CN114805624B - 一种牡丹皮多糖及其制备方法与应用 - Google Patents
一种牡丹皮多糖及其制备方法与应用 Download PDFInfo
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- CN114805624B CN114805624B CN202210478758.0A CN202210478758A CN114805624B CN 114805624 B CN114805624 B CN 114805624B CN 202210478758 A CN202210478758 A CN 202210478758A CN 114805624 B CN114805624 B CN 114805624B
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- polysaccharide
- cortex moutan
- water
- tree peony
- moutan
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- Medicines Containing Plant Substances (AREA)
Abstract
本发明公开了一种牡丹皮多糖及其制备方法和应用。本发明首先将牡丹皮使用乙醇浸提出去脂溶性色素和小分子物质。将处理过的牡丹皮进行水提醇沉,Sevag法除蛋白,透析后冷冻干燥制得牡丹皮粗多糖;牡丹皮粗多糖复溶,采用纤维素DEAE‑52柱层析分离纯化,并通过超滤离心获得牡丹皮纯化多糖MCP,其由半乳糖、阿拉伯糖和葡萄糖组成。本发明的牡丹皮多糖具有良好的抗炎活性,有望开发成抗炎药物或保健品。本发明对MCP的一级结构进行了研究,为进一步研究牡丹皮多糖结构和活性的构效关系建立基础。
Description
技术领域
本发明属于天然产物活性成分分离纯化技术领域,特别涉及一种牡丹皮多糖及其制备方法与应用。
背景技术
牡丹皮是芍药属多年生牡丹(Paeonia suffruticosaAndr.)的干燥根皮,作为药物或膳食补充剂在中国和其他亚洲国家的临床应用中有着悠久的历史。多糖因具有抗氧化、抗菌、抗炎、降血糖、抗糖化、抗肿瘤、调节肠道菌群、免疫调节和抗疲劳等多种重要的生物活性而备受关注。多糖是一类分子结构复杂且庞大的碳水化合物,含有很多不同单糖残基、糖苷键、支链、取代基。多糖的结构与其生物活性密切相关,其复杂的结构是其多样生物活性的基础。因此,开展多糖的研究首先要解析其结构,才能更好地阐释其生物活性机制,建立明确的构效关系,为挖掘以及拓展多糖的应用提供理论基础。此外,关于纯化后的牡丹皮多糖的抗炎活性及抗炎机制的研究报道比较少。
发明内容
为了克服上述现有技术的缺点与不足,本发明的首要目的在于提供一种纯化的牡丹皮多糖。
本发明另一目的在于提供上述纯化的牡丹皮多糖的制备方法。
本发明再一目的在于提供上述纯化的牡丹皮多糖的应用。
本发明的目的通过下述方案实现:
一种牡丹皮多糖,其重均分子量为86.74kDa,是一种由葡萄糖,半乳糖和阿拉伯糖组成的杂多糖。
优选的,所述的牡丹皮多糖由摩尔比为1.0:2.5:7.7的葡萄糖、半乳糖和阿拉伯糖组成。
更优选的,所述的牡丹皮多糖结构式如下所示:
一种上述的牡丹皮多糖的制备方法,其包括以下步骤:
(1)将牡丹皮粉碎后过筛,将过筛后的牡丹皮粉末用乙醇回流进行脱脂脱色,然后离心分离,收集沉淀物并烘干,得脱脂脱色的牡丹皮干粉;
(2)将步骤(1)制得的牡丹皮干粉,采用热水浸提法提取牡丹皮多糖,将水提液浓缩后进行醇沉,离心分离收集牡丹皮多糖沉淀物;
(3)对步骤(2)制得的牡丹皮多糖沉淀物采用Sevag法除蛋白,透析,冷冻干燥制得除蛋白牡丹皮粗多糖;
(4)将步骤(3)制得的牡丹皮粗多糖复溶于水中,配成多糖水溶液,采DEAE-52离子交换柱进行纯化,采用梯度洗脱法,依次用蒸馏水、不同浓度的NaCl溶液进行梯度洗脱,收集水洗脱组分,冷冻干燥得到牡丹皮多糖,并将其命名为MCP。
步骤(1)中所述的牡丹皮粉碎之前先清洗干净、烘干后再进行粉碎过筛,粉碎后过筛是指过100目的筛;
步骤(1)中所述的乙醇回流是指使用质量分数为80-95%的乙醇对牡丹皮干粉进行回流脱脂脱色,其中牡丹皮干粉每1质量份(g)固形物添加8-10体积份(mL)乙醇,回流时间为2-3h,回流温度为60~80℃,回流后置于40-50℃的鼓风干燥箱中烘干;回流脱脂脱色的操作可重复多次以便更好地脱脂脱色。
步骤(2)中所述的采用热水浸提法的温度为70-95℃,热水浸提的时间为0.5-3h;所述热水浸提的固液比为每1质量份(g)固形物添加10-30体积份(mL)水,优选为每1g固形物添加30mL水;
步骤(2)中所述热水浸提可重复多次,并将多次提取的水溶液合并再进行浓缩;为了得到更好纯度的牡丹皮多糖,合并水提液后优选为进行离心分离,收集上清液再进行浓缩。步骤(2)中所述的浓缩是指浓缩至原来体积的1/3-1/6;步骤(2)中所述的醇沉是指在浓缩后的水提液中添加乙醇,使乙醇最终体积分数为80-90%,4℃条件下静置12-48h(优选为24h)后,离心分离,收集牡丹皮多糖沉淀物。
步骤(3)中所述的采用Sevag法除蛋白是本领域公知的去除游离蛋白的有效方法。在粗多糖溶液中加入sevag试剂进行充分振摇,将游离蛋白变性成为不溶性物质,经离心分离去除,可到达去除蛋白的目的。具体的,其中粗多糖溶液与Sevag试剂的体积比为4:1,粗多糖溶液的浓度为20mg/mL。
步骤(3)中所述的透析的截留分子量为3500Da,透析时间为48~72h,期间换水4-8次;冷冻干燥的条件为压强为0.01~0.10MPa,温度为-50℃~-80℃。
步骤(3)中得到除蛋白牡丹皮粗多糖后还可以包括以下AB-8大孔树脂摇床脱色素的步骤,具体如下:将除蛋白牡丹皮粗多糖溶于水,加入AB-8大孔树脂,150rpm摇床室温脱色2h,得到除蛋白除色素的多糖水提液,再进行透析并冷冻干燥,其中透析的截留分子量为3500Da,冷冻干燥的温度为-80℃。
步骤(4)中所述的多糖水溶液的浓度为10~20mg/mL;步骤(4)中所述的依次用蒸馏水、不同浓度的NaCl溶液进行梯度洗脱是指依次用蒸馏水0.1mol/L、0.2mol/L和0.5mol/L的NaCl盐溶液进行洗脱,洗脱流速控制为1.0mL/min;
步骤(4)中在收集水洗脱组分后还包括进一步纯化的步骤,即选择分子量为30kDa的离心超滤管对其进行分离纯化,然后再冷冻干燥得到平均重均分子量均一的纯化牡丹皮多糖组分。
本发明将牡丹皮粗多糖采用热水浸提和纤维素DEAE-52柱层析的分离纯化方案能快速制备牡丹皮多糖MCP,其分子量为86.74kDa,由葡萄糖、半乳糖和阿拉伯糖组成。
本发明制备的牡丹皮多糖具有良好的抗炎活性,可作为天然抗炎保健食品或药物。
本发明相对于现有技术,具有如下的优点及有益效果:
本发明对MCP的一级结构进行了研究,为进一步研究牡丹皮多糖结构和活性的构效关系建立基础。而且本发明的牡丹皮多糖具有良好的抗炎活性,有望开发成抗炎药物或保健品。
附图说明
图1为实施案例1制备牡丹皮粗多糖的洗脱曲线。
图2为实施案例1制备牡丹皮多糖MCP的重均分子量分布图。
图3为实施案例1制备牡丹皮多糖MCP的单糖组成图(B)以及标准单糖的HPLC色谱图(A);其中Man:甘露糖;Rib:核糖;Rha:鼠李糖;GlcA :葡萄糖醛酸;GalA:半乳糖醛酸;Glc:葡萄糖;Gal:半乳糖;Xyl:木糖;Ara:阿拉伯糖;Fuc:岩藻糖。
图4为实施案例1制备牡丹皮多糖MCP的核磁氢谱图。
图5为实施案例1制备牡丹皮多糖MCP的核磁碳谱图。
图6为实施案例1制备牡丹皮多糖MCP的核磁COSY谱图。
图7为实施案例1制备牡丹皮多糖MCP的核磁HSQC谱图。
图8为实施案例1制备牡丹皮多糖MCP的核磁HMBC谱图。
图9为实施例1制备的牡丹皮多糖MCP的扫描电镜图。
图10为实施例2制备的牡丹皮多糖MCP对NO的产生示意图。
图11为实施例2制备的牡丹皮多糖MCP对IL-1β的影响示意图。
图12为实施例2制备的牡丹皮多糖MCP对IL-6的影响示意图。
图13为实施例2制备的牡丹皮多糖MCP对IFN-γ的影响示意图。
图14为实施例2制备的牡丹皮多糖MCP对TNF-α的影响示意图。
具体实施方式
下面结合实施例和附图对本发明作进一步详细的描述,但本发明的实施方式不限于此。实施例中未注明具体条件者,按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者,均为可以通过市售购买获得的常规产品。
实施例中所用试剂如无特殊说明均可从市场常规购得。
实施例1
牡丹皮多糖MCP的制备
(1)牡丹皮多糖的提取:
取1Kg牡丹皮样品,经粉碎机粉碎后过100目筛,加入8L 95%乙醇在70℃条件下回流2h,重复操作2次,离心分离,沉淀物在45℃下烘干。在脱脂脱色后的牡丹皮干粉中添加蒸馏水,按照固液比为每1质量份(g)固形物添加30体积份(mL)水添加的蒸馏水,在95℃的水浴中浸提3h,重复提取3次,过滤,离心,收集上清液,并将上清液减压浓缩至原体积的五分之一;缓慢往浓缩液中滴加无水乙醇,二者体积比为1:4,使乙醇最终体积分数为80%在4℃条件下静置24h,离心,收集沉淀物后烘干得知牡丹皮粗多糖73.5g。
(2)除蛋白和色素:
取30g牡丹皮粗多糖,用蒸馏水配成20mg/mL的多糖溶液,加入Sevag试剂除蛋白,其中粗多糖溶液与Sevag试剂的体积比为4:1,重复操作8次,透析(3500Da),冷冻干燥制得除蛋白的牡丹皮粗多糖。取除蛋白的牡丹皮粗多糖,用蒸馏水配成20mg/mL的多糖溶液,向脱蛋白多糖溶液中加入AB-8大孔树脂,150rpm摇床室温脱色2h,得到除蛋白除色素的多糖水提液,再使用分子量为3500Da的透析袋进行透析72h,将透析液于-80℃冷冻干燥,得到除杂后的牡丹皮粗多糖。
(3)DEAE-52柱层析:
为了制备牡丹皮分子量均一的多糖,将牡丹皮粗多糖用DEAE-52柱层析进一步纯化。称取300mg牡丹皮粗多糖溶于15mL蒸馏水中,离心(10000rpm,10min),取上清液沿壁缓慢加入DEAE-52层析柱(2.6×100cm)中,依次使用蒸馏水、0.1、0.2和0.5mol/L氯化钠(NaCl)水溶液进行洗脱,流速控制为1.0mL/min。收集洗脱液组分(10mL/管),并采用苯酚-硫酸法测试每管洗脱液在490nm处的吸光值,根据吸光值绘制洗脱曲线。将显示同一洗脱峰的样品合并,浓缩、透析除盐、选取分子量为30kDa的离心超滤管对总糖含量和洗脱得率最高的蒸馏水洗脱组分MCPs进行分离纯化,被截留的上层样品冷冻干燥后即为平均重均分子量均一的纯化多糖组分,将其命名为MCP。牡丹皮粗多糖的洗脱曲线如图1所示,MCP的重均分子量分布如图2所示。
(4)重均分子量测定:
采用凝胶排阻色谱测定MCP的重均分子量为86.74kDa,结果如图2所示。
(5)单糖组成分析:
采用1-苯基-3-甲基-5-吡唑啉酮(PMP)柱前衍生化法测定MCP的单糖组成。取10mg牡丹皮多糖MCP,用4mol/L三氟乙酸在110℃的油浴中水解4h,减压蒸干、PMP柱前衍生、二氯甲烷萃取三次除去多余的PMP、取水相过0.22μm滤膜后用高效液相进行分析。结果如图3所示,牡丹皮多糖MCP由葡萄糖、半乳糖和阿拉伯糖组成,摩尔比为1.0:2.5:7.7。
(6)甲基化分析:
取5.0mg干燥牡丹皮多糖MCP样品与1.5mL无水DMSO混合。将混合物超声直至样品完全溶解,然后在氮气保护下,快速加入100mg氢化钠颗粒,反应过夜后,冰水浴中往反应液中缓慢滴加2mL碘甲烷,反应2h后添加1mL蒸馏水中止反应,将该混合物用蒸馏水透析48h后冻干。重复甲基化过程,直到羟基(-OH)吸收峰(3200-3700cm-1)在红外光谱中消失。然后,将甲基化后多糖在120℃下用2mL的2M三氟乙酸水解6h,加入60mg NaBH4还原并在室温中过夜,用4mol/L甲酸中和溶液,并在65℃的真空旋转蒸发器中干燥,然后用甲醇洗涤产品三次,以去除乙酸。加入2.5mL乙酸酐于l00℃乙酸化l h,加入甲苯,蒸干,重复三次。加入2mL氯仿使其充分溶解,之后进行GC-MS分析。牡丹皮多糖MCP的甲基化结果见表1。甲基化结果表明牡丹皮多糖中主要包含5种连接方式,分别为T-Araf-(1→、→5)-Araf-(1→、→3,5)-Araf-(1→、→3,4)-Glcp-(1→和→4)-Galp-(1→五种糖苷键组成,其摩尔比为3.15:2.63:1.05:2.19:0.98。
表1 MCP的甲基化分析
(7)核磁解析:
取60mg牡丹皮多糖MCP用D2O置换2次后进行一维(氢谱和碳谱)和二维(COSY、HSQC和HMBC)核磁测试。根据MCP的单糖组成和甲基化结果,并结合MCP的1H、13C、1H-1H COSY、HSQC和HMBC(图4-8)信息,各糖苷键H和C的信号归属见表2,按照糖苷键分别标记为A、B、C、D和E,联合核磁氢谱、碳谱、COSY、HSQC、谱图和甲基化分析及文献报道,将各糖苷键的碳谱和氢谱化学位移值进行归类。综上所述,牡丹皮多糖的主链是以→5)-Araf-(1→与→3,4)-Glcp-(1→和→4)-Galp-(1→交叉链接组成的,侧链是T-Araf连接在→3,5)-Araf-(1→和→3,4)-Glcp-(1→的3号位上,由此可以推断出牡丹皮多糖结构简式如下所示:
表2 MCP的1H和13C化学位移
(8)扫描电镜分析:取适量干燥的MCP样品,放置于样品台,在真空喷镀仪中喷金处理后,进行扫描电镜分析,扫描电子显微镜为JSM-7001F(JEOL,Japan),在加速电压10.00kV下观察样品表面形貌,取适宜的放大倍数和具有代表性的视野进行拍照记录。牡丹皮多糖表面形貌如图9所示。
实施案例2:牡丹皮多糖的抗炎活性
通过ELISA试剂盒法检测MCP对炎症反应过程中NO的产生以及TNF-α,IL-6,IL-1β和INF-γ等促炎细胞因子的分泌量来评估MCP的抗炎活性。
NO:采用一氧化氮分析试剂盒(NO Assay Kit)在脂多糖(LPS)诱导的炎症模型下,研究不同浓度的牡丹皮多糖MCP对RAW264.7巨噬细胞产生NO的影响。将传代第5-9代RAW264.7细胞以6000细胞/孔接种于96孔板内,每组设置6个复孔。根据实验分组进行加药处理:空白对照(未加LPS和其他药物,只含有DMEM基础培养基、阴性对照(加1μg/mL LPS处理24h)、样品MCP(使用不同浓度的MCP先处理2h,浓度分别为25μg/mL、50μg/mL、100μg/mL、200μg/mL、400μg/mL、800μg/mL和1600μg/mL,再加1μg/mL的LPS处理24h),阳性对照(先使用地塞米松50μg/mL处理2h,再加1μg/mL的LPS处理24h)。收集上清液用Griess试剂检测上清中NO含量,用多功能酶标仪在540nm波长下测定反应产物的吸光值。按照一氧化氮(NO)试剂盒的测定方法,绘制NO标准曲线,根据测定的标准曲线计算各组细胞上清液的NO含量。结果见图10。一氧化氮(NO)是一种非正统的、最小的信使分子,由三种亚型的一氧化氮合酶产生,具有众多的分子靶点,可以通过测定LPS刺激RAW 264.7小鼠巨噬细胞中的NO的产生来评估MCP的抗炎活性。如图10所示,空白对照细胞NO分泌量为9.95μM,经过LPS刺激小鼠巨噬细胞24h后,可使NO分泌极显著升高至80.07μM(p<0.05),说明,LPS诱导细胞产生过度的炎症反应,产生大量NO。当细胞用地塞米松(DXMS)预处理后,细胞分泌的NO则下降至11.52μM(接近于空白对照细胞NO分泌量),极显著降低于LPS组(p<0.05);当细胞用不同浓度的MCP预处理,细胞分泌的NO量显著减少(P<0.05)。当MCP浓度在25~800μg/mL范围内,NO分泌量逐渐减少,当MCP浓度达到800μg/mL时,NO分泌量为19.18μM;当MCP浓度达到1600μg/mL时,NO分泌量为48.42μM,与MCP浓度为200μg/mL时NO分泌量(48.03μM)一致。说明,MCP在浓度25μg/mL时已经能明显减轻LPS刺激产生的炎症反应,在浓度为800μg/mL时,MCP的抗炎作用最好;在25~800μg/mL范围内,MCP抗炎作用呈现浓度依赖性增长,浓度过高时,MCP有一定的促炎作用。以上结果表明,一定浓度的MCP能够减少LPS刺激RAW 264.7小鼠巨噬细胞中的NO的产生,从而达到抗炎的作用。
炎症因子:采用ELISA试剂盒在脂多糖(LPS)诱导的炎症模型下,研究不同浓度的牡丹皮多糖MCP对RAW264.7巨噬细胞产生IL-1β、IL-6、IFN-γ和TNF-α四个炎症因子的影响。将传代第5-9代RAW264.7细胞以6000细胞/孔接种于96孔板内,每组设置6个复孔。根据实验分组进行加药处理:空白对照(未加LPS和其他药物,只含有DMEM基础培养基)、阴性对照(加1μg/mL LPS处理24h)、样品MCP(使用不同浓度的MCP先处理2h,浓度分别为25μg/mL、50μg/mL、100μg/mL、200μg/mL、400μg/mL、800μg/mL和1600μg/mL,再加1μg/mL的LPS处理24h),阳性对照(先使用地塞米松50μg/mL处理2h,再加1μg/mL的LPS处理24h)。收集上清液用于ELISA试剂盒测定。按每个ELISA试剂盒说明书进行操作来测定MCP对炎症细胞所分泌的IL-1β、IL-6、IFN-γ和TNF-α四个炎症因子的分泌水平,操作具体以订购的试剂盒说明书为准。结果见图11-图14。肿瘤坏死因子-α(TNF-α)和白细胞介素-1β和6(IL-1β和IL-6)在传递信息、激活与调节免疫细胞、介导T细胞和B细胞活化、增殖与分化及在炎症反应中起重要作用。作为体内重要的细胞因子,白细胞介素IL-1β可介导多种炎症反应。如图11-14所示,四种炎症因子IL-1β、IL-6、IFN-γ和TNF-α在正常细胞内的含量偏低,经LPS刺激后细胞内炎症因子的含量为呈现显著性增长,阳性对照处理后细胞内炎症因子的含量出现降低,一定浓度的MCP也能够抑制LPS刺激RAW 264.7细胞中的四种炎症因子的产生,这种抑制作用呈现浓度依赖性。这表明MCP可以通过抑制IL-1β、IL-6、IFN-γ和TNF-α四种炎症因子的产生来发挥抗炎活性。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
Claims (5)
1.一种牡丹皮多糖,其特征在于重均分子量为86.74 kDa,是一种由葡萄糖,半乳糖和阿拉伯糖组成的杂多糖;
所述的牡丹皮多糖由摩尔比为1.0 : 2.5 : 7.7的葡萄糖、半乳糖和阿拉伯糖组成;
所述的牡丹皮多糖结构式如下所示:
所述的牡丹皮多糖的制备方法,包括以下步骤:
(1)将牡丹皮粉碎后过筛,将过筛后的牡丹皮粉末用乙醇回流进行脱脂脱色,然后离心分离,收集沉淀物并烘干,得脱脂脱色的牡丹皮干粉;
(2)将步骤(1)制得的牡丹皮干粉,采用热水浸提法提取牡丹皮多糖,将水提液浓缩后进行醇沉,离心分离收集牡丹皮多糖沉淀物;
(3)对步骤(2)制得的牡丹皮多糖沉淀物采用Sevag法除蛋白,透析,冷冻干燥制得除蛋白牡丹皮粗多糖;
(4)将步骤(3)制得的牡丹皮粗多糖复溶于水中,配成多糖水溶液,采DEAE-52离子交换柱进行纯化,采用梯度洗脱法,依次用水、不同浓度的NaCl溶液进行梯度洗脱,收集水洗脱组分,冷冻干燥得到牡丹皮多糖,并将其命名为MCP;
步骤(3)中得到除蛋白牡丹皮粗多糖后还包括以下AB-8大孔树脂摇床脱色素的步骤,具体如下:将除蛋白牡丹皮粗多糖溶于水,加入AB-8大孔树脂,150rpm摇床室温脱色2h,得到除蛋白除色素的多糖水提液,再进行透析并冷冻干燥,其中透析的截留分子量为3500Da,冷冻干燥的温度为-80℃;
步骤(4)中所述的多糖水溶液的浓度为10~20 mg/mL;步骤(4)中所述的依次用蒸馏水、不同浓度的NaCl溶液进行梯度洗脱是指依次用蒸馏水0.1 mol/L、0.2 mol/L和0.5 mol/L的NaCl盐溶液进行洗脱,洗脱流速控制为1.0mL/min;
步骤(4)中在收集水洗脱组分后还包括进一步纯化的步骤,即选择分子量为30kDa的离心超滤管对其进行分离纯化,然后再冷冻干燥得到平均重均分子量均一的纯化牡丹皮多糖组分。
2.根据权利要求1所述的牡丹皮多糖,其特征在于:
步骤(1)中所述的牡丹皮粉碎之前先清洗干净、烘干后再进行粉碎过筛,粉碎后过筛是指过100目的筛;
步骤(1)中所述的乙醇回流是指使用质量分数为80-95%的乙醇对牡丹皮干粉进行回流脱脂脱色,其中牡丹皮干粉每1 g固形物添加8-10 mL乙醇,回流时间为2-3h,回流温度为60~80℃,回流后置于40-50℃的鼓风干燥箱中烘干。
3.根据权利要求2所述的牡丹皮多糖,其特征在于:
步骤(2)中所述的采用热水浸提法的温度为70-95℃,热水浸提的时间为0.5-3h;所述热水浸提的固液比为每1 g固形物添加10-30 mL水;
步骤(2)中所述的浓缩是指浓缩至原来体积的1/3-1/6;步骤(2)中所述的醇沉是指在浓缩后的水提液中添加乙醇,使乙醇最终体积分数为80-90%,4℃条件下静置12-48h后,离心分离,收集牡丹皮多糖沉淀物。
4.根据权利要求1所述的牡丹皮多糖,其特征在于:
步骤(3)中所述的采用Sevag法除蛋白是指在粗多糖溶液中加入sevag试剂进行充分振摇,将游离蛋白变性成为不溶性物质,经离心分离去除,可到达去除蛋白的目的,其中粗多糖溶液与Sevag试剂的体积比为4:1,粗多糖溶液的浓度为20mg/mL;
步骤(3)中所述的透析的截留分子量为3500Da,透析时间为48~72h,期间换水4-8次;冷冻干燥的条件为压强为0.01~0.10 MPa,温度为-50℃~-80℃。
5.根据权利要求1所述的牡丹皮多糖在制备天然抗炎药物中的应用。
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