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Article(id=1212693249723121696, tenantId=1146029695717560320, journalId=1190317699101192196, issueId=1212693246539649865, articleNumber=1001-2494(2024)19-1843-10, orderNo=null, doi=10.11669/cpj.2024.19.008, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1681056000000, receivedDateStr=2023-04-10, revisedDate=null, revisedDateStr=null, acceptedDate=null, acceptedDateStr=null, onlineDate=1767058201484, onlineDateStr=2025-12-30, pubDate=1728316800000, pubDateStr=2024-10-08, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1767058201484, onlineIssueDateStr=2025-12-30, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1767058201484, creator=13701087609, updateTime=1767058201484, updator=13701087609, issue=Issue{id=1212693246539649865, tenantId=1146029695717560320, journalId=1190317699101192196, year='2024', volume='59', issue='19', pageStart='1781', pageEnd='1880', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1767058200723, creator=13701087609, updateTime=1767059042003, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1212696775207600634, tenantId=1146029695717560320, journalId=1190317699101192196, issueId=1212693246539649865, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1212696775207600635, tenantId=1146029695717560320, journalId=1190317699101192196, issueId=1212693246539649865, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=1843, endPage=1852, ext={EN=ArticleExt(id=1212693250092220459, articleId=1212693249723121696, tenantId=1146029695717560320, journalId=1190317699101192196, language=EN, title=Optimization of Preparation Process and Quality Evaluation of Isovaleryl Shikonin Liposomes, columnId=null, journalTitle=Chinese Pharmaceutical Journal, columnName=null, runingTitle=null, highlight=null, articleAbstract=

OBJECTIVE To prepare liposome formulations encapsulating isovaleryl shikonin, to optimise the preparation process. METHODS The isovalerylshikonin-liposome (IsoSHK-lip) were prepared by the thin film dispersion method. The UV absorption, standard curve, precision, stability and recovery of IsoSHK-lip were investigated. A response surface optimization method was used to optimize a 3-factor, 3-level preparation scheme with A: lecithin-cholesterol mass ratio, B: lecithin-isovalerylshikonin mass ratio and C: volume of hydrated solvent as the three factors. The particle size, polymer dispersity index (PDI), Zeta potential, morphological characterisation and stability of IsoSHK-lip were also investigated for the optimal solution. RESULTS The response surface optimization predicted that the optimal preparation conditions for IsoSHK-lip were: lecithin-cholesterol mass ratio of 8.82∶1, lecithin-isovalerylshikonin mass ratio of 30.65∶1, and volume of hydrated solvent of 29.22 mL. Repeated preparation of the optimal IsoSHK-lip resulted in an average encapsulation rate of 90.03%, a mean particle size of 117.48 nm, a mean PDI of 0.246, and a mean Zeta potential of -13.59 mV. The stability experiments showed that the particle size, PDI and Zeta potential of IsoSHK-lip did not change significantly after 7 d at 4 ℃. Transmission electron microscopy showed that the IsoSHK-lip was subspherical with particle sizes in the range of 100-200 nm. CONCLUSION IsoSHK-lip is prepared by repeating the optimal results obtained by response surface methodology three times, resulting in a near spherical shape, smaller particle size, uniform particle size distribution and better stability of IsoSHK-lip, which provides the basis for subsequent pharmacological studies of dosage forms.

, correspAuthors=Dongmei QIN, authorNote=null, correspAuthorsNote=null, copyrightStatement=null, copyrightOwner=null, extLink=null, articleAbsUrl=null, sourceXml=null, magXml=null, pdfUrl=null, pdf=null, pdfFileSize=null, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=null, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=null, mapNumber=null, authorCompany=null, fund=null, authors=null, authorsList=Weijun CHEN, Dongmei QIN, Lingyu MENG, Yuefeng GAO), CN=ArticleExt(id=1212693252835295422, articleId=1212693249723121696, tenantId=1146029695717560320, journalId=1190317699101192196, language=CN, title=异戊酰紫草素脂质体的制备工艺优化及其质量评价, columnId=1190352405612040510, journalTitle=中国药学杂志, columnName=论著, runingTitle=null, highlight=null, articleAbstract=

目的 制备包载异戊酰紫草素的脂质体制剂,对其制备工艺进行优化,并研究其理化性质,为后期药理学研究提供基础。方法 采用薄膜分散法制备异戊酰紫草素脂质体(IsoSHK-lip),对异戊酰紫草素进行精密度、稳定性、回收率的考察并绘制标准曲线,采用响应面优化法,以卵磷脂-胆固醇质量比(A):卵磷脂-异戊酰紫草素质量比以及(B):水和介质体积(C)为三个因素,进行三因素三水平的制备方案优化,并对最优方案制得的IsoSHK-lip进行粒径、多分散系数(PDI)、Zeta电位、形态表征以及稳定性考察。结果 响应面优化预测IsoSHK-lip最优制备条件为:卵磷脂-胆固醇质量比为8.82∶1,卵磷脂-异戊酰紫草素质量比为30.65∶1,水和介质体积为29.22 mL。重复制备最优结果的IsoSHK-lip得到平均包封率为90.03%,粒径均值为117.48 nm,PDI均值为0.246,Zeta电位均值为-13.59 mV。并且稳定性实验表明,4 ℃放置7 d,IsoSHK-lip粒径、PDI、Zeta电位没有明显变化。透射电镜结果表明脂质体为近球形,粒径在100~200 nm之间。结论 IsoSHK-lip采用响应面法优化得到的最优结果重复制备3次后,得到了近球形、粒径较小、粒径分布均匀、稳定性较好的IsoSHK-lip,为后续的药理学研究提供了剂型研究基础。

, correspAuthors=秦冬梅, authorNote=null, correspAuthorsNote=
* 秦冬梅,女,博士,教授 研究方向:中药民族药分离及其药效作用机制研究
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陈卫军,男,硕士,副主任药师 研究方向:中药民族药对代谢性疾病机制及新药研究

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陈卫军,男,硕士,副主任药师 研究方向:中药民族药对代谢性疾病机制及新药研究

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陈卫军,男,硕士,副主任药师 研究方向:中药民族药对代谢性疾病机制及新药研究

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Chin Pharm J(中国药学杂志), 2022, 57(17): 1438-1446., articleTitle=Preparation of mitochondrial targeting curcumin TPP-PEG-PLGA iposomes and study on promoting hepatoma cell apoptosis, refAbstract=null)], funds=[Fund(id=1212795882714616364, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1212693249723121696, awardId=81860730, language=CN, fundingSource=国家自然科学基金项目资助(81860730), fundOrder=null, country=null)], companyList=[AuthorCompany(id=1212795876913893714, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1212693249723121696, xref=1, ext=[AuthorCompanyExt(id=1212795876926476628, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1212693249723121696, companyId=1212795876913893714, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=1 MOE Key Laboratory of Xinjiang Phytomedicine Resource and Utilization, School of Pharmacy, Shihezi University, Shihezi 832002, China), 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新疆医科大学第七附属医院药学部, 乌鲁木齐 830028)]), AuthorCompany(id=1212795877228466532, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1212693249723121696, xref=4, ext=[AuthorCompanyExt(id=1212795877236855140, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1212693249723121696, companyId=1212795877228466532, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=4 College of Applied Engineering, Henan University of Science and Technology, Sanmenxia 472000, China), AuthorCompanyExt(id=1212795877245243749, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1212693249723121696, companyId=1212795877228466532, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=4 河南科技大学应用工程学院, 河南 三门峡 472000)])], figs=[ArticleFig(id=1212795879862489548, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1212693249723121696, language=EN, label=Fig.1, caption=Comparison of full-wavelength scanning of IsoSHK, blank-lip and IsoSHK-lip, figureFileSmall=WEyEwtingljvi0QcSwl3wA==, figureFileBig=zKJzyvwQgd4+09nLT12aQA==, tableContent=null), ArticleFig(id=1212795879975735760, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1212693249723121696, language=CN, label=图1, caption=异戊酰紫草素(IsoSHK)、空白脂质体、异戊酰紫草素脂质体(IsoSHK-lip)全波长扫描对比图, figureFileSmall=WEyEwtingljvi0QcSwl3wA==, figureFileBig=zKJzyvwQgd4+09nLT12aQA==, tableContent=null), ArticleFig(id=1212795880235782616, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1212693249723121696, language=EN, label=Fig.2, caption=Effects of lecithin-cholesterol mass ratio on the encapsulation efficiency. n=3,$\stackrel{-}{x}$±s, figureFileSmall=22kxIosFv68x22F5EfA4OQ==, figureFileBig=/kyK36g2mrarQILEB9ginA==, tableContent=null), ArticleFig(id=1212795880315474397, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1212693249723121696, language=CN, label=图2, caption=卵磷脂-胆固醇(EPC-Chol)质量比对IsoSHK-lip包封率的影响。n=3,$\stackrel{-}{x}$±s, figureFileSmall=22kxIosFv68x22F5EfA4OQ==, figureFileBig=/kyK36g2mrarQILEB9ginA==, tableContent=null), ArticleFig(id=1212795880403554785, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1212693249723121696, language=EN, label=Fig.3, caption=Effects of isovalerylshikon lecithin mass ratio on the encapsulation efficiency of IsoSHK-lip.n=3,$\stackrel{-}{x}$±s, figureFileSmall=GmNkADnObZSA9h5bV/vQAQ==, figureFileBig=jOn61m5g38pmRjutlDd6Kg==, tableContent=null), ArticleFig(id=1212795880491635173, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1212693249723121696, language=CN, label=图3, caption=异戊酰紫草素-卵磷脂(IsoSHK-EPC)质量比对IsoSHK-lip包封率的影响。n=3,$\stackrel{-}{x}$±s, figureFileSmall=GmNkADnObZSA9h5bV/vQAQ==, figureFileBig=jOn61m5g38pmRjutlDd6Kg==, tableContent=null), ArticleFig(id=1212795880567132649, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1212693249723121696, language=EN, label=Fig.4, caption=Effects of the volume of hydrated solvent on the encapsulation efficiency of IsoSHK-lip. n=3,$\stackrel{-}{x}$±s, figureFileSmall=0yNLW6XqDJLavu/aMhzuPA==, figureFileBig=kLNbi0vBJweEota/APyDXw==, tableContent=null), ArticleFig(id=1212795880688767469, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1212693249723121696, language=CN, label=图4, caption=水合溶剂体积对IsoSHK-lip包封率的影响。n=3,$\stackrel{-}{x}$±s, figureFileSmall=0yNLW6XqDJLavu/aMhzuPA==, figureFileBig=kLNbi0vBJweEota/APyDXw==, tableContent=null), ArticleFig(id=1212795880755876334, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1212693249723121696, language=EN, label=Fig.5, caption=Contour map and 3D surface map of the effect of A and B on the encapsulation efficiency ratio (Y) of IsoSHK-lip, figureFileSmall=ZgvQdIpbD2V574OmXEXfUQ==, figureFileBig=BEx4TFQUNS6bUgwGBKsn1w==, tableContent=null), ArticleFig(id=1212795880852345328, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1212693249723121696, language=CN, label=图5, caption=EPC-Chol质量比(A)与EPC-IsoSHK质量比(B)对IsoSHK-lip包封率(Y)的等高线图和三维效应面图, figureFileSmall=ZgvQdIpbD2V574OmXEXfUQ==, figureFileBig=BEx4TFQUNS6bUgwGBKsn1w==, tableContent=null), ArticleFig(id=1212795880940425717, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1212693249723121696, language=EN, label=Fig.6, caption=Contour map and 3D surface map of the effect of A and C on the encapsulation efficiency ratio (Y) of IsoSHK-lip, figureFileSmall=kScO6YmwEWkhNw9CxL0Ypw==, figureFileBig=iEG8zjT3r6cWIpeZSmIxUQ==, tableContent=null), ArticleFig(id=1212795881041089016, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1212693249723121696, language=CN, label=图6, caption=EPC-Chol质量比(A)与HS体积(C)对IsoSHK-lip包封率(Y)的等高线图和三维效应面图, figureFileSmall=kScO6YmwEWkhNw9CxL0Ypw==, figureFileBig=iEG8zjT3r6cWIpeZSmIxUQ==, tableContent=null), ArticleFig(id=1212795881137558010, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1212693249723121696, language=EN, label=Fig.7, caption=Contour map and 3D surface map of the effect of B and C on the encapsulation efficiency ratio (Y) of IsoSHK-lip, figureFileSmall=Ygpl5j95cIMNHp4vlXtoOA==, figureFileBig=L6aNV3PBSuulUQm5dbSHHw==, tableContent=null), ArticleFig(id=1212795881204666877, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1212693249723121696, language=CN, label=图7, caption=EPC-IsoSHK质量比(B)与HS体积(C)对IsoSHK-lip包封率(Y)的等高线图和三维效应面图, figureFileSmall=Ygpl5j95cIMNHp4vlXtoOA==, figureFileBig=L6aNV3PBSuulUQm5dbSHHw==, tableContent=null), ArticleFig(id=1212795881271775744, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1212693249723121696, language=EN, label=Fig.8, caption=Electron photomicrograph of IsoSHK-lip

A-IsoSHK-lip morphology diagram(×10 000); B-IsoSHK-lip internal structure diagram(×50 000).

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A-IsoSHK-lip形态图(×10 000);B-IsoSHK-lip内部结构图(×50 000)。

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Stability assay of isovalerylshikonin

, figureFileSmall=null, figureFileBig=null, tableContent=
Parameters 0 h 2 h 4 h 6 h 8 h 10 h 12 h
A 0.673 0.670 0.667 0.668 0.670 0.674 0.669
ρ/μg·mL-1 42.64 42.45 42.26 42.33 42.43 42.71 42.39
ρ(Mean)/μg·mL-1 42.46
RSD/% 0.38
), ArticleFig(id=1212795881561182727, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1212693249723121696, language=CN, label=表1, caption=

IsoSHK稳定性检测实验

, figureFileSmall=null, figureFileBig=null, tableContent=
Parameters 0 h 2 h 4 h 6 h 8 h 10 h 12 h
A 0.673 0.670 0.667 0.668 0.670 0.674 0.669
ρ/μg·mL-1 42.64 42.45 42.26 42.33 42.43 42.71 42.39
ρ(Mean)/μg·mL-1 42.46
RSD/% 0.38
), ArticleFig(id=1212795881653457417, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1212693249723121696, language=EN, label=Tab.2, caption=

Precision assay of isovalerylshikonin

, figureFileSmall=null, figureFileBig=null, tableContent=
Parameters 1 2 3 4 5 6
A 0.134 0.135 0.136 0.137 0.135 0.138
ρ/μg·mL-1 8.34 8.40 8.46 8.53 8.38 8.59
ρ(Mean)/μg·mL-1 8.45
RSD/% 1.14
), ArticleFig(id=1212795881749926411, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1212693249723121696, language=CN, label=表2, caption=

IsoSHK精密度检测实验

, figureFileSmall=null, figureFileBig=null, tableContent=
Parameters 1 2 3 4 5 6
A 0.134 0.135 0.136 0.137 0.135 0.138
ρ/μg·mL-1 8.34 8.40 8.46 8.53 8.38 8.59
ρ(Mean)/μg·mL-1 8.45
RSD/% 1.14
), ArticleFig(id=1212795881825423885, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1212693249723121696, language=EN, label=Tab.3, caption=

Recovery assay for isovalerylshikonin

, figureFileSmall=null, figureFileBig=null, tableContent=
Sample
No.		 m(IsoSHK)
/μg		 m(Added)
/μg		 Abs ρ
/μg·mL-1		 ρ(Measured quantity)
/μg·4 mL-1		 Recovery
/%		 Mean recovery
/%		 RSD
/%
1 48 48 0.393 24.82 99.29 106.84 108.40 1.70
2 48 48 0.395 24.95 99.79 107.91
3 48 48 0.404 25.52 102.09 112.68
4 48 64 0.463 29.28 117.11 107.98
5 48 64 0.461 29.15 116.60 107.19
6 48 64 0.467 29.53 118.13 109.57
7 48 80 0.528 33.41 133.66 107.07
8 48 80 0.529 33.48 133.91 107.39
9 48 80 0.534 33.80 135.19 108.98
), ArticleFig(id=1212795881900921360, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1212693249723121696, language=CN, label=表3, caption=

IsoSHK回收率检测实验

, figureFileSmall=null, figureFileBig=null, tableContent=
Sample
No.		 m(IsoSHK)
/μg		 m(Added)
/μg		 Abs ρ
/μg·mL-1		 ρ(Measured quantity)
/μg·4 mL-1		 Recovery
/%		 Mean recovery
/%		 RSD
/%
1 48 48 0.393 24.82 99.29 106.84 108.40 1.70
2 48 48 0.395 24.95 99.79 107.91
3 48 48 0.404 25.52 102.09 112.68
4 48 64 0.463 29.28 117.11 107.98
5 48 64 0.461 29.15 116.60 107.19
6 48 64 0.467 29.53 118.13 109.57
7 48 80 0.528 33.41 133.66 107.07
8 48 80 0.529 33.48 133.91 107.39
9 48 80 0.534 33.80 135.19 108.98
), ArticleFig(id=1212795881972224531, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1212693249723121696, language=EN, label=Tab.4, caption=

Response surface design experimental factor level and coding

, figureFileSmall=null, figureFileBig=null, tableContent=
Factors Levels
-1 0 1
Lecithin-cholesterol mass ratio(A) 8∶1 9∶1 10∶1
Lecithin-isovalerylshikon in mass ratio(B) 3∶80 3∶90 3∶100
Volume of hydrated solvent/mL(C) 20 30 40
), ArticleFig(id=1212795882043527702, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1212693249723121696, language=CN, label=表4, caption=

响应曲面设计实验因素水平和编码

, figureFileSmall=null, figureFileBig=null, tableContent=
Factors Levels
-1 0 1
Lecithin-cholesterol mass ratio(A) 8∶1 9∶1 10∶1
Lecithin-isovalerylshikon in mass ratio(B) 3∶80 3∶90 3∶100
Volume of hydrated solvent/mL(C) 20 30 40
), ArticleFig(id=1212795882127413785, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1212693249723121696, language=EN, label=Tab.5, caption=

Box-Benhnken experimental design scheme and experimental results

, figureFileSmall=null, figureFileBig=null, tableContent=
No. Factors Encapsulation
efficiency(Y)/%		 Particle
size/nm		 PDI
index		 Zeta potential
/mV
EPC-Chol mass ratio(A) IsoSHK-EPC mass ratio(B) Volume of hydrated solvent(C)/mL
1 9∶1 80∶3 40 81.99 113.7 0.220 -12.63
2 9∶1 80∶3 20 83.85 113.8 0.246 -12.33
3 10∶1 80∶3 30 84.06 112.2 0.238 -11.74
4 8∶1 100∶3 30 87.69 113.4 0.225 -11.28
5 9∶1 30∶1 30 89.22 115.9 0.236 -11.85
6 9∶1 30∶1 30 89.03 118.6 0.237 -12.14
7 9∶1 100∶3 20 84.04 113.4 0.229 -12.24
8 10∶1 30∶1 20 84.36 114.8 0.235 -12.65
9 9∶1 30∶1 30 88.91 115.3 0.225 -12.74
10 10∶1 100∶3 30 81.23 124.6 0.267 -12.66
11 10∶1 30∶1 40 82.66 111.4 0.237 -13.00
12 9∶1 30∶1 30 90.04 113.4 0.229 -11.96
13 9∶1 100∶3 40 84.95 113.4 0.231 -12.32
14 9∶1 30∶1 30 90.60 115.1 0.229 -11.28
15 8∶1 30∶1 40 83.06 117.5 0.240 -12.36
16 8∶1 30∶1 20 84.98 107.9 0.241 -12.08
17 8∶1 80∶3 30 81.35 115.5 0.231 -11.86
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Box-Benhnken实验设计方案及实验结果

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No. Factors Encapsulation
efficiency(Y)/%		 Particle
size/nm		 PDI
index		 Zeta potential
/mV
EPC-Chol mass ratio(A) IsoSHK-EPC mass ratio(B) Volume of hydrated solvent(C)/mL
1 9∶1 80∶3 40 81.99 113.7 0.220 -12.63
2 9∶1 80∶3 20 83.85 113.8 0.246 -12.33
3 10∶1 80∶3 30 84.06 112.2 0.238 -11.74
4 8∶1 100∶3 30 87.69 113.4 0.225 -11.28
5 9∶1 30∶1 30 89.22 115.9 0.236 -11.85
6 9∶1 30∶1 30 89.03 118.6 0.237 -12.14
7 9∶1 100∶3 20 84.04 113.4 0.229 -12.24
8 10∶1 30∶1 20 84.36 114.8 0.235 -12.65
9 9∶1 30∶1 30 88.91 115.3 0.225 -12.74
10 10∶1 100∶3 30 81.23 124.6 0.267 -12.66
11 10∶1 30∶1 40 82.66 111.4 0.237 -13.00
12 9∶1 30∶1 30 90.04 113.4 0.229 -11.96
13 9∶1 100∶3 40 84.95 113.4 0.231 -12.32
14 9∶1 30∶1 30 90.60 115.1 0.229 -11.28
15 8∶1 30∶1 40 83.06 117.5 0.240 -12.36
16 8∶1 30∶1 20 84.98 107.9 0.241 -12.08
17 8∶1 80∶3 30 81.35 115.5 0.231 -11.86
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Response surface quadratic model regression and analysis of variance

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Source Sum of
squares		 Degree of
freedom		 Mean
square		 F P
Model 155.84 9 17.32 30.53 <0.000 12)
A 2.84 1 2.84 5.01 0.060 1
B 5.54 1 5.54 9.78 0.016 71)
C 2.61 1 2.61 4.60 0.069 1
AB 21.02 1 21.02 37.07 0.000 52)
AC 0.01 1 0.01 0.02 0.888 0
BC 1.92 1 1.92 3.38 0.108 5
A2 36.89 1 36.89 65.05 <0.000 12)
B2 38.34 1 38.34 67.60 <0.000 12)
C2 33.84 1 33.84 59.67 0.000 12)
Resdual 3.97 7 0.57
Lack of fit 1.84 3 0.61 1.15 0.430 9
Pure error 2.13 4 0.53
Cor total 159.81 16
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响应面二次模型回归和方差分析表

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Source Sum of
squares		 Degree of
freedom		 Mean
square		 F P
Model 155.84 9 17.32 30.53 <0.000 12)
A 2.84 1 2.84 5.01 0.060 1
B 5.54 1 5.54 9.78 0.016 71)
C 2.61 1 2.61 4.60 0.069 1
AB 21.02 1 21.02 37.07 0.000 52)
AC 0.01 1 0.01 0.02 0.888 0
BC 1.92 1 1.92 3.38 0.108 5
A2 36.89 1 36.89 65.05 <0.000 12)
B2 38.34 1 38.34 67.60 <0.000 12)
C2 33.84 1 33.84 59.67 0.000 12)
Resdual 3.97 7 0.57
Lack of fit 1.84 3 0.61 1.15 0.430 9
Pure error 2.13 4 0.53
Cor total 159.81 16
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Summary of stability results of different batches of IsoSHK-lip at 4 ℃ in 7 d

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No. Stable at 4 ℃ in 7 d
Sampling
time/d		 Particle
size/nm		 PDI
index		 Zeta potential
/mV
1 0 117.6 0.247 -13.6
118.7 0.234 -13.2
117.5 0.251 -13.9
7 118.7 0.242 -13.1
117.3 0.248 -13.0
117.9 0.248 -13.6
2 0 116.7 0.238 -13.9
117.6 0.255 -13.9
117.9 0.250 -13.3
7 117.9 0.249 -13.2
117.5 0.260 -13.0
118.6 0.252 -13.6
3 0 116.8 0.234 -13.6
116.5 0.257 -13.7
118.0 0.249 -13.2
7 117.9 0.238 -12.9
117.5 0.242 -13.3
117.2 0.254 -13.2
), ArticleFig(id=1212795882576204327, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1212693249723121696, language=CN, label=表7, caption=

不同批次IsoSHK-lip 7 d内4 ℃放置稳定性结果汇总表

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No. Stable at 4 ℃ in 7 d
Sampling
time/d		 Particle
size/nm		 PDI
index		 Zeta potential
/mV
1 0 117.6 0.247 -13.6
118.7 0.234 -13.2
117.5 0.251 -13.9
7 118.7 0.242 -13.1
117.3 0.248 -13.0
117.9 0.248 -13.6
2 0 116.7 0.238 -13.9
117.6 0.255 -13.9
117.9 0.250 -13.3
7 117.9 0.249 -13.2
117.5 0.260 -13.0
118.6 0.252 -13.6
3 0 116.8 0.234 -13.6
116.5 0.257 -13.7
118.0 0.249 -13.2
7 117.9 0.238 -12.9
117.5 0.242 -13.3
117.2 0.254 -13.2
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异戊酰紫草素脂质体的制备工艺优化及其质量评价
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陈卫军 2 , 秦冬梅 1, * , 孟凌宇 3 , 高月锋 4
中国药学杂志 | 论著 2024,59(19): 1843-1852
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中国药学杂志 | 论著 2024, 59(19): 1843-1852
异戊酰紫草素脂质体的制备工艺优化及其质量评价
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陈卫军2, 秦冬梅1, *, 孟凌宇3, 高月锋4
作者信息
  • 1 石河子大学药学院, 新疆植物药资源利用教育部重点实验室, 新疆 石河子 832002
  • 2 新疆第二医学院中医学院, 新疆 克拉玛依 834000
  • 3 新疆医科大学第七附属医院药学部, 乌鲁木齐 830028
  • 4 河南科技大学应用工程学院, 河南 三门峡 472000

    • 陈卫军,男,硕士,副主任药师 研究方向:中药民族药对代谢性疾病机制及新药研究

通讯作者:

* 秦冬梅,女,博士,教授 研究方向:中药民族药分离及其药效作用机制研究
Optimization of Preparation Process and Quality Evaluation of Isovaleryl Shikonin Liposomes
Weijun CHEN2, Dongmei QIN1, *, Lingyu MENG3, Yuefeng GAO4
Affiliations
  • 1 MOE Key Laboratory of Xinjiang Phytomedicine Resource and Utilization, School of Pharmacy, Shihezi University, Shihezi 832002, China
  • 2 School of Traditional Chinese Medicine, Xinjiang Second Medical Collage, Karamay 834000, China
  • 3 Department of Pharmacy, The Seventh Affiliated Hospital of Xinjiang Medical University, Urumuqi 830028, China
  • 4 College of Applied Engineering, Henan University of Science and Technology, Sanmenxia 472000, China
出版时间: 2024-10-08 doi: 10.11669/cpj.2024.19.008
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摘要
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目的 制备包载异戊酰紫草素的脂质体制剂,对其制备工艺进行优化,并研究其理化性质,为后期药理学研究提供基础。方法 采用薄膜分散法制备异戊酰紫草素脂质体(IsoSHK-lip),对异戊酰紫草素进行精密度、稳定性、回收率的考察并绘制标准曲线,采用响应面优化法,以卵磷脂-胆固醇质量比(A):卵磷脂-异戊酰紫草素质量比以及(B):水和介质体积(C)为三个因素,进行三因素三水平的制备方案优化,并对最优方案制得的IsoSHK-lip进行粒径、多分散系数(PDI)、Zeta电位、形态表征以及稳定性考察。结果 响应面优化预测IsoSHK-lip最优制备条件为:卵磷脂-胆固醇质量比为8.82∶1,卵磷脂-异戊酰紫草素质量比为30.65∶1,水和介质体积为29.22 mL。重复制备最优结果的IsoSHK-lip得到平均包封率为90.03%,粒径均值为117.48 nm,PDI均值为0.246,Zeta电位均值为-13.59 mV。并且稳定性实验表明,4 ℃放置7 d,IsoSHK-lip粒径、PDI、Zeta电位没有明显变化。透射电镜结果表明脂质体为近球形,粒径在100~200 nm之间。结论 IsoSHK-lip采用响应面法优化得到的最优结果重复制备3次后,得到了近球形、粒径较小、粒径分布均匀、稳定性较好的IsoSHK-lip,为后续的药理学研究提供了剂型研究基础。

异戊酰紫草素  /  脂质体  /  薄膜分散法  /  响应面优化法  /  包封率

OBJECTIVE To prepare liposome formulations encapsulating isovaleryl shikonin, to optimise the preparation process. METHODS The isovalerylshikonin-liposome (IsoSHK-lip) were prepared by the thin film dispersion method. The UV absorption, standard curve, precision, stability and recovery of IsoSHK-lip were investigated. A response surface optimization method was used to optimize a 3-factor, 3-level preparation scheme with A: lecithin-cholesterol mass ratio, B: lecithin-isovalerylshikonin mass ratio and C: volume of hydrated solvent as the three factors. The particle size, polymer dispersity index (PDI), Zeta potential, morphological characterisation and stability of IsoSHK-lip were also investigated for the optimal solution. RESULTS The response surface optimization predicted that the optimal preparation conditions for IsoSHK-lip were: lecithin-cholesterol mass ratio of 8.82∶1, lecithin-isovalerylshikonin mass ratio of 30.65∶1, and volume of hydrated solvent of 29.22 mL. Repeated preparation of the optimal IsoSHK-lip resulted in an average encapsulation rate of 90.03%, a mean particle size of 117.48 nm, a mean PDI of 0.246, and a mean Zeta potential of -13.59 mV. The stability experiments showed that the particle size, PDI and Zeta potential of IsoSHK-lip did not change significantly after 7 d at 4 ℃. Transmission electron microscopy showed that the IsoSHK-lip was subspherical with particle sizes in the range of 100-200 nm. CONCLUSION IsoSHK-lip is prepared by repeating the optimal results obtained by response surface methodology three times, resulting in a near spherical shape, smaller particle size, uniform particle size distribution and better stability of IsoSHK-lip, which provides the basis for subsequent pharmacological studies of dosage forms.

isovalerylshikonin  /  liposome  /  thin film dispersion  /  response surface optimization  /  encapsulation efficiency
陈卫军, 秦冬梅, 孟凌宇, 高月锋. 异戊酰紫草素脂质体的制备工艺优化及其质量评价. 中国药学杂志, 2024 , 59 (19) : 1843 -1852 . DOI: 10.11669/cpj.2024.19.008
Weijun CHEN, Dongmei QIN, Lingyu MENG, Yuefeng GAO. Optimization of Preparation Process and Quality Evaluation of Isovaleryl Shikonin Liposomes[J]. Chinese Pharmaceutical Journal, 2024 , 59 (19) : 1843 -1852 . DOI: 10.11669/cpj.2024.19.008
正文
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新疆紫草[Arnebia euchroma (Royle) Johnst.]又被称为新疆软紫草,是新疆地区的传统中药材。中医药将新疆软紫草用于治疗肿瘤、保肝抗炎、抗病毒及相关症状,并且其治疗病毒性肝炎、抗艾滋病病毒的效果显著[1-2]。新疆软紫草化学成分主要为脂溶性和水溶性,脂溶性以萘醌类化合物为主,水溶性化合物以酚酸类、黄酮类化合物以及多糖等为主[3]。大多数萘醌类化合物主要集中在紫草石油醚提取物中(紫草石油醚部位)[2],据本课题组前期研究表明[4],异戊酰紫草素(isovalerylshikonin,IsoSHK)为新疆软紫草的主要活性成分,但由于其存在水不溶性,不能缓释,以及生物利用率低等问题,因此,选取合适的药物载体包载异戊酰紫草素可以为后期的药理学研究提供研究基础。脂质体是由磷脂双分子层构成的囊泡,其亲水性头部基团朝向水性环境,脂溶性部分在脂质体内包载脂溶性药物[5],由于其独特的性质,包括生物相容性和生物降解性,降低包封药物毒性、脂质体膜的生物亲和性导致其可促进药物的吸收以及靶向病变部位及组织,被认为是一种非常先进的药物输送系统[6]。目前,脂质体作为抗癌药物的载体在临床应用广泛,脂质体在中药制剂的研发领域也越来越受人们关注。虽然脂质体一般被制备成注射剂来使用,但现在口服脂质体类制剂也被广泛研究,若能将中药活性成分与脂质体口服制剂结合到一起,不仅可以提高中药活性成分的稳定性,而且能实现药物靶向性,达到缓释、减毒的效果。
因此,基于课题组前期对于IsoSHK药理作用的研究基础[7],本研究将新疆软紫草主要活性成分IsoSHK包载于脂质体内部,并采用响应面优化法对异戊酰紫草素脂质体(isovalerylshikonin-liposome,IsoSHK-lip)的制备方案进行优化,并对IsoSHK-lip进行粒径、多分散系数(polymer dispersity index,PDI)、Zeta电位以及外观形态等表征实验,以期开发一种包载新疆软紫草脂溶性活性成分的制剂并后续开展新疆软紫草脂溶性活性成分的药理作用研究。
1 材料与仪器
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1.1 材料
IsoSHK,纯度≥98%,由本课题组前期提取分离制备得到,总量6.2 g。
1.2 试剂
蛋黄卵磷脂(批号:427D021)、胆固醇(批号:1170035)、磷钨酸负染色液(体积分数2%)(批号:20220620)均购自Solarbio公司;磷酸盐缓冲液(PBS) pH值7.2~7.4(1×)(批号:2021/12,北京博奥拓达科技有限公司);甲醇(批号:20200726)、氯仿(批号:20200726)均购自天津市风船化学试剂科技有限公司;普通碳支持膜(北京中镜科仪技术有限公司)。
1.3 仪器
旋转蒸发仪(上海爱朗仪器仪器有限公司,型号:EYELA N-1001);恒温水浴锅(上海爱朗仪器仪器有限公司,型号:EYELA SB-2000);循环水式真空泵(巩义市予华仪器有限责任公司,型号:SHZ-DⅢ);低温冷却液循环泵(郑州长城科工贸有限公司,型号:DLSB-5/20);数控超声波清洗器(昆山市超声仪器有限公司,型号:KQ-500DE);真空干燥仪(上海新苗医疗器械制造有限公司,型号:DZF-6050);电子天平(德国Sartorius公司,感量:0.01 mg,型号:BP211D);超纯水机(杭州永洁达净化科技有限公司,型号:UPWS-I-10T);激光粒度Zeta电位和绝对分子质量分析仪(英国马尔文仪器有限公司,型号:ZEN3600);紫外可见分光光度计(日本岛津公司,型号:UV-2600 22V CH);超声波细胞破碎仪(美国Sonics & Materials公司,型号:VCX130);恒温水浴振荡器(金坛市医疗仪器厂,型号:SHA-C);透射电子显微镜[日本日立公司,型号:HT7800(默认80 kV)]。
2 实验方法
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2.1 IsoSHK-lip的制备
IsoSHK-lip由薄膜分散法制备。首先精密称取一定比例蛋黄卵磷脂和胆固醇加入至50 mL甲醇和30 mL氯仿中溶解,再加入一定质量的IsoSHK,混匀转入圆底烧瓶中,溶解后在40 ℃下用旋转蒸发仪蒸干成蜂窝状膜,然后加入一定体积PBS(1×)溶液,40 ℃下使脂质膜旋转水合1 h得到IsoSHK-lip混悬液,用超声波细胞破碎仪探头在85%功率下冰浴超声破碎7.5 min 3次,0.45 和0.22 μm微孔滤膜过滤各3次,再超声1次、0.45和0.22 μm微孔滤膜过滤各3次,即得IsoSHK-lip。
2.2 IsoSHK-lip包封率的测定
脂质体的双分子层结构和细胞形态结构相似,常被报道为理想的药物载体。脂质体的包封率是评价脂质体质量的一个重要指标。
取IsoSHK-lip混悬液3 mL,与3 mL甲醇混匀后,经超声波细胞破碎仪超声溶解后静置10 min,使其破乳完全,将上述破乳完全溶液转移至离心管中,在4 ℃条件下12 000 r·min-1离心10 min,取上清液,以甲醇为空白,记录吸光度A,采用标准曲线计算IsoSHK浓度,进而可得出IsoSHK总含量。
另取3 mL IsoSHK-lip混悬液于离心管中,在5 500 r·min-1、4 ℃条件下离心10 min后取下层沉淀,重复操作3次,每次沉淀加2 mL甲醇溶解,以甲醇为空白,记录吸光度A,采用标准曲线计算IsoSHK浓度,进而计算游离的IsoSHK含量,然后根据公式1计算其包封率。
包封率(%)=(C总-C游)/C总×100%
公式1中,C总为IsoSHK总含量,C游为未包载进脂质体中游离的IsoSHK含量。
2.3 脂质体中IsoSHK的测定
2.3.1 检测波长的选择
准确称取IsoSHK对照品10 mg,置于10 mL量瓶中,加甲醇溶解并定容,配制成IsoSHK对照品溶液(1 mg·mL-1)。取适量IsoSHK-lip混悬液以及空白脂质体(Blank-liposome,Blank-lip)混悬液备用。精密移取IsoSHK-lip供试液、Blank-lip供试液以及IsoSHK对照品适量,用甲醇稀释,用紫外分光光度计在200~800 nm内扫描,得到IsoSHK、IsoSHK-lip以及Blank-lip最大吸收峰波长,将上述3个波长进行对比观察,判断脂质体材料是否对IsoSHK紫外测定存在影响。实验结果见图1,IsoSHK与IsoSHK-lip在518.5 nm波长处有最大吸收峰,而Blank-lip在此波长处无吸收。
2.3.2 IsoSHK标准曲线的绘制
准确吸取IsoSHK对照品溶液(1 mg·mL-1)2 mL于50 mL量瓶中,加甲醇定容,配制成IsoSHK储备液(40 μg·mL-1),于4 ℃冰箱保存备用。分别吸取IsoSHK储备液2、4、6、8、10 mL置10 mL量瓶中,加甲醇定容,摇匀,配制系列质量浓度为8、16、24、32、40 μg·mL-1对照品供试液,以甲醇为空白对照,在518.5 nm处测定吸光度A,并绘制标准曲线方程。IsoSHK的标准曲线为:Y=0.015 71X+0.003 056,r2=0.999 3(n=3),结果表明,IsoSHK在8~40 μg·mL-1内线性关系良好。
2.3.3 稳定性实验
精密吸取IsoSHK对照品供试液(40 μg·mL-1),分别在0、2、4、6、8、10、12 h用紫外分光光度计于518.5 nm进行测定,以甲醇为空白,测定并记录吸光度A,采用标准曲线计算相对标准偏差(relative standard deviation,RSD),确定对照品在12 h内的稳定性,结果见表1,表明IsoSHK对照品供试液稳定性良好。
2.3.4 精密度实验
精密吸取IsoSHK对照品供试液(8 μg·mL-1),以甲醇为空白,用紫外分光光度计重复测定6次,记录吸光度A,采用标准曲线计算RSD,结果见表2,表明IsoSHK对照品供试液精密度良好。
2.3.5 回收率实验
精密称取9份已知浓度对照品供试液(24 μg·mL-1)2 mL,每3份为一组,第一组中加2 mL 24 μg·mL-1 IsoSHK对照品溶液,第二组中加入2 mL 32 μg·mL-1 IsoSHK对照品溶液,第三组中加入2 mL 40 μg·mL-1 IsoSHK对照品溶液,以甲醇为空白,测定并记录吸光度A,采用标准曲线计算标品回收率及其RSD,实验结果见表3,结果表明,该标准曲线以及紫外测定IsoSHK含量的实验方法切实可行。
2.4 单因素实验考察
通过对比分析大量文献可知,脂胆比、药脂比、超声时间、水合介质类型及用量都对脂质体包封率有影响,基于单因素实验,本实验考察卵磷脂-胆固醇(lecithin-cholesterol,EPC-Chol)质量比(A)、卵磷脂-异戊酰紫草素(lecithin-isovalerylshikonin,EPC-IsoSHK)质量比(B)、水合介质(hydrated solvent,HS)体积(C)3个不同因素对包封率的影响。
2.4.1 不同EPC-Chol质量比单因素实验
精密称取90 mg EPC和不同质量Chol(10、13、18、30、90 mg)加入至50 mL甲醇和30 mL氯仿中溶解,随后置于圆底烧瓶中,最后加入3 mg IsoSHK,如“2.1”所述制备IsoSHK-lip,再按照“2.2”项下方法测量并计算其包封率(%)。
2.4.2 不同EPC-IsoSHK质量比单因素实验
精密称取90 mg EPC和10 mg Chol加入至50 mL甲醇和30 mL氯仿中溶解,随后置于圆底烧瓶中,最后将不同质量IsoSHK样品(1、3、5、7、9 mg)加入至圆底烧瓶,按“2.1”项下所述制备IsoSHK-lip,再按照“2.2”项下方法测量并计算其包封率(%)。
2.4.3 不同HS体积单因素实验
精密称取 90 mg EPC和 10 mg Chol至50 mL甲醇和30 mL氯仿中溶解,随后加入圆底烧瓶中,最后再加入3 mg IsoSHK,旋蒸成膜后加入不同体积的HS(10、20、30、40、50 mL),按“2.1”项下所述制备IsoSHK-lip,再按照“2.2”项下方法测量并计算其包封率(%)。
2.5 Box-Benhnken响应面法优化IsoSHK-lip制备工艺及验证实验
以单因素实验结果为依据,利用Design Expert 13.0软件设计了三因素三水平的响应面法实验,以EPC-Chol质量比(A)、EPC-IsoSHK质量比(B)、HS体积(C)为主要考察因素,分别用A、B、C来表示,以-1、0、1表示自变量低、中、高3个水平,以IsoSHK-lip的包封率(%)为响应值,共有17个实验点,响应面设计实验因素水平和编码表见表4。
由Box-Benhnken实验设计优化得到的最优IsoSHK-lip制备条件进行重复3次,验证软件分析所得条件的可靠性。
2.6 IsoSHK-lip平均粒径、PDI、Zeta电位的测定以及外观形态观察
取适量的IsoSHK-lip用PBS稀释后,用激光粒度Zeta电位和绝对分子质量分析仪测定平均粒径、PDI、Zeta电位,重复测定3次,求平均值。
在室温条件下,IsoSHK-lip采用负染法染色。取20 μL IsoSHK-lip混悬液于封口膜上,将碳支持膜正面放于IsoSHK-lip混悬液滴上,吸附脂质体混悬液5 min,之后将碳支持膜倒扣在滤纸上2 min吸干多余液体,稍干燥后在封口膜上滴加20 μL磷钨酸负染色液(2%),将碳支持膜正面扣在负染色液滴上,静置2 min后,将碳支持膜倒扣在滤纸上2 min吸干多余液体,放入1.5 mL EP管内保存,于透射电镜下观察并拍照。
2.7 IsoSHK-lip稳定性研究
将IsoSHK-lip封装于西林瓶中,于4 ℃进行保存,一周后取出,观察脂质体有无沉降,测定IsoSHK-lip粒径大小,PDI、Zeta电位,判断其稳定性。
2.8 数据处理与分析
“2.3.2”项下部分实验数据采用GraphPad Prism 9软件进行处理和分析,其他部分实验数据采用Origin 2018以及Excel 2019软件进行分析处理。
3 结果
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3.1 单因素实验对IsoSHK-lip包封率的影响
3.1.1 不同EPC-Chol质量比对IsoSHK-lip包封率的影响
如图2所示,当选取EPC-Chol质量比分别为1∶1、3∶1、5∶1、7∶1、9∶1制备脂质体时,IsoSHK-lip的包封率分别为81.29%、84.68%、88.05%、91.95%、93.79%。实验结果表明,当EPC-Chol质量比为9∶1时制备的脂质体包封率最大,综上所述,本实验选取EPC-Chol质量比为9∶1进行下一步单因素实验。
3.1.2 不同IsoSHK-EPC质量比对IsoSHK-lip包封率的影响
如图3所示,选取IsoSHK-EPC的比例为1∶90、1∶30、1∶18、7∶90、1∶9制备脂质体时,IsoSHK-lip的包封率分别为87.15%、92.65%、82.71%、64.29%、66.00%。实验结果表明,当IsoSHK-EPC质量比为1∶30时制备的脂质体包封率最大,综上所述,本实验选取IsoSHK-EPC质量比为1∶30进行下一步单因素实验。
3.1.3 不同HS体积对IsoSHK-lip包封率的影响
如图4所示,选取水合介质PBS体积为10、20、30、40、50 mL制备脂质体时,IsoSHK-lip的包封率分别为84.39%、88.10%、92.65%、91.48%、83.04%。实验结果表明,当水合介质PBS体积为30 mL时制备的脂质体包封率最大。综上所述,本实验选取水合介质体积30 mL进行下一步实验。
3.2 响应面法优化IsoSHK-lip制备工艺及验证实验
3.2.1 响应面实验结果
根据单因素实验结果,选取EPC-Chol质量比9∶1(A)、EPC-IsoSHK质量比30∶1(B)、HS体积为30 mL(C)设计响应面实验因素水平和编码,如表4所示,采用三因素三水平,选取了Box-Benhnken响应面设计的主要优化因素水平进行分析,Box-Benhnken实验设计方案及实验结果见表5。通过Design Expert 13.0软件分析,得到EPC-Chol质量比(A)、EPC-IsoSHK质量比(B)、HS体积(C)与包封率(Y,%)的响应面二次模型回归和方差分析表,见表6。
该模型的P值<0.000 1(P<0.01)(表6),F值为30.53,差异是极显著的,这表明该模型具有统计学意义,并且该模型失拟P值为0.430 9(P>0.05),F值为1.15,说明该模型失拟值不具有显著性,表明该回归模型具有很好的拟合效果,符合实验要求,可用该模型对实验结果进行统计分析,其中编码因素AB、A2、B2、C2差异极显著,编码因素B差异显著,编码因素A、C、AC、BC的影响并不明显。
该模型关于包封率(Y,%)与EPC-Chol质量比(A)、EPC-IsoSHK质量比(B)以及HS体积(C)的二次多项回归方程如下:Y=89.56-0.596 3A+0.832 5B-0.571 3C-2.29AB+0.055 0AC+0.692 5BC-2.96A2-3.02B2-2.84C2(r2=0.975 2),该模型的信噪比高(14.727 2),即可用于预测,方程的决定系数r2=0.975 2>0.95,表明实测值与预测值间有高度的相关性,能准确地预测试剂情况,Adjusted r2=0.943 2,表明模型响应值的变化有94.32%来源于所选自变量,该实验误差较小,上述拟合相关评估指标均符合实验要求,表明该回归方程比较符合实际操作情况,自变量与包封率之间的线性关系显著,可以用此模型对IsoSHK-lip的包封率进行分析预测。本模型中3个因素对包封率的影响大小顺序为:EPC-IsoSHK质量比(B)>EPC-Chol质量比(A)>HS体积(C)。
3.2.2 三因素对于IsoSHK-lip包封率的交互作用
现就EPC-Chol质量比(A)、EPC-IsoSHK质量比(B)、HS体积(C)三因素对IsoSHK-lip包封率的交互作用进行响应面可视化分析。
如图5所示,等高线呈现明显的椭圆形,并且3D曲面图呈现凸起状态,随着EPC-Chol质量比(A)和EPC-IsoSHK质量比(B)的增大,脂质体的包封率先变大后变小,如表5所示,AB的P值为0.000 5(P<0.01),表明EPC-Chol质量比(A)和EPC-IsoSHK质量比(B)对IsoSHK-lip包封率的交互作用极显著,当IsoSHK-lip包封率在A-B为8.82∶1时存在极大值,这是因为胆固醇可增加脂质体膜中双分子排列的紧密程度,起到稳定脂质体的作用,随着胆固醇的相对用量减少,在相同质量的脂质中,脂质体膜的刚性降低,总表面积增大,所包裹的药物增加,包封率增加,但随着磷脂的用量进一步增大,脂质双分子膜不对称型和通透性增大,药物渗漏较多,导致包封率下降。
如图6所示,等高线未呈现明显椭圆形,但3D曲面图呈现凸起状态,如表5所示,AC的P值为0.888 0(P>0.05),表明EPC-Chol质量比(A)和HS体积(C)对IsoSHK-lip包封率的交互作用并不显著。
如图7所示,等高线未呈现明显椭圆形,但3D曲面图呈现凸起状态;如表5所示,BC的P值为0.108 5(P>0.05),表明EPC-IsoSHK质量比(B)和HS体积(C)对IsoSHK-lip包封率的交互作用并不显著。
3.2.3 最优结果验证实验
通过Design Expert 13.0软件中的Numerical优化模块预测IsoSHK-lip最优制备条件为:EPC-Chol质量比为8.82∶1,EPC-IsoSHK质量比为30.65∶1,HS体积为29.22 mL。按比例换算,应称量EPC:90 mg,Chol:10.20 mg,IsoSHK:2.94 mg。按最优制备条件制备IsoSHK-lip,测得IsoSHK-lip的3次包封率为:90.06%、90.26%、89.75%,平均包封率为90.03%,包封率预测值为89.72%,指标的实测值与预测值RSD为0.24%,表明该模型预测的可靠性、可信度高,该工艺稳定可行。
3.3 IsoSHK-lip稳定性研究
采用激光粒度Zeta电位和绝对分子质量分析仪测定最优制备工艺重复3次制备的IsoSHK-lip的粒径、Zeta电位、PDI,留样一周后再次测定IsoSHK-lip的粒径、Zeta电位、PDI。如表7所示,不同批次的IsoSHK-lip的粒径基本稳定于117.48 nm左右,PDI均值为0.246,Zeta电位均值为-13.59 mV。由表7可看出,3批不同的IsoSHK-lip均具有相近的包封率,且稳定于90%左右,粒径和PDI较小。在4 ℃放置7 d后,不同批次IsoSHK-lip的粒径、PDI以及Zeta电位变化较小,较为稳定,表明IsoSHK-lip在4 ℃放置7 d后稳定性良好。
3.4 IsoSHK-lip的外观形态
IsoSHK-lip透射电镜观察结果见图8。图8A为透射电镜观察IsoSHK-lip整体形态图,制备的IsoSHK-lip大多数为近圆形的不规则形状,部分呈圆形或椭圆形,粒子大小并不均一,部分粒子间存在聚集的现象,可能是由于过滤时存在一定的问题,未使用脂质体挤出器进行过滤挤压,所以制备工艺中的过滤挤出有待加强,也有可能是因为透射电镜样品制样时存在问题,该问题需在后续研究中进行多方面的考察。
图8B为透射电镜观察IsoSHK-lip内部结构图,制备的IsoSHK-lip内部结构为微型泡囊体,并且箭头指向的脂质体为多室结构,该图表明脂质体制备成功,并且内部将IsoSHK包裹,结构为多室脂质体。
4 讨论
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由于新疆软紫草具有抗炎、抗肿瘤、抗菌等多种药理活性[2,8],在制剂领域的开发与研究是近年来热门的课题之一,各种新疆软紫草的制剂研究在临床上已经开始得到认可,主要包括油剂、栓剂、膏剂等治疗烧、烫伤的外用制剂等,现紫草剂型以复方制剂较为多见[9]。由于紫草素及其衍生物的低水溶性,水中分散性差,常常被制备成乳剂、膏剂、以及纳米制剂等。近几年也发现紫草素使用微囊包载后进行药理作用研究[10]。本实验将紫草素衍生物IsoSHK制备成脂质体,解决了IsoSHK的低水溶性这一缺点,有望提高IsoSHK的生物利用度。
脂质体制备方法的选择对于脂质体的粒径、结构会产生很大的影响,制备方法的选择一般会根据药物的性质以及用药目的来决定。Su等[11]采用乙醇注入法制备紫草素提取物脂质体以及碱化紫草素脂质体,其制备的脂质体放置长时间会发生凝聚、溶胀或者聚集的现象,并且存在渗漏的现象,稳定性较差。所以本研究采用薄膜分散法制备异戊酰紫草素脂质体,此种制备方法可获得多室脂质体,经超声处理后得到单室脂质体,显著提高脂溶性药物的溶解度,增加药物的靶向性和生物利用度,并且能够减少用药次数和给药剂量,提高疗效,并减少药物残留[12]。可通过脂质体挤出器过滤以及超声破碎等方法减小脂质体的粒径。
作为一种新型制剂,脂质体要想在临床上发挥出更好的疗效,首先需要有更高的包封率。因此,包封率不仅是脂质体制备工艺的重要的评价指标之一[13],而且也是区别于普通制剂发挥靶向、低毒性、缓释等特点的关键点[14]。本实验采用天然磷脂——蛋黄卵磷脂作为脂质体的制备材料,并且在制备过程中加入一定量的胆固醇,能加强IsoSHK-lip的稳定性,减少脂质体团聚,降低药物渗漏。
在脂质体制备过程中水合溶剂的选择也是一个很重要的因素,选择合适的水合溶剂会直接影响脂质体的包封率,本研究以脂质体的包封率为因变量,选取了A:EPC-Chol质量比、B:EPC-IsoSHK质量比以及C:HS体积作为实验的3个因素,采用响应面法对IsoSHK-lip的制备方法进行优化,最后确定最佳的制备工艺条件为:EPC-Chol质量比为8.82∶1,EPC-IsoSHK质量比为30.65∶1,HS体积为29.22 mL。按照上述最优比例重复制备3次脂质体得到的包封率均值为90.03%。以此方法制备的IsoSHK-lip粒径稳定在116~118 nm,Zeta电位为-13~-14 mV左右,PDI为0.23~0.25,实验结果表明IsoSHK-lip的粒径较小,PDI指数较低,并且IsoSHK-lip的包封率达到90%以上,表明该脂质体分散性良好,包封率较优。
综上所述,本研究先对异戊酰紫草素的检测波长、稳定性、精密度以及回收率进行考察,并对异戊酰紫草素脂质体、空白脂质体、异戊酰紫草素的全波长扫描进行检测,表明异戊酰紫草素在518.5 nm处有紫外吸收,并且脂质体材料对异戊酰紫草素的紫外吸收无影响,之后采用响应面优化法对IsoSHK-lip的制备方法进行优化,对优化后的制备工艺进行重复验证,并对IsoSHK-lip的粒径、PDI、Zeta电位以及外观形态进行表征,本研究优化后的IsoSHK-lip制备工艺为后续课题组新疆软紫草的药理作用研究提供了一定的研究基础。
基金
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  • 国家自然科学基金项目资助(81860730)
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2024年第59卷第19期
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doi: 10.11669/cpj.2024.19.008
  • 接收时间：2023-04-10
  • 首发时间：2025-12-30
  • 出版时间：2024-10-08
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  • 收稿日期：2023-04-10
基金
国家自然科学基金项目资助(81860730)
作者信息
    1 石河子大学药学院, 新疆植物药资源利用教育部重点实验室, 新疆 石河子 832002
    2 新疆第二医学院中医学院, 新疆 克拉玛依 834000
    3 新疆医科大学第七附属医院药学部, 乌鲁木齐 830028
    4 河南科技大学应用工程学院, 河南 三门峡 472000

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* 秦冬梅,女,博士,教授 研究方向:中药民族药分离及其药效作用机制研究
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鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
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