KR102603687B1 - Food Composition with Anti-obesity activity containing Stevia extract - Google Patents
Food Composition with Anti-obesity activity containing Stevia extract Download PDFInfo
- Publication number
- KR102603687B1 KR102603687B1 KR1020200144730A KR20200144730A KR102603687B1 KR 102603687 B1 KR102603687 B1 KR 102603687B1 KR 1020200144730 A KR1020200144730 A KR 1020200144730A KR 20200144730 A KR20200144730 A KR 20200144730A KR 102603687 B1 KR102603687 B1 KR 102603687B1
- Authority
- KR
- South Korea
- Prior art keywords
- stevia extract
- stevia
- extract
- food composition
- green tea
- Prior art date
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Classifications
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- A—HUMAN NECESSITIES
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- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L19/00—Products from fruits or vegetables; Preparation or treatment thereof
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
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Abstract
본 발명은 스테비아 추출물을 함유하는 식품조성물에 관한 것으로, 본 발명에 따르면, 신생혈관형성 억제기능을 통해 항비만 효과를 나타내는 스테비아 추출물을 함유하는 항비만 식품조성물을 제공할 수 있다.
또한 녹차 추출물을 더 함유하여 보다 효과가 향상된 항비만 식품조성물을 제공할 수 있다.The present invention relates to a food composition containing a stevia extract. According to the present invention, an anti-obesity food composition containing a stevia extract that exhibits an anti-obesity effect through an anti-angiogenesis function can be provided.
In addition, it is possible to provide an anti-obesity food composition with improved effectiveness by containing more green tea extract.
Description
본 발명은 스테비아 추출물을 함유하는 항비만 식품조성물에 관한 것으로, 더욱 자세하게는 신생혈관형성 억제기능을 가지는 스테비아 추출물 단독 또는 녹차 추출물과 혼합하여 사용함으로써, 우수한 항비만 효과를 나타내며 부작용이 없는 스테비아 추출물을 함유하는 항비만 식품조성물에 관한 것이다.The present invention relates to an anti-obesity food composition containing stevia extract. More specifically, the stevia extract, which has an anti-angiogenesis function, is used alone or mixed with green tea extract to produce a stevia extract that exhibits excellent anti-obesity effect and has no side effects. It relates to an anti-obesity food composition containing it.
현재 저출산과 생명공학기술 발달로 평균수명이 연장되고 있으며 우리나라 고령인구 비율이 계속 증가되고 있어 건강관리에 대한 관심 또한 높아지고 있는 추세이다.Currently, the average life expectancy is increasing due to low birth rates and the development of biotechnology, and as the proportion of the elderly population in Korea continues to increase, interest in health care is also increasing.
한편, 대사성질환을 관리하기 위해서는 여러 의약품이 사용되고 있으나 만성질환이라는 측면에서 식습관 및 운동을 통한 예방이 강조되고 있다. 이를 타깃으로 하여 여러 카테고리의 식품이 개발 유통되고 있다.Meanwhile, various medicines are used to manage metabolic diseases, but prevention through diet and exercise is emphasized in the context of chronic diseases. Several food categories are being developed and distributed targeting this.
식품 중 대사성질환을 타깃으로 한 식품에는 일부 대사성질환과 관련이 있는 기능성을 표현하는 건강기능식품, 대사성질환을 타깃으로 하는 체중조절용식품, 대사성질환의 유발을 저감화할 수 있는 일부 일반식품 등이 해당한다.Among foods, foods targeting metabolic diseases include health functional foods that express functionality related to some metabolic diseases, foods for weight control that target metabolic diseases, and some general foods that can reduce the occurrence of metabolic diseases. do.
국민건강영양조사 분석결과에 의하면 국내 30세 이상 성인 중 28.85가 대사증후군 유병율을 보이고 있으며, 대사증후군 5개의 구성 요소(허리둘레, 중성지방, 고밀도지방, 혈압, 공복혈당) 중 1개 이상 요소에서 기준치를 초과한 대상자는 73.7%에 해당한다고 나타났다.According to the results of the National Health and Nutrition Examination Survey, 28.85 of adults over 30 years of age in Korea have a prevalence of metabolic syndrome, and at least one of the five components of metabolic syndrome (waist circumference, neutral fat, high-density fat, blood pressure, and fasting blood sugar) is present. It was found that 73.7% of the subjects exceeded the standard.
이와 같은 대사성질환을 유발하는 원인 중 하나인 비만은 성장하는 소아청소년의 건강을 위협하는 원인이며, 특히 소아와 청소년 비만은 성인 비만으로 이행할 가능성이 높고, 대사성질환 외 심혈관질환, 위장질환, 폐질환, 골관절질환, 인슐린저항성 등의 합병증을 유발하여 성인기 건강에도 영향을 미칠 수 있다.Obesity, one of the causes of metabolic diseases, is a threat to the health of growing children and adolescents. In particular, obesity in children and adolescents has a high possibility of transitioning to adult obesity, and in addition to metabolic diseases, cardiovascular diseases, gastrointestinal diseases, and lung diseases. It can also affect health in adulthood by causing complications such as disease, bone and joint disease, and insulin resistance.
특히 우리나라 소아청소년에서 대사증후군의 유병율은 전반전으로 6.1에서 10.4%까지 다양하게 보고되고 있으며, 30세 이상에서 고혈압, 당뇨병 그리고 고콜레스테롤 혈증 유병율 모두 매우 높게 나타나고 있는 상황이다.In particular, the prevalence of metabolic syndrome in children and adolescents in Korea is reported to vary from 6.1 to 10.4%, and the prevalence of hypertension, diabetes, and hypercholesterolemia are all very high in people over 30 years of age.
이에 다양한 체지방 감소 관련 기능성 식품, 비만 치료제 등이 개발되고 있는 실정이며, 현재까지의 비만 치료제는 식용을 억제시키는 제제와 지방의 대사를 촉진시키거나 지방질의 합성을 억제시키거나 대사활성을 증가시키는 제제로 크게 나눌 수 있었다.Accordingly, various functional foods related to body fat reduction and obesity treatments are being developed, and the obesity treatments to date include agents that inhibit edibility and agents that promote fat metabolism, inhibit lipid synthesis, or increase metabolic activity. It could be broadly divided into
그러나, 이러한 식품과 치료제들은 합성제제로 이루어져 부작용이 많을 뿐만 아니라 습관성 문제도 대두되고 있다.However, these foods and treatments are made of synthetic products and not only have many side effects, but also have the problem of habituation.
이에 따라, 천연소재를 이용하여 부작용이 없으면서도 항비만 효과를 통해 체중 조절에 도움을 줄 수 있는 제제의 개발이 필요하다.Accordingly, there is a need to develop an agent using natural materials that has no side effects and can help control body weight through an anti-obesity effect.
상기와 같은 문제를 해결하고자, 본 발명은 신생혈관형성 억제기능을 가지는 스테비아 추출물 단독 또는 녹차 추출물과 혼합하여 사용함으로써, 우수한 항비만 효과를 나타내며 부작용이 없는 스테비아 추출물을 함유하는 항비만 식품조성물을 제공하는 데 목적이 있다.In order to solve the above problems, the present invention provides an anti-obesity food composition containing a stevia extract that exhibits excellent anti-obesity effects and has no side effects by using the stevia extract alone or mixed with green tea extract, which has an anti-angiogenic function. The purpose is to do this.
상기와 같은 과제를 해결하기 위하여, 본 발명의 실시예에 따른 스테비아 추출물을 함유하는 항비만 식품조성물을 제공할 수 있다.In order to solve the above problems, an anti-obesity food composition containing stevia extract according to an embodiment of the present invention can be provided.
여기서, 스테비아 추출물을 함유하는 항비만 식품조성물은 신생혈관억제 기능을 가지는 것을 특징으로 한다.Here, the anti-obesity food composition containing stevia extract is characterized by having an anti-angiogenic function.
또한, 상기 스테비아 추출물은 스테비아 잎으로부터 스테비아 추출물을 추출하는 추출단계 및 추출된 스테비아 추출물을 정제하는 정제단계를 통해 제조되는 것을 특징으로 한다.In addition, the stevia extract is characterized in that it is produced through an extraction step of extracting the stevia extract from stevia leaves and a purification step of purifying the extracted stevia extract.
또한, 상기 추출단계는 스테비아 잎을 분쇄하여 스테비아 분말로 제조하는 전처리단계; 상기 스테비아 분말에서 스테비아 추출물을 추출하는 추출물추출단계; 상기 스테비아 추출물을 농축하는 농축단계 및 농축된 스테비아 추출물을 동결건조시키는 동결건조단계를 포함할 수 있다.In addition, the extraction step includes a pretreatment step of grinding stevia leaves to produce stevia powder; An extract extraction step of extracting stevia extract from the stevia powder; It may include a concentration step of concentrating the stevia extract and a freeze-drying step of freeze-drying the concentrated stevia extract.
또한, 상기 전처리단계는 분쇄된 스테비아 분말을 n-헥산으로 탈지한 후 건조시키는 것을 특징으로 한다.In addition, the pretreatment step is characterized in that the pulverized stevia powder is degreased with n-hexane and then dried.
또한, 상기 추출물추출단계는 상기 스테비아 분말을 에탄올에 용해시켜 환류를 통해 스테비아 추출물을 추출하는 것을 특징으로 한다.In addition, the extract extraction step is characterized in that the stevia powder is dissolved in ethanol and the stevia extract is extracted through reflux.
또한, 상기 정제단계는 추출된 스테비아 추출물을 증류수에 용해시키고 컬럼(Column)에 충전시킨 후, 상기 컬럼에 수지를 첨가하여 비극성 물질을 분리시키는 제1 정제단계; 상기 컬럼에 수산화나트륨(NaOH)을 통과시켜 산성 물질을 용출시키는 제2 정제단계; 산성물질이 제거된 스테비아 추출물에 염화수소(HCl)을 첨가하여 중화시키는 중화단계 및 중화된 스테비아 추출물을 컬럼(Column)에 충전시킨 후, 에탄올을 통해 용출시켜 비극성 물질이 제거된 스테비아 추출물을 획득하는 제3 정제단계를 포함할 수 있다.In addition, the purification step includes a first purification step of dissolving the extracted stevia extract in distilled water and filling it in a column, and then adding resin to the column to separate non-polar substances; A second purification step of eluting acidic substances by passing sodium hydroxide (NaOH) through the column; A neutralization step of adding hydrogen chloride (HCl) to the stevia extract from which acidic substances have been removed, filling a column with the neutralized stevia extract, and then eluting with ethanol to obtain a stevia extract from which non-polar substances have been removed. It may include 3 purification steps.
또한, 상기 제3 정제단계에서 에탄올은 100% 에탄올인 것을 특징으로 한다.In addition, the ethanol in the third purification step is characterized in that it is 100% ethanol.
또한, 본 발명의 실시예에 따른 스테비아 추출물을 함유한 항비만 식품조성물은 녹차 추출물을 더 함유할 수 있다.Additionally, the anti-obesity food composition containing stevia extract according to an embodiment of the present invention may further contain green tea extract.
여기서, 상기 녹차 추출물은 녹차 사포닌을 포함하는 것을 특징으로 한다.Here, the green tea extract is characterized in that it contains green tea saponin.
또한 상기 녹차 사포닌은 녹차의 테아사포닌-E1(theasaponin E1)을 포함하는 것을 특징으로 한다.In addition, the green tea saponin is characterized in that it includes green tea theasaponin-E1 (theasaponin E1).
상기와 같은 본 발명의 실시예에 따른 스테비아 추출물을 함유하는 항비만 식품조성물은 신생혈관형성 억제기능을 가지는 스테비아 추출물을 함유함으로써, 항비만 효과를 나타내 체중조절에 도움을 줄 수 있다.The anti-obesity food composition containing the stevia extract according to the embodiment of the present invention as described above can exhibit an anti-obesity effect and help in weight control by containing the stevia extract with an anti-angiogenesis function.
특히, 녹차 추출물과 함께 사용하여 각 추출물을 단독으로 사용하는 것보다 우수한 항비만 효과를 나타낼 수 있다.In particular, when used together with green tea extract, it can exhibit a superior anti-obesity effect than when each extract is used alone.
또한 스테비아 추출물과 같은 천연소재를 사용함으로써, 합성제제인 기존의 항비만제제의 다양한 부작용 발생, 습관성 등의 문제를 해소할 수 있다.In addition, by using natural materials such as stevia extract, problems such as various side effects and habituation of existing synthetic anti-obesity drugs can be resolved.
도 1은 본 발명의 스테비아 추출물의 농도에 따른 세포독성 시험 결과 그래프.
도 2는 본 발명의 스테비아 추출물의 농도에 따른 신생혈관 형성 억제 시험 결과 그래프.
도 3의 (a) 내지 (f)는 본 발명의 스테비아 추출물의 농도에 따른 신생혈관 형성 억제 시험 결과 사진.
도 4는 본 발명의 스테비아 추출물의 농도에 따른 신호전달분자를 검출한 PCR 검사 사진.
도 5는 신생혈관 형성을 위한 신호전달 경로를 나타낸 개념도.
도 6의 (a) 내지 (f)는 3T3-L1 세포를 이용하여 본 발명의 스테비아 추출물의 농도에 따른 지방 축적 정도를 분석한 Oil Red 염색 결과 사진.
도 7은 3T3-L1 세포를 이용하여 본 발명의 스테비아 추출물과 스테비아 추출물 및 녹차 추출물의 농도에 따른 지방 축적율 분석 그래프.
도 8은 10개 그룹의 장간막 지방조직과 간 조직의 PCR 검사 사진.
도 9는 장간막의 지방형성 및 지방세포 형성 분자 경로를 나타낸 개념도.
도 10은 10개 그룹의 기간에 따른 체중 감소 결과 그래프.
도 11의 (a) 및 (b)는 10개 그룹의 Leptin 및 adiponectin 활성 측정 결과 그래프.Figure 1 is a graph of cytotoxicity test results according to concentration of the stevia extract of the present invention.
Figure 2 is a graph showing the results of a test for inhibiting angiogenesis according to the concentration of the stevia extract of the present invention.
Figure 3 (a) to (f) are photographs of the results of a test for inhibiting neovascularization according to the concentration of the stevia extract of the present invention.
Figure 4 is a photo of a PCR test detecting signaling molecules according to the concentration of the stevia extract of the present invention.
Figure 5 is a conceptual diagram showing the signal transduction pathway for new blood vessel formation.
Figures 6 (a) to (f) are photographs of Oil Red staining results obtained by analyzing the degree of fat accumulation according to the concentration of the stevia extract of the present invention using 3T3-L1 cells.
Figure 7 is a graph analyzing the fat accumulation rate according to the concentration of the stevia extract, stevia extract, and green tea extract of the present invention using 3T3-L1 cells.
Figure 8 is a PCR test photo of mesenteric fat tissue and liver tissue in 10 groups.
Figure 9 is a conceptual diagram showing the molecular pathways of mesenteric adipogenesis and adipocyte formation.
Figure 10 is a graph of weight loss results according to period in 10 groups.
Figures 11 (a) and (b) are graphs of the results of measuring Leptin and adiponectin activities in 10 groups.
이하, 도면을 참조한 본 발명의 설명은 특정한 실시 형태에 대해 한정되지 않으며, 다양한 변환을 가할 수 있고 여러 가지 실시예를 가질 수 있다. 또한, 이하에서 설명하는 내용은 본 발명의 사상 및 기술 범위에 포함되는 모든 변환, 균등물 내지 대체물을 포함하는 것으로 이해되어야 한다.Hereinafter, the description of the present invention with reference to the drawings is not limited to specific embodiments, and various changes may be made and various embodiments may be possible. In addition, the content described below should be understood to include all conversions, equivalents, and substitutes included in the spirit and technical scope of the present invention.
이하의 설명에서 제1, 제2 등의 용어는 다양한 구성요소들을 설명하는데 사용되는 용어로서, 그 자체에 의미가 한정되지 아니하며, 하나의 구성요소를 다른 구성요소로부터 구별하는 목적으로만 사용된다.In the following description, the terms first, second, etc. are terms used to describe various components, and their meaning is not limited, and is used only for the purpose of distinguishing one component from other components.
본 명세서 전체에 걸쳐 사용되는 동일한 참조번호는 동일한 구성요소를 나타낸다.Like reference numerals used throughout this specification refer to like elements.
본 발명에서 사용되는 단수의 표현은 문맥상 명백하게 다르게 뜻하지 않는 한, 복수의 표현을 포함한다. 또한, 이하에서 기재되는 "포함하다", "구비하다" 또는 "가지다" 등의 용어는 명세서상에 기재된 특징, 숫자, 단계, 동작, 구성요소, 부품 또는 이들을 조합한 것이 존재함을 지정하려는 것으로 해석되어야 하며, 하나 또는 그 이상의 다른 특징들이나, 숫자, 단계, 동작, 구성요소, 부품 또는 이들을 조합한 것들의 존재 또는 부가 가능성을 미리 배제하지 않는 것으로 이해되어야 한다.As used herein, singular expressions include plural expressions, unless the context clearly dictates otherwise. In addition, terms such as “comprise,” “provide,” or “have” used below are intended to designate the presence of features, numbers, steps, operations, components, parts, or a combination thereof described in the specification. It should be construed and understood as not excluding in advance the possibility of the presence or addition of one or more other features, numbers, steps, operations, components, parts or combinations thereof.
여기서, 반복되는 설명, 본 발명의 요지를 불필요하게 흐릴 수 있는 공지 기능, 및 구성에 대한 상세한 설명은 생략한다. 본 발명의 실시형태는 당 업계에서 평균적인 지식을 가진 자에게 본 발명을 보다 완전하게 설명하기 위해서 제공되는 것이다.Here, repeated descriptions, known functions that may unnecessarily obscure the gist of the present invention, and detailed descriptions of configurations are omitted. Embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art.
이하, 본 발명의 실시 예를 첨부한 도 1 내지 도 11을 참조하여 상세히 설명하기로 한다.Hereinafter, an embodiment of the present invention will be described in detail with reference to the accompanying FIGS. 1 to 11.
본 발명은 스테비아 추출물을 함유한 항비만 식품조성물에 관한 것으로, 스테비아 추출물이 신생혈관형성 억제기능을 갖는 것을 발견하였고, 이를 통해 스테비아 추출물을 사용하여 항비만 효과를 나타내며 부작용이 없는 항비만 식품조성물을 제공하고자 한다.The present invention relates to an anti-obesity food composition containing a stevia extract. It was discovered that the stevia extract has an anti-angiogenic function, and through this, an anti-obesity food composition that exhibits an anti-obesity effect and has no side effects using the stevia extract was developed. We would like to provide
또한, 추출 방법을 통해 신생혈관형성 억제기능 등이 보다 효과적으로 나타나도록 한다.In addition, the extraction method allows the anti-angiogenic function to be more effective.
또한, 스테비아 추출물과 함께 녹차 추출물을 함유함으로써, 신생혈관형성 억제기능이 보다 향상되어 항비만 효과가 우수한 식품조성물을 제공하고자 한다.In addition, by containing green tea extract along with stevia extract, the aim is to provide a food composition with excellent anti-obesity effect by improving angiogenesis inhibition function.
한편, 혈관신생(angiogenesis)은 기존의 혈관에서 새로운 혈관이 생성되는 일련의 과정을 일컫는 용어로서, 태아의 발생, 여성의 월경, 상처 치유과정 등의 정상적인 상황뿐 아니라 암세포의 성장과 전이, 비만세포의 성장, 류머티즘 관절염, 당뇨성 실명증 등의 여러 질병에서도 필수적으로 요구되는 것으로 알려져 있다. 즉, 정상적인 상태에서 생체는 혈관신생을 유도하는 인자들과 억제하는 인자들이 평형을 이루고 있으나, 질환이 유발되는 상황에서는 혈관성장을 촉진하는 인자들이 증가하거나, 억제하는 인자가 제대로 작용하지 못하여 혈관신생이 자율적으로 조절되지 못하고 계속 성장함으로써 질환으로 발전되게 된다. Meanwhile, angiogenesis is a term that refers to a series of processes in which new blood vessels are created from existing blood vessels. It refers to normal situations such as fetal development, female menstruation, and wound healing, as well as the growth and metastasis of cancer cells and mast cells. It is known to be essential for various diseases such as growth, rheumatoid arthritis, and diabetic blindness. In other words, in a normal state, the body maintains a balance of factors that induce and inhibit angiogenesis, but in a disease-induced situation, factors that promote vascular growth increase or factors that inhibit angiogenesis do not work properly, leading to angiogenesis. As this condition cannot be controlled autonomously and continues to grow, it develops into a disease.
따라서, 신생혈관의 형성을 억제함으로써 어떠한 조직에 새로운 혈관의 형성을 억제함으로써, 조직이 비대 해지는 것을 막을 수 있고 지방세포 형성 및 지방 형성의 억제가 가능하여, 항비만 효과를 가져올 수 있다고 사료된다.Therefore, it is believed that by suppressing the formation of new blood vessels in any tissue, it is possible to prevent tissue hypertrophy and to suppress adipocyte formation and fat formation, resulting in an anti-obesity effect.
본 발명의 실시예에 따른 스테비아 추출물을 함유하는 항비만 식품조성물은 스테비아 추출물을 함유할 수 있다.An anti-obesity food composition containing a stevia extract according to an embodiment of the present invention may contain a stevia extract.
스테비아 추출물은 스테비아로부터 추출된 것으로, Stevioside 및 Rebaudioside 등을 함유하고 있어 일반적으로 천연 감미료로 사용되고 있으나, 본 발명에서는 신생혈관형성 억제 기능, 지방세포 및 지방 형성 억제 기능을 갖는 것을 확인하였고, 이에 따라 스테비아 추출물은 항비만 식품조성물에 사용되어 항비만 효과를 나타낼 수 있다.Stevia extract is extracted from stevia and contains stevioside and rebaudioside, so it is generally used as a natural sweetener. However, in the present invention, it was confirmed that stevia has the function of inhibiting angiogenesis and adipocyte and fat formation. Accordingly, stevia The extract can be used in an anti-obesity food composition to exhibit an anti-obesity effect.
뿐만 아니라 본 발명의 추출방법을 통해 추출된 스테비아 추출물을 사용함으로써, 보다 우수한 신생혈관 억제 기능을 나타낼 수 있다. 이러한 스테비아 추출물의 제조방법에 대해 하기에서 자세히 설명하기로 한다.In addition, by using the stevia extract extracted through the extraction method of the present invention, it can exhibit a more excellent angiogenesis inhibition function. The manufacturing method of this stevia extract will be described in detail below.
스테비아 추출물은 추출단계 및 정제단계를 통해 제조될 수 있다.Stevia extract can be produced through extraction and purification steps.
구체적으로, 추출단계는 스테비아 잎으로부터 스테비아 추출물을 추출하는 단계로, 전처리단계, 추출물추출단계, 농축단계 및 동결건조단계를 포함할 수 있다.Specifically, the extraction step is a step of extracting stevia extract from stevia leaves and may include a pretreatment step, an extract extraction step, a concentration step, and a freeze-drying step.
먼저, 전처리단계는 건조된 스테비아 잎을 분쇄하여 스테비아 분말로 제조할 수 있다. 이는 추출 효율을 향상시키기 위해 건조된 스테비아 잎을 분말화하는 것이다.First, in the pretreatment step, dried stevia leaves can be pulverized to produce stevia powder. This involves powdering dried stevia leaves to improve extraction efficiency.
또한, 전처리단계는 스테비아 잎의 지용성 물질을 제거하기 위하여, 분쇄된 스테비아 분말을 n-헥산으로 탈지한 후 건조시킬 수 있다.Additionally, in the pretreatment step, the pulverized stevia powder may be degreased with n-hexane and then dried to remove fat-soluble substances in the stevia leaves.
즉, 식물인 스테비아 잎에는 오일, 지방 등 지용성 물질이 함유되어 있는데, 추출 시 이러한 성분은 필요없기 때문에 이를 제거함으로써, 극성성분만 추출하도록 할 수 있으며 추출이 더욱 용이하게 이루어지도록 할 수 있는 것이다. In other words, stevia leaves, which are plants, contain fat-soluble substances such as oil and fat. Since these components are not needed during extraction, by removing them, only the polar components can be extracted and extraction can be performed more easily.
추출물추출단계는 스테비아 분말에서 스테비아 추출물을 추출할 수 있는데, 스테비아 분말을 에탄올에 용해시키고 환류를 통해 스테비아 추출물을 추출할 수 있다.In the extract extraction step, the stevia extract can be extracted from the stevia powder. The stevia extract can be extracted by dissolving the stevia powder in ethanol and refluxing it.
이때, 에탄올은 에탄올의 농도가 70% 미만일 경우 극성 물질이 완전히 추출되지 못하는 우려가 있고, 70% 초과일 경우 알코올의 탈수작용에 따른 성분의 구조 변화가 일어날 가능성이 있어, 70% 에탄올을 사용하는 것이 바람직하나, 이에 한정되지 않는다.At this time, if the concentration of ethanol is less than 70%, there is a risk that polar substances may not be completely extracted, and if the concentration is more than 70%, there is a possibility that structural changes in the components may occur due to the dehydration effect of alcohol, so it is recommended to use 70% ethanol. This is preferable, but is not limited to this.
또한, 환류는 50 내지 60℃에서 5 내지 6.5시간 동안 이루어질 수 있으며, 60℃에서 6시간 동안 이루어지는 것이 가장 바람직하다.Additionally, reflux may be performed at 50 to 60°C for 5 to 6.5 hours, and is most preferably performed at 60°C for 6 hours.
이는 환류 온도가 50℃ 미만일 경우 추출이 용이하게 이루어지지 않아 추출 효율이 저하될 수 있으며, 60℃ 초과일 경우 성분이 변형되거나 파괴될 수 있기 때문이다.This is because if the reflux temperature is less than 50℃, extraction is not easy and extraction efficiency may decrease, and if it exceeds 60℃, the ingredients may be deformed or destroyed.
또한, 환류 시간이 5시간 미만일 경우 추출이 완전히 완료되지 못할 수 있고, 6.5시간 초과일 경우 이미 추출이 완료되었기 때문에 비효율적일 수 있다.Additionally, if the reflux time is less than 5 hours, extraction may not be completely completed, and if it is longer than 6.5 hours, extraction may have already been completed and may be inefficient.
이와 같은 환류를 통해 스테비아 분말에서 스테비아 추출물을 추출함으로써, 추출하고자 하는 극성 물질에 대한 추출이 더욱 잘 이루어질 수 있다.By extracting stevia extract from stevia powder through such reflux, the polar substance to be extracted can be better extracted.
농축단계는 추출물추출단계에서 추출된 스테비아 추출물을 감압 농축시킬 수 있다. In the concentration step, the stevia extract extracted in the extract extraction step may be concentrated under reduced pressure.
동결건조단계는 농축단계에서 농축된 스테비아 추출물을 동결건조시켜, 스테비아 추출물을 분말 형태로 만들 수 있다.In the freeze-drying step, the stevia extract concentrated in the concentration step can be freeze-dried to make the stevia extract in powder form.
이때, 동결건조는 -70 내지 -85℃에서 45 내지 55기압(atm)으로 23 내지 25시간동안 이루어질 수 있으며, -80℃에서 50기압(atm)으로 24시간 동안 이루어지는 것이 바람직하나, 이에 한정되지는 않는다.At this time, freeze-drying can be performed at -70 to -85°C at 45 to 55 atm for 23 to 25 hours, and is preferably performed at -80°C at 50 atm for 24 hours, but is not limited to this. does not
정제단계는 추출단계를 통해 추출되어 분말 형태로 제조된 스테비아 추출물을 정제하는 단계로, 정제를 통해 불순물을 제거하고 고농도화시켜 신생혈관 억제 기능 등이 보다 향상되도록 할 수 있다.The purification step is a step of purifying the stevia extract extracted through the extraction step and prepared in powder form. Through purification, impurities can be removed and the concentration can be increased to further improve the angiogenesis inhibition function.
이러한 정제단계는 제1 정제단계, 제2 정제단계, 중화단계 및 제3 정제단계를 포함할 수 있다.This purification step may include a first purification step, a second purification step, a neutralization step, and a third purification step.
먼저 제1 정제단계는 추출단계를 통해 추출된 스테비아 추출물을 증류수에 용해시키고, 칼럼(Column)에 충전시킨 후, 스테비아 추출물이 충전된 컬럼에 수지를 첨가하여, 스테비아 추출물에서 비극성 물질을 분리시킬 수 있다. First, in the first purification step, the stevia extract extracted through the extraction step is dissolved in distilled water, filled in a column, and then resin is added to the column filled with the stevia extract to separate non-polar substances from the stevia extract. there is.
이때, 비극성 물질은 수지에 흡착되어 스테비아 추출물에서 분리된 상태이긴 하나 아직 컬럼 안에서 섞여 있는 상태이고, 제3 정제단계를 통해 분리된 비극성 물질이 스테비아 추출물에서 완전히 제거될 수 있다.At this time, the non-polar substances are adsorbed on the resin and separated from the stevia extract, but are still mixed in the column, and the separated non-polar substances can be completely removed from the stevia extract through the third purification step.
이때, 수지는 non-polar macro resin를 사용함이 바람직하나, 이에 한정되지는 않는다.At this time, it is desirable to use non-polar macro resin as the resin, but is not limited to this.
여기서 스테비아 추출물은 40 내지 60중량부를 증류수 90 내지 110중량부에 용해시킬 수 있고, 50중량부를 증류수 100중량부에 용해시키는 것이 바람직하나, 이에 한정되지는 않는다.Here, 40 to 60 parts by weight of the stevia extract can be dissolved in 90 to 110 parts by weight of distilled water, and 50 parts by weight is preferably dissolved in 100 parts by weight of distilled water, but is not limited thereto.
제2 정제단계는 제1 정제단계에서 수지가 첨가된 컬럼에 수산화나트륨(NaOH)을 통과시켜 산성 물질을 용출시킬 수 있다. 이를 통해 스테비아 추출물에서 원하지 않는 물질인 산성 물질을 제거할 수 있다. In the second purification step, acidic substances can be eluted by passing sodium hydroxide (NaOH) through the column to which the resin was added in the first purification step. Through this, unwanted acidic substances can be removed from the stevia extract.
여기서 수산화나트륨(NaOH)은 0.4N NaOH를 사용하는 것이 바람직하나, 이에 한정되지는 않는다.Here, it is preferable to use 0.4N NaOH as sodium hydroxide (NaOH), but it is not limited to this.
중화단계는 제2 정제단계에서 산성물질이 제거된 스테비아 추출물을 컬럼에서 빼내 용기에 넣고 염화수소(HCl)을 첨가하여 중화시킬 수 있다. 이는 산성물질이 제거되어 염기성이 된 스테비아 추출물에 산성인 염화수소를 첨가하여 중화시키는 것이다. 재정제를 위해서는 스테비아 추출물의 중화가 필요하기 때문이다.In the neutralization step, the stevia extract from which acidic substances have been removed in the second purification step can be removed from the column, placed in a container, and hydrogen chloride (HCl) added to neutralize it. This is to neutralize the stevia extract from which acidic substances have been removed and become basic by adding acidic hydrogen chloride. This is because neutralization of the stevia extract is necessary for repurification.
제3 정제단계는 중화단계에서 중화된 스테비아 추출물을 새 컬럼(Column)에 충전시킨 후, 에탄올(용리액)을 통해 용출시켜 비극성 물질이 제거된 스테비아 추출물을 획득할 수 있다. 용출액이 최종적으로 얻어지는 스테비아 추출물이다.In the third purification step, the stevia extract neutralized in the neutralization step is filled into a new column, and then eluted with ethanol (eluent) to obtain a stevia extract from which non-polar substances have been removed. The eluate is the stevia extract that is ultimately obtained.
여기서 사용되는 에탄올은 100% 에탄올인 것이 바람직하다.The ethanol used here is preferably 100% ethanol.
이는 제3 정제단계가 극성 물질만을 온전히 용출해내기 위한 단계인데, 100% 에탄올을 사용하지 않을 경우, 분리 효율이 저하되기 때문이다.This is because the third purification step is to completely elute only polar substances, and if 100% ethanol is not used, the separation efficiency decreases.
이러한 스테비아 추출물은 3g/70kg-man/day 또는 100㎍/ml 이하의 농도로 항비만 식품조성물에 함유되는 것이 바람직하다. 또한 스테비아 추출물은 항비만 식품조성물 전체 중량%에 대하여 1 내지 3중량%가 함유될 수 있으며, 보다 바람직하게 3중량%가 함유될 수 있다.This stevia extract is preferably contained in the anti-obesity food composition at a concentration of 3g/70kg-man/day or 100㎍/ml or less. Additionally, the stevia extract may be contained in an amount of 1 to 3% by weight, more preferably 3% by weight, based on the total weight% of the anti-obesity food composition.
이러한 스테비아 추출물의 농도 또는 함량은 동물 실험을 바탕으로 하여 안전 용량을 결정한 것으로, 70Kg의 성인남자의 용량으로 치환 했을 때 3.0g/70Kg-man/day가 적당한 것으로 나타났다.The concentration or content of this stevia extract was determined as a safe dose based on animal experiments, and when substituted for the dose for a 70Kg adult male, 3.0g/70Kg-man/day was found to be appropriate.
또한, 본 발명의 실시예에 따른 스테비아 추출물을 함유한 항비만 식품조성물은 녹차 추출물을 더 함유할 수 있다.Additionally, the anti-obesity food composition containing stevia extract according to an embodiment of the present invention may further contain green tea extract.
녹차 추출물은 녹차 씨앗으로부터 추출된 추출물로써 녹차 사포닌을 포함하고, 신생혈관형성 억제 기능을 근간으로, 항비만(1. Anti-hyperlipidemic and Fat Pad Lowering Effect of Standardized Tea Seed Cake Extract in Mice Fed High-fat and High-carbohydrate Diet,KIM, 2015, BIOTECHNOLOGY AND BIOPROCESS ENGINEERING, 2. Theasaponin E1 as an effective ingredient for anti-angiogenesis and anti-obesity effects, KIM, 2014, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY) 효과뿐만 아니라, 고지혈증(한국등록특허 제10-1854322호, 녹차씨앗 추출물을 유효성분으로 포함하는 고지혈증 치료용 조성물) 및 항암 효과(HPLC fractionation and pharmacological assessment of green tea seed saponins for antimicrobial, anti-angiogenic, and hemolytic activities, KIM, 2016, Biotechnology and Bioprocess Engineering) 등을 나타낼 수 있다.Green tea extract is an extract extracted from green tea seeds, contains green tea saponin, and has anti-obesity (1. Anti-hyperlipidemic and Fat Pad Lowering Effect of Standardized Tea Seed Cake Extract in Mice Fed High-fat) based on its anti-angiogenic function. and High-carbohydrate Diet,KIM, 2015, BIOTECHNOLOGY AND BIOPROCESS ENGINEERING, 2. Theasaponin E1 as an effective ingredient for anti-angiogenesis and anti-obesity effects, KIM, 2014, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY) as well as hyperlipidemia (registered in Korea) Patent No. 10-1854322, Composition for treating hyperlipidemia containing green tea seed extract as an active ingredient) and anticancer effect (HPLC fractionation and pharmacological assessment of green tea seed saponins for antimicrobial, anti-angiogenic, and hemolytic activities, KIM, 2016, Biotechnology and Bioprocess Engineering), etc.
이와 같이 녹차의 사포닌(테아사포닌-E1(theasaponin E1) 등)을 함유하는 녹차 추출물을 스테비아 추출물과 함께 사용함으로써, 신생혈관 억제 기능, 지방세포 및 지방 형성 억제 기능이 보다 효과적으로 나타나도록 할 수 있다.In this way, by using green tea extract containing green tea saponins (theasaponin E1, etc.) together with stevia extract, the function of inhibiting new blood vessels and inhibiting adipocytes and fat formation can be displayed more effectively.
이러한 녹차 추출물은 항비만 식품조성물 전체 중량%에 대하여 0.005 내지 0.015중량%가 함유될 수 있으며, 보다 바람직하게 0.01중량%가 함유될 수 있다.This green tea extract may be contained in an amount of 0.005 to 0.015% by weight, more preferably 0.01% by weight, based on the total weight% of the anti-obesity food composition.
이와 같은 녹차 추출물의 함량은 항비만 효과 및 지방형성억제 작용 규명의 동물실험을 기반으로 결정한 것이다.The content of green tea extract was determined based on animal experiments to identify anti-obesity effects and anti-lipid formation effects.
상기에서 설명한 바와 같이, 본 발명의 실시예에 따른 스테비아 추출물을 함유하는 항비만 식품조성물은 신생혈관형성 억제기능을 가지는 스테비아 추출물을 함유함으로써, 항비만 효과를 나타내 체중조절에 도움을 줄 수 있다.As described above, the anti-obesity food composition containing a stevia extract according to an embodiment of the present invention contains a stevia extract that has an anti-angiogenesis function, thereby exhibiting an anti-obesity effect and helping in weight control.
특히, 녹차 추출물과 함께 사용하여 각 추출물을 단독으로 사용하는 것보다 우수한 항비만 효과를 나타낼 수 있다.In particular, when used together with green tea extract, it can exhibit a superior anti-obesity effect than when each extract is used alone.
또한 스테비아 추출물과 같은 천연소재를 사용함으로써, 합성제제인 기존의 항비만제제의 다양한 부작용 발생, 습관성 등의 문제를 해소할 수 있다.In addition, by using natural materials such as stevia extract, problems such as various side effects and habituation of existing synthetic anti-obesity drugs can be resolved.
이하 본 발명의 내용을 실시예, 비교예 및 시험예를 통하여 구체적으로 설명한다. 그러나, 이들은 이해를 돕기 위하여 제시되는 것으로서, 본 발명을 예시하는 것일 뿐 본 발명의 권리범위가 이들에 의해 한정되는 것은 아니다.Hereinafter, the contents of the present invention will be described in detail through examples, comparative examples, and test examples. However, these are presented to aid understanding, and only exemplify the present invention and do not limit the scope of the present invention.
[[ 실험예Experiment example 1] 세포독성 시험 1] Cytotoxicity test
본 발명의 추출방법을 통해 추출된 스테비아 추출물의 안전한 농도 범위를 결정하기 위하여, 세포독성 시험을 실시하였다.In order to determine the safe concentration range of the stevia extract extracted through the extraction method of the present invention, a cytotoxicity test was conducted.
HUVEC 라인을 한국 세포 라인 은행(서울, 한국)에서 구매하여, HUVEC를 EBM-2 배지(Clonetics??, USA)를 사용해 37℃, 5% CO2 인큐베이터(incubator)에서 배양하였다. 배양된 HUVEC를 96-웰 배양 플레이트에 넣고 스테비아 추출물을 농도별(0, 5, 15, 30, 50, 100, 200, 300 및 500㎍/ml)로 처리하고 37℃에서 24시간 동안 배양하였다. 그 후, 0.5%MTT 용액(Sigma, MO, USA)을 첨가하여 37℃에서 4시간동안 배양하였다. 마이크로플레이트 리더기(Biochrom Ltd., Cambridge, UK)를 이용하여 540㎛의 파장에서 흡광도를 측정하고, 생존율을 계산하였다.The HUVEC line was purchased from the Korean Cell Line Bank (Seoul, Korea), and the HUVEC were cultured in an incubator at 37°C and 5% CO 2 using EBM-2 medium (Clonetics®, USA). Cultured HUVECs were placed in a 96-well culture plate, treated with stevia extract at various concentrations (0, 5, 15, 30, 50, 100, 200, 300, and 500 μg/ml), and cultured at 37°C for 24 hours. Afterwards, 0.5% MTT solution (Sigma, MO, USA) was added and cultured at 37°C for 4 hours. Absorbance was measured at a wavelength of 540 μm using a microplate reader (Biochrom Ltd., Cambridge, UK), and survival rate was calculated.
그 결과는 도 1과 같다.The results are as shown in Figure 1.
도 1을 보면 알 수 있듯이, 스테비아 추출물 150㎍/ml부터 세포 생존율이 떨어지는 것을 확인할 수 있었다.As can be seen in Figure 1, it was confirmed that the cell viability decreased from 150 μg/ml of stevia extract.
이에 따라, 스테비아 추출물의 바람직한 농도는 100㎍/ml 이하인 것으로 선정하였다.Accordingly, the preferred concentration of stevia extract was selected to be 100 μg/ml or less.
[[ 실험예Experiment example 2] 신생혈관 형성 억제 시험 2] New blood vessel formation inhibition test
본 발명의 추출방법을 통해 추출된 스테비아 추출물의 신생혈관 억제 기능을 확인하기 위하여, 96- 웰 배양 플레이트에 Matrigel(BD Bioscience, MA, US)로 코팅 한 다음 37℃에서 1 시간 동안 배양시키고, HUVEC를 분리하여 Matrigel- 코팅 된 웰에 첨가하고 37℃에서 4 시간 동안 재배양 하였다. In order to confirm the angiogenesis inhibitory function of the stevia extract extracted through the extraction method of the present invention, a 96-well culture plate was coated with Matrigel (BD Bioscience, MA, US), cultured at 37°C for 1 hour, and HUVEC were separated and added to Matrigel-coated wells and re-cultured at 37°C for 4 hours.
스테비아 추출물을 농도별(5, 50, 100, 150 및 200㎍/ml)로 웰에 첨가하고 37℃에서 4 시간 동안 배양한 후, 형성된 튜브 망의 길이를 분석하였다.Stevia extract was added to the wells at various concentrations (5, 50, 100, 150, and 200 μg/ml) and incubated at 37°C for 4 hours, and then the length of the formed tube network was analyzed.
여기서, 튜브 망의 길이는 현미경(Nikon, Tokyo, Japan)하에서 플레이트의 웰을 불규칙적으로 5분야로 나누어 촬영하였고, 촬영된 이미지를 Scion Image 소프트웨어(NIH, ML, USA)를 사용하여 분석하였고, 각각의 처리 및 대조군에 대해 총 튜브 길이를 계산 하였다.Here, the length of the tube network was photographed by dividing the wells of the plate irregularly into 5 areas under a microscope (Nikon, Tokyo, Japan), and the captured images were analyzed using Scion Image software (NIH, ML, USA), respectively. Total tube length was calculated for the treatment and control groups.
그 결과는 도 2 및 도 3과 같다.The results are as shown in Figures 2 and 3.
도 2 및 도 3을 보면 알 수 있듯이, 스테비아 추출물 농도가 증가함에 따라 튜브 망의 길이가 짧은 것을 확인할 수 있었다. 이에 따라 스테비아 추출물은 신생혈관 형성 억제 기능을 가지는 것으로 판단된다.As can be seen in Figures 2 and 3, it was confirmed that the length of the tube network became shorter as the stevia extract concentration increased. Accordingly, it is believed that stevia extract has the function of inhibiting neovascularization.
[[ 실험예Experiment example 3] 신생혈관 형성의 신호전달 확인 3] Confirmation of signaling of new blood vessel formation
신생혈관형성억제의 기전을 파악하기 위하여, HUVECs을 EBM-2 배지에 배양한 후, 총 RNA의 추출은 TRI 용액을 사용하여 추출하였다. To determine the mechanism of inhibition of angiogenesis, HUVECs were cultured in EBM-2 medium, and total RNA was extracted using TRI solution.
DNA의 합성은 RevertAid Premium First Strand cDNA synthesis Kit를 사용하였고, 유전자의 발현은 PCR을 사용하였으며, 하기 표 1(신호전달분자의 유전자 primer)과 같은 primer를 사용하였다. 이때 스테비아 추출물을 농도별(5, 15, 30, 50 및 100㎍/ml)로 처리하였다.RevertAid Premium First Strand cDNA synthesis Kit was used for DNA synthesis, PCR was used for gene expression, and primers as shown in Table 1 below (signal transduction molecule gene primers) were used. At this time, the stevia extract was treated at different concentrations (5, 15, 30, 50, and 100 μg/ml).
또한, PCR 반응은 Takara ExTaq Hot Start Version (Takara, Japan)을 사용하였고, 반응은 94℃에서 3min, 94℃에서 30sec, 60℃에서 45sec, 그리고 72℃에서 1 분간, 또한, 72℃에서 7 분간 반응시켜 불완전하게 증폭된 것을 보완하였으며, 30cycle을 반응시킨 후 1.5% agarose gel로 전개하였다. Additionally, the PCR reaction used Takara ExTaq Hot Start Version (Takara, Japan), and the reaction was performed at 94°C for 3 min, 94°C for 30 sec, 60°C for 45 sec, and 72°C for 1 minute, and at 72°C for 7 minutes. Incomplete amplification was compensated for by reaction, and after 30 cycles of reaction, it was developed on a 1.5% agarose gel.
그 다음 신호전달분자의 PCR 검사를 진행하였다. 그 결과는 도 4와 같다.Next, PCR testing of signaling molecules was performed. The results are as shown in Figure 4.
한편, 도 5의 세포 표면의 VEGFR-2(VEGFR2), PI3K, β-catenin(β-cat) 및 VE-cadherin의 분자들의 신호가 핵내까지 신호가 전달되면 신생혈관이 형성되지만 도중에 신호가 차단되면 NF-kB까지 신호 전달이 일어나지 않아 신생혈관이 형성되지 못한다.On the other hand, when the signals of VEGFR-2 (VEGFR2), PI3K, β-catenin (β-cat), and VE-cadherin molecules on the cell surface shown in Figure 5 are transmitted to the nucleus, new blood vessels are formed, but if the signals are blocked along the way, new blood vessels are formed. Signal transmission to NF-kB does not occur, so new blood vessels are not formed.
도 4에 나타난 바와 같이, 스테비아 추출물의 농도 증가에 따라 VEGFR-2(VEGFR2), PI3K, β-catenin(β-cat), AKT-1(Akt), NF-kB가 감소되는 것으로 나타났다.As shown in Figure 4, VEGFR-2 (VEGFR2), PI3K, β-catenin (β-cat), AKT-1 (Akt), and NF-kB were found to decrease as the concentration of stevia extract increased.
즉, NF-kB의 발현이 감소됨에 따라 신생혈관 형성이 억제되는 것으로 판단된다.In other words, it is believed that neovascularization is suppressed as the expression of NF-kB is reduced.
[[ 실험예Experiment example 4] 3T3-L1 세포를 이용한 지방 축적 억제 효과 4] Effect of suppressing fat accumulation using 3T3-L1 cells
3T3-L1 세포를 분화 촉진한 후, 세포내 지방구 생성을 확인하기 위하여 지방적(lipid droplets)의 지방에 특이적으로 반응하여 염색되는 Oil Red O 염색법을 수행하여 축적 중인 지방을 염색된 지방구를 흡광도를 이용하여 분석하였다.After promoting differentiation of 3T3-L1 cells, Oil Red O staining, which specifically reacts to and stains fat in lipid droplets, was performed to confirm the production of intracellular fat globules, and the accumulated fat was stained with stained fat globules. Analyzed using absorbance.
3T3-L1 세포의 지방세포 유도는 세포 배양 웰(well)에 농도별 스테비아 추출물(0, 5, 15, 30, 50 및 100㎍/ml)과 농도별 스테비아 추출물 및 녹차 추출물의 혼합물(sp+st, 0, 5, 15, 30, 50 및 100㎍/ml)을 첨가한 후 100% 융합성(confluent)시키고 이틀 후 100μM 3-isobutyl-1-methylxanthine, Sigma I5879, 250μM dexamethasone, Sigma D4902, 170μM insulin 을 함유한 MDI 배지를 통해 지방세포로 유도하였으며, 3일 후에 10% FBS와 170nM insulin, Sigma I9278을 함유한 DMEM 배지로 각 실험 날짜까지 이틀에 한번 교환하였다.For adipocyte induction in 3T3-L1 cells, stevia extract at different concentrations (0, 5, 15, 30, 50, and 100㎍/ml) and a mixture of stevia extract and green tea extract at different concentrations (sp+st) were added to cell culture wells. , 0, 5, 15, 30, 50, and 100㎍/ml) were added to make it 100% confluent, and after two days, 100µM 3-isobutyl-1-methylxanthine, Sigma I5879, 250µM dexamethasone, Sigma D4902, and 170µM insulin. Adipocytes were induced through MDI medium containing, and after 3 days, the medium was replaced with DMEM medium containing 10% FBS, 170 nM insulin, and Sigma I9278 once every two days until the date of each experiment.
3T3-L1 세포 배양 중 각각 3, 6, 9일에 배양액 성분을 완전히 제거하기 위하여 phosphate buffered saline(PBS, pH 7.0)로 2번 세정하고 0.6%의 Oil Red O 용액을 준비한 후 약 2시간 동안 염색하고, PBS로 3번 세정한 후 isopropanol 1.5 mL을 넣어 oil red를 용출시켰다. 용출시킨 용액을 관찰하였으며 500nm에서 흡광도를 측정하여 지방 축적율을 계산하였다. 그 결과는 도 6 및 도 7과 같다.During 3T3-L1 cell culture, on days 3, 6, and 9, respectively, the cells were washed twice with phosphate buffered saline (PBS, pH 7.0) to completely remove culture media components, prepared with a 0.6% Oil Red O solution, and stained for about 2 hours. After washing three times with PBS, 1.5 mL of isopropanol was added to elute oil red. The eluted solution was observed, and the absorbance was measured at 500 nm to calculate the fat accumulation rate. The results are shown in Figures 6 and 7.
도 6은 지방 형성 억제 결과로, 5㎍/ml, 30㎍/ml, 50㎍/ml, 100㎍/ml의 스테비아 추출물과 스테비아 추출물(ST) 100㎍/ml 및 녹차 추출물(SP) 25㎍/ml를 혼합한 혼합물(ST+SP)을 처리한 oil red 염색 결과 사진이다.Figure 6 shows the results of inhibition of fat formation, 5 μg/ml, 30 μg/ml, 50 μg/ml, and 100 μg/ml of stevia extract, 100 μg/ml of stevia extract (ST), and 25 μg/ml of green tea extract (SP). This is a photo of the oil red staining result obtained by processing a mixture of ml (ST+SP).
도 6의 (a) 내지 (f)는 3T3-L1 세포를 이용하여 본 발명의 스테비아 추출물의 농도에 따른 지방 축적 정도를 분석한 Oil Red 염색 결과 사진.Figures 6 (a) to (f) are photographs of Oil Red staining results obtained by analyzing the degree of fat accumulation according to the concentration of the stevia extract of the present invention using 3T3-L1 cells.
도 6과 도 7을 보면 알 수 있듯이, 스테비아 추출물의 농도 증가에 따라 지방체가 현저히 감소되는 것을 확인할 수 있었다. 특히 도 6에서 혼합물(ST+SP)을 처리한 군에서는 거의 지방체가 나타나지 않아 스테비아 추출물 단독보다는 녹차 추출물과 혼합하여 사용하는 것이 보다 좋은 결과를 나타내는 것을 확인할 수 있었다.As can be seen in Figures 6 and 7, it was confirmed that fat body was significantly reduced as the concentration of stevia extract increased. In particular, in Figure 6, almost no fat bodies appeared in the group treated with the mixture (ST+SP), so it was confirmed that mixing with green tea extract gave better results than using stevia extract alone.
또한 도 7의 지방 축척율, 즉 지방 축적 억제 효과도 혼합물이 보다 높은 효과를 보여주는 것을 확인할 수 있었다.In addition, it was confirmed that the mixture showed a higher effect in the fat accumulation rate in Figure 7, that is, the effect of suppressing fat accumulation.
[[ 실험예Experiment example 5] 5] mRNAmRNA 발현에 관여하는 효소 및 전사인자에 대한 스테비아 추출물의 영향 Effect of stevia extract on enzymes and transcription factors involved in expression
지방 및 에너지 대사에 관련되는 경로의 biomaker에 스테비아 추출물이 어떠한 영향을 미치는가를 평가하기 위하여, 장간막 지방조직과 간 조직을 사용하여 PCR 검사를 진행하였다. 그 결과는 도 8과 같다.To evaluate the effect of stevia extract on biomakers of pathways related to fat and energy metabolism, PCR tests were performed using mesenteric fat tissue and liver tissue. The results are as shown in Figure 8.
실험은 8주령 암컷 ICR 마우스(Taconic, Chungbuk, Korea)는 1주간 환경 순응 과정 후 10 그룹으로 나누어 각 그룹별로 6 마리씩 무작위로 할당한 후 표준 조건(22℃에서 12시간 동안 광/암 주기 및 상대 습도 50%±5%)하에 관리되었고 임의로 먹이와 물을 공급하였다.In the experiment, 8-week-old female ICR mice (Taconic, Chungbuk, Korea) were divided into 10 groups after a 1-week environmental acclimatization process, 6 mice were randomly assigned to each group, and then subjected to standard conditions (light/dark cycle and relative light/dark cycle for 12 hours at 22°C). They were managed under humidity (50% ± 5%) and provided with food and water ad libitum.
여기서 10그룹은 HFC(고-지방 식이), HF-100(고-지방 식이+스테비아 추출물 100 mg/kg/mouse body weight/d), HF-300(고-지방 식이+스테비아 추출물 300 mg/kg/mouse body weight/d), HCC(고-탄수화물 식이), HC-100(고-탄수화물 식이+스테비아 추출물 100 mg/kg/mouse body weight/d), HC-300(고-탄수화물 식이+스테비아 추출물 300 mg/kg/mouse body weight/d), NC(정상 식이), PHF-300(4주간 고-지방 식이 후, 고-지방 식이+스테비아 추출물 300 mg/kg/mouse body weight/d), ORL(고-지방 식이+ Orlistat 50 mg/kg/mouse body weight/d), ST+SP(고-지방 식이+스테비아 추출물 및 녹차 추출물 350 mg/kg/mouse body weight/d)으로 이루어진다. 이때, ST+SP은 고-지방 식이에 스테비아 추출물 300 mg/kg/mouse body weight/d과 녹차 추출물 50 mg/kg/mouse body weight/d로 구성하였다.Here, the 10 groups are HFC (high-fat diet), HF-100 (high-fat diet + stevia extract 100 mg/kg/mouse body weight/d), and HF-300 (high-fat diet + stevia extract 300 mg/kg). /mouse body weight/d), HCC (high-carbohydrate diet), HC-100 (high-carbohydrate diet + stevia extract 100 mg/kg/mouse body weight/d), HC-300 (high-carbohydrate diet + stevia extract 300 mg/kg/mouse body weight/d), NC (normal diet), PHF-300 (after 4 weeks of high-fat diet, high-fat diet + stevia extract 300 mg/kg/mouse body weight/d), ORL (high-fat diet + Orlistat 50 mg/kg/mouse body weight/d), ST+SP (high-fat diet + stevia extract and green tea extract 350 mg/kg/mouse body weight/d). At this time, ST+SP consisted of a high-fat diet with 300 mg/kg/mouse body weight/d of stevia extract and 50 mg/kg/mouse body weight/d of green tea extract.
간 조직에서 ACC(acetyl-CoA carboxylase), GPAT(Glycerol-3-phosphate O-acyltransferase), FAS(fatty acid synthase) 및 MLYCD(malonyl-CoA decarboxylase) 등의 mRNA는 스테비아 추출물을 처리한 군들에서 감소되는 것을 확인할 수 있었다.In liver tissue, mRNAs such as ACC (acetyl-CoA carboxylase), GPAT (Glycerol-3-phosphate O-acyltransferase), FAS (fatty acid synthase), and MLYCD (malonyl-CoA decarboxylase) were decreased in the group treated with stevia extract. could be confirmed.
장간막 지방조직의 지방조직의 lipogenesis 및 adipogenesis에 관여하는 유전자의 발현은 도 9와 같이 이루어질 수 있는데, Pparg(peroxisome proliferator-activated receptor gamma), Srebp(Sterol regulatory element-binding protein 1), Cebpa(CCAAT/enhancer-binding protein alpha) 등은 스테비아 추출물을 처리한 군들에서 감소되는 것을 확인할 수 있었다.The expression of genes involved in lipogenesis and adipogenesis of mesenteric adipose tissue can be achieved as shown in Figure 9, including Pparg (peroxisome proliferator-activated receptor gamma), Srebp (Sterol regulatory element-binding protein 1), and Cebpa (CCAAT/ enhancer-binding protein alpha) was confirmed to be decreased in the group treated with stevia extract.
또한 장간막 WAT에 있어 lipogenesis 및 adipogenesis에 관련된 LPL(lipoprotein lipase), aP2(adipocyte protein 2) 및 leptin은 스테비아 추출물의 처리 농도 증가에 따라 감소되는 것으로 나타났다.In addition, lipoprotein lipase (LPL), adipocyte protein 2 (aP2), and leptin, which are involved in lipogenesis and adipogenesis in mesenteric WAT, were found to decrease with increasing concentration of stevia extract.
[[ 실험예Experiment example 6] 6] 항비만anti-obesity 효과 effect
스테비아 추출물의 항비만 효과를 확인하기 위하여 하기와 같이 실험을 진행하였고, 2일 마다 마우스의 체중을 확인하였다. 그 결과는 도 10과 같다.To confirm the anti-obesity effect of stevia extract, an experiment was conducted as follows, and the body weight of the mouse was checked every two days. The results are as shown in Figure 10.
실험은 8주령 암컷 ICR 마우스(Taconic, Chungbuk, Korea)는 1주간 환경 순응 과정 후 10 그룹으로 나누어 각 그룹별로 6 마리씩 무작위로 할당한 후 표준 조건(22℃에서 12시간 동안 광/암 주기 및 상대 습도 50%±5%)하에 관리되었고 임의로 먹이와 물을 공급하였다.In the experiment, 8-week-old female ICR mice (Taconic, Chungbuk, Korea) were divided into 10 groups after a 1-week environmental acclimatization process, 6 mice were randomly assigned to each group, and then subjected to standard conditions (light/dark cycle and relative light/dark cycle for 12 hours at 22°C). They were managed under humidity (50% ± 5%) and provided with food and water ad libitum.
여기서 10그룹은 HFC(고-지방 식이), HF-100(고-지방 식이+스테비아 추출물 100 mg/kg/mouse body weight/d), HF-300(고-지방 식이+스테비아 추출물 300 mg/kg/mouse body weight/d), HCC(고-탄수화물 식이), HC-100(고-탄수화물 식이+스테비아 추출물 100 mg/kg/mouse body weight/d), HC-300(고-탄수화물 식이+스테비아 추출물 300 mg/kg/mouse body weight/d), NC(정상 식이), PHF-300(4주간 고-지방 식이 후, 고-지방 식이+스테비아 추출물 300 mg/kg/mouse body weight/d), ORL(고-지방 식이+ Orlistat 50 mg/kg/mouse body weight/d), ST+SP(고-지방 식이+스테비아 추출물 및 녹차 추출물 350 mg/kg/mouse body weight/d)으로 이루어진다. 이때, ST+SP은 고-지방 식이에 스테비아 추출물 300 mg/kg/mouse body weight/d과 녹차 추출물 50 mg/kg/mouse body weight/d로 구성하였다.Here, the 10 groups are HFC (high-fat diet), HF-100 (high-fat diet + stevia extract 100 mg/kg/mouse body weight/d), and HF-300 (high-fat diet + stevia extract 300 mg/kg). /mouse body weight/d), HCC (high-carbohydrate diet), HC-100 (high-carbohydrate diet + stevia extract 100 mg/kg/mouse body weight/d), HC-300 (high-carbohydrate diet + stevia extract 300 mg/kg/mouse body weight/d), NC (normal diet), PHF-300 (after 4 weeks of high-fat diet, high-fat diet + stevia extract 300 mg/kg/mouse body weight/d), ORL (high-fat diet + Orlistat 50 mg/kg/mouse body weight/d), ST+SP (high-fat diet + stevia extract and green tea extract 350 mg/kg/mouse body weight/d). At this time, ST+SP consisted of a high-fat diet with 300 mg/kg/mouse body weight/d of stevia extract and 50 mg/kg/mouse body weight/d of green tea extract.
도 10을 보면 알 수 있듯이, 스테비아 추출물을 처리한 군에서 모두 체중이 감소되는 것을 확인할 수 있었다. 이것은 지방세포의 생성 및 지질 생성의 억제에 기인되는 것으로 판단된다.As can be seen in Figure 10, it was confirmed that body weight was reduced in all groups treated with stevia extract. This is believed to be due to the inhibition of fat cell production and lipid production.
또한 HF-300 및 HC-300의 체중은 양성 대조군 ORL(Orlistat)과 거의 동일하게 나타났으며, 인위적으로 고지방 식이로 체중을 증가 시킨 후에 스테비아를 투여한 경우인 PHF에서도 현저한 체중 감소 효과를 보이는 것을 확인할 수 있었다.In addition, the body weight of HF-300 and HC-300 was almost the same as that of the positive control ORL (Orlistat), and PHF, which was administered stevia after artificially increasing body weight with a high-fat diet, also showed a significant weight loss effect. I was able to confirm.
특히 스테비아 추출물(ST)+녹차 추출물(SP)에서 체중이 현저하게 감소되는 것을 확인할 수 있었으며, 양성대조군인 ORL(Orlistat)보다 스테비아 추출물(ST)+녹차 추출물(SP)가 현저한 효과를 나타내는 것을 확인할 수 있었다.In particular, it was confirmed that stevia extract (ST) + green tea extract (SP) significantly reduced body weight, and it was confirmed that stevia extract (ST) + green tea extract (SP) showed a more significant effect than the positive control ORL (Orlistat). I was able to.
[[ 실험예Experiment example 7] 7] 렙틴leptin (( LeptinLeptin ) 및 ) and 아디포넥틴Adiponectin (( adiponectinadiponectin ) 활성 평가) active evaluation
각 그룹(10개 그룹)에서 혈액 샘플을 채취하여, ELISA 키트(Abcam USA)를 사용하여 렙틴과 아디포넥틴의 혈청 농도를 측정하였다. 그 결과는 도 11과 같다.Blood samples were collected from each group (10 groups), and serum concentrations of leptin and adiponectin were measured using an ELISA kit (Abcam USA). The results are as shown in Figure 11.
각 그룹 샘플을 원심 분리하고 100㎕ 상등액을 키트 지침에 따라 모든 그룹의 샘플을 혼합하고, 키트 표준을 플레이트에 첨가한 후, 실온에서 30분 동안 배양한 다음, 96-웰 마이크로 플레이트 판독기를 사용하여 450nm에서 흡광도를 측정하였다.Centrifuge each group sample and 100 μl supernatant. Mix samples from all groups according to the kit instructions, add kit standards to the plate, incubate at room temperature for 30 minutes, and then plate using a 96-well microplate reader. Absorbance was measured at 450 nm.
렙틴은 체중이 증가하고 지방이 많을수록 증가는데, 도 11의 (a)에 나타난 바와 같이, 스테비아 추출물을 투여한 그룹들에서 렙틴의 활성이 낮게 나타나는 것을 확인할 수 있었다. 또한 ST+SP에서 ORL(Orlistat) 보다 렙틴이 낮게 나타나는 것을 확인할 수 있었다.Leptin increases as body weight increases and the amount of fat increases. As shown in (a) of Figure 11, leptin activity was confirmed to be low in the groups administered stevia extract. Additionally, it was confirmed that leptin appeared lower in ST+SP than in ORL (Orlistat).
아디포넥틴은 지방을 제어하는 중요한 인자로써, 도 11의 (b)를 보면, 스테비아 추출물을 투여한 그룹들에서 아디포넥틴의 활성이 훨씬 높게 나타나는 것을 확인할 수 있었고, 스테비아 추출물(ST)+녹차 추출물(SP)에서 가장 높게 나타나는 것을 확인할 수 있었다.Adiponectin is an important factor in controlling fat. Looking at Figure 11 (b), it was confirmed that the activity of adiponectin was much higher in the group administered stevia extract, and stevia extract (ST) + green tea extract (SP) It was confirmed that it appeared highest in .
이상으로 첨부된 도면을 참조하여 본 발명의 실시예를 설명하였으나, 본 발명이 속하는 기술분야에서 통상의 지식을 가진 자는 본 발명의 기술적 사상이나 필수적인 특징을 변경하지 않고 다른 구체적인 형태로 실시할 수 있다는 것을 이해할 수 있을 것이다. 따라서 이상에서 기술한 실시예는 모든 면에서 예시적인 것이며 한정적이 아닌 것이다.Although embodiments of the present invention have been described above with reference to the attached drawings, those skilled in the art will understand that the present invention can be implemented in other specific forms without changing the technical idea or essential features of the present invention. You will be able to understand it. Therefore, the embodiments described above are illustrative in all respects and are not restrictive.
Claims (11)
Anti-obesity activity containing 300 mg/kg/mouse body weight/d of stevia extract and 50 mg/kg/mouse body weight/d of green tea extract containing green tea saponin or 100 μg/ml of stevia extract and 25 μg/ml of the green tea extract. A food composition having.
상기 스테비아 추출물은,
신생혈관억제 기능을 가지는 것을 특징으로 하는 항비만 활성을 갖는 식품조성물.
According to paragraph 1,
The stevia extract is,
A food composition with anti-obesity activity, characterized by having an anti-angiogenic function.
상기 스테비아 추출물은,
스테비아 잎으로부터 스테비아 추출물을 추출하는 추출단계 및
추출된 스테비아 추출물을 정제하는 정제단계를 통해 제조되는 것을 특징으로 하는 항비만 활성을 갖는 식품조성물.
According to paragraph 1,
The stevia extract is,
Extraction step of extracting stevia extract from stevia leaves and
A food composition with anti-obesity activity, characterized in that it is manufactured through a purification step of purifying the extracted stevia extract.
상기 추출단계는,
스테비아 잎을 분쇄하여 스테비아 분말로 제조하는 전처리단계;
상기 스테비아 분말에서 스테비아 추출물을 추출하는 추출물추출단계;
상기 스테비아 추출물을 농축하는 농축단계 및
농축된 스테비아 추출물을 동결건조시키는 동결건조단계를 포함하는 항비만 활성을 갖는 식품조성물.
According to paragraph 3,
The extraction step is,
A preprocessing step of grinding stevia leaves to produce stevia powder;
An extract extraction step of extracting stevia extract from the stevia powder;
A concentration step of concentrating the stevia extract and
A food composition with anti-obesity activity comprising a freeze-drying step of freeze-drying concentrated stevia extract.
상기 전처리단계는,
분쇄된 스테비아 분말을 n-헥산으로 탈지한 후 건조시키는 것을 특징으로 하는 항비만 활성을 갖는 식품조성물.
According to paragraph 4,
The preprocessing step is,
A food composition with anti-obesity activity, characterized in that pulverized stevia powder is defatted with n-hexane and then dried.
상기 추출물추출단계는,
상기 스테비아 분말을 에탄올에 용해시켜 환류를 통해 스테비아 추출물을 추출하는 것을 특징으로 하는 항비만 활성을 갖는 식품조성물.
According to paragraph 4,
The extract extraction step is,
A food composition with anti-obesity activity, characterized in that the stevia powder is dissolved in ethanol and the stevia extract is extracted through reflux.
상기 정제단계는,
추출된 스테비아 추출물을 증류수에 용해시키고 컬럼(Column)에 충전시킨 후, 상기 컬럼에 수지를 첨가하여 비극성 물질을 분리시키는 제1 정제단계;
상기 컬럼에 수산화나트륨(NaOH)을 통과시켜 산성 물질을 용출시키는 제2 정제단계;
산성물질이 제거된 스테비아 추출물에 염화수소(HCl)을 첨가하여 중화시키는 중화단계 및
중화된 스테비아 추출물을 컬럼(Column)에 충전시킨 후, 에탄올을 통해 용출시켜 비극성 물질이 제거된 스테비아 추출물을 획득하는 제3 정제단계를 포함하는 항비만 활성을 갖는 식품조성물.
According to paragraph 3,
The purification step is,
A first purification step of dissolving the extracted stevia extract in distilled water and filling it in a column, then adding resin to the column to separate non-polar substances;
A second purification step of eluting acidic substances by passing sodium hydroxide (NaOH) through the column;
A neutralization step of adding hydrogen chloride (HCl) to the stevia extract from which acidic substances have been removed, and
A food composition with anti-obesity activity comprising a third purification step of filling a column with the neutralized stevia extract and eluting it with ethanol to obtain a stevia extract from which non-polar substances have been removed.
상기 제3 정제단계에서 에탄올은,
100% 에탄올인 것을 특징으로 하는 항비만 활성을 갖는 식품조성물.
In clause 7,
In the third purification step, ethanol is,
A food composition with anti-obesity activity, characterized in that it is 100% ethanol.
상기 녹차 사포닌은,
녹차의 테아사포닌-E1(theasaponin E1)을 포함하는 것을 특징으로 하는 항비만 활성을 갖는 식품조성물.
According to paragraph 1,
The green tea saponin is,
A food composition with anti-obesity activity, characterized in that it contains theasaponin E1 of green tea.
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Non-Patent Citations (2)
Title |
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KIM et al., Composition for Anti-Angiogenesis containing Tea-Saponin E 1 as an Effective Ingredient. 한국생물공학회, 생물공학의 동향. 2012년, p. 43 1부.* |
박정은 외 2인. C57BL/6J Mice에서 스테비아(Stevia rebaudiana bertoni) 잎 추출물의 항비만 효과. 한국식품과학회지. 2010년, Vol. 42, No. 5, pp. 586-592 1부.* |
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