JP6166592B2 - Agents containing a millet vinegar composition - Google Patents
Agents containing a millet vinegar composition Download PDFInfo
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- JP6166592B2 JP6166592B2 JP2013126594A JP2013126594A JP6166592B2 JP 6166592 B2 JP6166592 B2 JP 6166592B2 JP 2013126594 A JP2013126594 A JP 2013126594A JP 2013126594 A JP2013126594 A JP 2013126594A JP 6166592 B2 JP6166592 B2 JP 6166592B2
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- vinegar
- composition
- acne
- millet
- agent
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Description
本発明は、きび酢組成物に関する。より詳細には、本発明は、きび酢組成物を有効成分とする、脂肪細胞への分化の抑制、および／または内臓脂肪の蓄積の抑制のための剤に関する。本発明の剤は、肥満、メタボリックシンドローム、脂肪肝等の処置のために有用である。本発明は、食品および食品に用いる添加物の製造、ヘルスケア、保健指導および医療等の分野で有用である。 The present invention relates to a millet vinegar composition. More specifically, the present invention relates to an agent for suppressing differentiation into adipocytes and / or suppressing accumulation of visceral fat, which comprises an acne vinegar composition as an active ingredient. The agent of the present invention is useful for the treatment of obesity, metabolic syndrome, fatty liver and the like. INDUSTRIAL APPLICABILITY The present invention is useful in the fields of food and additives used for food, health care, health guidance and medical care.
生活習慣病の多くは、不適切な食生活や運動不足等の不健全な生活の積み重ねによって内臓脂肪型肥満となり、これが原因となって引き起こされる。内臓脂肪症候群は、メタボリックシンドロームとも称される。一方、肝臓の中性脂肪が異常に増加した状態を脂肪肝といい、成因はさまざまであるが、脂肪肝は肥満を伴うことも多く、アルコール過飲や栄養不適による脂肪肝は、脂肪性肝硬変の原因となることがある。また肥満を伴う脂肪肝は、肥満によって脂質異常症や高血圧、糖尿病などの生活習慣病に至るリスクが高まるため、放っておくと、それらの生活習慣病を引きおこす恐れがある。 Many lifestyle-related diseases are caused by visceral fat-type obesity due to unhealthy life such as inappropriate eating habits and lack of exercise. Visceral fat syndrome is also referred to as metabolic syndrome. On the other hand, the state in which the neutral fat in the liver is abnormally increased is called fatty liver, and the cause is various. Fatty liver is often accompanied by obesity, and fatty liver due to alcohol overdose or malnutrition is fatty cirrhosis. It may cause. Fatty liver with obesity increases the risk of obesity, hypertension, diabetes and other lifestyle-related diseases, and if left untreated, there is a risk of causing these lifestyle-related diseases.
他方、食品は、第一次機能としての栄養性、第二次機能としての嗜好性に加えて、第三次機能として、生体調節作用を有しており、当該機能性を利用した種々の製品がいわゆる健康食品等として、広く一般に受け入れられ、利用されてきている。食経験の比較的長い食品由来の物質は、食の安全に対する意識の高まりや天然物志向にも合致し、その機能に基づく種々の用途が研究され、様々な製品が開発されてきている。 On the other hand, in addition to nutrition as a primary function and palatability as a secondary function, foods have a bioregulatory action as a tertiary function, and various products using this functionality Have been widely accepted and used as so-called health foods. Substances derived from foods with a relatively long dietary experience match the growing awareness of food safety and natural products, and various applications based on their functions have been studied and various products have been developed.
非特許文献1は、黒酢による肥満抑制および糖尿病改善効果について報告している。ここでは、黒酢によるそれらの効果は、内臓脂肪型・糖尿病モデルラットOLETF (Otsuka Long-Evans Tokushima Fatty)には有効であるが、そのコントロールであるLETO (Otsuka Long-Evans Tokushima)ラットに対しては効果が低い旨が述べられている。 Non-Patent Document 1 reports on obesity suppression and diabetes improvement effects by black vinegar. Here, the effects of black vinegar are effective for the visceral fat type / diabetes model rat OLETF (Otsuka Long-Evans Tokushima Fatty), but against the control LETO (Otsuka Long-Evans Tokushima) rat. Is said to be less effective.
非特許文献2は、黒酢濃縮液の機能について検討したものである。ここでは、黒酢濃縮液を経口投与したラットにおいては、膵リパーゼの活性阻害による白色脂肪組織における細胞のサイズの減少、および脂肪細胞におけるPPARγとaP2とのmRNA発現レベルの低下が報告されている。 Non-Patent Document 2 examines the function of black vinegar concentrate. Here, in rats administered with black vinegar concentrate orally, a decrease in cell size in white adipose tissue due to inhibition of pancreatic lipase activity, and a decrease in mRNA expression levels of PPARγ and aP2 in adipocytes have been reported. .
特許文献1は、黒酢または黒酢のうち酢酸以外の成分（黒酢特有成分）を有効成分とする、抗肥満剤に関する。ここでは、この抗肥満作用は、黒酢特有成分の、脂肪組織内でのPPARγの穏やかなアンタゴニストとしての機能、脂肪酸合成酵素の発現を低下させる機能、アディポネクチンの分泌を促進／分泌の低下を抑制する機能に関与すると考えられる旨が述べられている。 Patent Document 1 relates to an anti-obesity agent containing black vinegar or a component other than acetic acid (black vinegar-specific component) as an active component. Here, this anti-obesity action is the function of black vinegar-specific components as a mild antagonist of PPARγ in adipose tissue, the function of decreasing the expression of fatty acid synthase, the promotion of adiponectin secretion and the suppression of the decrease in secretion It is stated that it is considered to be involved in the function to do.
本発明者は、食品由来の機能性の成分について鋭意検討してきた。きび酢は、奄美地方の特産品であるサトウキビを原料とし、天然の酵母および酢酸菌により醗酵生産されるが、一般的な米酢や、米・米麹・水を原料とする黒酢にはない成分組成を有すると考えられ、種々の機能が期待される。本発明者は今般、きび酢組成物について肥満に関連する種々の機能を発見し、本発明を為すに至った。 The inventor has intensively studied the functional ingredients derived from food. Although millet vinegar is made from sugarcane, a special product of the Amami region, and fermented and produced with natural yeast and acetic acid bacteria, it is not found in common rice vinegar or black vinegar made from rice, rice bran, and water. It is considered to have a component composition, and various functions are expected. The present inventor has recently discovered various functions related to obesity with respect to the acne vinegar composition, and has made the present invention.
本発明は以下の各発明を包含する。
[1] きび酢組成物を含む、脂肪細胞への分化抑制剤。
[2] 肥満またはメタボリックシンドロームの処置のための、[1]に記載の剤。
[3] きび酢組成物を含む、内臓脂肪の蓄積抑制剤。
[4] 肝臓への脂肪の蓄積抑制のためのものである、[3]に記載の剤。
[5] 脂肪肝リスクの低減のためのものである、[3]または[4]に記載の剤。
[6] 食事と共に摂取される、[3]〜[5]のいずれか一に記載の剤。
[7] きび酢組成物が、きび酢乾燥物または水溶性固形分量が12以上の濃縮物である、[1]〜[6]のいずれか一に記載の剤。
[8] きび酢組成物を、肥満もしくはメタボリックシンドロームである対象、または肥満もしくはメタボリックシンドロームのリスクのある対象に摂取させること含む、肥満またはメタボリックシンドロームの処置方法（医療行為を除く。）。
[9] きび酢組成物を、脂肪肝である対象、または脂肪肝のリスクのある対象に摂取させること含む、脂肪肝の処置方法（医療行為を除く。）。
[10] きび酢組成物を、食事と共に摂取させる、[9]に記載の方法。
[11] きび酢組成物が、きび酢乾燥物または水溶性固形分量が12以上の濃縮物である、[8]〜[10]のいずれか一に記載の剤。
The present invention includes the following inventions.
[1] An agent for inhibiting differentiation into adipocytes, comprising a millet vinegar composition.
[2] The agent according to [1] for the treatment of obesity or metabolic syndrome.
[3] A visceral fat accumulation inhibitor comprising a millet vinegar composition.
[4] The agent according to [3], which is for suppressing accumulation of fat in the liver.
[5] The agent according to [3] or [4], which is for reducing the risk of fatty liver.
[6] The agent according to any one of [3] to [5], which is taken with a meal.
[7] The agent according to any one of [1] to [6], wherein the millet vinegar composition is a dried millet vinegar or a concentrate having a water-soluble solid content of 12 or more.
[8] A method of treating obesity or metabolic syndrome (excluding medical practice) comprising ingesting a acne vinegar composition to a subject who is obese or metabolic syndrome, or at risk for obesity or metabolic syndrome.
[9] A method for treating fatty liver (excluding medical practice), comprising ingesting a acne vinegar composition into a subject with or at risk for fatty liver.
[10] The method according to [9], wherein the millet vinegar composition is taken with a meal.
[11] The agent according to any one of [8] to [10], wherein the millet vinegar composition is a dried millet vinegar or a concentrate having a water-soluble solid content of 12 or more.
本発明において数値範囲を「X〜Y」で表すときは、特に記載した場合を除き、その範囲は両端の数値XおよびYを含む。本発明で、組成物等おける成分の濃度に関し「%」で表すときは、特に記載した場合を除き、重量に基づく値である。 In the present invention, when a numerical range is represented by “X to Y”, the range includes numerical values X and Y at both ends unless otherwise specified. In the present invention, when the concentration of the component in the composition or the like is expressed by “%”, it is a value based on the weight unless otherwise specified.
本発明は、きび酢組成物を有効成分とする剤を提供する。 The present invention provides an agent comprising a millet vinegar composition as an active ingredient.
きび酢（「さとうきび酢」ということもある。）は、サトウキビ抽出物（搾汁液、黒糖）を糖質原料として、発酵により得られた食酢である。きび酢は、奄美大島において古来より調製されてきており、一般的には、サトウキビを圧搾し、搾汁を得て、得られた搾汁液を天然の酵母および酢酸菌により自然発酵させて得られる。 Acne vinegar (also referred to as “sugar cane vinegar”) is a vinegar obtained by fermentation using sugarcane extract (squeezed liquid, brown sugar) as a saccharide material. Acne vinegar has been prepared in Amami Oshima since ancient times, and is generally obtained by squeezing sugarcane to obtain juice and naturally fermenting the obtained juice with natural yeast and acetic acid bacteria.
本発明で「きび酢組成物」というときは、特に記載した場合を除き、きび酢から得られた濃縮物、乾燥物および粗精製物を含むが、きび酢自体は含まない。 In the present invention, the term “acne vinegar composition” includes a concentrate, a dried product and a crude product obtained from acne vinegar, except for the case specifically described, but does not include acne vinegar itself.
本発明の有効成分であるきび酢組成物の原料としてのきび酢は、サトウキビ抽出物（搾汁液、黒糖）を主たる糖質原料とし、酵母および酢酸菌により発酵させて得られたものであればよく、通常のきび酢を用いることができる。きび酢組成物を得るための濃縮、乾燥等の手段は、特に限定されず、食品分野で用いられる通常の手段を適用することができる。本発明においては、きび酢組成物として、きび酢濃縮物またはきび酢乾燥物を好適に用いることができる。きび酢濃縮物を用いる場合、濃縮の程度は特に限定されないが、例えば、水溶性固形分量（Brixということもある。）が12以上となるまで濃縮したものを用いることができ、好ましくは13以上となるまで濃縮したものを用いることができ、より好ましくは14以上となるまで濃縮したものを用いることができる。本発明で「水溶性固形分量（またはBrix）」というときは、特に記載した場合を除き、通常の意味で用いている。すなわち1gのショ糖が 20℃の水溶液100gに溶解しているとき、このショ糖溶液と同じ糖度屈折計の値を示す溶液のBrix値 が1と定義される。Brixの値は、食品分野で慣用されているBrix計により値をいう。なお、一般的なきび酢のBrixは、4.5〜5.0である。 The millet vinegar as a raw material of the millet vinegar composition which is an active ingredient of the present invention may be any sugarcane extract (squeezed liquid, brown sugar) as a main saccharide raw material, as long as it is obtained by fermentation with yeast and acetic acid bacteria, Normal millet vinegar can be used. Means such as concentration and drying for obtaining the acne vinegar composition are not particularly limited, and usual means used in the food field can be applied. In the present invention, as the acne vinegar composition, an acne vinegar concentrate or a dried acne vinegar can be suitably used. When the millet vinegar concentrate is used, the degree of concentration is not particularly limited. For example, a concentrated product can be used until the water-soluble solid content (sometimes referred to as Brix) is 12 or more, preferably 13 or more. What was concentrated until it became can be used, More preferably, what was concentrated until it became 14 or more can be used. In the present invention, the term “water-soluble solid content (or Brix)” is used in its ordinary meaning unless otherwise specified. That is, when 1 g of sucrose is dissolved in 100 g of an aqueous solution at 20 ° C., the Brix value of a solution showing the same sugar content refractometer value as this sucrose solution is defined as 1. The Brix value is a value obtained by a Brix meter commonly used in the food field. In addition, Brix of common millet vinegar is 4.5-5.0.
本発明者の検討によると、きび酢組成物は、マウス胎児由来前駆脂肪細胞3T3-L1細胞の脂肪細胞への分化を抑制した。したがって、本発明の剤は、脂肪細胞への分化を抑制するための剤として用いることができる。より具体的には、本発明の剤は、対象における肥満またはメタボリックシンドロームの処置のために用いることができる。なお、脂肪細胞への分化の抑制と肥満またはメタボリックシンドロームの処置との関係は、河田照雄の論文（Trace Nutrients Research 22: 1-5, 2005）等を参照することができる。 According to the study by the present inventor, the acne vinegar composition suppressed the differentiation of mouse embryo-derived preadipocytes 3T3-L1 cells into adipocytes. Therefore, the agent of the present invention can be used as an agent for suppressing differentiation into adipocytes. More specifically, the agents of the present invention can be used for the treatment of obesity or metabolic syndrome in a subject. For the relationship between inhibition of differentiation into adipocytes and treatment of obesity or metabolic syndrome, see Teruo Kawada's paper (Trace Nutrients Research 22: 1-5, 2005).
また本発明者の検討によると、きび酢組成物は、肝臓由来の細胞HepG2において長鎖脂肪酸の中性脂肪への取り込み量を減少させた。したがって、本発明の剤は、内臓脂肪の蓄積抑制剤として用いることができる。より具体的には、本発明の剤は、肝臓への脂肪の蓄積抑制のために、および／または脂肪肝の処置のため（好ましくは、脂肪肝リスクの低減のため）に、用いることができる。なお、HepG2細胞と内臓脂肪との関係は、M. Teresa Donato等の論文（Chemico-Biological Interactions 181: 417-423, 2009）等を参照することができる。 Further, according to the study of the present inventor, the acne vinegar composition decreased the amount of long-chain fatty acids incorporated into neutral fat in the liver-derived cell HepG2. Therefore, the agent of the present invention can be used as a visceral fat accumulation inhibitor. More specifically, the agent of the present invention can be used for suppressing accumulation of fat in the liver and / or for treating fatty liver (preferably for reducing the risk of fatty liver). . The relationship between HepG2 cells and visceral fat can be referred to a paper by M. Teresa Donato et al. (Chemico-Biological Interactions 181: 417-423, 2009).
ある成分または剤が、脂肪細胞への分化を抑制するか否かは、3T3-L1細胞等の適切な細胞を用いた系で、評価することができる。3T3-L1細胞はisobutyl-methylxanthine（IBMX）、dexamethasone（DEX）、インスリンによって効率良く脂肪細胞に分化する。また脂肪細胞の形成は、(1)幹細胞が脂肪細胞としての素地を獲得した脂肪芽細胞に決定される段階、(2)脂肪芽細胞が増殖する段階、(3)細胞の増殖停止段階、(4)前駆脂肪細胞が未成熟な脂肪細胞へ終末分化する段階、(5)未成熟な脂肪細胞に脂肪が蓄積し成熟する段階、そして(6)成熟脂肪細胞が分裂・増殖する段階に大別できるが、本発明者の検討によると、3T3-L1細胞を用いた場合は、IBMX、DEXおよびインスリンを含む分化誘導培地を用いた(4)の段階で、きび酢またはきび酢組成物を添加すると意図した効果が見られた。したがって、典型的な評価においては、少なくとも分化誘導培地で培養する際に評価の対象となる成分または剤を添加するとよい。そして培養後に得られた細胞群において、細胞数、1細胞当たりの脂肪滴数、1細胞当たりの脂肪滴面積を測定する。そして適切な対照群と比較して、分化抑制機能の有無およびその程度を評価することができる。 Whether a certain component or agent suppresses differentiation into adipocytes can be evaluated by a system using appropriate cells such as 3T3-L1 cells. 3T3-L1 cells are efficiently differentiated into adipocytes by isobutyl-methylxanthine (IBMX), dexamethasone (DEX), and insulin. In addition, the formation of adipocytes is (1) the stage where the stem cells are determined to be a lipoblast that has acquired a base as an adipocyte, (2) the stage where the adipocytes proliferate, (3) the stage of cell growth arrest, 4) Stages of preadipocyte differentiation into immature adipocytes (5) Stage of fat accumulation and maturation in immature adipocytes, and stage of (6) mature adipocyte division and proliferation However, according to the study of the present inventor, when 3T3-L1 cells are used, it is intended to add acne vinegar or an acne vinegar composition at the stage of (4) using differentiation induction medium containing IBMX, DEX and insulin. The effect was seen. Therefore, in a typical evaluation, it is advisable to add at least a component or an agent to be evaluated when culturing in a differentiation induction medium. Then, in the cell group obtained after the culture, the number of cells, the number of lipid droplets per cell, and the lipid droplet area per cell are measured. And the presence or absence of a differentiation suppression function and its extent can be evaluated compared with a suitable control group.
ある成分または剤が、肝臓細胞等、内臓組織を構成する細胞における、中性脂肪の合成、中性脂肪の取り込み、または蓄積を抑制するか否かは、HepG2細胞等の適切な細胞を用いた系で、評価することができる。より詳細には、長鎖脂肪酸（例えば、パルミチン酸）を添加した培地で細胞を培養する際に、またはそれ以外の適切な段階で、評価の対象となる成分または剤を添加して培養し、得られた細胞群において、細胞数、1細胞当たりの脂肪滴数、1細胞当たりの脂肪滴面積を測定する。そして適切な対照群と比較して、脂肪の有無およびその量を評価することができる。また肝毒性は創薬過程で評価すべき重要な項目の一つであり、異常な脂肪蓄積を起こさないかどうかを評価するための、HepG2細胞を用いる試験のためのキット（Stearosis Assay Kit, Colorimetric; ケイマンケミカル社製）が市販されている。脂肪肝を引き起こすことが知られ、このような評価においてポジティブコントロールとして使用される成分の一つに、クロロキン（chloroquine）がある。 Whether or not a certain component or agent suppresses the synthesis of triglyceride, triglyceride uptake or accumulation in cells constituting the visceral tissue such as liver cells was determined using appropriate cells such as HepG2 cells. The system can be evaluated. More specifically, when cells are cultured in a medium supplemented with a long-chain fatty acid (for example, palmitic acid), or at any other appropriate stage, the components or agents to be evaluated are added and cultured, In the obtained cell group, the number of cells, the number of lipid droplets per cell, and the lipid droplet area per cell are measured. The presence and amount of fat can then be assessed as compared to an appropriate control group. In addition, hepatotoxicity is one of the important items to be evaluated in the drug discovery process, and a kit for testing using HepG2 cells (Stearosis Assay Kit, Colorimetric) to evaluate whether abnormal fat accumulation occurs. ; Cayman Chemical Co.) is commercially available. One ingredient that is known to cause fatty liver and is used as a positive control in such assessments is chloroquine.
本発明で「肥満」というときは、特に記載した場合を除き、身長と体重から計算されるＢＭＩ（肥満指数）が、25以上である状態をいう。本発明で「肥満」というときは、医学的に減量を必要とする肥満、すなわち「肥満症」を含む。肥満症は、(1)ＢＭＩが25以上であり、かつ肥満に原因があるかもしくは肥満に関連していて、減量を必要とする健康障害を伴う場合、または(2)ＢＭＩが25以上であり、かつ(1)のような健康障害はなくても、検査によって内臓脂肪型肥満と診断された場合をいう。肥満に原因があるかもしくは肥満に関連していて、減量を必要とする健康障害は、耐糖能障害・2型糖尿病、脂質代謝異常（高コレステロール血症、低HDLコレステロール血症、高トリグリセリド血症を含む。）、高血圧、高尿酸血症・痛風、脂肪肝（non-alcoholic steatohepatitis (NASH)を含む）、冠動脈疾患（心筋梗塞、狭心症を含む。）、脳梗塞（脳血栓症、一過性脳虚血発作を含む。）、骨・関節疾患（変形性膝関節症、変形性股関節症、変形性脊椎症、腰痛症を含む。）、睡眠時無呼吸症候群・Pickwick症候群、月経異常（月経周期の異常、月経量と周期の異常、無月経月経随伴症状の異常を含む。）、肥満妊婦、心理的サポートが必要な肥満症を含む。 In the present invention, “obesity” refers to a state where the BMI (obesity index) calculated from height and weight is 25 or more, unless otherwise specified. In the present invention, “obesity” includes obesity requiring medical weight loss, that is, “obesity”. Obesity is (1) BMI is 25 or more and is associated with obesity or is associated with obesity and accompanied by a health disorder requiring weight loss, or (2) BMI is 25 or more In addition, it refers to a case where visceral fat type obesity is diagnosed by a test even if there is no health disorder as in (1). Health disorders that cause or are related to obesity and require weight loss include impaired glucose tolerance, type 2 diabetes, abnormal lipid metabolism (hypercholesterolemia, hypoHDL-cholesterolemia, hypertriglyceridemia) ), Hypertension, hyperuricemia / gout, fatty liver (including non-alcoholic steatohepatitis (NASH)), coronary artery disease (including myocardial infarction and angina), cerebral infarction (cerebral thrombosis, transient Cerebral ischemic attack), bone / joint diseases (including knee osteoarthritis, hip osteoarthritis, osteoarthritis, low back pain), sleep apnea syndrome / Pickwick syndrome, menstrual abnormalities ( Including menstrual cycle abnormalities, menstrual flow and cycle abnormalities, and abnormal menstrual symptoms.), Obese pregnant women, and obesity requiring psychological support.
また本発明で「メタボリックシンドローム（「内臓脂肪症候群」ということもある。）」というときは、特に記載した場合を除き、内臓に脂肪が蓄積した内臓脂肪型肥満に加えて、脂質異常、高血圧および高血糖のうち、いずれか2つ以上をあわせもった状態をいう。より具体的には、腹囲（へそ周り）が男性は85cm以上、女性は90cm以上（男女ともに、腹部CT検査の内臓脂肪面積が100cm2以上に相当）であって、脂質異常（中性脂肪 150mg/dL以上、HDLコレステロール 40mg/dL未満のいずれかまたは両方を満たす。）、高血圧（最高（収縮期）血圧 130mmHg以上、最低（拡張期）血圧 85mmHg以上のいずれかまたは両方を満たす。）および高血糖（空腹時血糖値 110mg/dL以上）のうち、いずれか2つ以上を満たす状態をいう。 Further, in the present invention, “metabolic syndrome” (also referred to as “visceral fat syndrome”) is, unless otherwise specified, in addition to visceral fat-type obesity in which fat has accumulated in the viscera, dyslipidemia, hypertension and A state in which any two or more of hyperglycemia are combined. More specifically, the circumference of the abdomen (around the navel) is 85 cm or more for men and 90 cm or more for women (both men and women correspond to an abdominal CT examination visceral fat area of 100 cm 2 or more), and lipid abnormalities (neutral fat 150 mg / dL or higher, HDL cholesterol less than 40 mg / dL or both, high blood pressure (highest (systolic) blood pressure 130 mmHg or higher, lowest (diastolic) blood pressure 85 mmHg or higher or higher) State that satisfies any two or more of blood glucose (fasting blood glucose level 110mg / dL or more).
本発明で疾患または状態について「処置」というときは、発症リスクの低減、発症の遅延、予防、治療、進行の停止、遅延を含む。処置には、医師が行う治療を目的とした行為と、医師以外の者、例えば栄養士等による非治療的行為とが含まれる。処置には、特定の食品の投与、摂取の推奨、食事指導、栄養指導、保健指導を含む。本発明における処置の対象は、人（個体）を含み、好ましくは、当該処置を施すことが望ましいか、または当該処置を施す必要のある人である。処置の対象は、人以外の動物であってもよく、この例としては、イヌ、ネコなどのコンパニオンアニマル、牛、豚などの家畜、実験動物、動物園等で飼育されている動物が挙げられる。 The term “treatment” for a disease or condition in the present invention includes reduction of risk of onset, delay of onset, prevention, treatment, stop of progression, and delay. The treatment includes an action for the purpose of treatment performed by a doctor and a non-therapeutic action by a person other than the doctor, such as a dietitian. Treatment includes administration of specific foods, intake recommendations, dietary guidance, nutritional guidance, and health guidance. The subject of the treatment in the present invention includes a person (individual), and is preferably a person who desirably or needs to perform the treatment. The subject of treatment may be an animal other than a human, and examples thereof include companion animals such as dogs and cats, domestic animals such as cows and pigs, laboratory animals, animals kept in zoos, and the like.
本発明で「剤」というときは、特に記載した場合を除き、きび酢組成物そのものである場合と、きび酢組成物およびそれ以外の成分を含む場合とがあるが、きび酢自体は含まず、またきび酢を含有する既存の食品は、含まない。 In the present invention, the term “agent”, unless otherwise specified, may be the acne vinegar composition itself or may contain the acne vinegar composition and other components, but does not include acne vinegar itself, Does not include existing foods containing.
本発明の剤による、有効成分であるきび酢組成物の摂取量は、目的の効果が発揮される量であればよい。例えば、きび酢は、従来技術の健康食品に含まれる黒酢よりも効果が高いとの観点からは、黒酢よりも少量での奏功が期待できるので、Brix 4.5のきび酢（酸度4.5）換算で、10mg/day以上とすることができ、11mg/dayとすることが好ましく、12mg/dayとすることがより好ましく、14mg/dayとすることがさらに好ましい。きび酢固形物換算では、0.3mg/day以上とすることができ、0.33mg/dayとすることが好ましく、0.36mg/dayとすることがより好ましく、0.42mg/dayとすることがさらに好ましい。また、肥満またはメタボリックシンドロームの処置において十分な効果が期待できるとの観点からは、Brix 4.5のきび酢（酸度4.5）換算で、10mg/day以上とすることができ、100mg/dayとすることが好ましく、500mg/dayとすることがより好ましく、1,000mg/dayとすることがさらに好ましい。きび酢固形物換算では、0.3mg/day以上とすることができ、3.0mg/dayとすることが好ましく、15mg/dayとすることがより好ましく、30mg/dayとすることがさらに好ましい。いずれの場合であっても、上限値は、Brix 4.5のきび酢（酸度4.5）換算で、5,000mg/day以下とすることができ、4,000mg/dayとすることがより好ましく、3,000mg/dayとすることがより好ましく、2,000mg/dayとすることがさらに好ましい。きび酢固形物換算では、150mg/day以下とすることができ、120mg/dayとすることがより好ましく、90mg/dayとすることがより好ましく、60mg/dayとすることがさらに好ましい。摂取は、一日に1〜数回に分けて行うことができる。 The intake of the acne vinegar composition, which is an active ingredient, according to the agent of the present invention may be an amount that exhibits the intended effect. For example, from the standpoint that kibine vinegar is more effective than black vinegar contained in conventional health foods, it can be expected to be effective in a smaller amount than black vinegar. It can be 10 mg / day or more, preferably 11 mg / day, more preferably 12 mg / day, and even more preferably 14 mg / day. In terms of acne vinegar solid matter, it can be 0.3 mg / day or more, preferably 0.33 mg / day, more preferably 0.36 mg / day, and still more preferably 0.42 mg / day. Further, from the viewpoint that a sufficient effect can be expected in the treatment of obesity or metabolic syndrome, it can be 10 mg / day or more, preferably 100 mg / day in terms of Brix 4.5 acne vinegar (acidity 4.5). 500 mg / day is more preferable, and 1,000 mg / day is even more preferable. In terms of acne vinegar solid matter, it can be 0.3 mg / day or more, preferably 3.0 mg / day, more preferably 15 mg / day, and even more preferably 30 mg / day. In any case, the upper limit can be 5,000 mg / day or less, more preferably 4,000 mg / day, in terms of Brix 4.5 acne vinegar (acidity 4.5), and 3,000 mg / day. More preferably, it is more preferably 2,000 mg / day. In terms of acne vinegar solid matter, it can be 150 mg / day or less, more preferably 120 mg / day, more preferably 90 mg / day, and still more preferably 60 mg / day. Ingestion can be divided into one to several times a day.
本発明の剤には、目的の効果を発揮しうる限り、きび酢組成物以外の他の成分を配合することができる。他の成分は、食品として許容される種々の添加剤、または医薬として許容される種々の添加剤であり得る。この例には、賦形剤、酸化防止剤抗（酸化剤）、香料、調味料、甘味料、着色料、増粘安定剤、発色剤、漂白剤、防かび剤、ガムベース、苦味料等、酵素、光沢剤、酸味料、乳化剤、強化剤、製造用剤、結合剤、緊張化剤（等張化剤）、緩衝剤、溶解補助剤、防腐剤、安定化剤、凝固剤等である。 In the agent of the present invention, other components than the acne vinegar composition can be blended as long as the desired effect can be exhibited. The other ingredients may be various food acceptable additives or various pharmaceutically acceptable additives. Examples include excipients, anti-oxidants (oxidants), flavorings, seasonings, sweeteners, colorants, thickeners, color formers, bleaches, fungicides, gum bases, bitters, etc. Enzymes, brighteners, acidulants, emulsifiers, reinforcing agents, production agents, binders, tonicity agents (isotonic agents), buffers, solubilizers, preservatives, stabilizers, coagulants, and the like.
他の成分は、きび酢組成物以外の機能性成分であってもよい。他の機能性成分に例としては、アミノ酸類（例えば、分岐鎖アミノ酸類、オルニチン）、不飽和脂肪酸類（例えば、EPA、DHA）、ビタミン類、微量金属類、ポリフェノール類、卵黄抽出物、はちみつ加工品、黒糖、オリゴ糖、食物繊維、グルコサミン、コンドロイチン類、CoQ10、フコイダン、フコキサンチン、アスタキサンチン、プラセンタ、酵母エキス、黒酢濃縮物、植物抽出物（ニンニク抽出物、イチョウ葉抽出物、茶抽出物、ビルベリー抽出物、ブルーベリー抽出物、各種人参抽出物、マカ抽出物、豆種皮抽出物、セントジョーンズワート抽出物、松樹皮抽出物、アサイー抽出物、ノニ抽出物）等が挙げられる。 The other component may be a functional component other than the acne vinegar composition. Examples of other functional ingredients include amino acids (eg, branched chain amino acids, ornithine), unsaturated fatty acids (eg, EPA, DHA), vitamins, trace metals, polyphenols, egg yolk extract, honey Processed products, brown sugar, oligosaccharide, dietary fiber, glucosamine, chondroitins, CoQ10, fucoidan, fucoxanthin, astaxanthin, placenta, yeast extract, black vinegar concentrate, plant extract (garlic extract, ginkgo biloba extract, tea extract Products, bilberry extract, blueberry extract, various carrot extracts, maca extract, bean seed coat extract, St. John's wort extract, pine bark extract, acai extract, noni extract) and the like.
本発明の剤が有効成分と有効成分以外の他の成分からなる場合、有効成分の含有量は、当業者であれば、製造し安さ、用いやすさ等の点から適宜設計でき、例えば、0.1〜99.9％とすることができ、また1〜95％とすることができ、また10％〜90％とすることができる。 When the agent of the present invention comprises an active ingredient and other components other than the active ingredient, the content of the active ingredient can be appropriately designed by those skilled in the art in terms of ease of manufacture, ease of use, and the like. It can be ˜99.9%, can be 1 to 95%, and can be 10% to 90%.
本発明の剤の形態は、上述したように、きび酢およびきび酢を含有する既存の食品を除き、種々の形態であり得る。例えば、経口医薬品、食品であり得る。本発明で「食品」というときは、特に記載した場合を除き、固形物のみならず、液状のもの、例えばスープ、飲料およびドリンク剤を含む。また、本発明で「食品」というときは、特に記載した場合を除き、ヒトを対象としたもののみならず、非ヒト動物を対象としたもの、例えば飼料およびペットフードを含む。さらに、本発明で「食品」というときは、特に記載した場合を除き、一般食品、健康食品、サプリメント、保健機能食品（栄養機能食品および特定保健用食品を含む。）を含み、また治療食（治療の目的を果たすもの。医師が食事箋を出し、それに従い栄養士等が作成した献立に基づいて調理されたもの。）、食事療法食、成分調整食、減塩食、介護食、減カロリー食、ダイエット食を含む。 As described above, the form of the agent of the present invention may be various forms except for acne vinegar and existing foods containing acne vinegar. For example, it can be an oral drug or food. The term “food” in the present invention includes not only solid substances but also liquid substances such as soups, beverages and drinks unless otherwise specified. In addition, the term “food” in the present invention includes not only human subjects but also non-human animal subjects such as feed and pet food, unless otherwise specified. Furthermore, the term “food” in the present invention includes general foods, health foods, supplements, health functional foods (including nutritional functional foods and foods for specified health use), and therapeutic foods (except for special cases). The one that fulfills the purpose of the treatment.The one prepared by the doctor who made a meal and was prepared based on the menu prepared by a dietitian etc.), dietary diet, ingredient adjustment diet, low salt diet, nursing diet, reduced calorie diet Including diet diet.
本発明の剤が、経口医薬品、健康食品、サプリメントの形態である場合、その剤形の例としては、軟カプセル剤、硬カプセル剤、錠剤、丸剤、粉末剤、顆粒剤、細粒剤、ゼリー剤、チューブ入り剤、ドリンク剤を挙げることができる。 When the agent of the present invention is in the form of oral pharmaceuticals, health foods, and supplements, examples of the dosage form include soft capsules, hard capsules, tablets, pills, powders, granules, fine granules, A jelly agent, a tube containing agent, and a drink agent can be mentioned.
軟カプセル剤、硬カプセル剤、錠剤または丸剤の形態とする場合、有効成分であるきび酢組成物の含量は、Brix 4.5のきび酢（酸度4.5）換算で、1剤あたり500mg、好ましくは600mg、より好ましくは750mg、さらに好ましくは1,000mgとすることができる。きび酢固形物換算では、1剤あたり1.5mg、好ましくは1.8mg、より好ましくは2.3 mg、さらに好ましくは3.0mgとすることができる。 In the form of soft capsules, hard capsules, tablets or pills, the content of the active ingredient acne vinegar composition is 500 mg per agent, preferably 600 mg, in terms of Brix 4.5 acne vinegar (acidity 4.5) Preferably it can be 750 mg, more preferably 1,000 mg. In terms of acne vinegar solids, it can be 1.5 mg per agent, preferably 1.8 mg, more preferably 2.3 mg, and even more preferably 3.0 mg.
本発明の剤が、一般食品の形態である場合、その具体的としては、飲料（例えば、清涼飲料、果汁飲料、野菜飲料、青汁）、調味料組成物（例えば、サラダドレッシング、マヨネーズ、トマトケチャップ、チキンコンソメ、ビーフコンソメ、化学調味料組成物、ウスターソース、とんかつソース、たれ、ハーブ塩、味噌、醤油、めんつゆ、だし）、スープまたはスープの素（例えば、コーンスープ、オニオンスープ、トマトスープ、ブイヤベース、コンソメスープ、味噌汁、吸い物）、ソース類（例えば、ホワイトソース、デミグラスソース、トマトソース、ミートソース、カレールウ、パスタソース）、菓子類（例えば、飴、チューインガム、錠菓、グミゼリー、チョコレート、ビスケット、スナック菓子、冷菓）、レトルトパウチ惣菜、チルド惣菜、冷凍惣菜、即席めん、保存食（例えば、酢漬け、塩漬け）、フライ類（例えば、フライドポテト、フライドチキン、フライドフィッシュ）、魚すり身製品、畜肉製品、チーズ、バター、発酵乳製品（例えば、ヨーグルト、乳酸菌飲料）、チーズ類（例えば、ナチュラルチーズ、プロセスチーズ、チーズスプレッド、発酵バター）、発酵植物製品（キムチ、すぐき、ぬか漬け、ピクルス、ザワークラウト）、野菜・果実製品を挙げることができる。 When the agent of the present invention is in the form of general food, specific examples thereof include beverages (for example, soft drinks, fruit juices, vegetable drinks, green juices), seasoning compositions (for example, salad dressings, mayonnaise, tomatoes) Ketchup, chicken consomme, beef consomme, chemical seasoning composition, Worcester sauce, tonkatsu sauce, sauce, herb salt, miso, soy sauce, noodle soup, soup stock, soup or soup base (eg corn soup, onion soup, tomato soup, Bouillabaisse, consomme soup, miso soup, soup), sauces (eg, white sauce, demiglace sauce, tomato sauce, meat sauce, curry roux, pasta sauce), confectionery (eg, salmon, chewing gum, tablet confectionery, gummy jelly, chocolate, biscuits, snacks) , Frozen dessert), retort pouch side dish, Ludo side dish, frozen side dish, instant noodles, preserved food (eg pickled, salted), fries (eg, fried potato, fried chicken, fried fish), fish surimi products, livestock meat products, cheese, butter, fermented dairy products (eg, , Yogurt, lactic acid bacteria beverages), cheeses (eg, natural cheese, processed cheese, cheese spread, fermented butter), fermented plant products (kimchi, taki, pickled, pickles, sauerkraut), vegetable / fruit products .
一般食品の形態とする場合、有効成分であるきび酢組成物の含量は、一食としての摂取量を勘案して設計することができる。一食としての一般食品の形態は、例えば80〜1,000ml（好ましくは、150〜750ml）の容器詰飲料がある。Brix 4.5のきび酢（酸度4.5）換算で、一食あたり500mg、好ましくは600mg、より好ましくは750mg、さらに好ましくは1,000mgとすることができる。きび酢固形物換算では、一食あたり1.5mg、好ましくは1.8mg、より好ましくは2.3mg、さらに好ましくは3.0mgとすることができる。 In the case of a form of general food, the content of the acne vinegar composition which is an active ingredient can be designed in consideration of the amount of intake as a single meal. The form of the general food as a meal is, for example, 80 to 1,000 ml (preferably 150 to 750 ml) of a packaged beverage. In terms of Brix 4.5 millet vinegar (acidity 4.5), it can be 500 mg per serving, preferably 600 mg, more preferably 750 mg, and even more preferably 1,000 mg. In terms of acne vinegar solids, it can be 1.5 mg per serving, preferably 1.8 mg, more preferably 2.3 mg, and even more preferably 3.0 mg.
本発明の剤は、食事と共に、例えば、食前、食事中、食後に、摂取することができる。また、本発明者の検討によると、きび酢組成物を用いたマウス胎児由来前駆脂肪細胞3T3-L1を用いた分化誘導試験では、分化誘導培地での培養段階、または分化誘導培地での培養段階およびインスリン培地での培養段階にきび酢組成物を添加した場合に、脂肪細胞への分化抑制効果が見られた。したがって、本発明の剤は、既に肥満症である段階、または肥満症に向かいつつある段階に摂取することが効果的であると考えられる。同様に、本発明の剤は、既にメタボリックシンドロームである段階、またはメタボリックシンドロームに向かいつつある段階に摂取することが効果的であると考えられる。 The agent of the present invention can be taken with a meal, for example, before, during, or after a meal. Further, according to the study of the present inventors, in the differentiation induction test using mouse fetal derived preadipocytes 3T3-L1 using the acne vinegar composition, the culture stage in the differentiation induction medium, or the culture stage in the differentiation induction medium and When the acne vinegar composition was added to the culture stage in the insulin medium, an effect of inhibiting differentiation into adipocytes was observed. Therefore, it is considered effective to take the agent of the present invention at a stage where the patient is already obese or is approaching obesity. Similarly, it is believed that the agent of the present invention is effective when taken at a stage where it is already in metabolic syndrome or is heading toward metabolic syndrome.
本発明の剤は、種々の公知の技術を用いて製造することができる。有効成分であるきび酢組成物を所定の比および／または濃度になるように調整する工程は、製造工程の種々の段階で適用できる。当業者であれば、各成分の溶解性、安定性、揮発性等を考慮して、本発明の剤のための製造工程を、適宜設計しうる。 The agent of the present invention can be produced using various known techniques. The process of adjusting the acne vinegar composition, which is an active ingredient, to a predetermined ratio and / or concentration can be applied at various stages of the production process. A person skilled in the art can appropriately design a production process for the agent of the present invention in consideration of solubility, stability, volatility, and the like of each component.
本発明の剤には、上述の疾患または状態の処置のために用いることができる旨を表示することができ、また上述の対象に対して摂取を薦める旨を表示することができる。より具体的には、本発明の剤には、体重または体脂肪の維持に適するまたは改善に役立つ旨；血圧または血糖値の良好な維持に適するまたは改善に役立つ旨；肥満、メタボリックシンドローム、内臓脂肪過多、または脂肪肝等のリスクの低減に資する旨を表示することができる。表示は、直接的にまたは間接的にすることができ、直接的な表示の例は、製品自体、パッケージ、容器、ラベル、タグ等の有体物への記載であり、間接的な表示の例は、ウェブサイト、店頭、展示会、看板、掲示板、新聞、雑誌、テレビ、ラジオ、郵送物、電子メール等の場所または手段よる、広告・宣伝活動を含む。 The agent of the present invention can display that it can be used for the treatment of the above-mentioned diseases or conditions, and can display that it is recommended to take the above-mentioned subject. More specifically, the agent of the present invention is suitable for maintaining or improving body weight or body fat; suitable for maintaining or improving blood pressure or blood glucose level; obesity, metabolic syndrome, visceral fat It can be displayed that it contributes to the reduction of the risk of excess or fatty liver. The labeling can be direct or indirect, and examples of direct labeling are descriptions on tangible objects such as the product itself, packages, containers, labels, tags, etc. Includes advertising and publicity activities by places or means such as websites, storefronts, exhibitions, signboards, bulletin boards, newspapers, magazines, television, radio, mailings, e-mails, etc.
以下、本発明を実施例を用いて説明するが、本発明の範囲は実施例に記載したものに限られない。 EXAMPLES Hereinafter, although this invention is demonstrated using an Example, the range of this invention is not restricted to what was described in the Example.
〔濃縮きび酢の製造および分析〕
下表の酢を原料として、濃縮きび酢（濃縮液体きび酢）および濃縮黒酢を製造した。
[Production and analysis of concentrated acne vinegar]
Concentrated cane vinegar (concentrated liquid cane vinegar) and concentrated black vinegar were produced using the vinegars in the table below.
濃縮は、大型ロータリーエバポレーターR-250（日本ビュッヒ株式会社）を用いて行った。詳細には、きび酢10kgをフラスコへ投入し、バス温度75℃、フラスコ回転数80rpmで120分減圧濃縮し、濃縮きび酢（brix 15.0）2.25kgを得た。また、黒酢10kgをフラスコへ投入し、バス温度75℃、フラスコ回転数80rpmで120分減圧濃縮し、濃縮黒酢（Brix 15.0）2.3kgを得た。 Concentration was performed using a large rotary evaporator R-250 (Nippon Buch Co., Ltd.). Specifically, 10 kg of acne vinegar was put into a flask and concentrated under reduced pressure at a bath temperature of 75 ° C. and a flask rotation speed of 80 rpm for 120 minutes to obtain 2.25 kg of concentrated acetic acid vinegar (brix 15.0). Further, 10 kg of black vinegar was put into the flask and concentrated under reduced pressure at a bath temperature of 75 ° C. and a flask rotation speed of 80 rpm for 120 minutes to obtain 2.3 kg of concentrated black vinegar (Brix 15.0).
得られた濃縮物を分析した（株式会社消費経済研究所による。）。結果を下表に示す。 The resulting concentrate was analyzed (according to Consumer Economic Research Institute). The results are shown in the table below.
〔脂肪細胞分化実験〕
材料および方法：
1. 細胞培養
脂肪細胞のモデルとしてヒューマンサイエンス研究資源バンクより購入した、マウス胎児由来前駆脂肪細胞3T3-L1（JCRB9014）を使用した。細胞は10％Fetal Bovine Serum (FBS, Hyclone, Central, USA)添加ダルベッコ変法イーグル培地（DMEM, Nissui, Tokyo, Japan）を用いて、37℃、5％ CO2存在下で経代培養した。DMEM培地は、1LのMilli-Q水に対して、DMEM粉末10 gを溶解し、これに硫酸ストレプトマイシン（明治製菓、東京）0.1 g力価、ペニシリンGカリウム（明治製菓）10万U、1 M HEPES（オートクレーブで滅菌）5 mL、10％ NaHCO3（オートクレーブで滅菌）10 mLを添加し、0.22μmフィルター滅菌したものをDMEM培地とし、4℃で保存した。
[Adipocyte differentiation experiment]
Materials and methods:
1. Cell culture Mouse embryonic preadipocytes 3T3-L1 (JCRB9014) purchased from Human Science Research Resource Bank were used as a model of adipocytes. Cells were subcultured using Dulbecco's modified Eagle's medium (DMEM, Nissui, Tokyo, Japan) supplemented with 10% Fetal Bovine Serum (FBS, Hyclone, Central, USA) in the presence of 5% CO2 at 37 ° C. DMEM medium is prepared by dissolving 10 g of DMEM powder in 1 L of Milli-Q water, 0.1 g titer of streptomycin sulfate (Meiji Seika, Tokyo), 100,000 U of penicillin G (Meiji Seika), 1 M 5 mL of HEPES (sterilized by autoclave) and 10 mL of 10% NaHCO3 (sterilized by autoclave) were added, and 0.22 μm filter sterilized was used as DMEM medium and stored at 4 ° C.
2. 脂肪細胞への分化誘導
(1) 分化誘導試薬の調製
i) 25 mM IBMX: 12 mgの3-isobutyl-1-methylxanthine (IBMX, Wako, Osaka)に3.5 M KOH 25μLを加えて溶解し、これを2.5 mLのDMEMに加えて、0.22μmのフィルター滅菌した。IBMXは用事調製して使用した。
ii) 25μM Dex: 1.72 mgのDexamethasone (Dex, Wako)に99%エタノール1 mLを加えて溶解し、これを10 mLのDMEMに加えて、0.22μmフィルター滅菌したものを25μM Dexとして-20℃で凍結保存した。
iii) 10 mg/mL インスリン：100 mgのインスリン(Wako)に10N HClを加えて溶解し、これをMilli-Q水で10 mLになるようにメスアップした後、0.22 mmフィルター滅菌したものを10 mg/mL Insulinとして-80℃で凍結保存した。
2. Induction of differentiation into adipocytes
(1) Preparation of differentiation-inducing reagent
i) 25 mM IBMX: Dissolve 25 mg of 3.5 M KOH in 12 mg of 3-isobutyl-1-methylxanthine (IBMX, Wako, Osaka), add it to 2.5 mL of DMEM, and sterilize by 0.22 μm filter . IBMX was prepared for use.
ii) 25 μM Dex: Dissolve 1.72 mg Dexamethasone (Dex, Wako) in 1 mL of 99% ethanol, add it to 10 mL of DMEM, and sterilize the 0.22 μm filter at 25 ° M Dex at -20 ° C. Cryopreserved.
iii) 10 mg / mL insulin: Add 10N HCl to 100 mg insulin (Wako), dissolve, make up to 10 mL with Milli-Q water, and then sterilize with 0.22 mm filter. It was stored frozen at −80 ° C. as mg / mL insulin.
(2) 分化誘導処理
i) コラーゲンコート処理：分化誘導処理を行う際は、コラーゲンコート処理を施したディッシュに細胞を播種した。コラーゲンは、コラーゲンＩ（BD, NY, USA）を使用し、0.02 Mの酢酸を用いて50μg/mLに希釈した後、0.22μmフィルター滅菌したものを4℃で保存した。細胞を播種する前のディッシュにコラーゲンＩ溶液を5μg/cm2になるようにコーティングし、37℃で1時間インキュベートした。コラーゲンＩ溶液を除去し、PBSで2回洗浄後、細胞を播種した。
(2) Differentiation induction processing
i) Collagen coating treatment: When differentiation induction treatment was performed, cells were seeded on a dish that had been subjected to collagen coating treatment. Collagen used was collagen I (BD, NY, USA), diluted to 50 μg / mL with 0.02 M acetic acid, and then sterilized with a 0.22 μm filter at 4 ° C. The dish before seeding the cells was coated with a collagen I solution at 5 μg / cm 2 and incubated at 37 ° C. for 1 hour. After removing the collagen I solution and washing twice with PBS, the cells were seeded.
ii) 分化誘導処理：3T3-L1前駆脂肪細胞を2×105 cell/mLで96ウェルプレートに播種し、10％FBS-DMEMで1-2日間培養した後(a)、コンフルエントに達したことを確認し、0.5 mM IBMX、0.25μM DEX、10μg/mLインスリン添加培地で72時間培養した（b）。その後、10μg/mLインスリンのみを含む培地で48時間培養した（c）。その後、10％FSB-DMEMのみの培地に戻して培養を行うことにより分化誘導を行った。
iii) サンプル添加：前掲の濃縮きび酢または濃縮黒酢は、10％FBS-DMEM培地での培養時（a）、分化誘導培地（0.5 mM IBMX、0.25μM DEX、10μg/mLインスリン添加培地）での培養時（b）、インスリン培地（10μg/mLインスリンのみを含む培地）での培養時（c）において、適宜、終濃度が0.5%、1%または3%となるように添加した。
ii) Differentiation-inducing treatment: 3T3-L1 preadipocytes were seeded in 96-well plates at 2 × 10 5 cells / mL and cultured in 10% FBS-DMEM for 1-2 days (a) and reached confluence. And cultured in a medium supplemented with 0.5 mM IBMX, 0.25 μM DEX, 10 μg / mL insulin for 72 hours (b). Thereafter, the cells were cultured for 48 hours in a medium containing only 10 μg / mL insulin (c). Thereafter, differentiation was induced by returning to a medium containing only 10% FSB-DMEM and culturing.
iii) Sample addition: Concentrated black vinegar or concentrated black vinegar listed above is cultured in 10% FBS-DMEM medium (a), differentiation induction medium (0.5 mM IBMX, 0.25 μM DEX, 10 μg / mL insulin-added medium). During culturing (b) and at the time of culturing in an insulin medium (medium containing only 10 μg / mL insulin) (c), the final concentration was appropriately adjusted to 0.5%, 1% or 3%.
(3) IN Cell Analyzer 1000を用いた脂肪細胞分化度合いの評価
i) 細胞内脂肪および核の染色：3T3-L1が脂肪細胞へと分化する際に細胞内に生じる脂肪滴を蛍光染色するためのBODIPY493/503 (D-3922, Invitrogen, San Diego, USA)を用いた。BODIPYは、細胞内の中性および非極性脂質を染色することが知られている。BODIPYは、エタノールで1 mg/mLに希釈し、-4℃で遮光保存した。使用する際は、PBSで25 ng/mLに希釈した。核の染色は、Hoechst33342 (Dojindo, Kumamoto, Japan)を使用した。Hoechst33342はDNA配列のAT選択的に結合するminor groove binderで、2本鎖DNA選択制を示し、細胞内の核を染色する。
(3) Evaluation of the degree of adipocyte differentiation using IN Cell Analyzer 1000
i) Staining of intracellular fat and nucleus: BODIPY493 / 503 (D-3922, Invitrogen, San Diego, USA) for fluorescent staining of lipid droplets generated in the cells when 3T3-L1 differentiates into adipocytes Using. BODIPY is known to stain intracellular neutral and nonpolar lipids. BODIPY was diluted to 1 mg / mL with ethanol and stored in the dark at -4 ° C. When used, it was diluted to 25 ng / mL with PBS. Hoechst33342 (Dojindo, Kumamoto, Japan) was used for nuclear staining. Hoechst33342 is a minor groove binder that selectively binds DNA sequences with AT, and exhibits a double-stranded DNA selection system, and stains intracellular nuclei.
ii) IN Cell Analyzer 1000による測定および解析：細胞をPBS100μLで洗浄し、10％ホルマリンを100μL添加後、室温で10分間インキュベートし、細胞の固定を行った。PBS 100μLで洗浄し、25 ng/mL BODIPY溶液を100μL添加後、暗所、室温で20分インキュベートし、細胞内の脂肪滴の染色を行った。BODIPY溶液を除去し、10μM Hoechst33342溶液を100μL添加後、暗所、室温で20分インキュベートし、核の染色を行った。Hoechst33342溶液を除去した後、PBS 100μLを添加し、IN Cell Analyzer 1000 (GE Healthcare, UK)で測定した。IN Cell Analyzer 1000に付属のソフトウェア（WorkstationあるいはDeveloper）を用いることで、細胞数、1細胞当たりの脂肪滴数、1細胞当たりの脂肪滴面積の測定を行った。 ii) Measurement and analysis using IN Cell Analyzer 1000: The cells were washed with 100 μL of PBS, added with 100 μL of 10% formalin, and incubated at room temperature for 10 minutes to fix the cells. After washing with 100 μL of PBS and adding 100 μL of a 25 ng / mL BODIPY solution, the cells were incubated in the dark at room temperature for 20 minutes to stain intracellular lipid droplets. The BODIPY solution was removed, and 100 μL of 10 μM Hoechst33342 solution was added, followed by incubation at room temperature in the dark for 20 minutes to stain the nucleus. After removing the Hoechst33342 solution, 100 μL of PBS was added, and measurement was performed with IN Cell Analyzer 1000 (GE Healthcare, UK). Using the software (Workstation or Developer) attached to IN Cell Analyzer 1000, the number of cells, the number of lipid droplets per cell, and the area of lipid droplets per cell were measured.
結果：
結果を図1に示した。また、サンプルを添加しないもの（ctrl）、およびきび酢または黒酢の代わりに、同濃度の酢酸を添加したもの（図中、例えば、「酢酸 0.5%」は、「きび酢 0.5%」と酢酸濃度が同じになるように、酢酸を添加した実験区を表す。）も同様に評価した。(b)段階、または(b)および(c)段階できび酢を添加した場合、高い効果（脂肪細胞への分化抑制効果）が観察された。図中、エラーバーは標準偏差を示す。*: p>0.05, **: p>0.01, ***: p>0.001（コントロールとの比較。n=3。student’s t-test）
result:
The results are shown in FIG. Also, the sample not added (ctrl), and the one with acetic acid of the same concentration instead of acne vinegar or black vinegar (in the figure, for example, "Acetic acid 0.5%" The experimental group to which acetic acid was added was also expressed in the same manner. When vinegar was added at stage (b) or at stage (b) and (c), a high effect (an effect of inhibiting differentiation into adipocytes) was observed. In the figure, error bars indicate standard deviation. *: p> 0.05, **: p> 0.01, ***: p> 0.001 (comparison with control, n = 3, student's t-test)
〔脂肪肝抑制実験〕
材料および方法：
1. サンプル調整
(1) 各種サンプル調整
前掲濃縮黒酢および濃縮液体キビ酢、ならびに固体キビ酢（きび酢乾燥物。収率約3%。日宝泉力本舗株式会社から提供された。）を用いた。固体キビ酢は10 mg/mLとなるようにPBSで溶かしサンプル溶液とした。各種サンプルは4℃で保存し、適宜転倒混和を行い実験に使用した。また、濃縮液体キビ酢の酢酸含有量が全体の6.87%であることから、6.87%酢酸（和光純薬, 大阪）溶液を各種サンプルのコントロールとして用いた。
[Fatty liver suppression experiment]
Materials and methods:
1. Sample adjustment
(1) Various sample preparations The above-mentioned concentrated black vinegar and concentrated liquid millet vinegar, and solid millet vinegar (dried millet vinegar. Yield about 3%. Provided by Nihosen Riki Honpo Co., Ltd.). Solid millet vinegar was dissolved in PBS so as to be 10 mg / mL to obtain a sample solution. Various samples were stored at 4 ° C. and mixed by inversion as appropriate for use in the experiment. Further, since the acetic acid content of the concentrated liquid millet vinegar was 6.87% of the total, a 6.87% acetic acid (Wako Pure Chemicals, Osaka) solution was used as a control for various samples.
(2) 各種サンプルのpHと浸透圧測定
3T3-L1細胞およびHepG2細胞に各種サンプルを添加する際、含まれる酢酸の細胞への影響をなくすため0.1もしくは1N NaOH溶液（和光純薬, 大阪）でpHを7.2付近にしたときの浸透圧を測定した。pHの測定にはCOMPACT pH METER twin pH（HORIBA, 京都）を、浸透圧測定にはOSMOMETER OM 802-D（VOGEL, BRD）を用いた。
(2) pH and osmotic pressure measurement of various samples
When adding various samples to 3T3-L1 cells and HepG2 cells, the osmotic pressure when the pH is adjusted to around 7.2 with 0.1 or 1N NaOH solution (Wako Pure Chemicals, Osaka) to eliminate the effect of acetic acid contained in the cells. It was measured. COMPACT pH METER twin pH (HORIBA, Kyoto) was used for pH measurement, and OSMOMETER OM 802-D (VOGEL, BRD) was used for osmotic pressure measurement.
2. HepG2細胞における脂肪酸処理およびサンプル処理
(1) パルミチン酸ナトリウム溶液の作製
10%脂肪酸不含BSA（Albumin, Bovine Serum, Fatty Acid-Free（CRL, Wiltshire, UK））/ Phosphate Buffered Saline （PBS）に、パルミチン酸ナトリウム（Chem Service, Inc., West Chester, USA）が最終濃度8 mMとなるように添加した。ソニケーションにより溶解させ、0.22μm PVDFシリンジフィルター（日本ミリポア, 東京）にてフィルトレーションを行い滅菌した。
2. Fatty acid treatment and sample treatment in HepG2 cells
(1) Preparation of sodium palmitate solution
10% fatty acid-free BSA (Albumin, Bovine Serum, Fatty Acid-Free (CRL, Wiltshire, UK)) / Phosphate Buffered Saline (PBS) and sodium palmitate (Chem Service, Inc., West Chester, USA) It added so that it might become a concentration of 8 mM. It was dissolved by sonication, filtered and sterilized with a 0.22 μm PVDF syringe filter (Nihon Millipore, Tokyo).
(2) サンプル処理
前日に、96 well plate（μClear Fluorescence Black Plate（Greiner bio-one , BRD））にHepG2細胞を2.0×104 cells/wellで播種し、ディッシュ底面に接着させておく。24時間後、1.5 mLエッペンチューブに入れた10%FBS/DMEM培地に、酢酸、黒酢、液体キビ酢は最終濃度0.5, 3%となるように各サンプルを添加し、そのサンプル溶液を1.0N NaOH溶液（和光純薬, 大阪）でpH7.2付近となるよう調整した。固体キビ酢は0.1、1、10%（固形物としての重量%）となるように10%FBS/DMEM培地に添加した。96well plateの培地を除去した後、これらの作製したサンプル溶液を100μL/wellで添加し、37℃、5% CO2存在下で24時間培養した。
また分画サンプルは、HepG2細胞を2.0×104 cells/wellで播種した24時間後、最終濃度10, 50μg/mLとなるようにサンプルを培地に添加した。
(2) Sample treatment On the day before, HepG2 cells are seeded at a density of 2.0 × 10 4 cells / well in a 96 well plate (μClear Fluorescence Black Plate (Greiner bio-one, BRD)) and allowed to adhere to the bottom of the dish. After 24 hours, add each sample to 10% FBS / DMEM medium in a 1.5 mL Eppendorf tube so that the final concentration of acetic acid, black vinegar, and liquid millet vinegar will be 0.5, 3%. The pH was adjusted to around 7.2 with NaOH solution (Wako Pure Chemicals, Osaka). Solid millet vinegar was added to 10% FBS / DMEM medium so as to be 0.1, 1, 10% (weight% as a solid). After removing the medium from the 96-well plate, these prepared sample solutions were added at 100 μL / well, and cultured for 24 hours at 37 ° C. in the presence of 5% CO 2 .
The fractional sample was added to the medium at a final concentration of 10, 50 μg / mL 24 hours after seeding HepG2 cells at 2.0 × 10 4 cells / well.
(3) パルミチン酸処理
サンプル処理を施す際に、パルミチン酸処理も行う。最終濃度0.1 mMとなるようにパルミチン酸ナトリウム溶液をwellの培地に添加し、サンプルと同様37℃、5% CO2存在下で24時間培養した。
(3) Palmitic acid treatment Palmitic acid treatment is also performed during sample treatment. A sodium palmitate solution was added to the well medium to a final concentration of 0.1 mM, and the cells were cultured in the presence of 5% CO 2 at 37 ° C. for 24 hours in the same manner as the sample.
3. HepG2細胞へのBODIPY染色
以下の操作は全て遮光条件下で行った。前日にサンプルおよびパルミチン酸処理したHepG2細胞をPBS 100μL/wellで洗浄した。37% formaldehyde（和光純薬, 大阪）をPBSで希釈し、10% formaldehyde溶液とした。これを100μL/wellで添加し10分間室温にて放置し固定した。その間にBODIPY493/503（Life Technologies, CA, USA）0.3μL/PBS 5 mLの脂肪滴染色液を作製した。その後、PBS 100μL/wellで２回洗浄した後、先ほど作製した脂肪滴染色液を100μL/wellで添加し20分室温にて放置し染色した。その間に、Hoechst33342（同仁化学, 熊本）1μL/PBS 1 mLを混合し細胞核染色液を作製した。脂肪滴染色液を除去し、PBS 100μL/wellで１回洗浄した後、細胞核染色液を100μL/wellで添加し15分室温にて放置し染色した。染色後、細胞核染色液を除去し、PBS 100μL/wellで１回洗浄した。さらにPBSを100μL/wellで添加し、IN Cell Analyzer 1000 Imaging Cytometer（GE Healthcare, Amersham ,UK）にて画像を取得した。画像はDeveloperで解析後、Spotfire DecisionSite Client 8.2 software (Spotfire Japan KK)によりグラフ化した。
3. BODIPY staining of HepG2 cells All the following operations were performed under light-shielded conditions. The sample and HepG2 cells treated with palmitic acid on the previous day were washed with 100 μL / well of PBS. 37% formaldehyde (Wako Pure Chemicals, Osaka) was diluted with PBS to make a 10% formaldehyde solution. This was added at 100 μL / well and allowed to stand at room temperature for 10 minutes for fixation. Meanwhile, a lipid droplet staining solution of BODIPY493 / 503 (Life Technologies, CA, USA) 0.3 μL / PBS 5 mL was prepared. Then, after washing twice with 100 μL / well of PBS, the lipid droplet staining solution prepared above was added at 100 μL / well and left to stand at room temperature for 20 minutes for staining. Meanwhile, 1 μL of Hoechst33342 (Dojin Chemical, Kumamoto) / 1 mL of PBS was mixed to prepare a cell nucleus staining solution. After removing the lipid droplet staining solution and washing once with 100 μL / well of PBS, the cell nucleus staining solution was added at 100 μL / well and allowed to stand at room temperature for 15 minutes for staining. After staining, the cell nucleus staining solution was removed and washed once with PBS 100 μL / well. PBS was further added at 100 μL / well, and images were acquired with an IN Cell Analyzer 1000 Imaging Cytometer (GE Healthcare, Amersham, UK). The images were analyzed with Developer and then graphed with Spotfire DecisionSite Client 8.2 software (Spotfire Japan KK).
結果：
細胞数の合計を図2に、脂肪滴面積および脂肪滴数を図3に、細胞当りの脂肪滴数の合計を図4に、細胞当りの脂肪面積の合計を図5に示した。図4および5から明らかであるように、固体きび酢を用いた実験区では、細胞当りの脂肪滴数および脂肪面積が抑制された。
result:
The total number of cells is shown in FIG. 2, the lipid droplet area and the number of lipid droplets are shown in FIG. 3, the total number of lipid droplets per cell is shown in FIG. 4, and the total fat area per cell is shown in FIG. As is clear from FIGS. 4 and 5, in the experimental group using solid acne vinegar, the number of fat droplets and the fat area per cell were suppressed.
〔製造例〕
1. きび酢エキス
前掲のきび酢1000mlを乾燥し、粉末化する。これに蒸留水を加え、再び乾燥処理する。この作業を数回繰り返し、酢酸を除去する。得られた粉末を蒸留水100mlに溶解し、10倍濃縮液とする。
[Production example]
1. Millet vinegar extract 1000ml of millet vinegar is dried and powdered. Distilled water is added to this and dried again. This operation is repeated several times to remove acetic acid. The obtained powder is dissolved in 100 ml of distilled water to obtain a 10-fold concentrated solution.
2. きび酢乾燥物
きび酢を減圧蒸留手段により乾燥させ、必要に応じ、殺菌して得る。
2. Dry millet vinegar Dry millet vinegar by vacuum distillation and sterilize if necessary.
3. きび酢飲料
きび酢に、同量の茶、および7〜10倍量の水を混合し、常法により加熱殺菌した後、これを200ｍＬずつ紙容器に無菌的に充填して製品とする。
3. Millet vinegar drink Mix millet vinegar with the same amount of tea and 7 to 10 times the amount of water, heat sterilize by conventional methods, and aseptically fill each 200 mL of this into a paper container.
4. 一般食品、健康食品
前掲のきび酢エキスまたはきび酢乾燥物を添加して、チューブ状食品、または前掲のきび酢エキスまたはきび酢乾燥物を内容物として、常法により軟カプセル剤または硬カプセル剤とする。
4. General foods and health foods Add the above-mentioned acetic acid vinegar extract or dried acetic acid vinegar, and use tube-shaped foods or the above-mentioned acetic acid vinegar extract or dried acetic acid vinegar as contents to make soft capsules or hard capsules by conventional methods. .
Claims (11)
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