CN116920018A - 一种山楂提取物及应用 - Google Patents
一种山楂提取物及应用 Download PDFInfo
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- CN116920018A CN116920018A CN202311105143.4A CN202311105143A CN116920018A CN 116920018 A CN116920018 A CN 116920018A CN 202311105143 A CN202311105143 A CN 202311105143A CN 116920018 A CN116920018 A CN 116920018A
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Abstract
本发明提供了一种山楂提取物及应用,所述山楂提取物包括0.21‑0.25%总黄酮、11‑14%%总三萜、2.0‑2.5%总酚,所述山楂提取物中鞣酸的含量不高于2.5%。本发明所述山楂提取物可最大限度保留活性成分,每克提取物相当于390g山楂果;大幅提高黄酮类、总酚、总三萜等活性成分的含量,同时使山楂提取物中鞣酸、重金属含量至理想水平,使山楂的大剂量服用成为可能。
Description
技术领域
本发明涉及食品技术领域,具体而言,涉及一种山楂提取物及应用。
背景技术
山楂属蔷薇科植物,其富含黄酮、三萜等功能成分而具有抗氧化、降血糖、降血脂等功能作用;若以山楂果作药用,性微温,味酸甘,入脾、胃、肝经,有消食健胃、活血化淤、收敛止痢之功效。
由于山楂中富含鞣酸且重金属含量偏高,鞣酸能引起蛋白质与钙、镁结合聚集为沉淀物并容易形成‘胃石’;在中药配方中山楂用量一般不超过10g,而且通常情况下,一个人每天摄入的山楂量不宜超过50g。而重金属尤其是砷的含量较高会存在安全性隐患,如何去除山楂中的鞣酸、重金属并大幅提高活性成分含量不仅可提高安全性且有利于开发新用途。
有鉴于此,特提出本发明。
发明内容
本发明解决的问题是获取活性成分含量高且鞣酸、重金属残留量低的山楂提取物。
为解决上述问题,本发明提供一种山楂提取物,包括0.21-0.25%总黄酮、11-14%%总三萜、2.0-2.5%总酚,所述山楂提取物中鞣酸的含量不高于2.5%。
优选的,包括0.22-0.24%总黄酮、12.5-13.5%总三萜、2.1-2.4%总酚,所述山楂提取物中鞣酸的含量不高于2.3%。
优选的,包括0.22-0.23%总黄酮、12.8-13.2%总三萜、2.2-2.3%总酚,所述山楂提取物中鞣酸的含量不高于2.2%。
优选的,所述总黄酮与鞣酸的含量之比不低于1:10。
优选的,所述山楂提取物溶解于超纯水体系中的平静平均粒径为103.18nm。
优选的,所述山楂提取物中砷的含量不高于0.06mg/kg,铅的含量不高于0.16mg/kg,汞的含量不高于0.02mg/kg。
优选的,所述山楂提取物中砷的含量不高于0.04mg/kg,铅的含量不高于0.07mg/kg,汞的含量不高于0.01mg/kg。
相对于现有技术,本发明所述山楂提取物具有下述有益效果:1)最大限度保留活性成分,每克提取物相当于390g山楂果;2)大幅提高黄酮类、总酚、总三萜等活性成分的含量,同时去除山楂提取物中鞣酸、重金属含量至理想水平,可以大量、长期服用而没有毒副作用,大大强化了山楂对人体的有效作用,表现出明显的保健和医疗效果;3)山楂提取物本身以纳米晶体形式存在,比单独的活性成分的生物利用度更高。
本发明还提供了一种山楂提取物的制备方法,包括:S1、将山楂果进行预处理得到初级山楂液;S2、初级山楂液进行微生物发酵,获取发酵上清液;
S3、利用弱碱阴离子交换树脂柱、弱极性大孔吸附树脂柱、阳离子交换树脂、色谱层析柱进行纯化;S4、浓缩干燥。
优选的,步骤S3中所述弱碱阴离子交换树脂柱的型号为LX6702、LX6702、LX67中的一种,所述弱极性大孔吸附树脂柱的型号为AB-8,所述阳离子交换树脂纯化的型号为LSC-200、LSC-AS、LCS-400中的至少一种,所述色谱层析柱的型号为LXB2000或LXB-20SS。
优选的,所述弱碱阴离子交换树脂柱、弱极性大孔吸附树脂柱的容量比为1:3-5。
优选的,步骤S3包括:
S31、将发酵上清液用0.5μm孔径的陶瓷膜过滤,滤液以1.5-3.0BV/h的流速上弱碱阴离子交换树脂柱进行过饱和吸附;流出液以1.5-2.5BV/h的流速上弱极性大孔吸附树脂柱,水洗至无色且pH值为7-8,用70%乙醇以0.5-0.7BV/h洗脱并收集洗脱液;
在上柱前期鞣酸及部分活性成分被吸附至弱碱阴离子交树脂柱,其中对鞣酸的吸附能力强于活性物质,因此当处于过饱和吸附状态时鞣酸可代替活性物质被吸附,而活性物质则随流出液流出,可尽可能保留活性成分而降低鞣酸的量;之后将流出液上大孔吸附树脂柱,此时大部分重金属离子在上柱过程中随流出液流出,再用去离子水水洗至流出液pH值为7~8,则大部分残留树脂间隙中的重金属会被洗掉。
S32、将洗脱混合液以1.2-2.0BV/h的流速上串联设置的阳离子交换树脂柱,之后水洗至无醇味,收集流出液及水洗液。
醇水溶液洗脱后,洗脱液中还会残留少量重金属,醇相直接上阳离子交换树脂,重金属离子会被阳离子交换树脂交换吸附,而活性成分处于醇相中基本不会被阳离子交换树脂所交换吸附,从而起着良好的脱除重金属的效果。
S33、将收集的流出液及水洗液真空减压浓缩至无醇味,加入磷酸调pH至3.4-3.6;按照8-12%柱体积进行上层析柱,洗脱并收集除单宁酸外的其他流出液。
经处理后的流出液减压浓缩至几乎无醇味,此时成分相对于初始的发酵上清液而言较为单一,通过色谱层析柱能够将残余的单宁酸分离并去除,合并其他流出液以进行后续处理。通过上述方法能够有效去除单宁酸及重金属,最大程度保留山楂中的活性成分,使大剂量口服成为可能。
优选的,步骤S1包括:挑选优质山楂果进行洗涤后投入破碎机中进行破碎,将山楂碎块中按照100g:200-400ml分别加入去离子水,加热至60-80℃保温10-30min;降温至35-45℃后按照100g:0.01-0.05g加入果胶酶,保温加热2.0-3.5h后固液分离,收集初级山楂液。
优选的,步骤S2为:将初级山楂液经巴斯德灭菌后接种4-6%巴斯德毕赤氏酵母,发酵培养23-27h,固液分离后收集发酵上清液。通过发酵能够大幅去除山楂中的糖类、果胶等成分
巴斯德毕赤酵母有很好的耐酸性,对生长培养条件要求很低;作为菌种利用初级山楂汁进行发酵,可最大限度消耗掉山楂汁中的单糖、寡糖、多糖、等成分,而其中的总黄酮、总酚酸类成分几乎没有减少,有利于山楂提取物中活性成分含量的大幅提高,使最终约1克山楂提取物等同于约390克山楂。
优选的,步骤S4为:收集的流出液在50-65℃下、真空压力-0.07~-0.095MPa下浓缩至固含量为18-25%,干燥得山楂提取物。通过上述工艺处理后得到的山楂提取物本身以纳米晶体形式存在,溶解于超纯水体系中的平均平均粒径为103.18nm,比单独的活性成分具有更高的生物利用度。
所述山楂提取物在制备治疗疾病的制品中的应用。所述疾病包括肿瘤、癌痛、各种急慢性炎症、红斑狼疮、心脑血管疾病等。优选的,所述疾病为癌痛,尤其是难治性癌痛。
附图说明
图1为本发明实施例1-3所述山楂提取物的制备工艺流程图;
图2为本发明实施例4中制备样品的粒径分布;
图3本发明实施例5中体外胃肠消化实验中样品的生物利用度结果,其中橙色、绿色、红色与蓝色分别代表黄酮标准品、多酚标准品、山楂提取物A(代表山楂提取物中多酚)、山楂提取物B(代表山楂提取物中黄酮)。
具体实施方式
为使本发明的上述目的、特征和优点能够更为明显易懂,下面结合附图对本发明的具体实施例做详细的说明。需要说明的是,在不冲突的前提下,本发明实施例中的技术特征可以相互组合。
作为药食同源类中药,传统中医药理论记载山楂具有消食健胃、行气散瘀、化浊降脂等功效,而现代临床药理也证实山楂具有降血脂、降血压、抗菌等多种健康促进的功效,已广泛应用于食品、保健品化妆品等领域;
由于重金属在山楂生长过程中会逐渐富集且不易代谢,当摄入受重金属污染的山楂时,重金属会被人体吸收并逐渐富集,严重时会出现重金属中毒而引起肝、肾、脾胃、神经等组织及器官损害;另外由于山楂中含有大量的有机酸、果酸、山楂酸、枸橼酸等,在空腹食用时会对胃黏膜产生刺激作用,使胃泛酸发胀。尤其是山楂中的鞣酸还能和胃酸结合形成胃石,胃石很难被消化掉且积累较多会引起胃溃疡、胃出血甚至胃穿孔。因此山楂食用或入药量有限,限制了其应用。为此申请人提出如下技术方案:
实施例1
一种山楂提取物的制备方法,包括:
S1、预处理挑选优质山楂果进行洗涤后投入破碎机中进行破碎,将山楂碎块中按照100g:300ml分别加入去离子水,加热至70℃保温20min;降温至50℃后按照100g:0.02g加入果胶酶,保温加热3.5h,再加入2.0%珍珠岩进行过滤,收集初级山楂液;
S2、发酵将初级山楂液经巴斯德灭菌后接种5%巴斯德毕赤氏酵母,发酵培养25h,固液分离后收集发酵上清液;
S3、大孔吸附树脂纯化将步骤S3所述的发酵上清液用0.5μm孔径的陶瓷膜,在膜压2MPa,20℃下,过滤除杂,滤液以4BV/h的流速上弱碱阴离子交换树脂柱(型号:LX6702,高径比2:1,树脂用量100ml);流出液以1.2BV/h的流速上弱极性大孔吸附树脂柱(型号:AB-8,高径比4:1,树脂用量330ml),以2BV/h的流速水洗至洗脱液无色且pH值约为8,再用70%的乙醇水溶液以0.5BV/h的流速洗脱并收集洗脱液,然后水洗至无醇味并收集水洗脱液,将收集的洗脱液混合;
S4、阳离子交换树脂纯化将步骤S3所得洗脱混合液以1BV/h的流速上阳离子交换树脂柱(型号:LSC-200,径高比为1:4,树脂用量133ml),之后用去离子水以2BV/h水洗至无醇味,收集流出液。
S5、色谱层析纯化将步骤S4所得的流出液置于单效浓缩器中,在60℃,压力-0.09MPa减压浓缩至无醇味,加入磷酸调pH至3.5;按照10%柱床体积上层析柱(型号LXB2000,高径比5:1,树脂用量1600ml),用50%丙酮以0.1BV/h进行洗脱,收集除单宁酸外的其他流出液;
S6、浓缩、干燥将步骤S5收集的流出液在60℃下、真空压力-0.09MPa下浓缩至固含量为20%,加入淀粉适量混匀,制粒干燥,整粒,加入羧甲淀粉钠、硬脂酸镁适量,混匀制片,包薄膜衣,即得。
经检测,本实施例所得山楂提取物中含0.234%总黄酮、11.3%总三萜、2.1%总酚,所述山楂提取物中鞣酸的含量为1.17%,去除率达99.4%,Pb的含量为:0.026mg/kg,砷的含量为0.05mg/kg,汞的含量不高于0.01mg/kg。
实施例2
一种山楂提取物的制备方法,如图1所示,包括:
S1、预处理挑选优质山楂果进行洗涤后投入破碎机中进行破碎,将山楂碎块中按照100g:400ml分别加入去离子水,加热至75℃保温15min;降温至40℃后按照100g:0.03g加入果胶酶,保温加热2.5h,再加入2.5%珍珠岩进行过滤,收集初级山楂液;
S2、发酵将初级山楂液经巴斯德灭菌后接种4.5%巴斯德毕赤氏酵母,发酵培养27h,固液分离后收集发酵上清液;
S3、大孔吸附树脂纯化将步骤S3所述的发酵上清液用0.5μm孔径的陶瓷膜,在膜压1.8MPa,25℃下过滤除杂,滤液以3.5BV/h的流速上弱碱阴离子交换树脂柱(型号:LX6703,高径比2.2:1,树脂用量80ml);流出液以1.2BV/h的流速上弱极性大孔吸附树脂柱(型号:AB-8,高径比4:1,树脂用量310ml),以1.8BV/h的流速水洗至洗脱液无色且pH值约为7.8,再用70%的丙酮水溶液以0.6BV/h的流速洗脱并收集洗脱液,然后水洗至无醇味并收集水洗脱液,将收集的洗脱液混合;
S4、阳离子交换树脂纯化将步骤S3所得洗脱混合液以1.2BV/h的流速依次上阳离子交换树脂柱(型号:LSC-200,径高比为1:3,树脂用量150ml),之后用去离子水以2BV/h水洗至无醇味,收集流出液。
S5、色谱层析纯化将步骤S4所得的流出液置于单效浓缩器中,在65℃,压力-0.08MPa减压浓缩至无醇味,加入磷酸调pH至3.6;按照8%柱床体积上层析柱(型号LXB-20SS,高径比3:1,树脂用量1200ml),用50%丙酮以0.1BV/h进行洗脱,收集除单宁酸外的其他流出液;
S6、浓缩、干燥将步骤S5收集的流出液在55℃下、真空压力-0.07MPa下浓缩至固含量为25%,加入淀粉适量混匀,制粒干燥,整粒,加入羧甲淀粉钠、硬脂酸镁适量,混匀制片,包薄膜衣,即得。
经检测,本实施例所得山楂提取物含中0.219%总黄酮、13.1%总三萜、2.4%总酚,所述山楂提取物中鞣酸的含量为1.55%,鞣酸去除率达99.5%,Pb的含量为:0.16mg/kg,砷的含量为0.04mg/kg,汞的含量不高于0.01mg/kg。
实施例3
一种山楂提取物的制备方法,包括:
S1、预处理挑选优质山楂果进行洗涤后投入破碎机中进行破碎,将山楂碎块中按照100g:200ml分别加入去离子水,加热至72℃保温24min;降温至45℃后按照100g:0.01g加入果胶酶,保温加热2.5h,再加入3%珍珠岩进行过滤,收集初级山楂液;
S2、发酵将初级山楂液经巴斯德灭菌后接种5.5%巴斯德毕赤氏酵母,发酵培养23h,固液分离后收集发酵上清液;
S3、大孔吸附树脂纯化将步骤S3所述的发酵上清液用0.5μm孔径的陶瓷膜,在膜压1.7MPa,30℃下,过滤除杂,滤液以3.0BV/h的流速上弱碱阴离子交换树脂柱(型号:LX67,高径比2.5:1,树脂用量90ml);流出液以1.5BV/h的流速上弱极性大孔吸附树脂柱(型号:AB-8,高径比3:1,树脂用量350ml),以2.5BV/h的流速水洗至洗脱液无色且pH值约为8,再用70%的乙醇水溶液以0.7BV/h的流速洗脱并收集洗脱液,然后水洗至无醇味并收集水洗脱液,将收集的洗脱液混合;
S4、阳离子交换树脂纯化将步骤S3所得洗脱混合液以1.2BV/h的流速上串联设置的阳离子交换树脂柱(型号:LSC-AS+LCS-400,高径比均为2:1,树脂用量各100ml),之后用去离子水以2.5BV/h水洗至无醇味,收集流出液。
S5、色谱层析纯化将步骤S4所得的流出液置于单效浓缩器中,在58℃,压力-0.07MPa减压浓缩至无醇味,加入磷酸调pH至3.4;按照12%柱床体积上层析柱(型号LXB200,高径比4:1,树脂用量1200ml),用60%乙醇以0.2BV/h进行洗脱,收集除单宁酸外的其他流出液;
S6、浓缩、干燥将步骤S5收集的流出液在58℃下、真空压力-0.07MPa下浓缩至固含量为22%,加入淀粉适量混匀,制粒干燥,整粒,加入羧甲淀粉钠、硬脂酸镁适量,混匀制片,包薄膜衣,即得。
经检测,本实施例所得山楂提取物中0.22%总黄酮、12.6%总三萜、2.3%总酚,所述山楂提取物中鞣酸为0.75%,鞣酸去除率达99.7%,Pb的含量为0.054mg/kg,砷的含量为0.03mg/kg,汞的含量不高于0.01mg/kg。
实施例4山楂提取物的表征
用超纯水将实施例2制备的山楂提取物进行稀释20倍,200rpm搅拌溶解30min,分两种方式进行处理:1)将样品在条件5000rpm,25℃下离心3min,吸取样品上清液,测定其粒径数据,记为A;再将上清液再在14000rpm,25℃下离心3min,取上清液测定粒径,记为B,将沉淀物使用相同的溶剂复溶,测定粒径记为C;2)直接在14000rpm,25℃下离心3min,分别测定样品上清液与沉淀物复溶的粒径,分别记为D和E。
分别取1ml样品心滴加至标准的样品池(石英池)通过ZetasizerNanoZS(英国马尔文仪器有限公司,仪器开机后预热半小时,使激光稳定)以动态光散射技术进行检测,测量温度为25.0℃,激光波长为633nm、测量角度θ=173°,使用前用滤过的溶剂淋洗样品池三次以上,重复测量3次以上,得到测定样本的水合粒径,结果见表1、图2。
表1稀释样品的粒径分布表
样品A、B的平均粒径分别为234.47nm、103.18nm,而样品B的光散射强度没有明显变化,属于动力学更稳定胶体粒子,说明再次高转速离心使山楂提取物对应纳米晶体的粒径明显减小。光散射强度值与颗粒粒径及数量呈正相关,说明离心过程中可能存在较大颗粒解体成更多小颗粒。若直接以14000rpm离心得到的上清液-即样品D对应的颗粒平均粒径为92.77nm,与样品B的颗粒粒径大小相近,但光散射强度值远低于样品B,而沉淀复溶得到的溶液-即样品E中光散度非常低,说明大部分颗粒留在了上清液中。
实施例5山楂提取物的生物利用度的测定
食品或者中草药的功效不仅取决于活性(药用)成分的剂量多少,更与活性成分的存在形式直接相关。以游离态或结合态形式存在的活性成分在胃肠道中的稳定性及最终吸收程度存在着显著差异,直接影响了活性成分的功效发挥。从鸡汤、咖啡、茶到中药汤剂,食品或中药汤剂中的的自组装微纳米颗粒(MNPs),由极性和非极性成分组成,并由加工过程中的化学反应和物理因素相互作用而成,一经发现即引起了人们的广泛关注。
5.1实验内容
模拟消化实验在动态仿生-模拟胃肠道消化系统AGDS中进行,其通过3D数字技术模拟人胃的真实形状和结构,该系统采用两组对称的滚轮和一组相反方向的滚轮来模拟人胃的蠕动,在整个消化过程中,使用pH-stat工作站(Titrando-Metrohm,Switzerland)控制pH值。
按照Minekus等人发布的国际标准模拟体外胃肠消化(INFOGEST标准协议)建立消化环境;模拟胃液(SGF)和模拟肠液(SIF)由每天新鲜的原液制备,使用前保持在37℃,其中猪胃粘膜胃蛋白酶在SGF的终浓度为2000U/mL,来自猪胰腺和胆汁盐(猪胆汁提取物)的胰酶溶解于SIF,最终浓度分别为100U/mL和10mM。
实验温度恒定在37℃,使用磁力搅拌器以95rpm的速度分别混合20mL的3种样品(山楂提取物及标准多酚儿茶素、标准黄酮槲皮素)和20mLSGF,并在整个消化过程中,使用pH-stat工作站将pH控制在3.0以模拟胃消化环境。2小时后,将SIF(pH7)以1:1(v/v)的比例添加到AGDS中,于相同条件下(37℃和95rpm)进行肠道相模拟2小时,全程控制pH为7.4。出于分析目的,每隔0.5h取等分消化物试样。
5.2检测方法
TPC通过改进的Folin-Ciocalteu方法通过紫外可见分光光度法测定,具体为在搅拌条件(200rpm)下用甲醇进行提取1小时,然后将混合物离心10分钟(10000rpm),再将0.125mL上清液转移到比色杯中,加入0.5mL蒸馏水和0.125mLFolinCiocalteu反应物,静置6分钟后,加入1.0mL蒸馏水和1.25mL碳酸钠。室温下孵育90分钟,随后于760nm波长处测定吸光度,并将吸光度与没食子酸(GA)的标准曲线进行比较,得到所测物质含量。
TFC的测定参照比色法,即将3.0g样品与3.0mL水混合,并将混合物以10,000rpm的速度离心10分钟(MedifrigerBL-S,P-Selecta)。之后,将0.25mL上清液与1mL蒸馏水和0.075mL5%亚硝酸钠溶液混合。静置6分钟后,加入0.15mL氯化铝溶液,再5分钟后,加入0.5mL氢氧化钠(1M)和2.0mL蒸馏水,使用分光光度计(JASCOV-630)在510nm处读取吸光度,类黄酮的总含量表示为每克干物质的儿茶素当量(mg)。
为了评估食物基质对多酚、类黄酮和总抗氧化活性在胃肠道体外消化过程中发生的变化的影响,按照生物可及性(Bioaccessibility)=x/y*100%来计算,其中X是透析阶段后样品中的(TPC(mgGA/g干物质)或FC(mg儿茶素当量/g干物质),Y是透析阶段后总消化样品中的TPC或TFC(mg),并以相同的单位表示。
5.3实验结果
多酚、黄酮与山楂提取物A、B在模拟胃肠道条件下的生物可及性释放动力学见图3。其中山楂提取物A曲线为通过TPC判断的生物可及性,而山楂提取物B曲线为通过TFC判断的生物可及性。可以看出山楂提取物A、B生物利用度显著高于黄酮与多酚。在SGF阶段的前半小时,山楂提取物A、B的生物利用度迅速增加(分别为18.0±1.7%和21.0±0.6%),在随后的1.5小时内逐渐上升且速率变慢。SGF期结束时,生物利用度分别达到33.4±0.9%和37.4±0.8%。相比之下,黄酮、多酚在SGF阶段释放缓慢,在SGF阶段结束时仅分别释放13.4±0.6%和11.5±0.9%。当暴露于pH值为7的SIF中时,所有样品中的生物利用度都突然增加。SIF结束时,山楂提取物A、B的生物利用度分别高达74.4±0.6%和81.4±1.5%,而黄酮、多酚的生物利用度仅为30.4±0.2%和25.8±1.6%。这表明山楂提取物的纳米晶体形式可有效提升其负载生物活性的生物利用度,山楂提取物中多酚黄酮的吸收率是游离纯化多酚黄酮的2倍以上。
虽然本发明披露如上,但本发明并非限定于此。任何本领域技术人员,在不脱离本发明的精神和范围内,均可作各种更动与修改,因此本发明的保护范围应当以权利要求所限定的范围为准。
Claims (9)
1.一种山楂提取物,其特征在于,包括0.21-0.25%总黄酮、11-14%%总三萜、2.0-2.5%总酚,所述山楂提取物中鞣酸的含量不高于2.5%。
2.根据权利要求1所述的山楂提取物,其特征在于,包括0.22-0.24%总黄酮、12.5-13.5%总三萜、2.1-2.4%总酚,所述山楂提取物中鞣酸的含量不高于2.3%。
3.根据权利要求2所述的山楂提取物,其特征在于,包括0.22-0.23%总黄酮、12.8-13.2%总三萜、2.2-2.3%总酚,所述山楂提取物中鞣酸的含量不高于2.2%。
4.根据权利要求3所述的山楂提取物,其特征在于,所述总黄酮与鞣酸的含量之比不低于1:10。
5.根据权利要求1所述的山楂提取物,其特征在于,所述山楂提取物溶解于超纯水体系中的平静平均粒径为103.18nm。
6.根据权利要求1-5任一项所述的山楂提取物,其特征在于,所述山楂提取物中砷的含量不高于0.06mg/kg,铅的含量不高于0.16mg/kg,汞的含量不高于0.02mg/kg。
7.根据权利要求6所述的山楂提取物,其特征在于,所述山楂提取物中砷的含量不高于0.04mg/kg,铅的含量不高于0.07mg/kg,汞的含量不高于0.01mg/kg。
8.权利要求1-7任一项所述山楂提取物在制备治疗疾病的制品中的应用。
9.根据权利要求8所述的应用,其特征在于,所述疾病为癌痛。
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