WO2018184526A1 - 植物萃取物在制备调控pdgfc、fgf2、igf1r、ptgis、nos3、edn1、plat、proc、vwf、f3、serpine1、il-8、icam1、vcam1及casp8基因的组合物中的用途 - Google Patents

植物萃取物在制备调控pdgfc、fgf2、igf1r、ptgis、nos3、edn1、plat、proc、vwf、f3、serpine1、il-8、icam1、vcam1及casp8基因的组合物中的用途 Download PDF

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WO2018184526A1
WO2018184526A1 PCT/CN2018/081719 CN2018081719W WO2018184526A1 WO 2018184526 A1 WO2018184526 A1 WO 2018184526A1 CN 2018081719 W CN2018081719 W CN 2018081719W WO 2018184526 A1 WO2018184526 A1 WO 2018184526A1
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Prior art keywords
extract
composition
grape seed
gene
tea
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PCT/CN2018/081719
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English (en)
French (fr)
Inventor
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 CN201880019606.6A priority Critical patent/CN110621329B/zh
Priority to US16/498,737 priority patent/US10918681B2/en
Publication of WO2018184526A1 publication Critical patent/WO2018184526A1/zh

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Definitions

  • the present invention relates to the use of a plant extract for the preparation of a composition for modulating PDGFC, FGF2, IGF1R, PTGIS, NOS3, EDN1, PLAT, PROC, VWF, F3, SERPINE1, IL-8, ICAM1, VCAM1 and CASP8 genes.
  • Cardiovascular disease is a major threat to human health. According to the information released by the Taiwan Health and Welfare Administration, among the top ten causes of death among Chinese people, cardiovascular disease accounts for three, including heart disease and cerebrovascular disease, respectively. 4. In the United States, cardiovascular disease is the leading cause of death for the American people. It can be seen that the threat of cardiovascular disease to human health has reached a level that cannot be ignored. At the beginning of the onset, there are usually no obvious symptoms, but once it appears In the case of complications, such as stroke, myocardial infarction, heart failure, renal failure or retinal hemorrhage, the patient's life has often been seriously threatened, and in order to treat cardiovascular diseases, the demand for cardiovascular drugs is also high, becoming a medical resource. The huge burden. Therefore, those skilled in the art are committed to developing products for protecting cardiovascular (including food products and pharmaceuticals) in response to the needs of a wide range of human health.
  • the object of the present invention is to provide a plant extract for the preparation of a composition for regulating PDGFC, FGF2, IGF1R, PTGIS, NOS3, EDN1, PLAT, PROC, VWF, F3, SERPINE1, IL-8, ICAM1, VCAM1 and CASP8 genes.
  • the extract is combined with Pu'er tea extract, and at least any one of the group consisting of grape seed extract and Pu'er tea extract.
  • the plant extract comprises at least 0.0039 mg/mL of grape seed extract, and at least 0.0039 mg/mL of four season spring tea extract, tomato extract, green coffee bean extract, or Pu'er Tea extract.
  • the plant extract comprises at least 0.0625 mg/mL of black tea extract and at least 0.0625 mg/mL of spinach extract.
  • the plant extract comprises at least 0.0156 mg/mL of red wine extract and at least 0.0156 mg/mL of Pu'er tea extract.
  • the composition has a cardiovascular protective effect.
  • the composition further comprises a pharmaceutically acceptable carrier.
  • the composition is in the form of a powder, granule, liquid, gel or paste.
  • the composition is prepared in the form of a pharmaceutical or food product.
  • the composition of the present invention comprises grape seed extract and four season spring tea extract, black tea extract and spinach extract, grape seed extract and spinach extract, grape seed extract and green coffee bean extract,
  • red wine extracts and Pu'er tea extracts, as well as plant extracts such as grape seed extract and Pu'er tea extract are through regulation of cardiovascular-related genes (including PDGFC, FGF2, IGF1R, PTGIS, NOS3, EDN1, PLAT).
  • cardiovascular-related genes including PDGFC, FGF2, IGF1R, PTGIS, NOS3, EDN1, PLAT.
  • PROC, VWF, F3, SERPINE1, IL-8, ICAM1, VCAM1 and CASP8 are expressed to achieve cardiovascular protection.
  • Figure 1 is a histogram showing the efficacy of a combination of plant extracts in the regulation of cardiovascular-related gene expression in an embodiment of the invention.
  • gene refers to a DNA sequence, including but not limited to: transcribed into mRNA [which can be translated into polypeptide chains], transcribed into rRNA or tRNA, or Enzymes and other proteins involved in DNA replication, transcription, and regulation serve as DNA sequences for recognition sites. This definition includes a variety of different sequence polymorphisms, mutations, and/or sequence variants, wherein such alternation does not affect the function of the gene product.
  • the term “gene” is intended to include regions that encode not only gene products but also regulatory regions including, for example, promoters, termination regions, translational regulatory sequences [such as ribosome binding sites and internal ribose).
  • gene further includes all introns and other DNA sequences that are spliced from mRNA transcripts, as well as variants resulting from alternative splice sites.
  • cardiovascular protection means capable of treating a cardiovascular disease, protecting the cardiovascular system, or reducing the risk of cardiovascular disease.
  • the pharmaceutical product can be manufactured into a dosage form suitable for parenterally or orally administration using techniques well known to those skilled in the art, including but not limited to: injectables (injection) [eg, sterile aqueous solution or dispersion], sterile powder, tablet, troche, lozenge ), pills, capsules, dispersible powders or granules, solutions, suspensions, emulsions, syrups, elixirs, Slurry and similar things.
  • injectables eg, sterile aqueous solution or dispersion
  • sterile powder sterile powder
  • tablet troche, lozenge
  • pills capsules
  • dispersible powders or granules solutions, suspensions, emulsions, syrups, elixirs, Slurry and similar things.
  • the pharmaceutical product according to the present invention may be selected from parenteral routes in a group consisting of: intraperitoneal injection, subcutaneous injection, intramuscular injection, and the like. Intravenous injection.
  • a pharmaceutically acceptable carrier may comprise one or more agents selected from the group consisting of: solvents, emulsifiers, suspending agents, decomposers (decomposer) ), a binding agent, an excipient, a stabilizing agent, a chelating agent, a diluent, a gelling agent, a preservative, Lubricants, absorption delaying agents, liposomes, and the like.
  • solvents solvents,
  • the pharmaceutically acceptable carrier may comprise a solvent selected from the group consisting of water, normal saline, phosphate buffered saline (PBS), a sugar-containing solution, An aqueous solution containing alcohol, and combinations thereof.
  • a solvent selected from the group consisting of water, normal saline, phosphate buffered saline (PBS), a sugar-containing solution, An aqueous solution containing alcohol, and combinations thereof.
  • the composition can be used as a food additive, added by a known method in the preparation of a raw material, or added during the production of a food, and formulated with any edible material for humans and Food products that are ingested by non-human animals.
  • types of food products include, but are not limited to, beverages, fermented foods, bakery products, health foods, and dietary supplements.
  • the grape seed extract of the embodiment of the present invention is obtained by extracting the seed of Vitis spp.
  • the extract can be purchased from Chenghong Biotechnology Co., Ltd.
  • the Puerh tea extract is the Pu'er tea.
  • the extract is obtained by extracting post-fermented leaves of Camellia sinensis, which can be purchased from Nanjing Zelang Biotechnology Co., Ltd.
  • the spinach extract is obtained by extracting spinach (Spinacia oleracea), which can be purchased from Hongxiang Agricultural Processing Factory
  • red wine extract is obtained by extracting red wine, which can be purchased from Shanghai Boyoutang Biotechnology Co., Ltd.
  • green coffee bean extract is for unroasted coffee (Coffea)
  • the spp.) seed is obtained by extraction, and the extract is commercially available from ARJUNA NATURAL EXTRACTS Co., Ltd. (India).
  • This example illustrates the preparation of the extract of Camellia sinensis leaves of the present invention.
  • the black tea is washed and dried, and the black tea is coarsely crushed by a pulverizer.
  • the crude black tea is extracted by using water as a solvent, and the solvent and the black tea coarsely mixed are uniformly mixed at a liquid to solid ratio of 5 to 20:1 to 5, and the extraction temperature is 50 to 100 ° C, preferably 75 to 95 ° C. °C.
  • the extraction time in this example is 0.5 to 3 hours.
  • the black tea extract obtained by the above extraction step was cooled to room temperature, it was filtered through a mesh of 400 mesh to remove residual solids.
  • the filtered black tea extract can be further concentrated under reduced pressure at 45 to 70 ° C to obtain a concentrated product.
  • This example illustrates the preparation of the Four Seasons Spring tea extract of the present invention.
  • the four seasons spring tea is washed and dried, and the four season spring tea is coarsely crushed by a pulverizer.
  • the coarse-grained material of the four seasons spring tea is extracted with water as a solvent, and the solvent is uniformly mixed with the coarse-grained material of the four seasons spring tea at a liquid-solid ratio of 5-20:1 to 5, and the extraction temperature is 50 ° C to 100 ° C, preferably 75 ° C ⁇ 95 ° C.
  • the extraction time in this example is 0.5 to 3 hours.
  • Example 2 Efficacy of a combination of plant extracts in regulating cardiovascular-related gene expression
  • human umbilical vein endothelial cells (HUVEC) [Biosource Collection and Research Center, purchased from the Food Industry Research and Development Institute (FIRDI) in Taiwan. , BCRC), BCRC number is H-UV001] cultured in EC medium (EC medium) [medium 200 (medium 200, M200) and 10% low serum growth supplement (LSGS) (Gibco; Cat.No.M-200-500)]. 2 mL of EC medium was added to each well of a 6-well culture plate to have 1.5 x 105 HUVEC cells per well.
  • EC medium EC medium
  • LSGS low serum growth supplement
  • HUVEC cells were divided into 13 groups including 7 comparison groups (i.e., comparison groups 1 to 7) and 6 experimental groups (i.e., experimental groups 1 to 6).
  • comparison groups i.e., comparison groups 1 to 7
  • 6 experimental groups i.e., experimental groups 1 to 6
  • an appropriate amount of each plant extract according to the above Example 1 was added to each group of cells, cultured in an incubator, and the culture solution was changed at 6, 24 hours and a plant extract or composition was added.
  • the types, concentrations, and/or dosage ratios of the plant extracts added to each group of cells are shown in Table 1 below.
  • each group of cell cultures was collected and subjected to gene expression analysis.
  • the target genes for analyzing cardiovascular related genes include platelet-derived growth factor C (PDGFC) gene, fibroblast growth factor 2 (FGF2) gene, Insulin-like growth factor 1 receptor (IGF1R) gene, prostaglandin I2 synthase (PTGIS) gene, nitric oxide synthase 3 (NOS3) gene, Endothelin 1 (EDN1) gene, plasminogen activator (tissue, PLAT) gene, protein C (protein C, PROC) gene, von Willebrand factor (VWF) gene, F3 gene, serpin peptidase inhibitor E1 (SERPINE1) gene, interleukin-8 (IL-8) gene, intercellular adhesion molecule 1 (ICAM1) Gene, vascular cell adhesion molecule 1 (VCAM1) gene, and cysteine-aspartate protease 8 (ca Spase 8, CASP8) gene.
  • PDGFC platelet-derived growth factor C
  • FGF2 fibroblast growth factor 2
  • IGF1R Insulin-like
  • RNA extraction was performed on each of the cell cultures obtained above using an RNA extraction kit (Geneaid). Take 2,000 ng of each group of RNA thus obtained and III reverse transcriptase III Reverse Transcriptase) (Invitrogen) reverse transcribes RNA into eDNA. Next, use eDNA as a template and use primer pairs to amplify the target gene [including PTGIS, NOS3, EDN1, PLAT, VWF, F3, SERPINE1, ICAM1, VCAM1, IL-8, CASP8, PDGFC, FGF2).
  • the relative expression levels of all target genes were quantified by the SCORE method, and the scores of cardiovascular expression-related genes in each group were calculated.
  • the SCORE method is calculated using the cycle threshold (Ct) value of the ACTB gene (as an internal control group) and a reference gene.
  • composition of the present invention has been confirmed to have cardiovascular protective effects by regulating cardiovascular-related gene expression, among which genes related to blood vessel growth include PDGFC gene, FGF2 gene, and IGF1R gene; genes related to vascular elasticity include PTGIS gene. , NOS3 gene, and EDN1 gene; genes involved in thrombosis include PLAT gene, PROC gene, VWF gene, F3 gene, and SERPINE1 gene; genes related to inflammatory factors include IL-8 gene, ICAM1 gene, and VCAM1 gene; And genes involved in apoptosis include the CASP8 gene.
  • genes related to blood vessel growth include PDGFC gene, FGF2 gene, and IGF1R gene
  • genes related to vascular elasticity include PTGIS gene. , NOS3 gene, and EDN1 gene
  • genes involved in thrombosis include PLAT gene, PROC gene, VWF gene, F3 gene, and SERPINE1 gene
  • genes related to inflammatory factors include IL-8 gene, ICAM1 gene, and VCAM1
  • Figure 1 is a histogram showing the efficacy of a combination of plant extracts in the regulation of cardiovascular-related gene expression in an embodiment of the invention.
  • Table 3 corresponds to the values of the cardiovascular expression gene expression scores calculated for each group of Fig. 1. Among them, the higher the score, the more protective cardiovascular effect.
  • Comparison group 4 8.9 Comparison group 5 18.22 Comparison group 6 11.44 Comparison group 7 3.99 Experimental group 1 16.66 Experimental group 2 19.5 Experimental group 3 22.7 Experimental group 4 16.51 Experimental group 5 32.76 Experimental group 6 25.17
  • the experimental group 1 ie, the grape seed extract is treated
  • the scores calculated by the combination of the four season spring tea extracts were significantly improved, and higher than the sum of the scores of the comparison group 3 and the comparison group 7; and the comparison group 1 (ie, the black tea extract) and the comparison group 2 ( In comparison with the spinach extract, the score calculated by the experimental group 2 (ie, the combination of the black tea extract and the spinach extract) was significantly improved, and was higher than that of the comparison group 1 and the comparison group 2.
  • the experimental group 5 ie, the combination of the treated red wine extract and the Pu'er tea extract
  • the calculated scores were significantly improved, and higher than the sum of the scores of the comparison group 5 and the comparison group 6; and compared with the comparison group 3 and the comparison group 6, the experimental group 6 (ie, the treatment of grape seed extract and Pu'er tea)
  • the scores calculated by the combination of the extracts were significantly improved and higher than the sum of the scores of the comparison group 3 and the comparison group 6.
  • compositions of the present invention are capable of regulating cardiovascular related genes (including PDGFC, FGE2, IGF1R, PTGIS, NOS3, EDN1, PLAT, PROC, VWF, F3, SERPINE1, IL-8, ICAM1). , VCAM1 and CASP8) performance to achieve cardiovascular protection.
  • cardiovascular related genes including PDGFC, FGE2, IGF1R, PTGIS, NOS3, EDN1, PLAT, PROC, VWF, F3, SERPINE1, IL-8, ICAM1). , VCAM1 and CASP8) performance to achieve cardiovascular protection.
  • the composition of the present invention can also be prepared in the form of a pharmaceutical or food product without limitation.

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Abstract

一种植物萃取物在制备调控PDGFC、FGF2、IGF1R、PTGIS、NOS3、EDN1、PLAT、PROC、VWF、F3、SERPINE1、IL-8、ICAM1、VCAM1及CASP8基因的组合物中的用途。植物萃取物包含选自由葡萄籽萃取物与四季春茶萃取物、红茶萃取物与菠菜萃取物、葡萄籽萃取物与菠菜萃取物、葡萄籽萃取物与青咖啡豆萃取物、红酒萃取物与普洱茶萃取物，及葡萄籽萃取物与普洱茶萃取物所组成的组。

Description

植物萃取物在制备调控PDGFC、FGF2、IGF1R、PTGIS、NOS3、EDN1、PLAT、PROC、VWF、F3、SERPINE1、IL-8、ICAM1、VCAM1及CASP8基因的组合物中的用途
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
本发明涉及一种植物萃取物在制备调控PDGFC、FGF2、IGF1R、PTGIS、NOS3、EDN1、PLAT、PROC、VWF、F3、SERPINE1、IL-8、ICAM1、VCAM1及CASP8基因的组合物中的用途。
背景技术
心血管疾病是人类健康的一大威胁，根据台湾卫生福利管理部门公布的资料，国人十大死因中，心血管疾病就占了三项，其中心脏疾病及脑血管疾病更分别高居第2及第4位。而在美国，心血管疾病更是美国人民的第一大死亡原因，由此可见，心血管疾病对人类健康的威胁已达不容忽视的程度，其发病之初通常没有明显症状，但等到一旦出现并发症时，例如脑中风、心肌梗塞、心衰竭、肾衰竭或视网膜出血等，患者生命通常已遭受严重的威胁，且为了治疗心血管疾病，心脏血管用药的需求也居高不下，成为医疗资源的庞大负担。因此，本领域的相关技术人员皆致力于开发用于保护心血管的产品(包括食品产品以及医药品)，以因应广大人类健康需求。
近年来，利用植物作为原料来开发具有疗效的医药品或用于预防保健的食品产品已逐渐受到市场重视。例如，某些植物常被用来做成食品或饮品的原料，包括茶叶或葡萄等。然而，本领域的技术人员目前并未将前述植物萃取物进行组合并拿来开发医药品或食品产品，因为若为某种植物萃取物，其必须施予相当高的剂量，在使用成本上以及效果上有其局限。因此，若能施 予多种萃取物的少量组分，即可产生单一成分的高剂量效果时，将会对本领域的技术带来相当大的突破。
发明内容
本发明的目的为提供一种植物萃取物在制备调控PDGFC、FGF2、IGF1R、PTGIS、NOS3、EDN1、PLAT、PROC、VWF、F3、SERPINE1、IL-8、ICAM1、VCAM1及CASP8基因的组合物中的用途，其中植物萃取物包含选自由葡萄籽萃取物与四季春茶萃取物、红茶萃取物与菠菜萃取物、葡萄籽萃取物与菠菜萃取物、葡萄籽萃取物与青咖啡豆萃取物、红酒萃取物与普洱茶萃取物，及葡萄籽萃取物与普洱茶萃取物所组成的组中的至少任一组合。
在本发明的一实施例中，植物萃取物包含至少0.0039mg/mL的葡萄籽萃取物、及至少0.0039mg/mL的四季春茶萃取物、波菜萃取物、青咖啡豆萃取物、或普洱茶萃取物。
在本发明的一实施例中，植物萃取物包含至少0.0625mg/mL的红茶萃取物及至少0.0625mg/mL的菠菜萃取物。
在本发明的一实施例中，植物萃取物包含至少0.0156mg/mL的红酒萃取物及至少0.0156mg/mL的普洱茶萃取物。
在本发明的一实施例中，组合物具有保护心血管的效果。
在本发明的一实施例中，组合物进一步包含医药上可接受的载体。
在本发明的一实施例中，组合物为粉末、颗粒、液体、胶状或膏状的剂型。
在本发明的一实施例中，组合物被制备成医药品或食品产品的形式。
综上所述，本发明的组合物包括葡萄籽萃取物与四季春茶萃取物、红茶萃取物与菠菜萃取物、葡萄籽萃取物与菠菜萃取物、葡萄籽萃取物与青咖啡豆萃取物、红酒萃取物与普洱茶萃取物、及葡萄籽萃取物与普洱茶萃取物等植物萃取物的功效在于：通过调控与心血管相关的基因(包括PDGFC、FGF2、IGF1R、PTGIS、NOS3、EDN1、PLAT、PROC、VWF、F3、SERPINE1、IL-8、ICAM1、VCAM1及CASP8)表达来达到保护心血管的功效。
附图说明
图1是一直方图，其显示本发明实施例中植物萃取物的组合在调控与心血管相关的基因表达上的功效。
具体实施方式
以下将配合附图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。
定义
本文中所使用数值为近似值，所有实验数据皆表示在20％的范围内，优选为在10％的范围内，最优选为在5％的范围内。
如本文中所使用的用语“基因(gene)”意指DNA序列，包括但不限于：可被转录成mRNA[它可被翻译成多肽链(polypeptide chains)]、被转录成rRNA或tRNA，或者供酶以及其他涉及DNA复制(DNA replication)、转录(transcription)以及调节(regulation)的蛋白质作为识别位点(recognition sites)的DNA序列。这个定义包括各种不同的序列多态性(sequence polymorphisms)、突变和/或序列变异体(sequence variants)，其中该等改变(alternation)不会影响基因产物(gene product)的功能。该用语“基因”意欲包括不只编码基因产物的区域还有调节区域包括，例如：启动子、终止区域(termination regions)、翻译调节序列(translational regulatory sequences)[诸如，核糖体结合位点以及内部核糖体进入位点(internal ribosome entry sites)]、增强子(enhancers)、沉默子(silencers)、绝缘子(insulators)、边界要素(boundary elements)、复制起点(replication origins)、基质附着部位(matrix attachment sites)，以及基因座控制区域(locus control regions)。该用语“基因”进一步包括所有内含子(intron)以及其他被剪接自mRNA转录本(mRNA transcripts)的DNA序列，还有由选择性剪接位点(alternative splice site)所导致的变异体。
如本文中所使用的用语“保护心血管(cardiovascular protection)”意指能够治疗心血管疾病、保护心血管系统，或降低心血管疾病发生的风险。
依据本发明，医药品可利用本领域技术人员所详知的技术而被制造成适合于非肠道(parenterally)或口服(orally)投药的剂型(dosage form)，这包括但不限于：注射品(injection)[例如，无菌的水性溶液(sterile aqueous solution)或分散液(dispersion)]、无菌的粉末(sterile powder)、锭剂(tablet)、片剂(troche)、口含锭(lozenge)、丸剂(pill)、胶囊(capsule)、分散性粉末(dispersible powder)或细颗粒(granule)、溶液、悬浮液(suspension)、乳剂(emulsion)、糖浆(syrup)、酏剂(elixir)、浓浆(slurry)以及类似的物。
依据本发明的医药品可以选自由下列所组成的组中的非肠道途径(parenteral routes)来投药：腹膜内注射(intraperitoneal injection)、皮下注射(subcutaneous injection)、肌肉内注射(intramuscular injection)以及静脉内注射(intravenous injection)。
依据本发明，医药上可接受的载体可包含一种或多种选自由下列所组成的组中的试剂：溶剂(solvent)、乳化剂(emulsifier)、悬浮剂(suspending agent)、分解剂(decomposer)、黏结剂(binding agent)、赋形剂(excipient)、安定剂(stabilizing agent)、螯合剂(chelating agent)、稀释剂(diluent)、胶凝剂(gelling agent)、防腐剂(preservative)、润滑剂(lubricant)、吸收延迟剂(absorption delaying agent)、脂质体(liposome)以及类似物。有关这些试剂的选用与数量是落在本领域技术人员的专业素养与例行技术范畴内。
依据本发明，医药上可接受的载体可包含选自由下列所组成的群组中的溶剂：水、生理盐水(normal saline)、磷酸盐缓冲生理盐水(phosphate buffered saline，PBS)、含糖溶液、含有醇的水性溶液(aqueous solution containing alcohol)、以及它们的组合。
依据本发明，组合物可被当作食品添加物(food additive)，通过公知方法在制备原料时添加，或是在食品的制作过程中添加，而与任一种可食性材料配制成供人类与非人类动物摄食的食品产品。
依据本发明，食品产品的种类包括但不限于：饮料(beverages)、发酵食品(fermented foods)、烘培产品(bakery products)、健康食品(health foods)以及膳食补充品(dietary supplements)。
实施例1.各种植物萃取物的来源或制备
本发明实施例的葡萄籽萃取物是对葡萄属(Vitis spp.)植物种子进行萃取而获得，该萃取物可购自诚鸿生物科技有限公司；普洱茶(Puerh tea)萃取物是对普洱茶茶叶(post-fermented leaves of Camellia sinensis)进行萃取而获得，该萃取物可购自南京泽朗生物科技有限公司；菠菜萃取物是对菠菜(Spinacia oleracea)进行萃取而获得，该萃取物可购自弘祥农产加工厂；红酒萃取物是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；青咖啡豆萃取物是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)。
本实施例举例说明本发明红茶(Camellia sinensis leaves)萃取物的制备方法。首先，将红茶洗净及干燥，以粉碎机将红茶粗碎。其次，以水为溶剂对红茶粗碎物进行萃取，该溶剂与红茶粗碎物以液固比5～20∶1～5均匀混合，萃取温度为50℃～100℃，优选为75℃～95℃。本实施例中萃取时间为0.5～3小时。
经上述萃取步骤所得红茶萃取物冷却至室温后，经由400目(mesh)的滤网过滤以移除残余固体物。该过滤后的红茶萃取物可进一步在45℃～70℃进行减压浓缩而获得浓缩产物。
本实施例举例说明本发明四季春茶(Four Seasons Spring tea)萃取物的制备方法。首先，将四季春茶洗净及干燥，以粉碎机将四季春茶粗碎。其次，以水为溶剂对四季春茶粗碎物进行萃取，该溶剂与四季春茶粗碎物以液固比5～20∶1～5均匀混合，萃取温度为50℃～100℃，优选为75℃～95℃。本实施例中萃取时间为0.5～3小时。
经上述萃取步骤所得四季春茶萃取物冷却至室温后，经由400目(mesh)的滤网过滤以移除残余固体物。该过滤后的四季春茶萃取物可进一步在45℃～70℃进行减压浓缩而获得浓缩产物。
实施例2.植物萃取物的组合在调控与心血管相关的基因表达上的功效
首先，将人类脐带静脉内皮细胞(human umbilical vein endothelial cells，HUVEC)[购自台湾的食品工业发展研究所(Food Industry Research and Development Institute，FIRDI)的生物资源保存及研究中心(Biosource Collection and Research Center，BCRC)，BCRC编号为H-UV001]培养于EC培养基(EC  medium)[添加有培养基200(medium 200，M200)及10％低血清生长添加剂(low serum growth supplement，LSGS)(Gibco；Cat.No.M-200-500)]中。在6孔培养盘的每孔中加入2mL的EC培养基，使每孔具有1.5x10 5个HUVEC细胞。
之后，将HUVEC细胞分成13组，其中包括7个比较组(即比较组1至7)以及6个实验组(即实验组1至6)。接着，将适量的依据上面实施例1的各个植物萃取物添加至各组细胞中，在培养箱中进行培养，并于6、24小时更换培养液且加入植物萃取物或组合物。有关各组细胞所添加的植物萃取物的种类、浓度，及/或用量比例是显示于下面表1中。
表1
Figure PCTCN2018081719-appb-000001
Figure PCTCN2018081719-appb-000002
在培养箱中进行培养之后，收集各组细胞培养物并拿来进行基因表达分析。
在本实施例中，用来分析与心血管相关的目标基因包括血小板衍生生长因子C(platelet-derived growth factor C，PDGFC)基因、纤维母细胞生长因子2(fibroblast growth factor 2，FGF2)基因、胰岛素样生长因子1受体(insulin-like growth factor 1 receptor，IGF1R)基因、前列腺素I2合成酶(prostaglandin I2 synthase，PTGIS)基因、一氧化氮合成酶3(nitric oxide synthase 3，NOS3)基因、内皮素1(endothelin 1，EDN1)基因、组织胞浆素原活化物(plasminogen activator，tissue，PLAT)基因、蛋白质C(protein C，PROC)基因、冯威里氏因子(von Willebrand factor，VWF)基因、F3基因、丝胺酸蛋白酶抑制蛋白E1(serpin peptidase inhibitor E1，SERPINE1)基因、介白素-8(interleukin-8，IL-8)基因、细胞间粘附分子1(intercellular adhesion molecule 1，ICAM1)基因、血管细胞粘附分子1(vascular cell adhesion molecule 1，VCAM1)基因、及半胱-天冬胺酸蛋白酶8(caspase 8，CASP8)基因。
以RNA萃取试剂盒(RNA extraction kit)(Geneaid)对上面所得到的各组细胞培养物进行RNA的萃取。对由此所得到的各组RNA取2,000ng并以
Figure PCTCN2018081719-appb-000003
III反转录酶(
Figure PCTCN2018081719-appb-000004
III Reverse Transcriptase)(Invitrogen)将RNA反转录为eDNA。接着，以eDNA作为模版(template)，并且使用用来扩增目标基因的引物对[包括PTGIS、NOS3、EDN1、PLAT、VWF、F3、SERPINE1、ICAM1、VCAM1、IL-8、CASP8、PDGFC、FGF2、IGF2BP3、IGF1R、及ACTB(作为内部对照组)，它们的核苷酸序列显示于下面表2]，在StepOne Plus实时PCR系统(StepOne Plus Real Time PCR system)(ABI)中利用KAPA SYBR FAST qPCR试剂盒(2x)(KAPA Biosystems)来进行定量实时聚合酶链反应 (quantitative real-time polymerase chain reaction，以下简称定量实时PCR)，以对目标基因进行扩增及定量。PCR产物的熔化曲线(melting curve)是在定量实时PCR反应期间进行确认。
表2
Figure PCTCN2018081719-appb-000005
Figure PCTCN2018081719-appb-000006
Figure PCTCN2018081719-appb-000007
全部目标基因的相对表达水平是以SCORE法来进行定量，而计算出各组与心血管相关的基因表达的分数。SCORE法是利用ACTB基因(作为内部对照组)及内参基因(reference gene)的循环阈值[cycle threshold(Ct)value]来计算。
目标基因的mRNA相对量是推导自方程式2 -ΔCt，其中ΔCt＝Ct 目标基因-Ct ACTB(β-肌动蛋白，beta-actin)，接着计算上述每个基因的表现量在该六组与空白对照组中的差值，并以此差值的合计作为心血管相关的基因表达的分数。
通过调控心血管相关的基因表达，已确认本发明的组合物具有保护心血管的功效，其中与血管生长有关的基因包括PDGFC基因、FGF2基因，及IGF1R基因；与血管弹性有关的基因包括PTGIS基因、NOS3基因、及EDN1基因；与血栓生成有关的基因包括PLAT基因、PROC基因、VWF基因、F3基因、及SERPINE1基因；与发炎因子有关的基因包括IL-8基因、ICAM1基因，及VCAM1基因；及与细胞凋亡有关的基因包括CASP8基因。
本实施例的结果是显示于图1及表3。图1是一直方图，其显示本发明实施例中植物萃取物的组合在调控与心血管相关的基因表达上的功效。表3对应显示图1的各组所计算出的与心血管相关的基因表达分数数值。其中，分数越高者，代表越具有保护心血管的功效。
表3
组别 与心血管相关的基因表达分数
比较组1 7.7
比较组2 9.1
比较组3 7.3
比较组4 8.9
比较组5 18.22
比较组6 11.44
比较组7 3.99
实验组1 16.66
实验组2 19.5
实验组3 22.7
实验组4 16.51
实验组5 32.76
实验组6 25.17
由图1及表3可见，与比较组3(即处理葡萄籽萃取物者)及比较组7(即处理四季春茶萃取物者)相较之下，实验组1(即处理葡萄籽萃取物及四季春茶萃取物的组合者)所计算出的分数有显著提升，并且高于比较组3及比较组7的分数总和；与比较组1(即处理红茶萃取物者)及比较组2(亦即处理菠菜萃取物者)相较之下，实验组2(即处理红茶萃取物及菠菜萃取物的组合者)所计算出的分数有显著提升，并且高于比较组1及比较组2的分数总和；与比较组2及比较组3相较之下，实验组3(即处理葡萄籽萃取物及菠菜萃取物的组合者)所计算出的分数有显著提升，并且高于比较组2及比较组3的分数总和；与比较组3及比较组4(即处理青咖啡豆萃取物者)相较之下，实验组4(即处理葡萄籽萃取物及青咖啡豆萃取物的组合者)所计算出的分数有显著提升，并且高于比较组3及比较组4的分数总和；与比较组5(即处理红酒萃取物者)及比较组6(亦即处理普洱茶萃取物者)相较之下，实验组5(即处理红酒萃取物及普洱茶萃取物的组合者)所计算出的分数有显著提升，并且高于比较组5及比较组6的分数总和；以及与比较组3及比较组6相较之下，实验组6(即处理葡萄籽萃取物及普洱茶萃取物的组合者)所计算出的分数有显著提升，并 且高于比较组3及比较组6的分数总和。
因此，本发明的实施例的实验结果显示：本发明各个植物萃取物的组合具有调控与心血管相关的基因表达的功效。因此，申请人认为，本发明的组合物能够藉由调控与心血管相关的基因(包括PDGFC、FGE2、IGF1R、PTGIS、NOS3、EDN1、PLAT、PROC、VWF、F3、SERPINE1、IL-8、ICAM1、VCAM1及CASP8)表现来达到保护心血管的功效。此外，本发明的组合物也能够制备成医药品或食品产品的形式，并无限制。
以上所述仅为举例性，而非为限制性者。任何未脱离本发明的精神与范畴，而对其进行的等效修改或变更，均应包含于后附的权利要求范围中。

Claims (8)

  1. 植物萃取物在制备调控PDGFC、FGF2、IGF1R、PTGIS、NOS3、EDN1、PLAT、PROC、VWF、F3、SERPINE1、IL-8、ICAM1、VCAM1及CASP8基因的组合物中的用途，其中所述植物萃取物包含选自由葡萄籽萃取物与四季春茶萃取物、红茶萃取物与菠菜萃取物、葡萄籽萃取物与菠菜萃取物、葡萄籽萃取物与青咖啡豆萃取物、红酒萃取物与普洱茶萃取物、及葡萄籽萃取物与普洱茶萃取物所组成的组中的至少任一组合。
  2. 如权利要求1所述的用途，其中所述植物萃取物包含至少0.0039mg/mL的葡萄籽萃取物、及至少0.0039mg/mL的四季春茶萃取物、波菜萃取物、青咖啡豆萃取物、或普洱茶萃取物。
  3. 如权利要求1所述的用途，其中所述植物萃取物包含至少0.0625mg/mL的红茶萃取物及至少0.0625mg/mL的菠菜萃取物。
  4. 如权利要求1所述的用途，其中所述植物萃取物包含至少0.0156mg/mL的红酒萃取物及至少0.0156mg/mL的普洱茶萃取物。
  5. 如权利要求1所述的用途，其中所述组合物具有保护心血管的效果。
  6. 如权利要求1所述的用途，其中所述组合物进一步包含医药上可接受的载体。
  7. 如权利要求1所述的用途，其中所述组合物为粉末、颗粒、液体、胶状或膏状的剂型。
  8. 如权利要求1所述的用途，其中所述组合物被制备成医药品或食品产品的形式。
PCT/CN2018/081719 2017-04-03 2018-04-03 植物萃取物在制备调控pdgfc、fgf2、igf1r、ptgis、nos3、edn1、plat、proc、vwf、f3、serpine1、il-8、icam1、vcam1及casp8基因的组合物中的用途 WO2018184526A1 (zh)

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