CN112386619A - 沙棘汁液的用途 - Google Patents
沙棘汁液的用途 Download PDFInfo
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- CN112386619A CN112386619A CN202010820586.1A CN202010820586A CN112386619A CN 112386619 A CN112386619 A CN 112386619A CN 202010820586 A CN202010820586 A CN 202010820586A CN 112386619 A CN112386619 A CN 112386619A
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- hippophae rhamnoides
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- lung
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Abstract
本发明公开一种沙棘汁液的用途,其用于制备一组合物。此组合物具有下列一种或多种改善下呼吸道状态的功能:修复肺损伤、改善肺功能及抑制下呼吸道发炎相关基因的表现量。
Description
技术领域
本发明是关于沙棘相关产品,特别是关于沙棘汁液用于制备改善下呼吸道状态的组合物的用途。
背景技术
自有机及天然的饮食概念兴起后,生技公司及食品业者积极投入关于天然植物的相关产品的研发。为使植物相关产品对身体健康帮助有科学验证的基础,植物的活性成分分析及功效评估成为产品开发的重点项目。
沙棘(Hippophae rhamnoides)又称海沙棘,其是胡颓子科沙棘属的植物。沙棘是一种原产于亚洲和欧洲的带有棘刺的耐寒落叶灌木,其果实呈现橘黄色。沙棘可防风固沙,并且沙棘果实具有很高的药用价值。因此,沙棘亦成为生技公司及食品业者积极研究开发的对象之一。
发明内容
有鉴于此,本发明提供一种沙棘汁液的用途,其是用于制备改善下呼吸道状态的组合物。其中,沙棘汁液具有下列一种或多种能力:修复肺损伤、改善肺功能及抑制下呼吸道发炎相关基因的表现量,以致沙棘汁液能改善下呼吸道状态。
在一实施例中,一种沙棘汁液的用途,其是用于制备修复肺损伤的组合物的用途。
在一些实施例中,沙棘汁液用以促进肺上皮细胞的创伤修复力,以致修复肺损伤。
在一实施例中,一种沙棘汁液的用途,其是用于制备改善肺功能的组合物的用途。
在一些实施例中,改善肺功能是指提升下列肺功能指针中的至少一种:用力肺活量及用力呼气中断流速。
在一些实施例中,沙棘汁液用以去痰及平喘,以致改善肺功能。
在一实施例中,一种沙棘汁液的用途,其是用于制备抑制下呼吸道发炎相关基因的表现量的组合物的用途。其中,下呼吸道发炎相关基因包括下列至少一者:白介素5受体α(interleukin-5receptor alpha;IL5RA)基因、粘膜相关淋巴组织淋巴瘤易位基因1(Mucosa associated lymphoid tissue lymphoma translocation gene 1;MALT1 gene)及瘤坏死因子受体超家族成员14(TNF superfamily member 14;TNFSF14)基因。
在一些实施例中,前述的组合物为医药品。
在一些实施例中,前述的组合物为食品产品或食品添加物。
在一些实施例中,前述的组合物为化妆品或保养品。
以下结合附图和具体实施例对本发明进行详细描述,但不作为对本发明的限定。
附图说明
图1是绘示控制组与实验组的伤口区域的相对愈合百分比的直方图。
图2是绘示沙棘汁液食用前与后的用力肺活量的变化的直方图。
图3是绘示沙棘汁液食用前与后的用力呼气中断流速的变化的直方图。
图4是绘示沙棘汁液食用前与后的呼吸道生痰情况的变化的直方图。
图5是绘示沙棘汁液食用前与后的易喘情况的变化的直方图。
图6是绘示沙棘汁液食用前与后的呼吸道及肺部发炎基因的表现量的变化的直方图。
具体实施方式
关于本文中所使用的浓度符号「wt%」通常是指重量百分浓度,而浓度符号「vol%」通常是指体积百分浓度。
在一些实施例中,由沙棘(Hippophae rhamnoides)的果实所获得的沙棘汁液可具有下列至少一种能力:修复肺损伤、改善肺功能及抑制下呼吸道发炎相关基因的表现量,以致沙棘汁液能改善下呼吸道状态。因此,沙棘汁液可用于制备具有下列至少一种能力的组合物:修复肺损伤、改善肺功能及抑制下呼吸道发炎相关基因的表现量。
在一些实施例中,一种沙棘汁液的用途,其是用于制备修复肺损伤的组合物的用途。在一些实施例中,沙棘汁液是借由促进肺上皮细胞的创伤修复力而修复肺损伤。
在一实施例中,一种沙棘汁液的用途,其是用于制备改善肺功能的组合物的用途。在一些实施例中,改善肺功能是指提升下列肺功能指针中的至少一种:用力肺活量及用力呼气中断流速。在一些实施例中,沙棘汁液是借由去痰及平喘而改善肺功能。
在一实施例中,一种沙棘汁液的用途,其是用于制备抑制下呼吸道发炎相关基因的表现量的组合物的用途。其中,下呼吸道发炎相关基因包括下列至少一者:白介素5受体α(Interleukin-5receptor alpha;IL5RA)基因、粘膜相关淋巴组织淋巴瘤易位基因1(Mucosa associated lymphoid tissue lymphoma translocation gene 1;MALT1 gene)及瘤坏死因子受体超家族成员14(TNF superfamily member 14;TNFSF14)基因。
在一些实施例中,前述的组合物可为医药品。换言之,此医药品包含有效含量的沙棘汁液。其中,此医药品可利用熟习此技艺者所详知的技术而被制造成适合于经肠地道、非经肠地道(parenterally)、口服地、或局部地(topically)投药剂型。
在一些实施例中,经肠道或口服的投药剂型可为,但不限于,锭剂(tablet)、片剂(troche)、口含锭(lozenge)、丸剂(pill)、胶囊(capsule)、分散性粉末(dispersiblepowder)或细颗粒(granule)、溶液、悬浮液(suspension)、乳剂(emulsion)、糖浆(syrup)、酏剂(elixir)、浓浆(slurry)或类似之物。在一些实施例中,非经肠地道或局部地投药剂型可为,但不限于,注射品(injection)、无菌的粉末(sterile powder)、外部制剂(externalpreparation)或类似之物。在一些实施例中,注射品的投药方式可为皮下注射(subcutaneous injection)、表皮内注射(intraepidermal injection)、皮内注射(intradermal injection)或病灶内注射(intralesional injection)。
在一些实施例中,含有沙棘汁液的医药品可更包含被广泛地使用于药物制造技术的医药上可接受的载剂(pharmaceutically acceptable carrier)。在一些实施例中,医药上可接受的载剂可为下列载剂中一种或多种:溶剂(solvent)、缓冲液(buffer)、乳化剂(emulsifier)、悬浮剂(suspending agent)、分解剂(decomposer)、崩解剂(disintegrating agent)、分散剂(dispersing agent)、黏结剂(binding agent)、赋形剂、安定剂(stabilizing agent)、螯合剂(chelating agent)、稀释剂(diluent)、胶凝剂(gelling agent)、防腐剂(preservative)、润湿剂(wetting agent)、润滑剂(lubricant)、吸收延迟剂(absorption delaying agent)、脂质体(liposome)以及类似之物。关于选用的载剂的种类与数量是落在熟习此项技术的人士的专业素养与例行技术范畴内。其中,作为医药上可接受的载剂的溶剂可为水、生理盐水(normal saline)、磷酸盐缓冲液(phosphatebuffered saline,PBS)或含有醇的水性溶液(alcohol containing aqueous solution)。
在一些实施例中,前述的组合物为食用组合物。换言之,食用组合物包含特定含量的沙棘汁液。其中,食用组合物的型态可为粉末、颗粒、溶液、胶体或膏体。
在一些实施例中,含有沙棘汁液的食用组合物可为食品产品或食品添加物(foodadditive)。
在一些实施例中,含有沙棘汁液的食品产品可为饮料(beverages)、发酵食品(fermented foods)、烘培产品(bakery products)、健康食品(health foods)或膳食补充品(dietary supplements)等。在一些实施例中,含有沙棘汁液的食品产品可更包括一佐剂。举例来说,佐剂可为麦芽糖糊精(Maltodextrin)、苹果酸、蔗糖素、柠檬酸、水果香料、蜂蜜香料、甜菊糖苷或其组合等。关于选用的载剂的种类与数量是落在熟习此项技术的人士的专业素养与例行技术范畴内。
在一些实施例中,含有沙棘汁液的食品添加物可为调味料、甜味料、香料、pH值调整剂、乳化剂、着色料或稳定剂等。
在一些实施例中,前述的组合物为化妆品或保养品。换言之,化妆品或保养品包含特定含量的沙棘汁液。
在一些实施例中,含有沙棘汁液的化妆品或保养品可为下列任一种型态:化妆水、凝胶、冻膜、泥膜、乳液、乳霜、唇膏、粉底、粉饼、蜜粉、卸妆油、卸妆乳、洗面奶、沐浴乳、洗发精、护发乳、防晒乳、护手霜、指甲油、香水、精华液及面膜。
在一些实施例中,含有沙棘汁液的化妆品或保养品可视需要更包含外用品可接受成分。在一些实施例中,外用品可接受成分可例如为乳化剂、渗透促进剂、软化剂、溶剂、赋型剂、抗氧化剂、或其组合。
在一些实施例中,沙棘汁液可由合适的浸提溶剂与沙棘果实进行一浸提程序所得。
举例来说,在浸提程序中,以水于50℃-100℃下浸提沙棘果实30分钟至90分钟适当时间以得到初提原液。其中,进行浸提的沙棘果实可为完整果实,或为经物理前处理而分离成碎片、颗粒或粉末等型态的果实。采用的物理前处理可包括下列至少一种:切碎、剪碎、捣碎、及研磨。在一些实施例中,在浸提程序中,初提原液可进一步进行过滤以去除杂质而得到滤液。在一些实施例中,在浸提程序中,滤液可进一步进行浓缩以得到浓缩液。其中,浓缩方式可采用减压浓缩。在一些实施例中,在浸提程序中,浓缩液可进一步干燥成粉末。其中,干燥方式可采用喷雾干燥。
也就是说,可依实际需求以浸提程序所得的初提原液、滤液、浓缩液或粉末作为沙棘汁液。
在一些实施例中,沙棘汁液亦可为市售的沙棘果實的原汁、浓缩原汁或原汁粉末。
例一:维生素C定量
秤取10mg的维生素C(L-Ascorbic Acid Sodium Salt,纯度≧98%,购自SIGMA-ALORICH)置于定量瓶中,然后以纯水(H2O)溶解并定量至10mL,以得到初始溶液(即含1000ppm的维生素C)。然后,依据下表一配置10ppm、25ppm、50ppm、100ppm及200ppm的标准溶液。
表一
标准溶液(ppm) | 10 | 25 | 50 | 100 | 200 |
初始溶液(mL) | 0.1 | 0.25 | 0.5 | 1.0 | 2.0 |
纯水(mL) | 9.9 | 9.75 | 9.5 | 9.0 | 8.0 |
总量(mL) | 10 | 10 | 10 | 10 | 10 |
秤取1.0g的沙棘果汁粉(其为100%的粉末状沙棘汁液)(购自Puredia)置于定量瓶中,然后以纯水(H2O)溶解并定量至50mL,以得到待测溶液。
接着,将待测溶液及标准溶液分别注入高效液相层析仪(HPLC)(机型:HitachiChromaster HPLC system,厂牌:Hitachi,Tokyo,Japan)以进行液相层析并就待测溶液与标准溶液所得波峰的滞留时间及吸收图谱比较鉴别之。其中,HPLC装设Ascentis ExpressC18层析管柱(厂牌:Supelco,内径2.1mm,长100mm,粒径2μm)。HPLC的设定值中,移动相0.1wt%草酸水溶液、流速为每分钟流0.2毫升(0.2mL/min),进样体积为2μL,进样次数为3重复,侦测波长为245nm,及管柱温度设定为30℃。
然后,依据下式(1)计算维生素C的含量。
维生素C的含量(ppm)=C×V÷M(1)
其中,C为由标准曲线求得待测溶液中维生素C的浓度(ppm)。V为待测溶液最后定量的体积,即50mL。M为取样分析的检体的重量,即1g。
于此,维生素C的滞留时间为1.8min(分钟)。并且,测得沙棘果汁粉中具有9334.03ppm的维生素C。
例二:肺损伤修复试验
于此,实验将会分为实验组以及控制组(未添加沙棘汁液)二组进行,各组分别进行三重复试验。
首先,以每孔1.5×105个细胞的细胞数,将人肺(支气管)上皮细胞株BEAS-2b(购自美国典型培养物保存中心(ATCC),编号CRL-9609)接种于含有支气管上皮细胞基础培养基(Bronchial epithelial cell basal Medium,BEME,品牌:Lonza)的24孔培养盘的各孔中,并置于37℃下培养过夜以形成细胞单层(cell monolayer)。
之后,使用200μL的吸管尖(pipette tip)于细胞单层创造伤口,然后移除培养基并以PBS(phosphate buffered saline,酸盐缓冲液,品牌:Gibco)润洗。然后,添加0.5mL无血清培养基至各孔中,并以显微镜(厂牌:ZEISS)观察及拍摄各孔中的细胞的照片(以下称0小时照片)。于此所使用的无血清培养基为Dulbecco的改良Eagle培养基(Dulbecco'sModified Eagle Medium,DMEM,品牌:Gibco)。
接着,将沙棘溶液添加至作为实验组的各孔中并移除等体积的无血清培养基,使实验组的无血清培养基含有0.25mg/ml的沙棘果汁粉。作为对照组的各孔则不做任何处理。其中,沙棘溶液为将2.23μL沙棘果汁添加至0.5mL无血清培养基中所形成的溶液,而沙棘果汁(0.056mg/μL)以沙棘果汁粉(其为100%的粉末状沙棘汁液)(购自Puredia)以纯水配置成糖度(Degrees Brix)为5.6的溶液。
接着,将各组的细胞于37℃下培养16小时。于16小时培养后,以显微镜(厂牌:ZEISS)观察及拍摄各孔中的细胞的照片(以下称16小时照片)。然后,使用Image J软件分析0小时照片与16小时照片,以得到各组的伤口愈合百分比。各组的伤口愈合百分比为各组在16小时的伤口区域的总细胞面积相对于0小时的伤口区域的总面积的百分比。因实验系进行三重复,故将各组的三重复实验的结果平均以得平均值,然后将控制组的平均值视为100%相对愈合百分比来将实验组的平均值换算成相对愈合百分比(%),如图1所示。在图1中,「*」代表在控制组比较下其p值小于0.05。
参照图1,相较于控制组,沙棘汁液的处理增加肺上皮细胞所修复伤口区域的面积达约36%。此结果说明沙棘汁液能促进肺上皮细胞的创伤修复力,因此具有修复肺损伤的功效,进而能有效阻隔肺部细胞纤维化的可能性。
例三:肺功能检测
令7位受试者(其年龄介于30岁至45岁且具有烟瘾、常接触二手烟、骑车通勤、肺部慢性发炎或呼吸道过敏等习惯)每日食用一包0.5g的沙棘果汁粉(其为100%的粉末状沙棘汁液)(购自Puredia),连续食用4周。于食用前(即第0周)及食用4周后(即第4周),以MirSpirobank功肺功能检测仪(厂牌:意大利Mir)搭配涡流气探头与吹嘴检测各受试者的下列肺功能指标:用力肺活量(Forced vital capacity,FVC)以及用力呼气中断流速(Forced Expiratory Flow in the middle 1/2of an expiration,FEF25-75),如图2及图3所示。其中,FVC为测定呼吸道阻力的指标。FEF25-75为测定气道阻塞的敏感指标,且其代表FVC中间50%用力吐气气流速度的平均。在图2及图3中,各长条是以7位受试者的对应量测结果的平均值绘示,并且第0周与第4周之间的统计学显著差异是借由学生t-试验来统计分析。在图2中,「*」代表在与第0周比较下其p值小于0.05。
并且,请各受试者于食用前(即第0周)及食用4周后(即第4周)进行肺功能问卷(如下表2所示)填写回馈并分析之,以评估肺功能改善的状况,如图4及图5所示。其中,在肺功能问卷中,评估项目A、B、E、F、G、H为生痰情况评估项目,评估项目C、D为易喘情况评估项目,评估项目I则依据受试回复的症况列入生痰情况评估项目和易喘情况评估项目中之一或不列入评估。在图4中,呼吸道生痰的严重情况是以7位受试者对生痰情况评估项目的评定分数的平均值呈现。在图5中,容易喘的严重情况是7位受试者对易喘情况评估项目的评定分数的平均值呈现。
表2
此外,于食用前(即第0周)及食用4周后(即第4周),还会搜集各受试者的血液,以供后续实验使用。
参照图2,相较于第0周,食用沙棘汁液4周后,受试者的用力肺活量显著提升0.14L(3.4%),换言之,沙棘汁液能提升肺活量。参照图3,相较于第0周,食用沙棘汁液4周后,受试者的用力呼气中断流速提升2.02%,换言之,沙棘汁液能使受试者呼吸更加畅通。参照图4及图5,食用沙棘汁液4周后,受试者呼吸道生痰及容易喘的情况皆有改善。此结果说明沙棘汁液具有改善肺功能的功效。
例四:抗下呼吸道发炎试验
于此,以例三搜集到的各受试者的第0周及第4周血液进行下呼吸道发炎相关基因(包括IL5RA基因、MALT1基因及TNFSF14基因)的表现量的检测。其中,IL5RA基因所表现的IL5RA会与多配体聚糖结合蛋白(Syndecan binding protein,SDCBP)相互作用活化与气管发炎相关的细胞激素IL5。MALT1基因所表现的MALT1蛋白质(Mucosa associated lymphoidtissue lymphoma translocation protein 1;MALT1 protein)为淋巴细胞抗原受体(如,B细胞抗原受体(B cell receptor,BCR)及/或T细胞抗原受体(T cell receptor,TCR))至核因子活化B细胞κ轻链增强子(Nuclear factor kappa-light-chain-enhancer ofactivated B cells,NF-κB)活化过程中重要的讯息传递分子。
首先,将搜集到的血液移至离心管中,并以300g的转速离心15分钟。于离心后,移除上层血浆,取2mL的肤色血球层(即,buffy coat)置于另一离心管中,再加入2mL的PBS均匀混和,以提供经稀释的buffy coat。将经稀释的buffy coat缓慢加入至内有3mL的Ficoll-Plague Plus溶液(购自Sigma)的另一离心管中,然后以400g的转速离心40分钟。于离心后,移除上层液体后,取出约2mL-3mL的中间层(即,单核细胞层)置于新的离心管中。将离心管中的单核细胞以PBS清洗3至5次后,再以300g的转速离心10分钟。于离心后,移除上清液,而所获得的沉淀物即为周边血单核球细胞(peripheral blood mononuclear cell,PBMC)。
获得的PBMC以细胞裂解液(购自Geneaid公司)破细胞,以得到细胞裂解液。再以RNA提取试剂套组(购自Geneaid公司)提取细胞裂解液内的RNA。接着,取2000奈克(ng)的提取到的RNA为模板,并利用III反转录酶(购自Invitrogene公司)将进行反转录作用,以产生相应的cDNA。后续,以cDNA作为模版并使用用来扩增标的基因的引子对(如下表3所示),在ABI StepOnePlusTM Real-Time PCR system(ABI StepOne Plus实时PCR系统,购自Thermo Fisher Scientific公司)中,利用KAPA SYBR FAST套组(购自Sigma公司)进行定量实时反转录聚合酶连锁反应(quantitative real-time reverse transcriptionpolymerase chain reaction),以定量IL5RA基因、MALT1基因与TNFSF14基因的mRNA表现量,进而推断各基因编码的蛋白质的表现量。
其中,ABI StepOne Plus实时PCR系统的设定条件为95℃反应1秒,60℃反应20秒,总共40个循环,并使用2-ΔCt方法进行基因定量。IL5RA基因对应的引子对为IL5RA-F与IL5RA-R。MALT1基因对应的引子对为MALT1-F与MALT1-R。TNFSF14基因对应的引子对为TNFSF14-F与TNFSF14-R。
表3
于此,将7位受试者对应基因的定量结果取平均以得到平均值,然后透过将第0周的平均值视为1(相对表现量)来将第4周的平均值换算成相对表现量(%),如图6所示。并且,第0周的量测结果与第4周的量测结果之间的统计学显著差异是借由学生t-试验来统计分析。在图6中,「***」代表在与第0周比较下其p值小于0.001。
参照图6,相比第0周,食用沙棘汁液4周后,呼吸道及肺部发炎基因(即MALT1基因、TNSF14F基因与IL5RA基因)的表现量皆显著降低。此结果说明沙棘汁液具有减缓肺部发炎情况的功效。
综上所述,根据本发明任一实施例的沙棘汁液的用途,其用于可制备改善下呼吸道状态的组合物。换言之,前述的组合物具有下列一种或多种功能:修复肺损伤、改善肺功能及抑制下呼吸道发炎相关基因的表现量,以致能改善下呼吸道状态。
当然,本发明还可有其它多种实施例,在不背离本发明精神及其实质的情况下,熟悉本领域的技术人员可根据本发明作出各种相应的改变和变形,但这些相应的改变和变形都应属于本发明权利要求的保护范围。
序列表
<110> 大江生医股份有限公司
<120> 沙棘汁液用于改善下呼吸道状态的组合物的用途
<130> N/A
<150> US62886653
<151> 2019-08-14
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> forward primer for IL5RA gene
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<210> 2
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<212> DNA
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<220>
<223> reverse primer for IL5RA gene
<400> 2
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<210> 3
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<212> DNA
<213> Artificial Sequence
<220>
<223> forward primer for MALT1 gene
<400> 3
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<210> 4
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> reverse primer for MALT1 gene
<400> 4
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<210> 5
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> forward primer for TNFSF14 gene
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<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> reverse primer for TNFSF14 gene
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ccgacatcac catccttcct 20
Claims (9)
1.一种沙棘汁液的用途,其特征在于,其为沙棘汁液用于制备修复肺损伤的组合物的用途。
2.根据权利要求1所述的用途,其特征在于,该沙棘汁液用以促进肺上皮细胞的创伤修复力。
3.一种沙棘汁液的用途,其特征在于,其为沙棘汁液用于制备改善肺功能的组合物的用途。
4.根据权利要求3所述的用途,其特征在于,改善该肺功能是指提升下列肺功能指标中的至少一种:用力肺活量及用力呼气中断流速。
5.根据权利要求3所述的用途,其特征在于,该沙棘汁液用以去痰及平喘。
6.一种沙棘汁液的用途,其特征在于,其为沙棘汁液用于制备抑制下呼吸道发炎相关基因的表现量的组合物的用途,其中该下呼吸道发炎相关基因包括下列至少一者:白介素5受体α基因、粘膜相关淋巴组织淋巴瘤易位基因1及瘤坏死因子受体超家族成员14基因。
7.根据权利要求1至6中任一项所述的用途,其特征在于,该组合物为医药品。
8.根据权利要求1至6中任一项所述的用途,其特征在于,该组合物为食品产品或食品添加物。
9.根据权利要求1至6中任一项所述的用途,其特征在于,该组合物为化妆品或保养品。
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