이용하였다. ABTS+ 라디칼 소거능은 다음과 같은 식으로 계산하였다.ABTS + radical scavenging ability was determined by Re et al. [1. Re, R., Pellegrini, N., Proteggente, A., Pannala, A. and Yang, M, Rice-Evans C. 1999. Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radical Bio. Med. 26: 1231-1237.]. ABTS was dissolved in distilled water to a concentration of 7.4 mM, and then 2.6 mM potassium persulfate was mixed therein in a volume ratio of 1:1, and reacted for 24 hr in the dark at room temperature. The mixed ABTS solution was diluted with PBS solution and adjusted to 0.7 (±0.02) at 732 nm. Then, 180 μl of the ABTS solution and 20 μl of the sample were mixed, reacted for 5 minutes, and absorbance was measured at 732 nm. In addition, Vitmin C was used as a control. ABTS + radical scavenging ability was calculated as follows.
도 5는 대마 추출물의 ABTS+ 라디칼 소거능 측정 결과를 나타낸 그래프이다. ABTS+ 라디칼 소거능 실험은 DPPH 라디칼 소거능 실험과 함께 대표적인 항산화 활성 측정 방법 중 하나로 알려져 있다. 도 5에서 보이는 바와 같이 대마 줄기 추출물 및 대마 뿌리 추출물은 2,500 ㎍/㎖ 농도에서 각각 86% 및 79%의 ABTS+ 라디칼 소거능을 나타내었고, 대마 줄기 추출물이 대마 뿌리 추출물보다 다소 높은 결과를 나타내었다. 다만, 표준품으로 사용한 Vitamin C와 동일한 농도인 50 ㎍/㎖에서 비교하였을 때 대마 줄기 추출물과 대마 뿌리 추출물 모두 현저히 낮은 ABTS+ 라디칼 소거능 값을 나타내고 있으므로 DPPH 라디칼 소거능 결과와 동일하게 항산화 효능은 미비한 것으로 사료된다.5 is a graph showing the measurement result of ABTS + radical scavenging ability of cannabis extract. The ABTS + radical scavenging activity experiment is known as one of the representative antioxidant activity measurement methods along with the DPPH radical scavenging activity experiment. As shown in FIG. 5 , the hemp stem extract and hemp root extract exhibited 86% and 79% ABTS + radical scavenging activity at 2,500 μg/ml concentration, respectively, and the hemp stem extract showed somewhat higher results than the hemp root extract. However, when compared at 50 μg/ml, which is the same concentration as Vitamin C used as a standard product, both the hemp stem extract and hemp root extract showed significantly lower ABTS + radical scavenging activity values, so it is considered that the antioxidant efficacy was insufficient as the DPPH radical scavenging activity results. do.
3. 대마 추출물의 항비만 활성 평가3. Evaluation of anti-obesity activity of hemp extract
(1) 3T3-L1 Cell에 대한 세포 독성 평가(MTT assay)(1) Cytotoxicity evaluation for 3T3-L1 cells (MTT assay)
96-well plate에 3T3-L1 지방전구세포를 1×106 cells/well로 동일하게 분주하고 24 hr 동안 배양하였다. 이후, 기존의 배지를 제거하고 새로운 배지를 넣어준 후 시료를 농도 의존적으로(50, 200 ㎍/㎖)을 넣고 4 hr 동안 배양한 후 상등액을 제거하고 well에 형성된 formazan은 DMSO 100 ㎕를 첨가하여 용해시킨 후 570 nm에서 흡광도를 측정하였다. 시료를 처리하지 않은 음성대조군을 100%의 생존율로 간주하고 이를 기준으로 세포 생존율을 비교하였다. 3T3-L1 preadipocytes were equally distributed at 1×10 6 cells/well in a 96-well plate and cultured for 24 hr. Then, after removing the old medium and adding a new medium, the sample was added concentration-dependently (50, 200 μg/ml), incubated for 4 hr, the supernatant was removed, and formazan formed in the well was added with 100 μl of DMSO. After dissolution, absorbance was measured at 570 nm. A negative control group that was not treated with the sample was regarded as 100% viability, and cell viability was compared based on this.
도 6은 대마 추출물의 3T3-L1 Cell에 대한 세포 독성 평가(MTT assay) 결과를 나타낸 그래프이다. 지방전구세포인 3T3-L1 세포는 여러 호르몬과 다양한 전사인자들에 의해 지방세포로 분화되면서 세포 내 지방을 축적한다. 지방분화 세포에 대한 대마 줄기 추출물 및 대마 뿌리 추출물의 세포독성을 측정한 결과, 도 6에서 보이는 바와 같이 모든 추출물에서 200㎍/㎖ 농도까지 세포독성을 나타내지 않았다.6 is a graph showing the results of cytotoxicity evaluation (MTT assay) for 3T3-L1 cells of cannabis extract. 3T3-L1 cells, which are preadipocytes, accumulate intracellular fat as they differentiate into adipocytes by various hormones and various transcription factors. As a result of measuring the cytotoxicity of the hemp stem extract and hemp root extract against adipogenic cells, as shown in FIG. 6 , all extracts showed no cytotoxicity up to a concentration of 200 μg/ml.
(2) 3T3-L1 Cell에 대한 지방세포로의 분화 억제능 측정(2) Measurement of the ability of 3T3-L1 cells to inhibit differentiation into adipocytes
대마 추출물의 3T3-L1 세포에 대한 지방세포로의 분화 억제능을 알아보기 위해 1×105 cells/mL의 농도로 24 well plate에 분주(seeding)한 후 48 hr 뒤에 배지를 교환하고, 48 hr 후에 MDI(IBMX, dexametason, insulin)와 시료를 처리하였다. MDI 처리 48 hr 후에 insulin과 시료를 처리하고, 다시 48 hr 후에 시료를 처리하고, 다시 48 hr 후에 시료를 처리하고, 48 hr 후에 분화된 세포를 관찰하기 위해서 배양된 세포를 10% formalin in PBS로 10분간 실온에서 고정하고 60% isopropanol을 이용하여 1회 세척 후 건조시켰다. 이후, Oil red-o를 이용하여 15분 간 염색하고 증류수로 4번 세척한 후 증류수를 제거하자마자 바로 fat droplets를 현미경으로 관찰하였다. 현미경 관찰 후 99% isoprophyl alcohol을 넣고 180 rpm에서 5분간 녹인 뒤 520 nm에서 흡광도를 측정하였다.To examine the ability of the hemp extract to inhibit the differentiation of 3T3-L1 cells into adipocytes, the medium was exchanged after 48 hr after seeding in a 24 well plate at a concentration of 1×10 5 cells/mL, and MDI after 48 hr. (IBMX, dexametason, insulin) and samples were treated. After 48 hr of MDI treatment, the sample was treated with insulin, the sample was processed again after 48 hr, the sample was processed after another 48 hr, and the cultured cells were treated with 10% formalin in PBS to observe the differentiated cells after 48 hr. It was fixed at room temperature for 10 minutes, washed once with 60% isopropanol, and dried. Thereafter, after staining with Oil red-o for 15 minutes, washing with distilled water 4 times, and immediately after removing the distilled water, fat droplets were observed under a microscope. After microscopic observation, 99% isoprophyl alcohol was added, dissolved at 180 rpm for 5 minutes, and absorbance was measured at 520 nm.
도 7은 대마 추출물의 3T3-L1 Cell에 대한 지방세포로의 분화 억제능 측정 결과를 나타낸 그래프이다. 도 7에서 보이는 바와 같이 대마 줄기 추출물 및 대마 뿌리 추출물을 50, 200ppm으로 희석하여 전지방 세포의 지방세포로의 분화 억제능을 측정한 결과 지방세포로의 분화를 유도한 MDI 처리군과 비교할 때 200 ㎍/㎖ 농도에서 대마 줄기 추출물과 대마 뿌리 추출물은 모두 약 13% 억제하는 효능을 나타내었지만 그 효능은 미비한 것으로 확인되었다.7 is a graph showing the measurement results of the inhibition of differentiation into adipocytes for 3T3-L1 cells of cannabis extract. As shown in FIG. 7 , the ability to inhibit pre-adipocyte differentiation into adipocytes was measured by diluting the hemp stem extract and hemp root extract at 50 and 200 ppm. Compared to the MDI-treated group that induced differentiation into adipocytes, 200 μg/ml At the concentration, both the hemp stem extract and the hemp root extract showed an inhibitory effect of about 13%, but the efficacy was confirmed to be insignificant.
4. 대마 추출물의 미백 활성 평가4. Evaluation of whitening activity of hemp extract
(1) Tyrosinase 효소 저해활성(Tyrosinase enzyme inhibition activity) 평가(1) Tyrosinase enzyme inhibition activity evaluation
대마 추출물의 Tyrosinase 효소 저해활성(Tyrosinase enzyme inhibition activity) 평가를 Laskin 등의 방법[Laskin, D. L., Pendino, K. J. 1995. Macrophages and inflammatory mediators in tissue injury. Annu. Rev. Pharmacol. Toxicol. 35: 655-677.]에 준하여 DOPA oxidase의 방법으로 진행하였다. 시료 10 ㎕에 110 Unit/㎖ Tyrosinase enzyme 20 ㎕를 첨가하고 1.5mM L-DOPA(3,4-Dihydroxyl phenylalanine) 170 ㎕를 혼합한 후 37℃에서 10분간 반응시킨 다음 450 nm에서 흡광도로 측정하였다. 대조군으로 Arbutin(Sigma co., USA)을 이용하였다. Tyrosinase 효소 저해활성은 다음과 같은 식으로 계산하였다.Tyrosinase enzyme inhibition activity of hemp extract was evaluated by Laskin et al. [Laskin, D. L., Pendino, K. J. 1995. Macrophages and inflammatory mediators in tissue injury. Annu. Rev. Pharmacol. Toxicol. 35: 655-677.], followed by the method of DOPA oxidase. 20 μl of 110 Unit/ml Tyrosinase enzyme was added to 10 μl of sample, and 170 μl of 1.5 mM L-DOPA (3,4-Dihydroxyl phenylalanine) was mixed, reacted at 37° C. for 10 minutes, and then absorbance was measured at 450 nm. Arbutin (Sigma co., USA) was used as a control. Tyrosinase enzyme inhibitory activity was calculated as follows.
도 8은 대마 추출물의 Tyrosinase 효소 저해활성 측정 결과를 나타낸 그래프이다. Tyrosinase는 멜라닌 합성 초기단계에 필수적으로 관여하는 산화효소이다. 멜라닌은 자외선으로부터 피부를 보호하기 위해 생성되지만 과잉 생성될 경우, 기미, 주근깨, 검버섯과 같은 피부 트러블 문제가 생기고 색소 침착으로 인해 피부색이 어두워지며 더 나아가 피부암을 유발하는 원인이 되는 것으로 알려져 있다. Tyrosinase 효소를 저해하게 되면 최종적으로 멜라닌의 과잉 생성을 막을 수 있기 때문에 피부트러블, 피부미백, 피부암 등의 문제를 예방할 수 있기 때문에 미백 효능 평가 방법 중 하나로 Tyrosinase 효소 저해활성 실험을 진행하였다. 도 8에서 보이는 바와 같이 대마 줄기 추출물 및 대마 뿌리 추출물은 5,000 ㎍/㎖ 농도에서 각각 26% 및 43%의 Tyrosinase 효소 저해활성을 보였으나, 500 ㎍/㎖ 농도에서 대조군으로 사용한 Vitmin C의 경우 37%의 저해활성을 보인 반면, 대마 줄기 추출물 및 대마 뿌리 추출물은 500 ㎍/㎖ 농도에서 각각 4% 및 16%의 저해활성을 보여 그 효능은 미비한 것으로 확인되었다.8 is a graph showing the results of measuring the tyrosinase enzyme inhibitory activity of the hemp extract. Tyrosinase is an oxidative enzyme essential in the initial stage of melanin synthesis. Melanin is produced to protect the skin from UV rays, but when it is excessively produced, skin trouble problems such as spots, freckles, and age spots occur, and the skin color becomes dark due to pigmentation. It is known that it causes skin cancer. Tyrosinase enzyme inhibitory activity was tested as one of the methods for evaluating the efficacy of whitening, because inhibition of the tyrosinase enzyme can prevent the excessive production of melanin, thereby preventing problems such as skin troubles, skin whitening, and skin cancer. As shown in FIG. 8 , the hemp stem extract and hemp root extract showed tyrosinase enzyme inhibitory activity of 26% and 43%, respectively, at a concentration of 5,000 μg/ml, but 37% in the case of Vitmin C used as a control at a concentration of 500 μg/ml. On the other hand, the hemp stem extract and hemp root extract showed inhibitory activity of 4% and 16%, respectively, at a concentration of 500 μg/ml, confirming that the efficacy was insignificant.
(2) B16F10 Cell에 대한 세포 독성 평가(MTT assay)(2) Cytotoxicity evaluation for B16F10 cells (MTT assay)
96-well plate에 B16F10 멜라닌세포를 1×106 cells/well로 동일하게 분주하고 24 hr 동안 배양하였다. 이후, 기존의 배지를 제거하고 새로운 배지를 넣어준 후 시료를 농도 의존적으로(50, 200 ㎍/㎖)을 넣고 4 hr 동안 배양한 후 상등액을 제거하고 well에 형성된 formazan은 DMSO 100 ㎕를 첨가하여 용해시킨 후 570 nm에서 흡광도를 측정하였다. 시료를 처리하지 않은 음성대조군을 100%의 생존율로 간주하고 이를 기준으로 세포 생존율을 비교하였다. B16F10 melanocytes were equally distributed at 1×10 6 cells/well in a 96-well plate and cultured for 24 hr. Then, after removing the old medium and adding a new medium, the sample was added concentration-dependently (50, 200 μg/ml), incubated for 4 hr, the supernatant was removed, and formazan formed in the well was added with 100 μl of DMSO. After dissolution, absorbance was measured at 570 nm. A negative control group that was not treated with the sample was regarded as 100% viability, and cell viability was compared based on this.
도 9는 대마 추출물의 B16F10 Cell에 대한 세포 독성 평가(MTT assay) 결과를 나타낸 그래프이다. B16F10 Cell은 멜라닌 세포로서 표피 기저층에 존재하며, 세포질내의 멜라노좀에서 멜라닌이 합성된다. 멜라닌 합성은 자외선, cytokine, 성장인자 및 호르몬 등에 의해 조절되고 세포내의 다양한 기전을 통해 이루어진다. 도 9에서 보이는 바와 같이, 피부 멜라닌 세포에 대한 대마 줄기 추출물 및 대마 뿌리 추출물의 세포독성을 측정한 결과 50, 200 ㎍/㎖ 농도까지 세포독성을 나타내지 않았다.9 is a graph showing the results of cytotoxicity evaluation (MTT assay) for B16F10 cells of cannabis extract. B16F10 Cells are melanocytes that exist in the basal layer of the epidermis, and melanin is synthesized in melanosomes in the cytoplasm. Melanin synthesis is regulated by ultraviolet rays, cytokines, growth factors and hormones, and is accomplished through various mechanisms within cells. As shown in FIG. 9 , as a result of measuring the cytotoxicity of the hemp stem extract and hemp root extract to skin melanocytes, cytotoxicity was not shown up to concentrations of 50 and 200 μg/ml.
(3) B16F10 Cell에 대한 멜라닌 생성 저해활성 평가(3) Evaluation of melanin production inhibitory activity against B16F10 Cell
B16F10 cell을 12-well plate에 0.5×105 cells/mL이 되도록 접종하고 24 hr 동안 배양한 후, 각 well에 시료와 200 nM의 melanin stimulating hormone(α-MSH)을 48 hr 동안 처리하였다. 이후, phosphate buffered saline (PBS)로 2회 세척한 후 lysis buffer(0.1 M sodium phosphate buffer, 1% triton X-100, 0.2 mM PMSF)를 이용하여 세포를 용해시켰다. 이후, Cell scraper를 이용하여 cell을 긁어내고 원심분리하여 세포 침전물을 만든 후 10% dimethyl sulfoxide(DMSO)가 첨가된 1 N NaOH 용액을 200 μL 첨가하고 80℃에서 1 hr 동안 용해한 후 405 nm에서 흡광도를 측정하였다. 멜라닌 양은 합성 멜라닌을 사용하여 작성된 표준 직선으로로부터 구하고, 실험군의 멜라닌 양을 정상군의 멜라닌 양에 대한 백분율로 계산하였다. 또한, 양성대조군으로는 arbutin(100 μg/mL)을 사용하였다.B16F10 cells were inoculated into a 12-well plate so that 0.5×10 5 cells/mL was inoculated and cultured for 24 hr, and then a sample and 200 nM melanin stimulating hormone (α-MSH) were treated in each well for 48 hr. Then, after washing twice with phosphate buffered saline (PBS), cells were lysed using a lysis buffer (0.1 M sodium phosphate buffer, 1% triton X-100, 0.2 mM PMSF). Then, after scraping the cells using a cell scraper and centrifuging to make a cell precipitate, 200 µL of 1 N NaOH solution with 10% dimethyl sulfoxide (DMSO) was added, dissolved at 80 °C for 1 hr, and absorbance at 405 nm was measured. The amount of melanin was obtained from a standard straight line prepared using synthetic melanin, and the amount of melanin in the experimental group was calculated as a percentage of the amount of melanin in the normal group. In addition, arbutin (100 μg/mL) was used as a positive control.
도 10은 대마 추출물의 B16F10 Cell에 대한 멜라닌 생성 저해활성 평가 결과를 나타낸 그래프이다. 멜라닌은 자외선이나 외부자극으로부터 피부를 보호하는 긍정적인 기능을 하고 있지만 과도한 멜라닌 생성과 비정상적인 분포는 비정상적인 과색소침착의 원인이 되며 피부에 흑색종 및 색소침착을 일으켜 심할 경우 피부암의 원인이 되기도 한다. α-MSH에 의해 멜라닌 합성이 활성화된 B16F10 세포에 대해 대마 줄기 추출물 및 대마 뿌리 추출물이 멜라닌 생성에 미치는 억제 효과를 알아보았다. 도 10에서 보이는 바와 같이 대마 줄기 추출물 및 대마 뿌리 추출물은 200 ㎍/㎖ 농도에서 각각 약 19% 및 약 15% 정도 멜라닌 생성을 억제하는 것을 확인할 수 있었지만 그 효능은 미비한 것으로 사료된다.10 is a graph showing the evaluation results of melanin production inhibitory activity against B16F10 Cells of cannabis extract. Melanin has a positive function to protect the skin from UV rays or external stimuli, but excessive melanin production and abnormal distribution cause abnormal hyperpigmentation, causing melanoma and pigmentation in the skin, which in severe cases can cause skin cancer. The inhibitory effect of cannabis stem extract and hemp root extract on melanin production on B16F10 cells in which melanin synthesis was activated by α-MSH was investigated. As shown in FIG. 10 , it was confirmed that the cannabis stem extract and the hemp root extract inhibit melanin production by about 19% and about 15%, respectively, at a concentration of 200 μg/ml, but the efficacy is considered to be insignificant.
5. 대마 추출물의 항염증 활성 평가5. Evaluation of anti-inflammatory activity of hemp extract
대마 추출물의 항염증 활성은 대마 추출물의 RAW 264.7 Cell에 대한 NO 생성 저해활성을 통해 평가하였다. 96-well plate에 1×106 cells/well이 되도록 분주하고 5% CO2와 37℃가 유지되는 배양기에서 24 hr 동안 배양하여 세포를 well에 부착시켰다. 이후, 그 후 기존 배지를 제거하고, 시료를 농도 의존적으로(25, 50, 100, 200 ㎍/㎖) 처리하고 1 hr 동안 배양하고, LPS(1 ㎍/㎖)를 처리한 후 23 hr 동안 배양하였다. 배양 완료 후 상등액 100 ㎕를 취하고, 여기에 동량의 Griess 시약을 첨가하고 10분간 반응시킨 후 540 nm에서 흡광도를 측정하였다.The anti-inflammatory activity of the hemp extract was evaluated through the NO production inhibitory activity of the hemp extract against RAW 264.7 Cells. Dispense to 1×10 6 cells/well in a 96-well plate, and incubate for 24 hr in an incubator maintained at 5% CO 2 and 37° C. to attach the cells to the wells. After that, the old medium is removed, and the sample is treated concentration-dependently (25, 50, 100, 200 μg/ml) and cultured for 1 hr, and then cultured for 23 hr after treatment with LPS (1 μg/ml). did. After completion of the culture, 100 μl of the supernatant was taken, the same amount of Griess reagent was added thereto, and the absorbance was measured at 540 nm after reacting for 10 minutes.
도 11은 대마 추출물의 RAW 264.7 Cell에 대한 NO 생성 저해활성 평가(NO assay) 결과를 나타낸 그래프이다. NO는 면역반응, 세포독성, 신경전달계 및 혈관이완 등 여러 가지 생물학적인 과정에 관여하는 것으로 알려져 있으며, LPS 자극에 의해 발현된 iNOS는 많은 양의 NO를 생성하게 되며 이에 대한 세포독성은 염증반응, 세포의 돌연변이 및 종양 발생 등에도 관여하는 것으로 알려져 있는바, 대마 추출물의 항염증 효능을 NO 생성 저해 활성 평가를 통해 진행하였다. 도 11에서 보이는 바와 같이 대마 줄기 추출물, 대마 뿌리 추출물 및 대마 종자 추출물은 200 ㎍/㎖의 농도에서 각각 약 54%, 약 45% 및 약 약 50% 수준으로 NO 생성을 억제하는 것으로 나타났고, 대마 줄기 추출물이 가장 우수한 항염증 효능을 보였으나, 대마 종자 추출물과 큰 차이를 보이지 않았다.11 is a graph showing the results of evaluation of NO production inhibitory activity (NO assay) for RAW 264.7 Cells of cannabis extract. NO is known to be involved in various biological processes such as immune response, cytotoxicity, neurotransmission and vascular relaxation. As it is known to be involved in cell mutation and tumor development, the anti-inflammatory efficacy of the hemp extract was evaluated through the NO production inhibitory activity evaluation. As shown in FIG. 11 , the hemp stem extract, hemp root extract and hemp seed extract were shown to inhibit NO production at a concentration of 200 μg/ml, respectively, at about 54%, about 45%, and about 50%, and hemp Although the stem extract showed the best anti-inflammatory effect, there was no significant difference from the hemp seed extract.
6. 대마 추출물의 항당뇨 활성 평가6. Evaluation of antidiabetic activity of hemp extract
대마 추출물의 α-glucosidase 효소 저해활성(α-glucosidase enzyme inhibition activity)을 Choe 등의 방법에 따라 측정하고 대마 추출물의 항당뇨 활성을 평가하였다. 시료 25 ㎕에 2.5 mM ρNPG(ρ-nitrophenol glucoside) 50 ㎕를 첨가하고 여기에 0.2 Unit/㎖ α-glucosidase enzyme 25 ㎕를 혼합한 후 37℃에서 20분간 반응시켰다. 이후, 1 M NaOH 50 ㎕를 첨가하여 반응을 정지시킨 후 405 nm에서 흡광도를 측정하였다. 또한, 대조군으로 Acarbose(Sigma co., USA)를 이용하였다. α-glucosidase 효소 저해활성은 다음과 같은 식으로 계산하였다.The α-glucosidase enzyme inhibition activity of the hemp extract was measured according to the method of Choe et al., and the antidiabetic activity of the hemp extract was evaluated. To 25 μl of the sample, 50 μl of 2.5 mM ρNPG (ρ-nitrophenol glucoside) was added, and 25 μl of 0.2 Unit/ml α-glucosidase enzyme was mixed thereto, followed by reaction at 37° C. for 20 minutes. Then, 50 μl of 1 M NaOH was added to stop the reaction, and absorbance was measured at 405 nm. In addition, Acarbose (Sigma co., USA) was used as a control. The α-glucosidase enzyme inhibitory activity was calculated as follows.
도 12는 대마 추출물의 α-glucosidase 효소 저해활성(α-glucosidase enzyme inhibition activity) 측정 결과를 나타낸 그래프이다. α-glucosidase는 소장점막의 미세융모막에 존재하는 효소로써 다당류의 탄수화물을 단당류로 분해하기 때문에 탄수화물의 소화와 흡수에 필수적인 효소이다. 소장내의 점막에서 α-glucosidase의 효소 활성을 저해하면 다당류의 분해를 방해하고, 소장에서 glucose의 흡수를 지연시켜 식후 혈당의 급격한 상승을 제어할 수 있다. 도 12에서 보이는 바와 같이 대마 줄기 추출물 및 대마 뿌리 추출물은 100 ㎍/㎖ 농도에서 각각 59% 및 65%의 α-glucosidase 효소 저해활성을 나타내었고, 동일 처리 농도의 대마 종자 추출물과 비교하였을 때 약 10배 이상의 효능을 보였다. 또한, 표준품으로 사용한 Acarbose가 500㎍/㎖ 농도에서 36%의 α-glucosidase 효소 저해활성을 나타낸 것과 비교하였을 때에도 대마 줄기 추출물 및 대마 뿌리 추출물은 낮은 처리 농도에서도 현저하게 높은 저해활성 값을 나타내고 있는바, 항당뇨 효능이 매우 우수한 것으로 사료된다.12 is a graph showing the measurement results of α-glucosidase enzyme inhibition activity of the cannabis extract. α-glucosidase is an enzyme that exists in the microchorionic membrane of the small intestine mucosa and is essential for digestion and absorption of carbohydrates because it breaks down carbohydrates from polysaccharides into monosaccharides. Inhibiting the enzymatic activity of α-glucosidase in the mucous membrane of the small intestine interferes with the decomposition of polysaccharides and delays the absorption of glucose in the small intestine, thereby controlling the rapid rise in postprandial blood glucose. As shown in FIG. 12 , the hemp stem extract and hemp root extract exhibited 59% and 65% of α-glucosidase enzyme inhibitory activity at a concentration of 100 μg/ml, respectively, and compared to the hemp seed extract at the same treatment concentration, about 10 showed more than double the efficacy. In addition, even when Acarbose used as a standard product showed 36% α-glucosidase enzyme inhibitory activity at a concentration of 500 μg/ml, the hemp stem extract and hemp root extract showed significantly higher inhibitory activity values even at low treatment concentrations. , is considered to have very good antidiabetic effect.
7. 대마 줄기 추출물을 포함하는 약학 조성물의 제조7. Preparation of Pharmaceutical Composition Containing Hemp Stem Extract
하기의 약학 조성물 제조에서 대마 줄기 추출물은 상기의 제조예 1에서 수득한 것이다.In the preparation of the following pharmaceutical composition, the hemp stem extract was obtained in Preparation Example 1 above.
<7-1> 산제의 제조<7-1> Preparation of powder
대마 줄기 추출물 20 ㎎Hemp Stem Extract 20 mg
유당 100 ㎎
탈크 10 ㎎
상기의 성분을 혼합하고 기밀포에 충진하여 산제를 제조하였다.The above ingredients were mixed and filled in an airtight cloth to prepare a powder.
<7-2> 정제의 제조<7-2> Preparation of tablets
대마 줄기 추출물 10 ㎎Hemp Stem Extract 10 mg
옥수수전분 100 ㎎
유 당 100 ㎎
스테아린산 마그네 2 ㎎Magne stearate 2 mg
상기의 성분을 혼합한 후, 통상의 정제의 제조방법에 따라서 타정하여 정제를 제조하였다.After mixing the above ingredients, tablets were prepared by tableting according to a conventional manufacturing method of tablets.
<7-3> 캡슐제의 제조<7-3> Preparation of capsules
대마 줄기 추출물 10 ㎎Hemp Stem Extract 10 mg
결정성 셀룰로오스 3 ㎎3 mg of crystalline cellulose
유 당 15 ㎎Lactose 15 mg
스테아린산 마그네슘 0.2 ㎎Magnesium stearate 0.2 mg
상기의 성분을 혼합한 후, 통상의 캡슐제의 제조방법에 따라서 젤라틴 캡슐에 충전하여 캡슐제를 제조하였다.After mixing the above ingredients, the capsules were prepared by filling in gelatin capsules according to a conventional manufacturing method of capsules.
<7-4> 환의 제조<7-4> Preparation of the ring
대마 줄기 추출물 10 ㎎Hemp Stem Extract 10 mg
유당 150 ㎎Lactose 150 mg
글리세린 100 ㎎100 mg of glycerin
자일리톨 50 ㎎
상기의 성분을 혼합한 후, 통상의 방법에 따라 1환 당 4 g이 되도록 제조하였다.After mixing the above components, it was prepared so as to be 4 g per ring according to a conventional method.
<7-5> 과립의 제조<7-5> Preparation of granules
대마 줄기 추출물 15 ㎎Hemp Stem Extract 15 mg
대두추출물 50 ㎎Soybean extract 50 mg
포도당 200 ㎎
전분 600 ㎎Starch 600 mg
상기의 성분을 혼합한 후, 30% 에탄올 100 ㎎을 첨가하여 섭씨 60 ℃에서 건조하여 과립을 형성한 후 포에 충진하였다.After mixing the above components, 100 mg of 30% ethanol was added and dried at 60° C. to form granules, and then filled in a bag.
<7-6> 주사제의 제조<7-6> Preparation of injection
대마 줄기 추출물 10 ㎎Hemp Stem Extract 10 mg
소디움 메타비설파이트 3.0 ㎎Sodium metabisulfite 3.0 mg
메틸파라벤 0.8 ㎎Methylparaben 0.8 mg
프로필파라벤 0.1 ㎎Propylparaben 0.1 mg
주사용 멸균증류수 적량Proper amount of sterile distilled water for injection
상기의 성분을 혼합한 후, 이중 2㎖를 앰플에 충전하고 멸균하여 주사제를 제조하였다.After mixing the above ingredients, 2 ml of them were filled in an ampoule and sterilized to prepare an injection.
8. 대마 줄기 추출물을 포함하는 식품 조성물의 제조8. Preparation of food composition comprising hemp stem extract
하기의 식품 조성물 제조에서 대마 줄기 추출물은 상기의 제조예 1에서 수득한 것이다.In the preparation of the following food composition, the hemp stem extract was obtained in Preparation Example 1 above.
<8-1> 밀가루 식품의 제조<8-1> Manufacturing of wheat flour food
밀가루 100 중량부에 대마 줄기 추출물 0.5 중량부를 밀가루에 첨가하고, 이 혼합물을 이용하여 빵, 케이크, 쿠키, 크래커 및 면류를 제조하였다.0.5 parts by weight of hemp stem extract was added to 100 parts by weight of wheat flour, and breads, cakes, cookies, crackers and noodles were prepared using this mixture.
<8-2> 유제품(dairy products)의 제조<8-2> Production of dairy products
우유 100 중량부에 대마 줄기 추출물 0.5 중량부를 우유에 첨가하고, 상기 우유를 이용하여 버터 및 아이스크림과 같은 다양한 유제품을 제조하였다.0.5 parts by weight of hemp stem extract was added to 100 parts by weight of milk, and various dairy products such as butter and ice cream were prepared using the milk.
<8-3> 선식의 제조<8-3> Manufacture of wire type
현미, 보리, 찹쌀, 율무를 공지의 방법으로 알파화시켜 건조시킨 것을 배전한 후 분쇄기로 입도 60 메쉬의 분말로 제조하였다.Brown rice, barley, glutinous rice, and barley radish were pregelatinized by a known method and dried, and then roasted and prepared as a powder having a particle size of 60 mesh with a grinder.
검정콩, 검정깨, 들깨도 공지의 방법으로 쪄서 건조시킨 것을 배전한 후 분쇄기로 입도 60 메쉬의 분말로 제조하였다.Black soybeans, black sesame, and perilla were also steamed and dried by a known method, and then roasted and prepared into powder having a particle size of 60 mesh with a grinder.
상기에서 제조한 곡물류, 종실류 및 대마 줄기 추출물을 다음의 비율로 배합하여 제조하였다.It was prepared by mixing the grains, seeds and hemp stem extracts prepared above in the following ratios.
곡물류(현미 30 중량부, 율무 17 중량부, 보리 20 중량부),Grains (30 parts by weight of brown rice, 17 parts by weight of barley, 20 parts by weight of barley),
종실류(들깨 7 중량부, 검정콩 8 중량부, 검정깨 7 중량부),Seeds (7 parts by weight of perilla, 8 parts by weight of black beans, 7 parts by weight of black sesame),
대마 줄기 추출물(1 중량부),Hemp stem extract (1 part by weight),
영지(0.5 중량부),Reishi (0.5 parts by weight),
지황(0.5 중량부)Rehmannia (0.5 parts by weight)
<8-4> 건강음료의 제조<8-4> Manufacturing of health drinks
액상과당(0.5 g), 올리고당(4 g), 설탕(2 g), 식염(0.5 g), 물(77 g)과 같은 부재료와 대마 줄기 추출물 1 g을 균질하게 배합하여 순간 살균을 한 후 이를 유리병, 패트병 등 소포장 용기에 포장하여 제조하였다.Sub-materials such as high fructose (0.5 g), oligosaccharide (4 g), sugar (2 g), salt (0.5 g), and water (77 g) and 1 g of hemp stem extract are homogeneously mixed and instantaneously sterilized. It was manufactured by packaging in small packaging containers such as glass bottles and plastic bottles.
<8-5> 야채 주스의 제조<8-5> Preparation of vegetable juice
대마 줄기 추출물 2 g을 토마토 또는 당근 주스 1,000 ㎖에 가하여 야채 주스를 제조하였다.Vegetable juice was prepared by adding 2 g of hemp stem extract to 1,000 ml of tomato or carrot juice.
<8-6> 과일 주스의 제조<8-6> Preparation of fruit juice
대마 줄기 추출물 1 g을 사과 또는 포도 주스 1,000 ㎖ 에 가하여 과일 주스를 제조하였다.Fruit juice was prepared by adding 1 g of hemp stem extract to 1,000 ml of apple or grape juice.
이상에서와 같이 본 발명을 상기의 실시예를 통해 설명하였지만 본 발명이 반드시 여기에만 한정되는 것은 아니며 본 발명의 범주와 사상을 벗어나지 않는 범위 내에서 다양한 변형실시가 가능함은 물론이다. 따라서, 본 발명의 보호범위는 본 발명에 첨부된 특허청구의 범위에 속하는 모든 실시 형태를 포함하는 것으로 해석되어야 한다.As described above, the present invention has been described through the above embodiments, but the present invention is not necessarily limited thereto, and various modifications are possible without departing from the scope and spirit of the present invention. Accordingly, the protection scope of the present invention should be construed to include all embodiments falling within the scope of the claims appended hereto.
Claims (9)
상기 대마 줄기 추출물의 추출 용매는 에탄올 농도가 55~95%(w/w)인 함수 에탄올인 것을 특징으로 하는 항당뇨용 조성물.
As a composition comprising a hemp stem extract or comprising a hemp stem extract as an active ingredient,
The extract solvent of the hemp stem extract is an antidiabetic composition, characterized in that the ethanol concentration is 55 to 95% (w / w) hydrous ethanol.
1) 대마 줄기를 에탄올 농도가 55~95%(w/w)인 함수 에탄올로 추출하여 추출액을 얻는 단계;
2) 상기 추출액을 여과하여 잔사를 제거하고 여과된 추출액을 얻는 단계; 및
3) 상기 여과된 추출액을 농축하고 건조하여 고상의 추출물을 얻는 단계.
According to claim 1, wherein the cannabis stem extract is antidiabetic composition, characterized in that obtained through the following steps:
1) extracting the hemp stem with hydrous ethanol having an ethanol concentration of 55 to 95% (w/w) to obtain an extract;
2) filtering the extract to remove residues and obtaining a filtered extract; and
3) Concentrating and drying the filtered extract to obtain a solid extract.
상기 대마 줄기 추출물의 추출 용매는 에탄올 농도가 55~95%(w/w)인 함수 에탄올인 것을 특징으로 하는 당뇨병 예방 또는 치료용 약학 조성물.
As a composition comprising a hemp stem extract or comprising a hemp stem extract as an active ingredient,
The extraction solvent of the hemp stem extract is a pharmaceutical composition for preventing or treating diabetes, characterized in that the ethanol concentration is 55 to 95% (w/w) hydrous ethanol.
1) 대마 줄기를 에탄올 농도가 55~95%(w/w)인 함수 에탄올로 추출하여 추출액을 얻는 단계;
2) 상기 추출액을 여과하여 잔사를 제거하고 여과된 추출액을 얻는 단계; 및
3) 상기 여과된 추출액을 농축하고 건조하여 고상의 추출물을 얻는 단계.
The pharmaceutical composition according to claim 4, wherein the cannabis stem extract is obtained through the following steps:
1) extracting the hemp stem with hydrous ethanol having an ethanol concentration of 55 to 95% (w/w) to obtain an extract;
2) filtering the extract to remove residues and obtaining a filtered extract; and
3) Concentrating and drying the filtered extract to obtain a solid extract.
상기 대마 줄기 추출물의 추출 용매는 에탄올 농도가 55~95%(w/w)인 함수 에탄올인 것을 특징으로 하는 당뇨병 예방 또는 개선용 식품 조성물.
As a composition comprising a hemp stem extract or comprising a hemp stem extract as an active ingredient,
The extraction solvent of the hemp stem extract is a food composition for preventing or improving diabetes, characterized in that the ethanol concentration is 55 to 95% (w/w) hydrous ethanol.
1) 대마 줄기를 에탄올 농도가 55~95%(w/w)인 함수 에탄올로 추출하여 추출액을 얻는 단계;
2) 상기 추출액을 여과하여 잔사를 제거하고 여과된 추출액을 얻는 단계; 및
3) 상기 여과된 추출액을 농축하고 건조하여 고상의 추출물을 얻는 단계.The food composition according to claim 7, wherein the hemp stem extract is obtained through the following steps:
1) extracting the hemp stem with hydrous ethanol having an ethanol concentration of 55 to 95% (w/w) to obtain an extract;
2) filtering the extract to remove residues and obtaining a filtered extract; and
3) Concentrating and drying the filtered extract to obtain a solid extract.
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US20070099987A1 (en) * | 2004-02-16 | 2007-05-03 | Lola Weiss | Treating or preventing diabetes with cannabidiol |
JP2011509989A (en) | 2008-01-21 | 2011-03-31 | ジーダブリュー・ファーマ・リミテッド | New uses for cannabinoids |
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