CN115919909A - 杨梅枝条醇提物在制备抗肝癌药物方面的应用 - Google Patents
杨梅枝条醇提物在制备抗肝癌药物方面的应用 Download PDFInfo
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Abstract
本发明属于天然药物技术领域，具体涉及一种杨梅枝条醇提物在制备抗肝癌药物方面的应用。所述的杨梅枝条醇提物由新鲜去叶的杨梅枝条干燥、粉碎后经70％乙醇提取获得。所述杨梅枝条醇提物能抑制肝癌细胞增殖，且呈浓度依赖关系；所述杨梅枝条醇提物能将肝癌细胞阻滞在细胞周期的S期；另外，所述杨梅枝条醇提物能促使肝癌细胞凋亡。因此，本发明为抗肝癌药物的研制提供了技术支持，且本发明能变废为宝，充分利用资源。
Description
技术领域
本发明涉及天然药物技术领域，尤其涉及杨梅枝条醇提取在制备抗肝癌药物方面的应用。
背景技术
肝细胞性肝癌(hepatocellularcarcinoma，HCC)，是最常见的原发性肝脏恶性肿瘤。根据《2021年全球癌症统计》，男性肝癌发病率已趋于稳定；然而在女性中呈稳步上升趋势(每年2％)，主要是由于肥胖、乙型肝炎病毒和丙型肝炎病毒等因素导致。在肝癌早期，化疗、手术切除等治疗方法提高了患者的生存率，但5年内肝癌复发率高达70％，大多数HCC患者被确诊时为肝癌晚期，生存期仅6～8个月。由于HCC晚期症状表现和有限的治疗选择，HCC时刻威胁着人类的生命健康。因此，寻找和开发有效抑制肝癌细胞生长的药物，延长肝癌患者的生存时间，降低死亡率具有极其重要的意义。
杨梅(Myrica rubra Sieb.et Zucc)是中国南方的特色果树，其果实色泽鲜艳，甜酸适口，风味独特，广受大众喜爱。杨梅树四季常绿，枝繁叶茂，长期放任树体生长，会消耗大量营养，且枝条过多会导致果园通风不良，进一步影响果实的产量和质量，另外，果树太高也不利于采摘和管理。为节约养分，提高杨梅产量和质量，需要对枝条进行修剪。杨梅在我国南方地区栽植面积广，每年修剪产生的大量杨梅枝条多被当作废弃物，未能得到有效利用。
发明内容
为了克服上述问题，本发明提供一种杨梅枝条醇提物在制备抗肝癌药物方面的应用。
本发明的目的是通过以下技术方案予以实现：
本发明提供了一种杨梅枝条醇提物在制备抗肝癌药物方面的应用。
该杨梅枝条醇提物的制备过程包括以下步骤：
(1)取杨梅枝条，去除叶片；
(2)60℃烘干，粉碎得样品粉末；
(3)用70％乙醇超声提取步骤(2)中的样品粉末，过滤得提取液，重复提取4次，合并滤液；旋蒸去除提取液中乙醇，剩余部分冷冻干燥得杨梅枝条醇提物。
在制备过程，上述步骤中的烘干温度、乙醇比例和提取次数等参数均可做适当调整。
优选地，所述的杨梅枝条包括杨梅修剪产生的废弃枝条。杨梅修剪每年都会产生大量废弃枝条，选用废弃枝条可变废为宝，充分利用资源。
进一步地，所述杨梅枝条醇提物将HepG2细胞阻滞在细胞周期的S期，其上调HepG2细胞p21基因表达，下调CyclinD和CDK6基因表达，可通过p21-CyclinD/CDK6信号通路将HepG2细胞阻滞在细胞周期的S期。
进一步地，所述杨梅枝条醇提物促使HepG2细胞凋亡，其下调HepG2细胞Raf、MEK1、ERK1、Bcl-2基因表达，可通过Raf-MEK1-ERK1信号通路和Bcl-2促使HepG2细胞凋亡。
所述杨梅枝条醇提物中含有多种酚类化合物，其中含量最高的5种为没食子酸，杨梅苷、没食子儿茶素、2,5-二羟基苯甲酸、表没食子儿茶素没食子酸酯。酚类化合物具有抗肿瘤、抗氧化等多种活性，杨梅枝条醇提物种酚类化合物种类多，含量高，可能是其抗肝癌的主要活性成分。
进一步地，所述肝癌为肝细胞性肝癌。
本发明的优势在于：(1)本发明公开了杨梅枝条醇提物在制备抗肝癌药物方面的用途，为抗肝癌药物的开发提供了一个新的选择。
(2)本发明的杨梅枝条醇提物制备简单，成本低，且能充分利用废弃杨梅枝条，变废为宝，充分利用资源。
附图说明
图1不同浓度杨梅枝条醇提物作用HepG2细胞48h后对细胞增殖的抑制作用，**表示p＜0.01。
图2不同浓度杨梅枝条醇提物作用HepG2细胞48h后对细胞形态的影响。
图3不同浓度杨梅枝条醇提物作用HepG2细胞48h后对细胞周期的影响。
图4不同浓度杨梅枝条醇提物作用HepG2细胞48h后的细胞周期分布，*表示p<0.05，**表示p<0.01。
图5不同浓度杨梅枝条醇提物作用HepG2细胞48h后对细胞凋亡的影响。
图6不同浓度杨梅枝条醇提物作用HepG2细胞48h后的细胞凋亡率，**表示p<0.01。
图7杨梅枝条醇提物作用HepG2细胞48h后对细胞周期调控相关基因的影响，*表示p<0.05，**表示p<0.01。
图8杨梅枝条醇提物作用HepG2细胞48h后对细胞凋亡调控相关基因的影响，*表示p<0.05，**表示p<0.01。
具体实施方式
下面结合实施例对本发明作进一步的说明。
实施例1杨梅枝条醇提物的制备
取修剪废弃后的新鲜杨梅枝条，去除叶片等多余部分。在60℃条件下将枝条烘干至恒重，粉碎、研磨得样品粉末。取样品粉末，在常温下用70％乙醇超声提取，过滤取滤液，重复4次。合并滤液，旋蒸去除乙醇，再冻干得杨梅枝条醇提物(MRBE)。1200g样品粉末可获得280g左右MRBE，约占干重23％。
实施例2杨梅枝条醇提物精准靶向-黄酮-酚类检测
(1)试验方法
取冻干后MRBE样品约50mg，加入600μL水/甲醇(V/V＝1/2，含琥珀酸-2,2,3,3-d450ng/mL)，再加入400μL氯仿；加入2颗钢珠，研磨机研磨(60Hz，2min)；冰水浴超声提取20min；离心10min(4℃，13000rpm)，取上清500μL装入EP管中；向残渣中继续加入400μL水：甲醇(V/V＝1/2，含琥珀酸-2,2,3,3-d4 50ng/mL)，涡旋1min，超声提取20min；离心10min(4℃，13000rpm)，取300μL上清，跟之前的500μL上清合并，共计800μL；取200μL上清挥干，然后用200μL水/甲醇(V/V＝18/7，含内标L-2-氯苯丙氨酸10ng/mL)复溶，涡旋30s，超声2min，-20℃下静置2h；离心5min(4℃，13000rpm)，用注射器吸取200μL的上清液，使用0.22μm的有机相针孔过滤器过滤后，转移到棕色LC进样小瓶，进UPLC-ESI-MS/MS(AB Sciex)分析。
每个色谱峰的峰面积(Peak Area)代表对应代谢物的相对含量，将代谢物的积分峰面积带入标准曲线线性方程进行计算，进一步代入计算公式计算后，最终得到实际样本中各代谢物的绝对含量数据。
样品代谢物含量(ng/g)＝C*V/M*N
公式中各字母含义：
C：样本中代谢物峰面积带入标准曲线计算得到的浓度值(ng/mL)；
V：定容体积(0.2mL)；
M：称取的样本质量(g)；
N：稀释倍数(5倍)。
(2)试验结果
通过MRBE精准靶向-黄酮-酚类检测，试验结果如表1显示，MRBE中有77种黄酮-酚类化合物，其中没食子酸含量最高为1122478.47ng/g，接着依次是杨梅苷659828.52ng/g、没食子儿茶素205632.70ng/g、2,5-二羟基苯甲酸189624.39ng/g、表没食子儿茶素没食子酸酯186575.14ng/g等。该77种化合物分为13类，包括：苯甲酸及其衍生物(1725324.76ng/g)、黄酮醇(923095.94ng/g)、儿茶素及其衍生物(598237.09ng/g)、花色素(54521.99ng/g)、苯丙素类(15770ng/g)、黄酮(10052.5ng/g)等。
已有大量酚类抗肿瘤作用报道，例如：没食子酸通过细胞毒作用、诱导肿瘤细胞凋亡、抑制肿瘤血管形成、抗氧化作用等机制抗肿瘤【李沐涵等，没食子酸抗肿瘤作用研究进展，中医药信息，2011年第28卷第一期】；杨梅苷显著降低HepG2细胞的存活性，促进其凋亡【甜艳花等，君迁子叶杨梅苷诱导HepG2细胞凋亡极其作用机制，食品科学，2021，Vol.42，No.11】；表没食子儿茶素没食子酸酯能抑制细胞凋亡和细胞周期停滞，具有抗肺癌、乳腺癌、肝癌等作用【罗可望等，表没食子儿茶素没食子酸酯抗肿瘤作用的研究进展】；等等。另外，上述试验结果表明，MRBE中酚类种类多，含量高，因此该类成分可能是MRBE抗肝癌作用的主要活性组分。
表1杨梅枝条醇提物中黄酮、多酚类代谢物质及含量
实施例3杨梅枝条醇提物对HepG2细胞(人肝癌细胞)的抑制作用
(1)试验方法
取对数生长期细胞经胰酶消化后，用DMEM培养基(美国GIBCO BRL公司)吹打配成1×105个/mL细胞悬液，加入96孔培养板，每孔100μL，在5％CO2、37℃培养箱中培养24h。待细胞完全贴壁后，实验组(含细胞100μL)分别加入0μg/mL、100μg/mL、200μg/mL、300μg/mL、400μg/mL样品液100μL，每组8孔；正常对照孔(含细胞100μL)加入含0.1％DMSO的无血清培养液100μL；空白对照孔(不含细胞)加入等量的相对应多糖无血清培养液，用于MTT测定调零。培养箱中孵育48h后，加入5g/L MTT 20μL，继续培养4h，去上清液，加入DMSO 150μL，在摇床上震动摇匀，在酶标仪570nm波长下测得各孔吸光度值D。按下式计算细胞的抑制率：
抑制率＝(正常对照组D570nm–试验组D570nm)/正常对照组A570nm×100％(n＝6)
(2)试验结果
经MRBE处理HepG2细胞48h后，100μg/mL、200μg/mL、300μg/mL、400μg/mL 4个浓度的MRBE对HepG2细胞增殖均表现出显著抑制作用。在100μg/ml浓度下，抑制率达到5.57％±0.06％；在200μg/ml浓度下，抑制率达到21.34％±0.17％；在300μg/ml浓度下，抑制率达到45.52％±1.15％；在400μg/ml浓度下，抑制率达到65.51％±1.73％。抑制率随提取物浓度的增加而增加，呈剂量依赖关系，与空白对照组比较，差异有统计学意义(p<0.01)(图1)。另外，经过不同浓度MRBE处理48h后的细胞贴壁性均减弱，形态由圆梭状变得皱缩，细胞碎片增多(图2)。
该试验结果说明MRBE抑制人肝癌细胞的增殖，且呈浓度依赖关系。
实施例4 PI流式细胞术分析细胞周期分布
(1)试验方法
取对数生长期HepG2细胞，以密度为1×105个/mL接种于6孔板内，每孔2mL。37℃、5％CO2培养箱中培养24h。换成含0.5％血清的DMEM，继续孵育12h，使细胞进入静止期，弃上清。实验分组：细胞正常对照组、200μg/mL MRBE组、400μg/mL MRBE组，培养48h后，收集培养的细胞于1500r/min离心10min，悬浮于4℃预冷的200μL PBS中，缓慢加入预冷的纯乙醇600μL(70％)，悬浮固定细胞，4℃过夜。检测前用PBS洗涤细胞2次。每一样本中加入100μLRNase，于37℃中孵育15min。再加入200μL碘化丙啶，室温下避光保存5min。利用流式细胞仪分析(美国BD公司，FACSCalibor流式细胞仪)细胞周期中各时期的细胞百分数，实验重复3次。
(2)试验结果
结果显示(图3和图4)，不同浓度MRBE(200μg/mL、400μg/mL)作用48h均影响HepG2细胞周期各时相的分布。当提取物浓度为200μg/mL，G0/G1期细胞比率(42.84％±1.16％)与对照组(45.65％±1.15％)无显著差异；S期细胞比率(50.03％±1.73％)极显著高于对照组(12.13％±0.58％)；G2/M期细胞比率(7.12％±0.35％)极显著低于对照组(42.22％±1.15％)。当提取物浓度为400μg/mL，G0/G1期细胞比率(39.36％±1.15％)显著低于对照组(45.65％±1.15％)；S期细胞比率(59.73％±1.73％)极显著高于对照组(12.13％±0.58％)；G2/M期细胞比率(0.94％±0.06％)极显著低于对照组(42.22％±1.15％)。
哺乳动物细胞周期按顺序分为五个阶段(G0期、G1期、S期、G2期、M期)，其正常运行是在一系列称为限制点(checkpoint)的严格检控下进行的，分为G1/S限制点、S期限制点、G2/M限制点、中后期检验点(纺锤体组装限制点)。只有前一个阶段完全完成，后一个阶段才能正常开始。上述试验结果中与对照组相比，不同浓度MRBE(200μg/mL、400μg/mL)组S期细胞比率均及显著增高，说明MRBE促使HepG2细胞停滞在细胞周期的S期。
实施例5 Annexin V-PI双染流式细胞术检测细胞凋亡
(1)试验方法
取对数生长期HepG2细胞，以密度为1×105个/mL接种于6孔板内，每孔2mL。37℃、5％CO2培养箱中培养24h。换成含0.5％血清的DMEM，37℃继续孵育12h，使细胞进入静止期，弃上清。实验分组：细胞正常对照组、200μg/mL MRBE组、400μg/mL MRBE组，培养48h后，收集培养的细胞。细胞染色：加入500μL Binding Buffer悬浮细胞，加入5μLAnnexin V/FITC混匀后，加入5μL Propidium Iodide，混匀。室温避光反应15min，流式细胞仪上机分析。流式细胞仪检测，采用15mW氩离子激光，激发光波长为488nm，发射波长为530nm，每样品用CellQuest软件自动获取5×103个细胞，计算Annexin V-FITC及PI双染阳性细胞百分含量，试验重复3次。
(2)试验结果
流式细胞分析仪检测结果显示(图5和图6)，浓度为200μg/mL和400μg/mL的MRBE作用48h，均可显著促进Hepg2细胞凋亡：早期凋亡率200μg/mL MRBE组(47.52％±2.31％)、400μg/mL MRBE组(46.83％±1.73％)均极显著高于对照组(3.46％±0.12％)；早期凋亡率200μg/mL MRBE组(23.56％±0.88％)、400μg/mL MRBE组(23.56％±0.88％)均极显著高于对照组(3.90％±0.12％)；坏死细胞200μg/mL MRBE组(2.36％±0.12％)、400μg/mL MRBE组(4.67％±0.29％)均显极著高于对照组(0.83％±0.06％)。
实施例6实时荧光定量PCR实验检测目的基因mRNA的转录水平
(1)试验方法
实验分组如下：细胞正常对照组、400μg/mL MRBE组。培养6h和48h后收集各组细胞。细胞用预冷的PBS清洗后，按照RNA提取试剂盒(日本TaKaRa公司)说明书提取总RNA，然后用反转录试剂盒(日本TaKaRa公司)反转录合成cDNA。采用SYBER Premix Ex Taq试剂盒(RR420A，Takara)在Real-time PCR仪器(StepOnePlus，ABI公司)上进行扩增。总反应体积为20μl，反应体系各试剂的量按照试剂盒说明书加入。扩增体系为95℃预热30s，40个循环的95℃反应5s和60℃反应30s，内参基因为β-actin。每个样品重复测三次。测定结果用ΔΔCt法计算得的基因表达的相对倍数变化表示。
(2)试验结果
①如图7所示，与对照组相比，400μg/mL MRBE作用HepG2细胞48h后：TGF-β(转化生长因子-β)在转录水平上无显著差异；Smads基因家族(转录激活因子)中的Smad4在转录水平显著下调；p21在转录水平上显著上调，p53在转录水平显著下调，p27在转录水平上无显著影响；CDK1、CDK2、CDK3、CDK5、CDK7在转录水平显著下调，CDK4、CDK6在转录水平无显著差异；细胞周期蛋白CyclinA、CyclinB、CyclinC、CyclinE在转录水平显著下调，CyclinD在转录水平无显著影响；E2F基因在转录水平上显著下调；Rb和DP1在转录水平上无显著性差异。
细胞周期是细胞复制的基础过程，细胞周期中各时期受到多种基因协同调控。本试验涉及了转化生长因子β(transforming growth factor，TGF-β)家族、Smads家族、细胞周期蛋白依赖性激酶(cyclin-dependent kinases，CDK)、细胞周期蛋白依赖性激酶抑制剂(CKI)、细胞周期蛋白(Cyclin)、E2F家族、DP家族等细胞周期相关基因。TGF-β家族的成员控制着细胞增殖、凋亡、分化和迁移等功能，TGF-β信号参与了从最初肝损伤到HCC的所有疾病进展阶段。Smad蛋白是TGF-β家族信号的主要细胞内介体。TGF-β先磷酸化下游介质Smad2和Smad3，磷酸化的Smad2和Smad3与Smad4结合进入细胞核。一旦位于细胞核中，Smad复合物直接与DNA相互作用，或与其他DNA结合蛋白复合，并控制靶基因的转录。p21、p27是一种细胞周期蛋白依赖性激酶(CDK)抑制剂，属于CIP/Kip家族，对细胞周期单独或者协同起到负调控作用。p53是一种肿瘤抑制因子，当细胞遭受压力并发生不受控制的分裂和增殖时p53被激活，诱导下游靶基因p21表达，导致细胞周期停滞。p53功能的失活是癌细胞用来逃避凋亡的方法之一。p21在多种癌症中被下调，通过直接与G1/S转换相关的激酶结合在多个细胞过程中发挥关键作用。就哺乳动物而言，在一般细胞周期调节中，CDK4和CDK6与CycD形成激酶复合体使Rb家族蛋白Rb磷酸化促进细胞从G0期进展到G1期，同时使E2F1、CycA、CycE从Rb蛋白抑制中释放得以转录，之后CDK2通过与CycA和CycE形成激酶复合物促进细胞从G1期进展到S期，CDK1与CycA和CycB形成激酶复合物促进细胞从S期进展到M期。p27与p21一样可通过对Cyclin/CDK复合物活性的抑制诱导基因表达的变化，当p27表达水平增加2～3倍可完全抑制Cyclin/CDK复合物，p21也可直接与E2F1结合并抑制其转录活性，独立于CDK2或Rb发挥作用。
上述试验结果中MRBE在转录水平上显著上调抑癌基因p21的表达，同时显著下调CyclinD和CDK6的表达，因此MRBE可通过p21-CyclinD/CDK6信号通路使HepG2细胞在S期停滞。
②如图8所示，与对照组相比，400μg/mL MRBE作用HepG2细胞48h后：抑凋亡蛋白基因Bcl-2、Bcl-xl在转录水平上显著下调，Bcl-w在转录水平上无显著影响；促凋亡蛋白基因Bad、Bid在转录水平上显著下调，Bik、Bax、Bim、Bak在转录水平上无显著影响；Ras和MEK1在转录水平上显著上调，Raf在转录水平上显著下调，ERK1在转录水平上极显著下调。
细胞凋亡是指为维持内环境的稳定,由基因调控的细胞自主、有序的死亡,又称程序性细胞死亡，其过程涉及一系列基因的激活、表达以及调控等。本试验涉及了Bcl-2家族和Ras/Raf/MEK/ERK信号通路相关基因。主要细胞凋亡途径包括外源性(死亡受体介导凋亡)信号通路、内源性(细胞线粒体介导凋亡)信号通路、穿孔素/颗粒酶介导信号通路。细胞线粒体介导凋亡的关键在于线粒体外膜透化(MOMP)过程，即在线粒体膜上形成允许细胞色素C通过的通道，使细胞色素C从线粒体内部释放到胞质。该过程主要受Bcl-2家族调控，根据Bcl-2家族各成员细胞凋亡中的功能和它们拥有的Bcl-2同源域(BH)的数量分为三组，第1组即为具有抗凋亡效应的Bcl-w、Bcl-xl和Bcl-2蛋白等，通过抑制促凋亡蛋白阻碍细胞凋亡，第2组即促凋亡的Bax和Bak蛋白分子，被激活后结合到线粒体外膜并在线粒体外膜上形成圆形孔道引发一系列细胞凋亡反应，第3组即促凋亡的BH3-only蛋白，只含有1个BH3同源区域，包括Bik、Bid、Bim、Bad等，可直接结合并激活Bax或Bak，促进MOMP进程，同时抑制第1组Bcl-2蛋白的作用，最终促进细胞凋亡的发生。Ras/Raf/MEK/ERK信号通路的异常激活在HCC细胞增殖、分化、存活和凋亡中起主要作用。Hoffmann等人证明Ras、MEK、ERK和的mRNA分别在33％、40％、50％和50％的HCC患者中过表达。
上述试验结果中MRBE对Raf、MEK1、ERK1、Bcl-2蛋白的表达有明显下调作用，因此MRBE癌细胞通过Raf-MEK1-ERK1信号通路和Bcl-2的共同作用促使HepG2细胞凋亡。Bcl-2在HCC进展中充当促肿瘤因子，癌细胞通过上调抗凋亡Bcl-2家族成员从而逃避凋亡。
上述实施例仅用于解释和说明本发明的技术思想及特点，不能以此限定本发明的保护范围，本发明还可以有许多变形，凡是本领域的普通技术人员能从本发明公开的内容直接导出或联想到的所有变形，均应认为是本发明的保护范围。
Claims (9)
1.杨梅枝条醇提物在制备抗肝癌药物方面的应用。
2.根据权利要求1所述的杨梅枝条醇提物在制备抗肝癌药物方面的应用，其特征在于，所述杨梅枝条醇提物的制备过程包括以下步骤：
(1)取杨梅枝条，去除叶片；
(2)60℃烘干，粉碎得样品粉末；
(3)用70％乙醇超声提取步骤(2)中的样品粉末，过滤得提取液，重复提取4次，合并滤液；旋蒸去除提取液中乙醇，剩余部分冷冻干燥得杨梅枝条醇提物。
3.根据权利要求1所述的杨梅枝条醇提物在制备抗肝癌药物方面的应用，其特征在于，所述的杨梅枝条包括杨梅修剪产生的废弃枝条。
4.根据权利要求1所述的杨梅枝条醇提物在制备抗肝癌药物方面的应用，其特征在于，所述杨梅枝条醇提物将HepG2细胞阻滞在细胞周期的S期。
5.根据权利要求4所述的杨梅枝条醇提物在制备抗肝癌药物方面的应用，其特征在于，所述杨梅枝条醇提物上调HepG2细胞p21基因表达，下调CyclinD和CDK6基因表达，通过p21-CyclinD/CDK6信号通路将HepG2细胞阻滞在细胞周期的S期。
6.根据权利要求1所述的杨梅枝条醇提物在制备抗肝癌药物方面的应用，其特征在于，所述杨梅枝条醇提物促使HepG2细胞凋亡。
7.根据权利要求6所述的杨梅枝条醇提物在制备抗肝癌药物方面的应用，其特征在于，所述杨梅枝条醇提物下调HepG2细胞Raf、MEK1、ERK1、Bcl-2基因表达，通过Raf-MEK1-ERK1信号通路和Bcl-2促使HepG2细胞凋亡。
8.根据权利要求1所述的杨梅枝条醇提物在制备抗肝癌药物中的用途，其特征在于，所述杨梅枝条醇提物中含有没食子酸，杨梅苷、没食子儿茶素、2,5-二羟基苯甲酸、表没食子儿茶素没食子酸酯。
9.根据权利1所述的杨梅枝条醇提物在制备抗肝癌药物方面的应用，其特征在于，所述肝癌为肝细胞性肝癌。
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| CN102040576A (zh) * | 2010-09-27 | 2011-05-04 | 南京泽朗医药科技有限公司 | 一种从杨梅枝叶中提取杨梅素的方法 |
| CN103099291A (zh) * | 2011-11-15 | 2013-05-15 | 浙江海洋学院 | 一种以杨梅叶为原料的天然防腐剂的制备方法 |
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| CN102040576A (zh) * | 2010-09-27 | 2011-05-04 | 南京泽朗医药科技有限公司 | 一种从杨梅枝叶中提取杨梅素的方法 |
| CN103099291A (zh) * | 2011-11-15 | 2013-05-15 | 浙江海洋学院 | 一种以杨梅叶为原料的天然防腐剂的制备方法 |
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