中国药学杂志

Serial Number	Inclusion yield/%	RSD/%
1	96.64	0.71
2	95.82
3	97.18

Serial Number	Inclusion yield/%	RSD/%
1	96.64	0.71
2	95.82
3	97.18

Serial Number	Amount	Consumption volume of Na₂S₂O₃ standard solution/mL	Peroxide value/g·100 g^-1	Mean value/g·100 g^-1	RSD/%
1	2.010 1	124.8	7.88	7.98	1.50
2	2.003 9	128.1	8.11
3	2.003 1	125.3	7.94

Serial Number	Amount	Consumption volume of Na₂S₂O₃ standard solution/mL	Peroxide value/g·100 g^-1	Mean value/g·100 g^-1	RSD/%
1	2.010 1	124.8	7.88	7.98	1.50
2	2.003 9	128.1	8.11
3	2.003 1	125.3	7.94

Serial number	Amount	Consumption volume of Na₂S₂O₃ standard solution/mL	Peroxide value/g·100 g^-1	Mean value/g·100 g^-1	RSD/%
1	1.019 7	6.1	0.76	0.76	2.02
2	1.005 4	6.1	0.77
6	1.008 9	5.9	0.74

Serial number	Amount	Consumption volume of Na₂S₂O₃ standard solution/mL	Peroxide value/g·100 g^-1	Mean value/g·100 g^-1	RSD/%
1	1.019 7	6.1	0.76	0.76	2.02
2	1.005 4	6.1	0.77
6	1.008 9	5.9	0.74

Influence factor test	Placement time/d	Appearance	pH	Viscosity /Pa·s	Peroxide value /g·100 g^-1
High temperature	0	Clear, no graininess, shiny, without layering, oil floating, thinning and oil-water separation	5.49±0.01	46.62±0.14	0.146±0.002
	5	Clear, no graininesst, shiny, without layering, oil floating and thinning	5.48±0.02	33.25±0.11	0.114±0.003
	10	Clear, no graininess, shiny, without layering, oil floating and thinning	5.91±0.02	30.27±0.15	0.048±0.003
High humidity	0	Clear, no graininess, shiny, without layering, oil floating, thinning and oil-water separation	5.49±0.01	46.32±0.17	0.146±0.002
	5	Clear, no graininess, shiny, without layering, oil floating, thinning and oil-water separation	5.50±0.02	46.78±0.13	0.143±0.003
	10	Clear, no graininess, shiny, without layering, oil floating, thinning and oil-water separation	5.51±0.01	46.53±0.19	0.141±0.008
Strong light	0	Clear, no graininess, shiny, without layering, oil floating, thinning and oil-water separation	5.49±0.01	46.70±0.16	0.146±0.002
	5	Clear, no graininess, shiny, without layering, oil floating, thinning and oil-water separation	5.48±0.03	46.56±0.18	0.145±0.005
	10	Clear, no graininess, shiny, without layering, oil floating, thinning and oil-water separation	5.50±0.01	46.89±0.13	0.141±0.007

Influence factor test	Placement time/d	Appearance	pH	Viscosity /Pa·s	Peroxide value /g·100 g^-1
High temperature	0	Clear, no graininess, shiny, without layering, oil floating, thinning and oil-water separation	5.49±0.01	46.62±0.14	0.146±0.002
	5	Clear, no graininesst, shiny, without layering, oil floating and thinning	5.48±0.02	33.25±0.11	0.114±0.003
	10	Clear, no graininess, shiny, without layering, oil floating and thinning	5.91±0.02	30.27±0.15	0.048±0.003
High humidity	0	Clear, no graininess, shiny, without layering, oil floating, thinning and oil-water separation	5.49±0.01	46.32±0.17	0.146±0.002
	5	Clear, no graininess, shiny, without layering, oil floating, thinning and oil-water separation	5.50±0.02	46.78±0.13	0.143±0.003
	10	Clear, no graininess, shiny, without layering, oil floating, thinning and oil-water separation	5.51±0.01	46.53±0.19	0.141±0.008
Strong light	0	Clear, no graininess, shiny, without layering, oil floating, thinning and oil-water separation	5.49±0.01	46.70±0.16	0.146±0.002
	5	Clear, no graininess, shiny, without layering, oil floating, thinning and oil-water separation	5.48±0.03	46.56±0.18	0.145±0.005
	10	Clear, no graininess, shiny, without layering, oil floating, thinning and oil-water separation	5.50±0.01	46.89±0.13	0.141±0.007

Review factors	Appearance	pH	Viscosity /Pa·s	Peroxide value /g·100 g^-1
High-speed centrifuge	Clear, no graininess, shiny, without layering, oil floating, thinning and oil-water separation	5.49±0.01	46.68±0.12	0.149±0.03
Low temperature experiment	Clear, no graininess, shiny, without layering, oil floating, thinning and oil-water separation	5.50±0.01	46.71±0.13	0.153±0.03

Review factors	Appearance	pH	Viscosity /Pa·s	Peroxide value /g·100 g^-1
High-speed centrifuge	Clear, no graininess, shiny, without layering, oil floating, thinning and oil-water separation	5.49±0.01	46.68±0.12	0.149±0.03
Low temperature experiment	Clear, no graininess, shiny, without layering, oil floating, thinning and oil-water separation	5.50±0.01	46.71±0.13	0.153±0.03

Sample	Diameter of bacteriostatic ring of test strain
a	14	16	9
b	9	9	9
c	5	5	5

Sample	Diameter of bacteriostatic ring of test strain
a	14	16	9
b	9	9	9
c	5	5	5

Test strain	Average bacteriostatic rate of different time/%	Number of positive control colonies
Candida albicans	77.82	83.24	88.69	89.10	1.26×10³
Staphylococcus aureus	95.97	97.30	98.38	99.18	1.25×10⁴
Escherichia coli	89.40	91.14	94.52	98.92	2.67×10³

Test strain	Average bacteriostatic rate of different time/%	Number of positive control colonies
Candida albicans	77.82	83.24	88.69	89.10	1.26×10³
Staphylococcus aureus	95.97	97.30	98.38	99.18	1.25×10⁴
Escherichia coli	89.40	91.14	94.52	98.92	2.67×10³

Reaction	Score	Reaction	Score
Epithele		Vascular hyperemia
Normal, intact	0	Nothing	0
Cell degeneration or flattening	1	Few	1
Tissue deformation	2	Light	2
Local erosion	3	Moderate	3
Extensive erosion	4	Severe vascular rupture	4
Leukocyte infiltration (each high power field)		Edema
Nothing	0	Nothing	0
Very few (less than 25)	1	Few	1
Mild (26-50)	2	Light	2
Moderate (51-100)	3	Moderate	3
Weight (more than 100)	4	Severe	4

Reaction	Score	Reaction	Score
Epithele		Vascular hyperemia
Normal, intact	0	Nothing	0
Cell degeneration or flattening	1	Few	1
Tissue deformation	2	Light	2
Local erosion	3	Moderate	3
Extensive erosion	4	Severe vascular rupture	4
Leukocyte infiltration (each high power field)		Edema
Nothing	0	Nothing	0
Very few (less than 25)	1	Few	1
Mild (26-50)	2	Light	2
Moderate (51-100)	3	Moderate	3
Weight (more than 100)	4	Severe	4

Average score	Reaction degree
0	Nothing
1-4	Extremely light
5-8	Light
9-11	Moderate

Average score	Reaction degree
0	Nothing
1-4	Extremely light
5-8	Light
9-11	Moderate

Group	Animal number	Microscopic scoring	Average score
Control group	1	0	0	0	0	0
	2	0	0	0	0
	3	0	0	0	0
Ozone oil gel group	1	0	0	0	0	0
	2	0	0	0	0
	3	0	0	0	0
Ozone oil group	1	2	1	2	2	7
	2	2	1	2	2
	3	2	1	2	2

Group	Animal number	Microscopic scoring	Average score
Control group	1	0	0	0	0	0
	2	0	0	0	0
	3	0	0	0	0
Ozone oil gel group	1	0	0	0	0	0
	2	0	0	0	0
	3	0	0	0	0
Ozone oil group	1	2	1	2	2	7
	2	2	1	2	2
	3	2	1	2	2

Application test reaction	Grade
No obvious change	0
Sporadic or spotted erythema	1
Moderate confluent erythema	2
Severe erythema and edema	3

Application test reaction	Grade
No obvious change	0
Sporadic or spotted erythema	1
Moderate confluent erythema	2
Severe erythema and edema	3

Group	Application test reaction		Grade
Control group	No obvious change	No obvious change	0
Ozone oil gel group	No obvious change	No obvious change	0

Group	Application test reaction		Grade
Control group	No obvious change	No obvious change	0
Ozone oil gel group	No obvious change	No obvious change	0

臭氧油包合物凝胶的制备与评价

PDF下载

吴玉欢 ¹ , 彭颖 ¹ , 雷晓萌 ¹ , 魏鑫华 ¹ , 张观生 ¹ , 王堂勋 ¹ , 李东勲 ¹^,^* , 张国松 ¹^,²^,^*

中国药学杂志 | 论著 2024,59(18): 1725-1734

收起

中国药学杂志 | 论著 2024, 59(18): 1725-1734

臭氧油包合物凝胶的制备与评价

全屏

吴玉欢¹, 彭颖¹, 雷晓萌¹, 魏鑫华¹, 张观生¹, 王堂勋¹, 李东勲¹^,^*, 张国松¹^,²^,^*

作者信息

¹ 江西中医药大学中药固体制剂制造技术国家工程研究中心, 南昌 330006

² 江西中医药大学中西医结合儿童健康研究院(儿童创新药物研究中心), 南昌 330006

吴玉欢,女,硕士研究生研究方向:药物新剂型与新技术

通讯作者:

^* 张国松,男,博士,教授研究方向:药物新剂型与新技术研究 Tel:(0791)87119617;

李东勲,男,博士,副教授研究方向:新药开发及仿制药一致性评价研究 Tel:(0791)87119617

Preparation and Evaluation of Ozone Oil Inclusion Compound Gel

Yuhuan WU¹, Ying PENG¹, Xiaomeng LEI¹, Xinhua WEI¹, Guansheng ZHANG¹, Tangxun WANG¹, Dongxun LI¹^,^*, Guosong ZHANG¹^,²^,^*

Affiliations

¹ National Engineering Research Center of Chinese Medicine Solid Preparation Manufacturing Technology, Jiangxi University of Chinese Medicine, Nanchang 330006, China

² Integrated Chinese and Western Medicine Institute for Children Health & Drug Innovation, Jiangxi University of Chinese Medicine, Nanchang 330006, China

出版时间: 2024-09-22 doi: 10.11669/cpj.2024.18.008

文章导航

摘要

收起

目的研究臭氧油包合物凝胶的处方与制备工艺,并对其进行评价。方法以羟丙基-β-环糊精(hydroxypropyl-β-cyclodextrin,HP-β-CD)为包合材料,卡波姆-940为基质制备臭氧油包合物凝胶,并对其外观性状、pH值、黏度、过氧化值、保湿率、流变学性质、稳定性、抑菌性能和生物安全性进行评价。结果最终得到外观为澄清、无颗粒感、均匀细腻、有光泽、黏度适中、无油腻性的半固体凝胶;pH值为(5.48±0.01),黏度为 (46.78±0.18) Pa·s,保湿率在放置24、36、48 h后分别为 (96.69±0.75)%、 (95.84±0.44)%、 (94.12±0.89)%;流变学性质良好;稳定性试验表明臭氧油凝胶不宜放在高温保存。抑菌结果显示,臭氧油凝胶对金黄色葡萄球菌、白色念珠菌、大肠埃希菌均有抑菌效果,金黄色葡萄球菌和大肠埃希菌抑菌率达到95%以上,白色念珠菌抑菌率达到80%以上;生物安全性评价试验表明,臭氧油凝胶无阴道黏膜刺激性、无皮肤致敏反应和无急性全身毒性反应。结论此方法制备臭氧油包合物凝胶的工艺简便可行、易于操作,在药品及化妆品领域中具有一定的推广价值和应用前景,为臭氧油的进一步工业化应用奠定了实验基础。

关键词

臭氧油 / 羟丙基-β-环糊精 / 包合物 / 凝胶 / 卡波姆940

Abstract

收起

OBJECTIVE To study and evaluate the formulation and preparation process of ozone oil inclusion compound gel. METHODS Hydroxypropyl- β-cyclodextrin (HP-β-CD) was the inclusion material and Carbome-940 was the matrix to prepare the ozone oil inclusion compound gel, and its appearance, pH value, viscosity, peroxide value, moisture retention rate, rheological properties, stability, bacteriostatic performance and biological safety were evaluated. RESULTS Finally, the semi solid gel with clear appearance, no particle feeling, uniform, fine, shiny, moderate viscosity and no greasy property was obtained. The pH value was (5.48±0.01), the viscosity was (46.78±0.18) Pa·s, and the moisture retention rates were (96.69±0.75)%, (95.84±0.44)%, and (94.12±0.89)% after 24, 36, and 48 hours of storage, respectively, with good rheological properties. The stability test showed that ozone oil gel should not be stored at high temperature. The bacteriostatic results showed that the ozone oil gel had bacteriostatic effects on Staphylococcus aureus, Candida albicans and Escherichia coli. The bacteriostatic rate of Staphylococcus aureus and Escherichia coli reached more than 95%, and the bacteriostatic rate of Candida albicans reached more than 80%. The biosafety evaluation test showed that the ozone oil gel had no vaginal mucosa irritation, skin sensitization and acute systemic toxicity. CONCLUSION The process of preparing ozone oil inclusion compound gel by this method is simple, feasible and easy to operate. It has certain promotion value and application prospect in drugs and cosmetics, which lays an experimental foundation for further industrial application of ozone oil.

Key words

ozone oil / hydroxypropyl-β-cyclodextrin / inclusion compound / gel / carbomer 940

引用本文

吴玉欢, 彭颖, 雷晓萌, 魏鑫华, 张观生, 王堂勋, 李东勲, 张国松. 臭氧油包合物凝胶的制备与评价. 中国药学杂志, 2024 , 59 (18) : 1725 -1734 . DOI: 10.11669/cpj.2024.18.008

Yuhuan WU, Ying PENG, Xiaomeng LEI, Xinhua WEI, Guansheng ZHANG, Tangxun WANG, Dongxun LI, Guosong ZHANG. Preparation and Evaluation of Ozone Oil Inclusion Compound Gel[J]. Chinese Pharmaceutical Journal, 2024 , 59 (18) : 1725 -1734 . DOI: 10.11669/cpj.2024.18.008

正文

收起

臭氧油是一种天然的抗菌剂,由臭氧与植物油中不饱和双键反应而保存在不饱和脂肪酸中得到的,已被广泛应用于临床,如皮肤科、口腔科、妇科和眼科等,均有显著疗效,在妇科可以用于阴道炎、盆腔炎、急性宫颈炎、子宫内膜炎等疾病的治疗^[1]。臭氧油的作用机制完全不同于抗菌药物,根据Criegee所描述的臭氧化机制^[2],臭氧与植物油中不饱脂肪酸的碳碳双键在不同的反应条件下会形成臭氧化合物和不同的含氧物质,如醛、酮、氢过氧化物、过氧化物衍生物等^[3]。Criegee机制主要分为三步,第一步是臭氧与不饱和碳碳双键发生1,3-偶极环加成及其逆转反应,反应产物为初级臭氧化物;第二步是初级臭氧化物分解反应,产物为羰基氧化物和羰基化合物;第三步是羰基氧化物与羰基化合物发生反应,形成稳定、可分离的次级臭氧化物(图1)^[3]。上述生成的臭氧化产物在病变处与伤口面接触时会释放活性氧,如过氧化物,超氧化物,羟基自由基等。这些活性氧溶解在水中,会产生过氧化氢,通过过氧化氢介导的氧化作用消除病原体,从而达到抗菌的效果^[4],作用之后产生氧气和水,不会产生耐药性。但臭氧油在使用过程中存在稳定性差、水溶性差、气味难闻、刺激性强等缺陷,限制了其推广应用^[1]。因此,开发一种适宜的臭氧油剂型,在保持其抗菌性能的同时,克服上述缺陷,对于其推广应用具有重要意义。

包合技术是解决挥发油类物质稳定性差、水溶性差等问题的常用方法。羟丙基-β-环糊精 (HP-β-CD) 是β-CD的羟丙基衍生物,其克服了分子内氢键的形成,不仅保留了β-CD的包合能力,溶解度也提高了20~50倍,已成为挥发油类物质的主要包合材料^[5]。凝胶剂因其具有生物相容性好、水溶性强、渗透性强、刺激性小、使用方便等优点,在阴道黏膜给药系统中得到了广泛应用^[6]。因此,本实验针对臭氧油应用存在的诸多缺陷,综合应用HP-β-CD包合和凝胶剂两项制剂技术的优势,设计并构建以HP-β-CD包载的臭氧油包合物为中间体,以生物相容性好的卡波姆为基质的阴道黏膜给药系统,以达到保持臭氧油抑菌能力、提高稳定性、增加水溶性、降低刺激性和掩盖不良气味等目的,为进一步研发臭氧油产品奠定实验基础。

1 仪器与材料

收起

BSA2202S型电子天平(赛多利斯科学仪器有限公司);恒温加热磁力搅拌器(巩义市予华仪器有限责任公司);RE-2000B型旋转蒸发仪(郑州方圆仪器科技有限公司);JCM-7000型冷场发射扫描电镜—能谱仪(日本JEOL公司);尼科莱 iS50型傅立叶红外变换光谱仪(美国赛默飞世尔科技公司);DSC 8000型差示扫描量热仪(美国 PE公司);AV 600 Mhz型核磁共振氢谱仪(瑞士布鲁克);101-3-BS-Ⅱ电热恒温鼓风干燥箱(上海跃进医疗器械厂);KQ-250V型超声波清洗器(昆山市超声仪器有限公司);LS-3000UV低温光照仪(北京天星科仪科技有限公司);HC-3016R型高速冷冻离心机(安徽中科中佳科学仪器有限公司);生物安全柜、旋涡震荡仪(山东省济南市平阴县安城镇欧莱博智能制造产业园);恒温培养箱(济南鑫贝西生物技术有限公司);高温高压蒸汽灭菌锅(上海博讯实业有限公司医疗设备厂);生物组织石蜡包埋机(湖北亚光医用电子技术有限公司);DM2500型光学显微镜、石蜡切片机(徕卡公司);数显恒温水浴锅(巩义市予华仪器有限公司)。

臭氧油(徐州金源臭氧设备有限公司,批号:20211026,含量:70 g·L^-1);HP-β-CD(西安德立生物化工有限公司,批号:20211104);卡波姆940(广州康乔汉普药业有限公司);甘油(南昌白云药业有限公司);三乙醇胺(成都华邑药用辅料制造有限责任公司);可溶性淀粉、碘化钾(国药集团化学试剂有限责任公司);无水碳酸钠(天津大茂化学试剂厂);硫代硫酸钠(江陵奔达制药有限公司);无水乙醇、石油醚、冰醋酸、三氯甲烷为分析纯。

新西兰兔[体质量(2~2.5)kg,雌性,9只]购于赣州市畜牧水产研究所,实验动物许可证号为SCXK(赣)2018-0009;白化豚鼠[雄性,体质量(300~500) g,雄性,15只]购于湖南太平生物科技有限公司,实验动物许可证号为SCXK(湘)2020-0005;初成年KM小鼠[体质量 (18~22) g,雄性,10只]购于湖南斯莱克景达实验动物有限公司,实验动物许可证号为SCXK(湘)2019-0004。

金黄色葡萄球菌 (CMCC26003)、大肠埃希菌 (CMCC44102)、白色念珠菌(CMCC98001)(中国医学细菌保藏管理中心);沙氏葡萄糖液体培养基、胰酪大豆胨液体培养基、营养琼脂培养基、沙氏琼脂培养基(北京奥博星生物技术有限公司)。

2 方法与结果

收起

2.1 臭氧油凝胶的制备

2.1.1 臭氧油包合物的制备^[7]

按臭氧油和HP-β-CD质量比1∶10,称取HP-β-CD 10 g,加入无水乙醇40 mL,置恒温磁力搅拌器上,45 ℃搅拌溶解,制成HP- β-CD无水乙醇溶液;另称取臭氧油1 g,加入3 mL无水乙醇稀释,制成臭氧油无水乙醇溶液,随后将臭氧油无水乙醇溶液缓慢地边搅拌边加入到HP- β-CD无水乙醇溶液中,继续搅拌1.5 h,回收无水乙醇,干燥,再用适量石油醚洗涤2次,挥干,得臭氧油包合物。由公式1计算出包合物产率为(96.55±0.68)%,结果见表1。

公式(1)包合物产率(%) =包合物的实际质量/(臭氧油的投入质量+HP-β-CD的投入质量)×100%

2.1.2 过氧化值的测定

臭氧油过氧化值的测定:过氧化值是评估臭氧油抗菌活性的重要指标,其过氧化值越大,抗菌活性越强^[8]。按照GB 5009.227-2016过氧化值的测定要求^[9],用过氧化物相当于碘的质量分数表示过氧化值,按公式(2)计算:

公式(2)X=(V-V₀)×c×0.126 9/M×100

式中:X为过氧化值(g·100 g^-1);c为硫代硫酸钠标准溶液的浓度,单位为mol·L^-1;V为样品消耗硫代硫酸钠滴定液的体积(mL);V₀为空白试验消耗硫代硫酸钠滴定液的体积(mL);M为样品的质量(g);0.126 9为与1.00 mL硫代硫酸钠标准滴定溶液[c(Na₂S₂O₃)=1.000 mol·L^-1]相当的碘的质量;100为换算系数。测定结果见表2。

包合物过氧化值的测定:精密称定经石油醚洗涤、挥干后的包合物1 g,加适量三氯甲烷-冰乙酸混合液(2∶3) 超声处理40 min,按照“臭氧油过氧化值的测定”项下测定其过氧化值,求出包合物中臭氧油的过氧化值。测定结果见表3。

2.1.3 包合物的鉴定

扫描电镜观察(SEM):分别取HP-β-CD、物理混合物和臭氧油包合物适量,采用扫描电子显微镜在100×、200×放大倍数下,对样品进行观察,其中加速电压为5 kV。观察到HP-β-CD呈空心的圆球状,物理混合物中臭氧油分散在HP-β-CD的表面及四周,且HP-β-CD的结构也没有发生变化,而臭氧油包合物在形态上明显发生了变化,转变成了块状结构,说明臭氧油成分进入到HP-β-CD分子空腔内,使HP-β-CD形态上发生了变化,表明包合物形成,见图2。

傅里叶红外光谱 (FTIR):分别取臭氧油、HP-β-CD、物理混合物和臭氧油包合物适量,臭氧油采用KBr涂片法,其余采用KBr压片法,在分辨率为4 cm^-1,光谱范围为4 000~400 cm^-1条件下全谱扫描。结果见图3A,臭氧油在2 925和2 854 cm^-1分别为臭氧油中的不饱和键的亚甲基(C=C-CH₂-C=C)基团的不对称和对称伸缩振动峰,1 745 cm^-1为羰基(C=O)的特征吸收峰, 与Uzun等^[10]描述的一致;图3B中3 363 cm^-1处为HP-β-CD的-OH伸缩振动峰,2 975和2 927 cm^-1为-CH₃及-CH₂-中C-H的伸缩振动峰,1 028 cm^-1为葡萄糖单位中C-O的伸缩振动峰^[11];臭氧油和HP-β-CD的特征吸收峰同时出现在物理混合物的红外光谱中,这表明HP-β-CD与臭氧油没有形成包合物,只是简单地混合物;而臭氧油包合物(图3D)的红外图谱显示,臭氧油在2 925、2 854 cm^-1处C-H的伸缩振动峰消失,在1 745 cm^-1处醛基的吸收峰发生轻微偏移(蓝移);HP-β-CD在3 363 cm^-1处的-OH伸缩振动峰和1 028 cm^-1处的C-O-C伸缩振动峰也发生了偏移(红移),进一步说明了臭氧油和HP-β-CD之间存在相互作用,说明臭氧油与HP-β-CD形成的了臭氧油包合物。

差示扫描量热分析 (DSC):以空白坩埚为参比,检测空气为氮气,升温范围25~250 ℃,升温速度10 ℃·min^-1,气体流速20 mL·min^-1,对HP-β-CD、物理混合物、臭氧油包合物进行DSC分析。臭氧油在150 ℃出现放热峰(图4A),HP-β-CD在99 ℃出现吸热峰(图4B),在物理混合物图谱中(图4C),同时出现臭氧油与HP-β-CD特征峰,表明这2种成分之间没有相互作用。然而,包合物的DSC热谱图(图4D)与HP-β-CD相似,且臭氧油的特征峰消失,DSC热谱图表明臭氧油包合物的形成。

核磁共振氢谱 (¹HNMR):通过¹HNMR可以获取关于客体分子在HP-β-CD腔内空间位置的有价值的信息^[12]。图5是臭氧油、物理混合物、臭氧油包合物的核磁共振氢谱,物理混合物表现出与臭氧油极度相似的峰,而形成包合物后,臭氧油中H的化学位移在9.76、2.76发生了一定程度的变化,并且在化学位移3.72出现了新的信号峰,这是臭氧油成功进入HP-β-CD分子的表现,表明臭氧油包合物成功形成。

2.1.4 臭氧油凝胶的制备^[13]

称取卡波姆940粉末0.4 g,少量多次均匀撒于纯化水中,磁力搅拌,使其充分溶胀后,再取甘油8 g,边搅拌边加入到已溶胀的卡波姆940溶液中,即得空白凝胶溶液。然后取臭氧油包合物粉末10 g,将其边搅拌边缓慢加入空白凝胶中,充分搅拌使完全溶解,采用三乙醇胺调节pH至5.0~5.5,再加纯化水至100 g,充分搅拌,混合均匀,离心脱气,即得。

2.2 臭氧油凝胶的质量评价

2.2.1 外观性状

外观为澄清、无颗粒感、均匀细腻、有光泽、黏度适中、无油腻性,铺展性良好的半固体凝胶。

2.2.2 pH的测定

取3批臭氧油凝胶适量,分别测定pH值,结果臭氧油凝胶pH值分别为5.49、5.50、5.49,符合局部外用制剂对pH值的要求。

2.2.3 黏度的测定

取3批臭氧油凝胶适量,分别采用旋转流变仪MCR101(安东帕,奥地利),测定温度(25±0.1) ℃,剪切速率1 s^-1,转子PP50连续剪切,每隔10 s记录1次黏度值,求平均值。测得臭氧油凝胶的黏度分别为46.92、46.98、46.84 Pa·s,RSD为0.15% (n=3)。

2.2.4 保湿率测定

取3批臭氧油凝胶适量,精密称定其质量(M₀),均匀涂布于载玻片上,将该载玻片置于室温(25±1) ℃、相对湿度为42.76%(饱和碳酸钾溶液)的恒温、恒湿密封干燥器中。经放置24、36、48 h后取出,分别精密称定放置不同时间的质量(M),根据公式(3)计算其保湿率:

公式(3)保湿率%=M/M₀×100%

结果显示3批臭氧油凝胶放置24、36、48 h后的保湿率(96.69±0.75)%、(95.84±0.44)%、(94.12±0.89)%,表明凝胶的保湿能力较好。

2.2.5 臭氧油凝胶过氧化值测定

取3批臭氧油凝胶适量,精密称定2 g,加适量三氯甲烷-冰乙酸混合液 (2∶3) 超声处理40 min,按照“2.1.2”项下方法测定其过氧化值,测得结果每100 g为(0.149±0.003) g。

2.3 臭氧油凝胶的稳定性考察

按照《中国药典》2020年版四部附录指导原则,开展高温(60 ℃)、高湿[25 ℃、相对湿度(90±5)%]、强光照射试验(4 500±500)lx,并参考文献[14]开展了高速离心(10 000 r·min^-1,离心30 min)及低温试验(4 ℃放置24 h),考察不同时间点外观性状、pH值、黏度及过氧化值的变化。试验结果表明臭氧油凝胶在高湿、强光照、高速离心及低温试验条件下稳定,在高温条件下不稳定,具体见表4~5。

2.4 臭氧油凝胶的流变学性质考察

采用MCR101旋转流变仪,选择转子CP50-1(直径为50 mm,间隙0.05 mm的不锈钢几何板),温度为 (25±0.1) ℃进行测试。

2.4.1 稳态剪切实验

在剪切速率为(0~100)s^-1范围内,测定凝胶的黏度并绘制黏度扫描曲线,结果见图6。由图6可见,随着臭氧油凝胶的剪切速率的增加,黏度降低,呈现出剪切变稀现象,属于假塑性流体。在假塑区,随着剪切速率的增加,卡波姆分子相互之间缠绕的线团状也会随之发生形态的变化和拉长,使得占据的空间缩小,凝胶流动阻力减少^[15]。因此,随着剪切速率的增大,凝胶的黏度降低。

2.4.2 动态振荡实验

凝胶线性黏弹区:通过凝胶线性黏弹区测试结果可得到凝胶的内部三维网状结构被破坏所能承受的最大形变,在频率为1Hz,应变 (γ) 为0.01%~100%内进行扫描,结果见图7。由图7可见,G'的曲线在0.01%~10%内变化较为平稳,为凝胶的线性黏弹区,且G'大于G″,弹性占主体,表现出凝胶结构,在G″和G'交点之后,凝胶结构遭到破坏,G'小于G″,黏性占主体,表现出流体性质。因此对于该凝胶的动态振荡实验应控制γ在10%以内。

频率扫描:频率扫描旨在描述样品在非破坏性变形范围内的时间相关特性,在γ为1%,频率为0.01~100 Hz内进行振荡频率扫描,测定臭氧油凝胶的黏弹曲线。由图8可见,无论在低频区还是在高频区,凝胶体系G'一直大于G″,故该体系是以弹性效应为主导地位的。当剪切频率逐渐增大时,G'基本保持不变,没有出现明显的频率依赖性,表明凝胶始终具有稳定的三维网状结构。

2.4.3 触变性实验

触变性是指物体受到剪切时黏度变小,停止剪切时黏度又增加或受到剪切时黏度变大,停止剪切时黏度又变小的性质,即一“触”即“变”的性质^[15]。在0~60 s内和在60~120 s内分别执行剪切速率上升和剪切速率下降两个程序,并分别记录黏度值,结果见图9。随着剪切速率的增加,臭氧油凝胶黏度呈降低现象,剪切速率逐渐降低时,臭氧油凝胶的黏度不断升高。凝胶在剪切率上升和下降过程的黏度曲线基本重合,说明无触变环,凝胶无触变性。因为当剪切停止时,分子之间的结构又会迅速恢复,从而出现了剪切速率下降时的曲线与上升时的曲线发生重合的现象^[15]。该特性有利于阴道给药,当使用外力挤压凝胶至药用部位时,黏度下降而易于流动,凝胶进入药用部位后在很短的时间即可恢复原有的黏度,使得其易于黏附于阴道黏膜表面,不易从中泄漏。

2.4.4 凝胶温度的测定

在频率为1 Hz、γ为1%、升温速度为2 ℃·min^-1条件下,将测试温度从4 ℃匀速上升至40 ℃,对凝胶进行黏度-温度扫描。结果见图10,随着温度增加,样品黏度值基本保持平稳,说明该凝胶的热稳定性强,对温度的耐受性较好。

2.5 臭氧油凝胶的抑菌性能评价

2.5.1 菌悬液制备

将大肠埃希菌、金黄色葡萄球菌和白色念珠菌的24 h斜面培养物用磷酸盐缓冲液(PBS)洗下,制成菌悬液(要求浓度为:用100 μL滴于对照样片上,回收菌数为每片1×10⁴~9×10⁴ cfu)。

2.5.2 抑菌环实验^[16]

将直径5 mm圆形滤纸片高压蒸汽灭菌,干燥,放入试样溶液中浸泡50 min,取出待用。将供试菌均匀涂布接种于营养琼脂(大肠埃希菌、金黄色葡萄球菌)、沙氏琼脂(白色念珠菌)培养基平板上,再用无菌镊将处理好的纸片间隔贴于其上,贴放好后,用无菌镊轻压纸片,使其紧贴于平板表面,盖好平板,置(36±1)℃培养18~24 h观察结果,测量抑菌环的直径(包括贴片)并记录,实验重复3次。

抑菌环结果显示(图11,表6),臭氧油凝胶对白色念珠菌、金黄色葡萄球菌和大肠杆菌均有抑菌效果。

2.5.3 定量抑菌实验^[17]

采用GB15979-2002溶出性抑菌试验方法进行定量抑菌试验,结果判定:抑菌率≥50%~90%,有抑菌作用;抑菌率≥90%,有较强抑菌作用。结果显示臭氧油凝胶对金黄色葡萄球菌和大肠埃希菌抑菌率都达到95%以上,白色念珠菌抑菌率达到80%以上,说明产品有较强抑菌作用,具体见表7。

2.6 臭氧油凝胶的生物安全性评价

2.6.1 阴道黏膜刺激实验^[18-19]

选取阴道无损伤的雌性白化兔9只,臭氧油组3只、臭氧油凝胶组3只、空白对照组3只。提起动物尾巴暴露出阴道口,分别将臭氧油、臭氧油凝胶、氯化钠注射液1 g注入阴道内,间隔24 h重复上述步骤,连续5 d。末次接触后24 h无痛处死动物,完整切下阴道后纵向切开,肉眼检查上皮组织层的刺激、损伤以及坏死情况,并对阴道组织进行组织学检查和评分,计算刺激指数(表8~10)。

结果显示,末次给药24 h后,肉眼观察到实验兔上皮组织层无损伤、坏死等异常情况。组织学评价见图12,臭氧油组可见黏膜层、肌层、纤维层结构都发生变形,刺激指数平均记分为7分,说明臭氧油对实验兔阴道黏膜有刺激性。对照组可见黏膜层、肌层、纤维层结构都正常,并且没有出现炎症、充血、水肿等现象。臭氧油凝胶组结果和对照组基本一致,黏膜层、肌层、纤维层结构也正常,无炎症、充血、水肿,两组平均记分为0,结果表明该凝胶无阴道刺激反应。

2.6.2 皮肤致敏实验^[18]

诱导阶段:将豚鼠随机分为臭氧油凝胶组10只、空白对照组5只。实验前24 h,剃除豚鼠背部脊柱两侧被毛,取吸水性纱布(约3 cm×3 cm)浸透臭氧油凝胶,贴敷于每只动物左上背部位的除毛皮肤上,然后用封闭性包扎带加以固定,(6±0.5)h后除去包扎带和敷贴片。1周中连续3 d重复该步骤,同法操作3周。对照组以氯化钠注射液同法操作。

激发阶段:最后一次诱导贴敷后14 d,将浸透臭氧油凝胶的敷贴片单独局部贴敷于每只动物去毛的未试部位。(6±0.5)h后除去包扎带和敷贴片。于激发后24及48h对实验部位进行评分(表11)。

结果显示,各组动物皮肤均未出现红斑水肿等现象,并均未出现死亡、休克等严重全身性过敏反应。实验组和对照组反应分级均为0,结果表明臭氧油凝胶无皮肤致敏反应(表12)。

2.6.3 急性全身毒性实验^[19-20]

以生理盐水为浸提介质,按0.2 g·mL^-1制备臭氧油凝胶溶液,37 ℃浸提72 h,取上清液作为待测溶液。以初成年KM小鼠为研究对象,将初成年KM小鼠随机分为臭氧油凝胶组和空白对照组,每组5只,臭氧油凝胶组和空白对照组按50 mL·kg^-1腹腔分别注射待测溶液、生理盐水。记录观察到的毒性症状,包括开始的时间、程度和持续时间。在72 h观察期内,臭氧油凝胶组动物的反应不大于空白对照组动物,则判定供试品无急性全身毒性反应。结果显示,在72 h观察期内,臭氧油凝胶组和空白对照组动物未见异常症状,判定臭氧油凝胶组无急性全身毒性反应。

3 讨论

收起

臭氧油凝胶制剂属于外用制剂,具有给药便利,患者依从性高等优势。本文先将臭氧油用HP-β-CD包合制备成包合物,包合物通过SEM、FTIR、¹HNMR、DSC等方法进行鉴定与表征,结果表明臭氧油可被包合到HP-β-CD空腔里,再以包合物为中间体,以卡波姆940为基质制备臭氧油凝胶,结果表明制得的臭氧油凝胶稳定性好、黏度适中、流变学性质良好,适合于阴道黏膜给药。

本实验通过HP-β-CD包合臭氧油,再将其制备成包合物凝胶之后,不仅提高了臭氧油的稳定性、增加水溶性、掩盖不良气味等,还保持其原有的抑菌能力,抑菌环实验结果显示,臭氧油凝胶具有抑菌效果;定量实验结果显示,臭氧油凝胶对金黄色葡萄球菌、大肠埃希菌有较强的抑菌作用,抑菌率达到95%以上;对白色念珠菌也有抑菌作用,抑菌率达到80%;该研究结果与Burcu等^[21]研究的臭氧橄榄油β-CD包合物和Anna等^[22]研究的臭氧油脂质体抑菌结果描述一致,为进一步研发臭氧油产品奠定实验依据。臭氧油对阴道黏膜有明显的刺激性,而将其制备成臭氧油凝胶后,对阴道黏膜没有明显的刺激性,且没有出现明显皮肤致敏反应和无急性全身毒性反应,安全性实验结果与Anna等^[22]研究的臭氧油脂质体制剂描述结果一致,为臭氧油剂型临床应用及推广提供了实验依据。

综上所述,本实验采用的凝胶制备工艺步骤明晰、操作简单,所制备的凝胶稳定性好、抑菌效果显著、安全有效,为臭氧油给药新剂型的开发提供实验基础。

基金

收起

国家自然科学基金项目资助(81960720)
国家自然科学基金项目资助(82260771)
江西中医药大学创新药物研究与开发创新团队项目资助(CXTD22001)

参考文献

收起

文献

收起

参考文献引证文献

排序方式：

[1]

UGAZIO

, TULLIO

, BINELLO

, et al. Ozonated oils as antimicrobial systems in topical applications. their characterization, current applications, and advances in improved delivery techniques[J]. Molecules, 2020, 25(2): 1-24.

[2]

HASSAN

, STAHLBERGER

, ROSENBAUM

, et al. Criegee intermediates beyond ozonolysis: synthetic and mechanistic insights[J]. Angew Chem Int Ed Engl, 2021, 60(28): 15138-15152.

[3]

CATALDO, FRANCO. Chemical and thermochemical aspects of the ozonolysis of ethyl oleate: decomposition enthalpy of ethyl oleate ozonide[J]. Chem Phys Lipids, 2013, 175-176: 41-49.

[4]

OUF

S A

, MOUSSA

T A A

, ABD ELMEGEED

A S M

, et al. Anti-fungal potential of ozone against some dermatophytes[J]. Braz J Microbiol, 2016, 47(3): 697-702.

[5]

SUN

J Y

, PANG

F P

, LIU

M X

. Hydroxypropyl-β-Progress in the application of cyclodextrins in natural drugs[J]. Chin Tradit Pat Drugs(中成药), 2015, 37 (2): 388-391

[6]

WANG

Y H

, LI

H J

, YANG

, et al. Research progress of vaginal mucosal drug delivery system[J]. Chin J Exp Tradit Med Form(中国实验方剂学杂志), 2019, 25 (17): 219-225

[7]

HAO

J X

, ZHA

L C

, FAN

, et al. Optimization of hydroxypropyl in volatile oil of Xiangguo Jianxiao tablets based on information entropy weight method- β- Cyclodextrin inclusion process and its characterization[J]. Chin Tradit Herb Drugs(中草药), 2022, 53 (13): 3962-3971.

[8]

KOGAWA

N R D A

, ARRUDAB

E J D

, CAMILA

, et al. Synthesis, characterization, thermal behavior, and biological activity of ozonides from vegetable oils[J]. Rsc Adv, 2015, 5(80): 65427-65436.

[9]

National Health and Family Planning Commission of the People's Republic of China, National Standards of the People's Republic of China National food safety standard Determination of peroxide value in food: GB 5009.227-2016[S]. 2016.

[10]

UZUN

, KAYNAK

E G

, IBANOGLU

, et al. Chemical and structural variations in hazelnut and soybean oils after ozone treatments[J]. Grasasy Aceites, 2018, 69(2): 2531-2532.

[11]

WANG

Y L

, YUAN

, LIU

Y W

. Characterization and component analysis of cinnamon essential Oil/Hydroxypropyl-β-cyclodextrin complex[J]. China Food Addit (中国食品添加剂)[J], 2018, 177 (11): 56-61

[12]

ZHANG

G J

, GU

L B

, ZHOU

M S

, et al. Squalene/γ- Preparation and molecular simulation of cyclodextrin inclusion complex[J]. Food Sci(食品科学), 2019, 40 (20): 28-33

[13]

JIANG

L L

, JIANG

C Z

, MA

, et al. Preparation and pharmacodynamic evaluation of hydrogel containing loratadine inclusion compound[J]. Chin J Pharm(中国医药工业杂志), 2021, 52(6):804-812.

[14]

X X

, PAN

T T

, HONG

Y Q

, et al. Preparation and quality evaluation of tea tree oil microemulsion gel[J]. Chin Tradit Herb Drugs(中草药), 2015, 46 (13): 1892-1900

[15]

CHEN

Q Q

, LIU

, XU

, et al. Effect of different addition ratios of Carbomer 980 and Carbomer 981 on rheological properties of Bizotai gel[J]. Chin Pharm(中国药师), 2019, 22 (1): 57-60

[16]

National Health Commission of the People's Republic of China, the health industry standard of the People's Republic of China Evaluation method of antibacterial and bacteriostatic effect: WS/T 650-2019[S]. 2019.

[17]

General Administration of Quality Supervision, Inspection and Quarantine of the People's Republic of China, China National Standardization Administration Hygienic standard for disposable sanitary articles: GB15979-2002[S]. 2002.

[18]

General Administration of Quality Supervision, Inspection and Quarantine of the People's Republic of China, China National Standardization Administration National Standard of the People's Republic of China Biological Evaluation of Medical Devices Part 10: Irritation and Skin Sensitization Test: GB/T16886.10-2017[S]. 2017.

[19]

X C

, WANG

L F

, FENG

R D

. Biosafety evaluation of silver ion gynecological repair gel[J]. China Med Dev Inf(中国医疗器械信息), 2020, 26 (19): 15-16, 21.

[20]

General Administration of Quality Supervision, Inspection and Quarantine of the People's Republic of China, China National Standardization Administration Biological evaluation of medical devices Part 11: Systemic toxicity test: GB/T1688.11-2011[S]. 2011.

[21]

BALCI

, GUNESOGLU

, BESEN

B S

. Antibacterial finishing of 100% cotton fabric with β-Cyclodextrin-Ozonated olive oil inclusion complex[J]. AATCC J Res, 2016, 3(6): 12-18.

[22]

CUTARELLI

, CARLINI

, SARNO

, et al. The role of ozone carried by liposomes in the therapy of infectious and skin-regenerating ocular surface[J]. J Biomed Sci Eng, 2019, 12(7):347-353.

2024年第59卷第18期

PDF下载

引用本文

BibTeX

文章信息

doi: 10.11669/cpj.2024.18.008

接收时间：2024-04-03
首发时间：2025-12-30
出版时间：2024-09-22

补充材料

相关文章

文章信息

作者

出版历史

收稿日期：2024-04-03

基金

国家自然科学基金项目资助(81960720)

国家自然科学基金项目资助(82260771)

江西中医药大学创新药物研究与开发创新团队项目资助(CXTD22001)

作者信息

¹ 江西中医药大学中药固体制剂制造技术国家工程研究中心, 南昌 330006

² 江西中医药大学中西医结合儿童健康研究院(儿童创新药物研究中心), 南昌 330006

通讯作者:

^* 张国松,男,博士,教授研究方向:药物新剂型与新技术研究 Tel:(0791)87119617;

李东勲,男,博士,副教授研究方向:新药开发及仿制药一致性评价研究 Tel:(0791)87119617

参考文献

分享链接

https://castjournals.cast.org.cn/joweb/zgyxzz/CN/10.11669/cpj.2024.18.008

分享至

全文二维码

扫描看全文

引用本文

BibTeX

本文的引用情况

2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

关闭全屏

BibTeX
EndNote
RefWorks
TxT

Serial Number	Inclusion yield/%	RSD/%
1	96.64	0.71
2	95.82
3	97.18

Serial Number

Inclusion yield/%

RSD/%

96.64

0.71

95.82

97.18

Serial Number	Amount	Consumption volume of Na₂S₂O₃ standard solution/mL	Peroxide value/g·100 g^-1	Mean value/g·100 g^-1	RSD/%
1	2.010 1	124.8	7.88	7.98	1.50
2	2.003 9	128.1	8.11
3	2.003 1	125.3	7.94

Serial Number

Amount

Consumption volume of Na₂S₂O₃ standard solution/mL

Peroxide value/g·100 g^-1

Mean value/g·100 g^-1

RSD/%

2.010 1

124.8

7.88

7.98

1.50

2.003 9

128.1

8.11

2.003 1

125.3

7.94

Serial number	Amount	Consumption volume of Na₂S₂O₃ standard solution/mL	Peroxide value/g·100 g^-1	Mean value/g·100 g^-1	RSD/%
1	1.019 7	6.1	0.76	0.76	2.02
2	1.005 4	6.1	0.77
6	1.008 9	5.9	0.74

Serial number

Amount

Consumption volume of Na₂S₂O₃ standard solution/mL

Peroxide value/g·100 g^-1

Mean value/g·100 g^-1

RSD/%

1.019 7

6.1

0.76

2.02

1.005 4

6.1

0.77

1.008 9

5.9

0.74

Influence factor test	Placement time/d	Appearance	pH	Viscosity /Pa·s	Peroxide value /g·100 g^-1
High temperature	0	Clear, no graininess, shiny, without layering, oil floating, thinning and oil-water separation	5.49±0.01	46.62±0.14	0.146±0.002
	5	Clear, no graininesst, shiny, without layering, oil floating and thinning	5.48±0.02	33.25±0.11	0.114±0.003
	10	Clear, no graininess, shiny, without layering, oil floating and thinning	5.91±0.02	30.27±0.15	0.048±0.003
High humidity	0	Clear, no graininess, shiny, without layering, oil floating, thinning and oil-water separation	5.49±0.01	46.32±0.17	0.146±0.002
	5	Clear, no graininess, shiny, without layering, oil floating, thinning and oil-water separation	5.50±0.02	46.78±0.13	0.143±0.003
	10	Clear, no graininess, shiny, without layering, oil floating, thinning and oil-water separation	5.51±0.01	46.53±0.19	0.141±0.008
Strong light	0	Clear, no graininess, shiny, without layering, oil floating, thinning and oil-water separation	5.49±0.01	46.70±0.16	0.146±0.002
	5	Clear, no graininess, shiny, without layering, oil floating, thinning and oil-water separation	5.48±0.03	46.56±0.18	0.145±0.005
	10	Clear, no graininess, shiny, without layering, oil floating, thinning and oil-water separation	5.50±0.01	46.89±0.13	0.141±0.007

Influence factor
test

Placement
time/d

Appearance

Viscosity
/Pa·s

Peroxide value
/g·100 g^-1

High temperature

Clear, no graininess, shiny, without layering, oil floating, thinning and oil-water separation

5.49±0.01

46.62±0.14

0.146±0.002

Clear, no graininesst, shiny, without layering, oil floating and thinning

5.48±0.02

33.25±0.11

0.114±0.003

Clear, no graininess, shiny, without layering, oil floating and thinning

5.91±0.02

30.27±0.15

0.048±0.003

High humidity

Clear, no graininess, shiny, without layering, oil floating, thinning and oil-water separation

5.49±0.01

46.32±0.17

0.146±0.002

Clear, no graininess, shiny, without layering, oil floating, thinning and oil-water separation

5.50±0.02

46.78±0.13

0.143±0.003

Clear, no graininess, shiny, without layering, oil floating, thinning and oil-water separation

5.51±0.01

46.53±0.19

0.141±0.008

Strong light

Clear, no graininess, shiny, without layering, oil floating, thinning and oil-water separation

5.49±0.01

46.70±0.16

0.146±0.002

Clear, no graininess, shiny, without layering, oil floating, thinning and oil-water separation

5.48±0.03

46.56±0.18

0.145±0.005

Clear, no graininess, shiny, without layering, oil floating, thinning and oil-water separation

5.50±0.01

46.89±0.13

0.141±0.007

Review factors	Appearance	pH	Viscosity /Pa·s	Peroxide value /g·100 g^-1
High-speed centrifuge	Clear, no graininess, shiny, without layering, oil floating, thinning and oil-water separation	5.49±0.01	46.68±0.12	0.149±0.03
Low temperature experiment	Clear, no graininess, shiny, without layering, oil floating, thinning and oil-water separation	5.50±0.01	46.71±0.13	0.153±0.03

Review
factors

Appearance

Viscosity
/Pa·s

Peroxide value
/g·100 g^-1

High-speed centrifuge

Clear, no graininess, shiny, without layering, oil floating, thinning and oil-water separation

5.49±0.01

46.68±0.12

0.149±0.03

Low temperature experiment

Clear, no graininess, shiny, without layering, oil floating, thinning and oil-water separation

5.50±0.01

46.71±0.13

0.153±0.03

Sample	Diameter of bacteriostatic ring of test strain
a	14	16	9
b	9	9	9
c	5	5	5

Sample

Diameter of bacteriostatic ring of test strain

Candida
albicans

Staphylococcus
aureus

Escherichia
coli

Test strain	Average bacteriostatic rate of different time/%	Number of positive control colonies
Candida albicans	77.82	83.24	88.69	89.10	1.26×10³
Staphylococcus aureus	95.97	97.30	98.38	99.18	1.25×10⁴
Escherichia coli	89.40	91.14	94.52	98.92	2.67×10³

Test
strain

Average bacteriostatic
rate of different time/%

Number of
positive control
colonies

2 min

5 min

10 min

20 min

Candida albicans

77.82

83.24

88.69

89.10

1.26×10³

Staphylococcus aureus

95.97

97.30

98.38

99.18

1.25×10⁴

Escherichia coli

89.40

91.14

94.52

98.92

2.67×10³

Reaction	Score	Reaction	Score
Epithele		Vascular hyperemia
Normal, intact	0	Nothing	0
Cell degeneration or flattening	1	Few	1
Tissue deformation	2	Light	2
Local erosion	3	Moderate	3
Extensive erosion	4	Severe vascular rupture	4
Leukocyte infiltration (each high power field)		Edema
Nothing	0	Nothing	0
Very few (less than 25)	1	Few	1
Mild (26-50)	2	Light	2
Moderate (51-100)	3	Moderate	3
Weight (more than 100)	4	Severe	4

Reaction

Score

Reaction

Score

Epithele

Vascular hyperemia

Normal, intact

Nothing

Cell degeneration or flattening

Few

Tissue deformation

Light

Local erosion

Moderate

Extensive erosion

Severe vascular rupture

Leukocyte infiltration (each high power field)

Edema

Nothing

Very few (less than 25)

Few

Mild (26-50)

Light

Moderate (51-100)

Moderate

Weight (more than 100)

Severe

Average score	Reaction degree
0	Nothing
1-4	Extremely light
5-8	Light
9-11	Moderate

Average score

Reaction degree

Nothing

1-4

Extremely light

5-8

Light

9-11

Moderate

Group	Animal number	Microscopic scoring	Average score
Control group	1	0	0	0	0	0
	2	0	0	0	0
	3	0	0	0	0
Ozone oil gel group	1	0	0	0	0	0
	2	0	0	0	0
	3	0	0	0	0
Ozone oil group	1	2	1	2	2	7
	2	2	1	2	2
	3	2	1	2	2

Group

Animal
number

Microscopic scoring

Average
score

Epithele

Leukocyte infiltration

Vascular hyperemia

hydroncus

Control group

Ozone oil gel group

Ozone oil group

Application test reaction	Grade
No obvious change	0
Sporadic or spotted erythema	1
Moderate confluent erythema	2
Severe erythema and edema	3

Application test reaction

Grade

No obvious change

Sporadic or spotted erythema

Moderate confluent erythema

Severe erythema and edema

Group	Application test reaction		Grade
Control group	No obvious change	No obvious change	0
Ozone oil gel group	No obvious change	No obvious change	0

Group

Application test reaction

Grade

Spiloplaxia

Hydroncus

Control group

No obvious change

Ozone oil gel group

No obvious change