CN117482049B - 一种抗肿瘤组合物 - Google Patents
一种抗肿瘤组合物 Download PDFInfo
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- CN117482049B CN117482049B CN202311852245.2A CN202311852245A CN117482049B CN 117482049 B CN117482049 B CN 117482049B CN 202311852245 A CN202311852245 A CN 202311852245A CN 117482049 B CN117482049 B CN 117482049B
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- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
本发明涉及一种抗肿瘤组合物,其为包含甲氨蝶呤和3‑羰基‑23‑羟基‑羽扇豆‑20(29)‑烯‑28‑酸两种药效成分的注射乳剂;将3‑羰基‑23‑羟基‑羽扇豆‑20(29)‑烯‑28‑酸和甲氨蝶呤联合注射到肿瘤小鼠模型中可发现,甲氨蝶呤和3‑羰基‑23‑羟基‑羽扇豆‑20(29)‑烯‑28‑酸对肿瘤有协同抑制功效,可提高低剂量下的肿瘤抑制效果,减少高毒性甲氨蝶呤的用量,降低由甲氨蝶呤所导致的毒副作用。3‑羰基‑23‑羟基‑羽扇豆‑20(29)‑烯‑28‑酸被证实可提高低剂量甲氨蝶呤诱导肿瘤细胞凋亡的能力,可提高对肝肾病患者的用药安全性。
Description
技术领域
本发明涉及医药技术领域,具体涉及一种抗肿瘤组合物。
背景技术
恶性淋巴瘤是具有相当异质性的一大类肿瘤,虽然好发于淋巴结,但是由于淋巴系统的分布特点,使得淋巴瘤属于全身性疾病,几乎可以侵犯到全身任何组织和器官,严重威胁着人类生命健康。甲氨蝶呤的化学名称为L-(+)-N-[4-[[(2,4-二氨基-6-蝶啶基]甲基]甲氨基]苯甲酰基)谷氨酸。四氢叶酸是在体内合成嘌呤核苷酸和嘧啶脱氧核苷酸的重要辅酶,甲氨蝶呤作为一种叶酸还原酶抑制剂,主要抑制二氢叶酸还原酶而使二氢叶酸不能还原成有生理活性的四氢叶酸,甲氨蝶呤还能使嘌呤核苷酸和嘧啶核苷酸的生物合成过程中一碳基团的转移作用受阻,导致DNA的生物合成受到抑制。甲氨蝶呤被证实可用于各型急性白血病,特别是急性淋巴细胞白血病、恶性淋巴瘤、非何杰金氏淋巴瘤和蕈样肉芽肿、多发性骨髓病等。然而,甲氨蝶呤毒性很大,包括胃肠道反应、肝功能损害,包括黄疸、丙氨酸氨基转移酶、碱性磷酸酶、γ谷氨酰转肽酶等增高。大剂量应用时,由于本品和其他代谢产物沉积在肾小管而致高尿酸血症肾病。此时可出现血尿、蛋白质血症甚至尿毒症。长期用药还可引起咳嗽、气短、肺炎或肺纤维化,骨髓抑制等,主要引起白细胞和血小板减少,引起明显骨髓抑制,贫血和血小板下降而致皮肤或内脏出血等。其次是肿瘤对甲氨蝶呤产生的耐药性,剂量越大产生耐药性越快。为提高低剂量甲氨蝶呤的治疗疗效,研发与甲氨蝶呤具有协同抗肿瘤作用的药物成为首要任务。
3-羰基-23-羟基-羽扇豆-20(29)-烯-28-酸是一种五环三萜类化合物,是毛茛科植物白头翁中主要活性成分之一。3-羰基-23-羟基-羽扇豆-20(29)-烯-28-酸,在抗肿瘤方面对多种肿瘤均有抑制作用,对肺癌、肝癌、黑色素瘤、乳腺癌以及卵巢癌等具有治疗作用,可促进人血癌细胞、人早幼粒白血病细胞的凋亡,增强阿霉素抗肿瘤的作用,保护阿霉素所导致的心脏毒性等。然而,目前尚未见到将甲氨蝶呤和3-羰基-23-羟基-羽扇豆-20(29)-烯-28-酸组合在一起用于抗肿瘤治疗的相关报道,更未见到将3-羰基-23-羟基-羽扇豆-20(29)-烯-28-酸用于抗淋巴癌的报道。
发明内容
(一)要解决的技术问题
鉴于现有技术的上述缺点、不足,本发明提供一种抗肿瘤组合物,所述组合物为包含甲氨蝶呤和3-羰基-23-羟基-羽扇豆-20(29)-烯-28-酸两种药效成分的注射乳剂,本发明通过将3-羰基-23-羟基-羽扇豆-20(29)-烯-28-酸与甲氨蝶呤联合使用,有效提高低剂量甲氨蝶呤的治疗疗效,减小甲氨蝶呤对人体肝肾毒性和降低耐药性。
(二)技术方案
第一方面,本发明提供一种抗肿瘤组合物,所述组合物为包含甲氨蝶呤和3-羰基-23-羟基-羽扇豆-20(29)-烯-28-酸两种药效成分的注射乳剂。
优选地,3-羰基-23-羟基-羽扇豆-20(29)-烯-28-酸与甲氨蝶呤质量比为20-60:10。
优选地,3-羰基-23-羟基-羽扇豆-20(29)-烯-28-酸与甲氨蝶呤质量比为30-50:10。
优选地,所述抗肿瘤组合物为抗淋巴肿瘤药物。
优选地,所述抗肿瘤组合物还包含植物油、乳化剂、甘油、抗氧剂和注射用水。
优选地,所述植物油为玉米油、大豆油或芝麻油;所述乳化剂为豆磷脂或蛋黄卵磷脂;所述抗氧剂为亚硫酸钠与维生素E或维生素C的组合。
优选地,所述组合物的制备方法为:
S1、将植物油加热至温度60-80℃,在保温条件下,加入乳化剂、3-羰基-23-羟基-羽扇豆-20(29)-烯-28-酸、甲氨蝶呤后,在10000-15000rpm剪切10-30min使溶解;
S2、将注射用水加入水相罐,温度60-80℃,加入甘油,搅拌均匀,保温;
S3、剪切条件下,将S1得到的油相缓慢加入S2制得的水相中,加完后继续剪切15-30min;加入抗氧剂后,再剪切搅拌3-10min,之后加注射用水定容,搅拌均匀;再转移至高压均质机中,1600-2000bar均质5-7次,控制乳剂粒径D80在0.1-0.3μm,然后经精滤、充氮灌装、密封;121℃旋转灭菌12-30min。
优选地,所述抗肿瘤组合物含有1-2.5%甲氨蝶呤、2.5-15%的3-羰基-23-羟基-羽扇豆-20(29)-烯-28-酸、12.5-15%植物油、1-3%乳化剂、2-5%抗氧剂、5-10%甘油、余量为注射用水。
第二方面,本发明还提供3-羰基-23-羟基-羽扇豆-20(29)-烯-28-酸在制备抗淋巴肿瘤药物中的应用,将甲氨蝶呤和3-羰基-23-羟基-羽扇豆-20(29)-烯-28-酸共同作为抗淋巴肿瘤治疗药物的药效成分。
(三)有益效果
本发明的主要技术效果在于:采用淋巴癌模型小鼠及细胞(人淋巴癌细胞RAJI或U937)实验证明:通过将甲氨蝶呤和3-羰基-23-羟基-羽扇豆-20(29)-烯-28-酸组合用于抗肿瘤治疗,对淋巴癌细胞有协同抑制功效,利用低毒性的3-羰基-23-羟基-羽扇豆-20(29)-烯-28-酸联合治疗,可增强低剂量甲氨蝶呤诱导的淋巴细胞凋亡作用,进而减少甲氨蝶呤的施药量和肝肾毒副作用及耐药性的形成。
附图说明
图1为3-羰基-23-羟基-羽扇豆-20(29)-烯-28-酸(2.5-15mg/kg)联合甲氨蝶呤(1mg/kg)对淋巴癌小鼠肿瘤生长的影响实验结果。
图2为3-羰基-23-羟基-羽扇豆-20(29)-烯-28-酸(2.5-15mg/kg)联合甲氨蝶呤(2.5mg/kg)对淋巴癌小鼠肿瘤生长的影响实验结果。
图3为3-羰基-23-羟基-羽扇豆-20(29)-烯-28-酸增强甲氨蝶呤诱导的凋亡作用的实验结果图。
具体实施方式
为了更好的解释本发明,以便于理解,下面结合附图,通过具体实施方式,对本发明作详细描述。
在本发明接下来的实施例中3-羰基-23-羟基-羽扇豆-20(29)-烯-28-酸联合甲氨蝶呤对小鼠淋巴癌的协同抑制效果采用金正均Q值法验证,对肿瘤细胞增殖协同抑制效果采用联合指数(combined index,CI)法验证。两种效果评价方法中,统计学分析采用SPSS21.0统计软件进行分析,多组均数比较采用单因素分析,P<0.05为差异有统计学意义。
动物实验药物协同抑制效果评价方法:采用金正均Q值法:
Q=Ea+b/(Ea+Eb-Ea*Eb);
其中,Ea+b为A药和B药联合时的抑制率,Ea和Eb分别是A药和B药单独用药时的抑制率。Q<0.85为拮抗作用,0.85≤Q<1.15为相加作用,Q≥1.15为协同作用。
实施例1
本实施例验证3-羰基-23-羟基-羽扇豆-20(29)-烯-28-酸联合甲氨蝶呤在淋巴癌小鼠中的协同抗肿瘤作用。
实验方法为:将L5178Y细胞(小鼠淋巴癌细胞)在体外培养两代后,以5×106个/只接种于实验动物雄性balb/c(体重在25g左右)小鼠右腋部皮下。当平均肿瘤体积约为100mm3时,将荷瘤小鼠随机分为四组进行后续治疗或干预,其中一组为空白组。治疗方式包括:每日腹腔注射单独的3-羰基-23-羟基-羽扇豆-20(29)-烯-28-酸和甲氨蝶呤的乳剂,或每日腹腔注射包含3-羰基-23-羟基-羽扇豆-20(29)-烯-28-酸和甲氨蝶呤两种有效成分的乳剂,空白组注射等量空白乳剂。注射乳剂的制备方法如下:
(1)将植物油加热至温度65℃,在保温条件下,加入乳化剂和甲氨蝶呤(或3-羰基-23-羟基-羽扇豆-20(29)-烯-28-酸或两种一起加入)后,在10000rpm剪切30min使溶解;
(2)将注射用水加入水相罐,温度65℃,加甘油,搅拌均匀,保温;
(3)剪切条件下,将(1)制备的油相缓慢加入(2)制备的水相中,加完后继续在10000rpm剪切20min;加入抗氧剂后,再剪切搅拌5min,之后加注射用水定容,搅拌均匀;再转移至高压均质机中,1600bar均质5次,控制乳剂粒径D80在0.15μm,然后经精滤、充氮灌装、密封;121℃旋转灭菌20min。
其中,植物油为玉米油,在注射乳剂中含量为13%,乳化剂为蛋黄卵磷脂,在注射乳剂中含量为2%,抗氧剂为亚硫酸钠与维生素C按质量2:1的组合,在注射乳剂中含量为2.5%;甘油在注射乳剂中含量为6%,其余量为注射用水。抗氧剂亚硫酸钠的pH为9-9.5,该pH范围有利于保持甲氨蝶呤在注射乳剂中的稳定。空白组注射的乳剂载药量为0。
每组的实验动物均为10只,每3天测定动物体重。所有小鼠均在无特定病原体(SPF)条件下饲养,自由饮水和进食。每两天测量一次肿瘤体积。18天后,称量小鼠体重后脱颈处死,剥取瘤块组织及肝脏、肾脏进行称重,抑瘤率的测定按计算公式:肿瘤抑制率(%)=(阴性对照组瘤重一实验组瘤重)/阴性对照组瘤重×100%。
评价两药协同作用采用金正均Q值法: Q = Ea+b/(Ea+Eb-Ea*Eb) 。
实验结果见表1-3所示及图1-2所示。
图1为3-羰基-23-羟基-羽扇豆-20(29)-烯-28-酸(2.5-15mg/kg)联合甲氨蝶呤(1mg/kg)对L5178Y淋巴癌小鼠肿瘤生长的影响,图2为3-羰基-23-羟基-羽扇豆-20(29)-烯-28-酸(2.5-15mg/kg)联合甲氨蝶呤(2.5mg/kg)对L5178Y淋巴癌小鼠肿瘤生长的影响。
表1:3-羰基-23-羟基-羽扇豆-20(29)-烯-28-酸联合甲氨蝶呤(1mg/kg)对L5178Y淋巴癌小鼠瘤重的影响(x±s,n=10)
组别 | 瘤重(g) | 抑瘤率(%) | 金正均Q值 |
模型组 | 2.69±0.93 | \ | \ |
甲氨蝶呤 (1mg/kg) ip 组 | 1.89±0.23** | 29.74% | \ |
3-羰基-23-羟基-羽扇豆-20(29)-烯-28-酸 (2.5mg/kg) ip组 | 2.34±0.56 | 13.01% | \ |
3-羰基-23-羟基-羽扇豆-20(29)-烯-28-酸 (5mg/kg) ip组 | 2.21±0.51 | 17.84% | \ |
3-羰基-23-羟基-羽扇豆-20(29)-烯-28-酸 (10mg/kg) ip 组 | 2.17±0.47* | 19.33% | \ |
3-羰基-23-羟基-羽扇豆-20(29)-烯-28-酸 (15mg/kg) ip 组 | 2.08±0.24** | 22.68% | \ |
3-羰基-23-羟基-羽扇豆-20(29)-烯-28-酸(2.5mg/kg)+甲氨蝶呤(1mg/kg) ip组 | 1.41±0.29** | 47.58% | 1.223813755 |
3-羰基-23-羟基-羽扇豆-20(29)-烯-28-酸(5mg/kg)+甲氨蝶呤(1mg/kg) ip组 | 1.23±0.15** | 54.28% | 1.283799686 |
3-羰基-23-羟基-羽扇豆-20(29)-烯-28-酸(10mg/kg)+甲氨蝶呤(1mg/kg) ip组 | 1.16±0.06** | 56.88% | 1.312906725 |
3-羰基-23-羟基-羽扇豆-20(29)-烯-28-酸(15mg/kg)+甲氨蝶呤(1mg/kg) ip组 | 1.02±0.04** | 62.08% | 1.359284698 |
注:*与模型组比较,*p<0.05,**p<0.01。
表2:3-羰基-23-羟基-羽扇豆-20(29)-烯-28-酸联合甲氨蝶呤(2.5mg/kg)对L5178Y淋巴癌小鼠瘤重的影响(x±s,n=10)
组别 | 瘤重(g) | 抑瘤率(%) | 金正均Q值 |
模型组 | 2.81±0.73 | \ | \ |
甲氨蝶呤 (2.5mg/kg) ip组 | 1.60±0.19* | 43.06% | \ |
3-羰基-23-羟基-羽扇豆-20(29)-烯-28-酸 (2.5mg/kg) ip组 | 2.44±0.66 | 13.17% | \ |
3-羰基-23-羟基-羽扇豆-20(29)-烯-28-酸 (5mg/kg) ip组 | 2.28±0.61 | 18.86% | \ |
3-羰基-23-羟基-羽扇豆-20(29)-烯-28-酸 (10mg/kg) ip 组 | 2.18±0.49* | 22.42% | \ |
3-羰基-23-羟基-羽扇豆-20(29)-烯-28-酸 (15mg/kg) ip 组 | 2.13±0.25** | 24.20% | \ |
3-羰基-23-羟基-羽扇豆-20(29)-烯-28-酸(2.5mg/kg)+甲氨蝶呤(2.5mg/kg)ip组 | 1.17±0.68* | 58.36% | 1.1543799 |
3-羰基-23-羟基-羽扇豆-20(29)-烯-28-酸(5mg/kg)+甲氨蝶呤(2.5mg/kg)ip组 | 1.03±0.45** | 63.35% | 1.177420494 |
3-羰基-23-羟基-羽扇豆-20(29)-烯-28-酸(10mg/kg)+甲氨蝶呤(2.5mg/kg)ip组 | 0.88±0.24** | 68.68% | 1.230303305 |
3-羰基-23-羟基-羽扇豆-20(29)-烯-28-酸(15mg/kg)+甲氨蝶呤(2.5mg/kg)ip组 | 0.67±0.34** | 76.16% | 1.339854281 |
注:*与模型组比较,*p<0.05,**p<0.01。
表3:3-羰基-23-羟基-羽扇豆-20(29)-烯-28-酸联合甲氨蝶呤对L5178Y淋巴癌小鼠脏器指数的影响(x±s, n=10);
组别 | 肝脏指数 | 肾脏指数 |
正常组 | 3.10±0.33** | 1.21±0.05 |
模型组 | 8.16±0.86 | 1.87±0.07 |
3-羰基-23-羟基-羽扇豆-20(29)-烯-28-酸(5mg/kg)ip组 | 3.65±0.47 | 1.27±0.07 |
3-羰基-23-羟基-羽扇豆-20(29)-烯-28-酸(10mg/kg)ip组 | 3.76±0.54 | 1.31±0.06 |
3-羰基-23-羟基-羽扇豆-20(29)-烯-28-酸(15mg/kg)ig组 | 4.05±0.26 | 1.33±0.05 |
甲氨蝶呤(2.5mg/kg)组 | 5.01±0.12** | 1.45±0.08* |
甲氨蝶呤(2.5mg/kg)+3-羰基-23-羟基-羽扇豆-20(29)-烯-28-酸(15mg/kg)组 | 4.91±0.56** | 1.37±0.06** |
注:*与模型组比较,*p<0.05,**p<0.01。其中,肝脏指数(Liver index,LI)=肝脏重量(mg)/体重(g);肾脏指数(Kidney index,KI)=肾脏重量(mg)/体重(g)。
以上实验结果提示:与甲氨蝶呤组比较,联合用药组的瘤重显著降低,且联合用药组的金正均Q值≥1.15,表明两药具有协同抗肿瘤作用。同时,根据肝脏指数和肾脏指数可看出,联合用药组与单独注射甲氨蝶呤组相比,肝脏指数和肾脏指数无显著变化,表明联用组未见有任何毒性增强的趋势。此外,细胞试验结果也明显地支持了所述两种药物具有协同抗淋巴瘤的功效。
实施例2
本实施例验证3-羰基-23-羟基-羽扇豆-20(29)-烯-28-酸与甲氨蝶呤联合使用对RAJI细胞 (人淋巴癌细胞)的协同抑制作用。验证方法如下:取对数生长期RAJI细胞,调整密度为2×105个/mL,每孔 l00 μL接种于96孔板中,继续培养24h后,加入不同浓度的药物,每组设置3个复孔。对照组加入终浓度为0.5%的DMSO,空白组不加RAJI细胞和DMSO。
将96孔板置 37℃,5% CO2培养箱培养48h后,每孔加入10 μL的CCK-8溶液,37℃、5%CO2培养箱内孵育1h,酶标仪于450 nm处测定其OD值并计算抑制率。计算抑制率:抑制率=[1-(实验组平均OD值-空白组平均OD值)/(对照组平均OD值-空白组平均OD值)]×100%。验结果见表4、5。
细胞实验药物协同抑制效果评价方法:
联合指数(combined index,CI)计算方法:
;
其中,(D)1、(D)2为两种药物联用使增殖抑制率达X时的药物浓度;(Dx)1、(Dx)2是两种药物单独使用使增殖抑制率达X时的药物浓度。运用Calcμsyn2.0软件分析药物协同作用:CI<1表示两药相互协同;CI=1表示两药作用相加;CI>1表示两药作用拮抗;CI<0.3表示两药协同作用强。
表4:3-羰基-23-羟基-羽扇豆-20(29)-烯-28-酸和甲氨蝶呤单独对RAJI细胞增殖作用的抑制效应(x±s,n=6)
表5:3-羰基-23-羟基-羽扇豆-20(29)-烯-28-酸和甲氨蝶呤联合对RAJI细胞增殖作用的协同抑制效应(x±s,n=6)
以上实验结果提示:3-羰基-23-羟基-羽扇豆-20(29)-烯-28-酸与甲氨蝶呤联合使用比单独使用甲氨蝶呤更能抑制肿瘤细胞生长,并且联合抑制指数CI值均小于1,表明3-羰基-23-羟基-羽扇豆-20(29)-烯-28-酸与甲氨蝶呤两种药物之间具有协同作用。
实施例3
本实施例验证3-羰基-23-羟基-羽扇豆-20(29)-烯-28-酸可以增强甲氨蝶呤诱导的凋亡作用。本实施例将Annexin V与PI匹配使用,可将处于不同凋亡时期的细胞区分开来。Annexin V细胞凋亡检测试剂盒购自联科生物科技有限公司,按照试剂盒说明书进行操作,步骤如下:
①接种细胞于6孔细胞培养板中;
②37℃环境5%CO2,浓度的培养箱中培养24h;
③药物处理细胞,处理方式分为以下五组:
第一组:3-羰基-23-羟基-羽扇豆-20(29)-烯-28-酸(5μg/mL);
第二组:甲氨蝶呤(1.8μg/mL);
第三组:3-羰基-23-羟基-羽扇豆-20(29)-烯-28-酸(5μg/mL)+甲氨蝶呤(1.8μg/mL);
第四组:甲氨蝶呤(5μg/mL);
第五组:PBS处理的空白组(Ctrl组)。
④37℃环境,5%CO2浓度的培养箱中继续培养48h;
⑤收集细胞上清液,并用PBS清洗细胞洗2-3次,胰酶消化2min;
⑥在1000rpm转速下,4℃离心5min;
⑦采用PBS清洗细胞1次;
⑧在1000rpm转速下,4℃离心5min;
⑨弃上清,细胞重悬于300μl的Binding Bμffer结合缓冲液中;
⑩加入10μlAnnexinV-FITC和5μlPI,轻轻混匀;
⑪避光室温反应5min;
⑫最后进行流式细胞仪测定;
结果判定:细胞凋亡早期会发生PS外漏,但其细胞膜完整,因此为FITC标记的AnnexinV染色阳性,即FITC+/PI-,位于流式细胞检测图的右下象限;晚期凋亡或者死亡细胞的细胞膜崩解,AnnexinV不能结合于细胞膜上,表现为FITC-/PI+,位于右上象限;而左下象限为非凋亡的活细胞,表现为FITC-/PI-。结果如图3所示:3-羰基-23-羟基-羽扇豆-20(29)-烯-28-酸能够显著增强甲氨蝶呤对人淋巴癌U937细胞诱导凋亡的作用,使用低剂量(1.8μg/mL)的甲氨蝶呤联合低毒性的3-羰基-23-羟基-羽扇豆-20(29)-烯-28-酸,在体外诱导人淋巴癌U937细胞的凋亡小效果优于高剂量(4.0μg/mL)的甲氨蝶呤,由此可增强低剂量甲氨蝶呤诱导的淋巴细胞凋亡作用,进而减少甲氨蝶呤的施药量和肝肾毒副作用及耐药性的形成。图示为在3-羰基-23-羟基-羽扇豆-20(29)-烯-28-酸的干预下,甲氨蝶呤对人淋巴癌U937细胞凋亡的影响 (x±s, n=8);与正常组比较,**P<0.01;与低剂量甲氨蝶呤组比较,## P<0.01;与高剂量(4.0μg/mL)甲氨蝶呤组比较,***P<0.01。
最后应说明的是:以上各实施例仅用以说明本发明的技术方案,而非对其限制;尽管参照前述各实施例对本发明进行了详细的说明,本领域的普通技术人员应当理解:其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分或者全部技术特征进行等同替换;而这些修改或者替换,并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。
Claims (3)
1.一种抗肿瘤组合物,其特征在于,所述抗肿瘤组合物为抗淋巴肿瘤的注射乳剂,所述组合物的药效成分为甲氨蝶呤和3-羰基-23-羟基-羽扇豆-20(29)-烯-28-酸两种药效成分;其中,3-羰基-23-羟基-羽扇豆-20(29)-烯-28-酸与甲氨蝶呤质量比为20-60:10;所述抗肿瘤组合物还包含植物油、乳化剂、甘油、抗氧剂和注射用水;所述植物油为玉米油,所述乳化剂为蛋黄卵磷脂,所述抗氧剂为亚硫酸钠与维生素C的组合;
所述组合物的制备方法为:
S1、将植物油加热至温度60-80℃,在保温条件下,加入乳化剂、3-羰基-23-羟基-羽扇豆-20(29)-烯-28-酸、甲氨蝶呤后,在10000-15000rpm剪切10-30min使溶解;
S2、将注射用水加入水相罐,温度60-80℃,加入甘油,搅拌均匀,保温;
S3、剪切条件下,将S1得到的油相缓慢加入S2制得的水相中,加完后继续剪切15-30min;加入抗氧剂后,再剪切搅拌3-10min,之后加注射用水定容,搅拌均匀;再转移至高压均质机中,1600-2000bar均质5-7次,控制乳剂粒径D80在0.1-0.3μm,然后经精滤、充氮灌装、密封;121℃旋转灭菌12-30min。
2.根据权利要求1所述的抗肿瘤组合物,其特征在于,3-羰基-23-羟基-羽扇豆-20(29)-烯-28-酸与甲氨蝶呤质量比为30-50:10。
3.根据权利要求1所述的抗肿瘤组合物,其特征在于,所述抗肿瘤组合物按质量百分比计,其组成为:1-2.5%甲氨蝶呤、2.5-15%的3-羰基-23-羟基-羽扇豆-20(29)-烯-28-酸、12.5-15%植物油、1-3%乳化剂、2-5%抗氧剂、5-10%甘油,余量为注射用水。
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