WO2018184525A1 - 包含植物萃取物的组合物及其用途 - Google Patents

包含植物萃取物的组合物及其用途 Download PDF

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WO2018184525A1
WO2018184525A1 PCT/CN2018/081717 CN2018081717W WO2018184525A1 WO 2018184525 A1 WO2018184525 A1 WO 2018184525A1 CN 2018081717 W CN2018081717 W CN 2018081717W WO 2018184525 A1 WO2018184525 A1 WO 2018184525A1
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composition
extract
food
green tea
expression
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PCT/CN2018/081717
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English (en)
French (fr)
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林咏翔
陈怡卉
甘恺雯
刘复诚
陈巧婷
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大江生医股份有限公司
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Priority to CN201880021297.6A priority Critical patent/CN110545833B/zh
Priority to US16/499,473 priority patent/US10933107B2/en
Publication of WO2018184525A1 publication Critical patent/WO2018184525A1/zh

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Definitions

  • the present invention relates to a composition comprising a plant extract and uses thereof, and more particularly to a composition for promoting blood glucose efficiency and a use thereof for promoting expression of a GLUT4 gene.
  • Blood sugar refers to glucose in the blood, which is derived from the digestion of the small intestine and can be further transported by blood to various cells in the body as the main source of energy for the cells.
  • concentration of blood sugar is strictly regulated and controlled, and the general concentration is about 900 mg/L (ie, 5 mmol/L or 90 mg/dl), and the normal range is between 4-6 mmol/L, which is maintained at a constant level.
  • concentration range but with diet and rest, there will be large fluctuations, generally one to two hours after eating, the blood sugar concentration will increase, and in the morning will be the lowest.
  • Deregulation of blood glucose levels can lead to a variety of diseases, especially when persistent blood glucose levels are too high.
  • Diabetes can be classified into type 1 diabetes, in which insulin cannot produce or produce insufficient, and type 2 diabetes, in which cells respond abnormally or unresponsive to insulin. Regardless of the type of diabetes, if left untreated, it will cause many complications, such as: diabetic ketoacidemia, hyperosmolar hyperglycemia, non-ketoacidic coma, and cardiovascular disease, chronic kidney disease, retinopathy, diabetic foot , strokes, etc. According to statistics, the number of people with diabetes in the world was about 422 million in 2016. Many patients suffered from complications, their quality of life declined, and their life risks increased. Therefore, the regulation of blood sugar has always been the goal that scientists are trying to solve.
  • Glucose transporter 4 (GLUT4), which is involved in the transport of glucose, is mainly found in muscle and cartilage tissue. When the protein is in the cytoplasm, it can bind to insulin. Once the blood glucose level is increased, it will stimulate. The glucose carrier protein 4 is displaced to the cell membrane, releasing insulin to rapidly regulate blood sugar, and can also bind glucose to carry it back into the cell. Therefore, if the gene expression of glucose carrier protein 4 can be increased, more glucose carrier protein 4 will be produced, and the sensitivity to insulin can be improved to achieve the purpose of adjusting or lowering blood sugar.
  • One of the objects of the present invention is to provide a composition comprising a plant extract, by itself, or as a pharmaceutical composition or a food composition, which substantially promotes the expression of the GLUT4 gene in combination with the action of two plant extracts.
  • a composition comprising a plant extract, by itself, or as a pharmaceutical composition or a food composition, which substantially promotes the expression of the GLUT4 gene in combination with the action of two plant extracts.
  • the above composition comprises a citrus extract and a green tea extract.
  • the ratio of the citrus extract to the green tea extract is from 0.8 to 1.2: 1.2 to 0.8, preferably 1:1.
  • the dosage of the citrus extract and the green tea extract may be from 0.03125 to 0.25 mg/ml, respectively.
  • a pharmaceutical composition for promoting expression of a GLUT4 gene is provided, which can be prepared into a pharmaceutical composition.
  • the pharmaceutical composition may include the aforementioned composition, and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition may be in the form of a solution, capsule or lozenge.
  • a food composition for promoting expression of a GLUT4 gene is provided.
  • the food composition may include the aforementioned composition, and a food ingredient, and the food ingredient may be a component of a general food, a health food, a dietary supplement, or a drink.
  • composition comprising a plant extract, the composition being used to promote expression of a GLUT4 gene, and the composition may be the aforementioned composition.
  • the plant extract-containing composition may be further used for the production of a pharmaceutical composition that promotes expression of the GLUT4 gene, or for the manufacture of a food, health food, dietary supplement or drink that promotes expression of the GLUT4 gene. .
  • Figure 1 is a graph comparing the effects of different plant extracts or combinations on GLUT4 gene expression in the examples of the present invention.
  • the green tea is washed and dried, and the green tea is coarsely crushed by a pulverizer, and then the solvent is mixed with the green tea coarse powder at a liquid-solid ratio of 5 to 20:1 to 5 in water as a solvent.
  • the green tea coarse fraction is extracted at 50 ° C to 100 ° C for 0.5 to 3 hours.
  • the extraction temperature is preferably from 75 ° C to 95 ° C.
  • the green tea extract obtained by the above extraction step was cooled to room temperature, it was filtered through a mesh mesh to remove residual solids.
  • the filtered green tea extract can be further concentrated under reduced pressure at 45 ° C to 70 ° C to obtain a concentrated product.
  • the citrus extract of the examples of the present invention is obtained by extracting Citrus reticulata fruit, which is commercially available from Lawton Trading Co., Ltd.
  • the red wine extract is obtained by extracting red wine, which can be purchased from Shanghai Boyoutang Biotechnology Co., Ltd.
  • the green coffee bean extract is obtained by extracting unroasted coffee (Coffea spp.) seeds, which are commercially available from ARJUNA NATURAL EXTRACTS Co., Ltd. (India).
  • the blueberry extract is obtained by extracting the fruit of North American blueberry (Vaccinium Cyanococcus), which can be purchased from Yuanhong Herbal Research Co., Ltd. These extracts are formulated in water at appropriate concentrations for use.
  • HepG2 cells purchased from ATCC HB8065 were prepared and cultured in a cell culture medium [Dulbecco's modified Eagle's culture solution (Gibco) containing 1% penicillin/streptomycin, 10% fetal bovine serum]. 2 ml of the cell culture medium was added to each well of a 6-well plate to have 1.5 ⁇ 10 5 HepG2 cells per well.
  • a cell culture medium [Dulbecco's modified Eagle's culture solution (Gibco) containing 1% penicillin/streptomycin, 10% fetal bovine serum]. 2 ml of the cell culture medium was added to each well of a 6-well plate to have 1.5 ⁇ 10 5 HepG2 cells per well.
  • the samples were then divided into two groups, of which group A was a blank control group and group B was a test group.
  • group B according to the plant extracts and component types listed in Table 1, respectively, the dose was 0.03125 mg/ml to 0.5 mg/ml, and the individual extracts or the combination of the two extracts were added in a ratio of 1:1.
  • the extract prepared in the above Example 1 or 2 was reacted at 37 ° C for 6 hours and 24 hours, after which the analysis of the amount of GLUT4 gene expression was carried out.
  • the doses shown in the table are the doses of the individual extracts, or the components of the combined extract.
  • RNA was extracted with an RNA extraction kit (Genemark), and the RNA (2000 ng) was reverse-transcribed into cDNA by reverse transcriptase (SuperScript III Reverse Transcripatase, Invitrogen). Then, qPCR (KAPA CYBR FAST qPCR Kits, KAPA Biosystems) was performed using the primer pairs listed in Table 2 using the ABI Step One Plus Real-Time PCR system to quantify the expression of the GLUT4 gene [to ACTB] The expression of the ( ⁇ -actin, beta-actin) gene was used as an internal control group]. During the real-time polymerase chain reaction, the melting curve was analyzed, and the SCORE method was used.
  • the cycle threshold (Ct) of the aforementioned ACTB gene was used as the internal control group, and the blank control group was used as the control.
  • the glycemic gene index corresponding to gene expression is shown in Table 1 and Figure 1, and Figure 1 is a histogram graphically shown in Table 1. The higher the index value, the better the promotion of GLUT4 gene expression.
  • the index of the promoting effect of the citrus extract alone (0.25 mg/ml) on the expression of the GLUT4 gene was 1.10
  • the index of the promoting effect of the green tea extract alone (0.25 mg/ml) was 0.
  • the index of the promoting effect can be as high as 2.84, which is greatly increased to 258% compared with the original citrus effect.
  • the citrus extract is combined with the blueberry extract to act, the index of the promoting effect is reduced to zero. Therefore, the effect of the combination between the extract components is not necessarily the multiplying effect, and in the examples of the present invention, the promoting effect obtained by combining the citrus extract with the green tea extract is preferred.
  • compositions for promoting GLUT4 gene expression in the embodiment of the present invention when combined with the two extracts of citrus extract and green tea extract, can produce an unexpected multiplication effect, so that the GLUT4 gene is The expression increased significantly. Accordingly, these compositions can be utilized to prepare related pharmaceutical or food compositions that allow the applicator or consumer to achieve an adjustment in blood glucose concentration, thereby improving hyperglycemia.
  • composition for promoting expression of the GLUT4 gene may be further added to a food, a health food, a dietary supplement or a drink.
  • the pharmaceutical composition may be further added to a carrier or other adjuvant well known in the art.
  • the dosage form of the pharmaceutical composition can be, but is not limited to, a solution, a capsule, or a lozenge.

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Abstract

一种包含植物萃取物的组合物，所述植物萃取物包括：柑橘萃取物与绿茶萃取物。该萃取物组合更能够有效提升GLUT4基因的表达。

Description

包含植物萃取物的组合物及其用途
相关申请的交叉引用
本申请主张在2017年04月03日在美国提交的美国专利申请号No.62/480,860、以及在2017年05月08日在美国提交的美国专利申请号No.62/503,185的优先权，其全部内容通过引用包含于此。
技术领域
本发明涉及一种包含植物萃取物的组合物及其用途，尤其涉及一种可促进GLUT4基因表达而具有调节血糖功效的组合物及其用途。
背景技术
血糖，指血液中的葡萄糖，其来自于小肠的消化，并可进一步通过血液将其输送到身体中的各个细胞，作为细胞主要的能量来源。在人体中，血糖的浓度是被严格调节与控制的，一般浓度约为900mg/L(即5mmol/L或90mg/dl)左右，正常范围在4-6mmol/L之间，其维持在恒定的浓度范围中，但随着饮食与作息，会有较大的波动产生，一般进食后一到两个小时血糖浓度会升高，而在早晨时则会降到最低。血糖浓度失调会导致多种疾病，特别是持续血糖浓度过高时就可能引发糖尿病。
糖尿病可分为胰岛素无法产生或产生不足的1型糖尿病，以及细胞对于胰岛素的反应不正常或没有反应的2型糖尿病。不论是哪一型糖尿病，如果不进行治疗，将引发诸多并发症，例如：糖尿病酮酸血症、高渗透压高血糖非酮酸性昏迷，以及心血管疾病、慢性肾脏病、视网膜病变、糖尿病足、中风等。据统计，全世界糖尿病患人口于2016年时约有4.22亿，众多患者苦于并发症，生活质量下降，生命风险更是提升。因此，关于血糖的调控一直是科学家们汲汲营营努力所欲解决的目标。
葡萄糖载体蛋白4(glucose transporter 4，GLUT4)，与葡萄糖的运送相关，主要存在于肌肉及软骨组织中，该蛋白在细胞质中时就可结合装载胰岛素，一旦血液中葡萄糖浓度升高，就会刺激葡萄糖载体蛋白4位移至细胞膜上， 释放出胰岛素以迅速调节血糖，同时也可结合葡萄糖，将其携带回细胞中。因此，葡萄糖载体蛋白4的基因表达若能增加，将可产生更多的葡萄糖载体蛋白4，而提升对胰岛素的敏感度，达到调整或降低血糖的目的。
在国人日常所常食用或饮用的植物或保健成分中，例如：红酒、柑橘、蓝莓、绿茶等，并未知悉其单独或组合是否具有促进GLUT4基因表达的效果，若又能从复方的组合物中，找寻出可大幅提升GLUT4基因表达的组份组合，对于相关糖尿病患者而言将是一大福音。
发明内容
本发明目的之一在于提供一种包含植物萃取物的组合物，通过其本身，或制备成医药组合物或食品组合物，在结合两种植物萃取物作用的情况下，大幅促进GLUT4基因的表达，达到使个体在血糖浓度上能获得适度调节或降低的目的。
在本发明的一实施例中，上述组合物包含柑橘萃取物与绿茶萃取物。
在本发明的一实施例中，其中该柑橘萃取物相对于绿茶萃取物的比例为0.8～1.2：1.2～0.8，优选为1：1。在一方式中，该柑橘萃取物与该绿茶萃取物的剂量可分别为0.03125～0.25mg/ml。
在本发明的另一实施例中，提供一种促进GLUT4基因表达的医药组合物，可将前述组成分制备成医药组合物。
在本发明的前述实施例中，该医药组合物可包括前述的组合物、及药学上可接受的载体。
在本发明的前述实施例中，该医药组合物的剂型可为溶液、胶囊或锭剂。
本发明的再一实施例中，提供一种促进GLUT4基因表达的食品组合物。
在本发明的前述实施例中，该食品组合物可包括前述的组合物、及食品成分，而该食品成分可为一般食品、保健食品、膳食补充品或饮品的成分。
本发明的另一实施例中，提供一种包含植物萃取物的组合物的用途，利用该组合物来促进GLUT4基因表达，而该组合物可为前述的组合物。
在本发明的前述实施例中，该包含植物萃取物的组合物可进一步用于制造促进GLUT4基因表达的医药组合物，或用于制造促进GLUT4基因表达的食品、保健食品、膳食补充品或饮品。
附图说明
图1为本发明实施例中不同植物萃取物或组合对于GLUT4基因表达影响的结果比较图。
具体实施方式
以下将进一步说明本发明的实施方式，下述所列举的实施例用以阐明本发明的发明特点及应用，而非以限定本发明的范围，对于本领域技术人员来说，在不脱离本发明的精神和范围内，还可以做出若干改进和润饰，因此本发明的保护范围应当以本申请权利要求书的内容为准。
实施例1绿茶萃取物的制备
将绿茶(green tea)洗净及干燥，用粉碎机将绿茶粗碎，之后以水为溶剂，以该溶剂与绿茶粗碎物以液固比5～20：1～5的比例混合后，在50℃～100℃下对绿茶粗碎物进行萃取0.5～3小时。萃取温度优选为75℃～95℃。
经上述萃取步骤所得绿茶萃取物冷却至室温后，以400目(mesh)滤网过滤以移除残余固体物。过滤后的绿茶萃取物可进一步在45℃～70℃下进行减压浓缩而获得浓缩产物。
实施例2其他植物萃取物的制备或来源
本发明实施例的柑橘萃取物通过对柑橘(Citrus reticulata)果实进行萃取而获得，该萃取物可购自罗顿贸易有限公司。红酒萃取物通过对红葡萄酒进行萃取而获得，该萃取物可购自上海博优堂生物技术有限公司。青咖啡豆萃取物通过对未经烘焙的咖啡属植物(Coffea spp.)种子进行萃取而获得，该萃取物可购自ARJUNA NATURAL EXTRACTS有限公司(印度)。蓝莓萃取物通过对北美蓝莓(Vaccinium Cyanococcus)果实进行萃取而获得，该萃取物可购自元弘草本科研有限公司。将这些萃取物以水配制成适当浓度后备用。
实施例3血糖调控基因的检测
准备HepG2细胞(购自ATCC HB8065)，在细胞培养液[含有1％盘尼西林/链霉素(Penicillin/streptomycin)、10％胎牛血清的Dulbecco's modified Eagle's培养液(Gibco)]中培养。于6孔板每孔中加入2ml细胞培养液，使每孔具有1.5×10 5个HepG2细胞。
之后将样本分为二组，其中，A组为空白对照组，B组则为试验组。试验组B中分别依照表1所列的植物萃取物、组份种类，分别以0.03125mg/ml至0.5mg/ml的剂量，以个别萃取物，或两萃取物组合以1：1的比例加入由前述实施例1或2中所制备的萃取物后，于37℃下反应6与24小时，之后进行GLUT4基因表达量状况的分析。表中所示剂量为个别萃取物、或组合萃取物中各组份的剂量。
将上述各组细胞回收，以RNA萃取试剂盒(Genemark)萃取其RNA，并以反转录酶(SuperScript III Reverse Transcripatase,Invitrogen)将该些RNA(2000ng)反转录为cDNA。之后利用实时聚合酶链式反应系统(ABI Step One Plus Real-Time PCR system)，利用表2所列引物对进行qPCR(KAPA CYBR FAST qPCR Kits，KAPA Biosystems)，以定量GLUT4基因的表达[以ACTB(β-肌动蛋白，beta-actin)基因的表达做为内部对照组]。实时聚合酶链式反应过程中，分析其解离曲线(melting curve)，并采用SCORE法，以前述ACTB基因的循环阀值(cycle threshold，Ct)做为内部对照组，以空白对照组为对照基因，进行基因表达相对定量分析。以SCORE法分析时，目标基因(GLUT4)的mRNA相对量推导自方程式2 -△Ct，其中△Ct＝Ct 目标基因-Ct ACTB，接着计算GLUT4基因的表达量在各组与空白对照组中的差值，并以此差值的总和作为血糖基因指数。基因表达所对应的血糖基因指数，其结果如表1与图1所示，图1为表1图示化的柱状图。指数数值越高，代表对于GLUT4基因表达的促进效果越好。
表1
Figure PCTCN2018081717-appb-000001
Figure PCTCN2018081717-appb-000002
表2
Figure PCTCN2018081717-appb-000003
由表1与图1的结果可知，单独柑橘萃取物(0.25mg/ml)对于GLUT4基因表达的促进效果的指数为1.10，单独绿茶萃取物(0.25mg/ml)的促进效果的指数为0，但将二者组合作用后，其促进效果的指数可高达2.84，相对于原来柑橘的效果，大幅提高至258％。相较之下，若将柑橘萃取物与蓝莓萃取物组合后加以作用，反而使促进效果的指数降为0。因此，关于萃取物组份间的组合效果，不一定是相乘效果，在本发明的实施例中，以柑橘萃取物结合绿茶萃取物所获得的促进效果为优选。
通过上述试验可知，本发明实施例的促进GLUT4基因表达的组合物，在结合柑橘萃取物与绿茶萃取物两种萃取物的情况下，可产生超乎预期的相乘促进效果，使GLUT4基因的表达显着增加。因此，可利用这些组合物制备相关的医药或食品组合物，使施用者或食用者在血糖浓度获得调节，从而改善血糖过高的情形。
此外，本发明实施例的促进GLUT4基因表达的组合物，也可进一步添加于食品、保健食品、膳食补充品或饮品中。将本发明实施例的促进GLUT4基因表达的组合物制备为医药组合物时，该医药组合物也可进一步加入所属技术领域所熟知的载体或其他辅剂。医药组合物的剂型，可为但不限于溶液、胶囊、或锭剂。

Claims (10)

  1. 一种包含植物萃取物的组合物，其包含柑橘萃取物与绿茶萃取物。
  2. 如权利要求1所述的组合物，其中所述柑橘萃取物相对于该绿茶萃取物的比例为0.8～1.2：1.2～0.8。
  3. 如权利要求2所述的组合物，其中所述柑橘萃取物相对于该绿茶萃取物的比例为1：1。
  4. 如权利要求1所述的组合物，其中所述柑橘萃取物与绿茶萃取物的剂量分别为0.03125～0.25mg/ml。
  5. 一种医药组合物，包含如权利要求1所述的组合物、及药学上可接受的载体。
  6. 如权利要求5所述的医药组合物，其中所述医药组合物的剂型为溶液、胶囊或锭剂。
  7. 一种食品组合物，包括如权利要求1所述的组合物、及食品成分，该食品成分为一般食品、保健食品、膳食补充品或饮品的成分。
  8. 权利要求1所述的组合物用于促进GLUT4基因的表达的用途。
  9. 如权利要求8所述的用途，其中权利要求1所述的组合物是进一步用于制造促进GLUT4基因表达的医药组合物。
  10. 如权利要求8所述的用途，其中权利要求1所述的组合物是进一步用于制造促进GLUT4基因表达的食品、保健食品、膳食补充品或饮品。
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