WO2016150355A1 - 提高绿原酸生物利用度的绿原酸酰化物及应用 - Google Patents

提高绿原酸生物利用度的绿原酸酰化物及应用 Download PDF

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WO2016150355A1
WO2016150355A1 PCT/CN2016/076820 CN2016076820W WO2016150355A1 WO 2016150355 A1 WO2016150355 A1 WO 2016150355A1 CN 2016076820 W CN2016076820 W CN 2016076820W WO 2016150355 A1 WO2016150355 A1 WO 2016150355A1
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chlorogenic acid
acylate
bioavailability
acid acylate
group
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PCT/CN2016/076820
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French (fr)
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张洁
黄望
张亮
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四川九章生物科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/216—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acids having aromatic rings, e.g. benactizyne, clofibrate

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  • the invention belongs to the field of biomedicine, and particularly relates to a chlorogenic acid acylate and application thereof for improving the bioavailability of chlorogenic acid.
  • Chlorogenic acid (English: Chlorogenic acid) is also known as caffeic acid. That is, 3-caffeoylquinic acid, which is a polyphenolic compound, is widely found in higher dicotyledons and ferns, and is mainly found in Lonicera and Artemisia plants of Lonicerae. The higher content plants are mainly Eucommia, Honeysuckle, Sunflower, Wood, Coffee, Cocoa and the like.
  • Chlorogenic acid has anti-inflammatory, anti-viral, anti-tumor, immune-enhancing effects, and is unstable under acid, alkali, light and high temperature conditions.
  • Zhou Hongqi, Olthof MR and Gonthier MP reported that chlorogenic acid is unstable. Phenolic compounds, and are susceptible to factors such as digestive flora, and the bioavailability of oral administration is low.
  • Margreet et al. further confirmed in the rat ileostomy test that rats administered orally were absorbed into the bloodstream in the small intestine only one-third of the dose administered. This experimental phenomenon is related to the intestinal flora. Metabolism is closely related; therefore, chlorogenic acid is difficult to exert its pharmacological effects by oral administration.
  • the present inventors provide a chlorogenic acid acylate which improves the bioavailability of chlorogenic acid and an application thereof, and improves the bioavailability and antitumor effect of oral administration in antitumor.
  • R 1 , R 2 , R 3 , R 4 , and R 5 are RCO + functional groups or H + ions, and the RCO + functional group is at any one of R 1 , R 2 , R 3 , R 4 , and R 5 positions or In the RCO + functional group, R is an alkyl group having 1 to 6 carbon atoms.
  • the R may be a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, a hexyl group, an isopropyl group, an isobutyl group or the like.
  • R 1 , R 2 and R 3 are H + ions, wherein R 4 and R 5 are C 2 H 3 O + .
  • the chlorogenic acid acylate of such a structure can improve bioavailability and enhance drug efficacy.
  • the medicament is an oral preparation prepared by adding chlorogenic acid acylate as an active ingredient and adding one or more pharmaceutically acceptable pharmaceutical excipients.
  • the oral preparation is a tablet, a capsule, a granule, a powder, a pill, or an oral solution.
  • the unit preparation of the oral preparation contains 1-10 mg of chlorogenic acid acylate, and the unit preparation contains 10-200 mg of chlorogenic acid acylate in an optimized scheme.
  • the preparation method of the chlorogenic acid acylate in the present invention is as follows:
  • the chlorogenic acid and the solvent are stirred and mixed in a vessel, and the catalyst is added dropwise, and then an acetylating agent is added to react, dried, dissolved in a solvent, washed with water to remove other impurities, and dried again to obtain a chlorogenic acid acylate.
  • dimethylformamide (DMF) is stirred magnetically in a container, chlorogenic acid is added, triethylamine is added dropwise, then acetic anhydride is added, dried in vacuum to dryness, dissolved in ethanol, and poured into high-speed stirred water to precipitate. After filtration and drying, the acylated chlorogenic acid, ie, chlorogenic acid acylate is obtained; or DMF is electromagnetically stirred, chlorogenic acid is added, pyridine is added dropwise, and then an appropriate amount of acetic anhydride is added to react, and the mixture is dried to dryness in ethyl acetate. After dissolving, it was washed three times with distilled water, and the ethyl acetate was separated and dried to obtain chlorogenic acid in which the phenolic hydroxyl group was acetylated.
  • DMF dimethylformamide
  • the chlorogenic acid acylate in the invention can increase the blood concentration of chlorogenic acid in the blood after oral administration, that is, can improve the bioavailability of chlorogenic acid in the body, and solve the chlorogenic acid in the intestine when administered orally.
  • the effect of the flora is low, and the absorption rate is low; at the same time, the inhibitory effect of chlorogenic acid on the tumor is significantly improved.
  • Figure 1 is a graph showing the concentration of chlorogenic acid in canine serum of the present invention
  • Figure 2 is a graph showing tumor growth curve in the present invention
  • a chlorogenic acid acylate for improving the bioavailability of chlorogenic acid which has the following structural formula:
  • R 1 , R 2 , R 3 , R 4 , and R 5 are RCO + functional groups or H + ions, and the RCO + functional group is at any one of R 1 , R 2 , R 3 , R 4 , and R 5 positions or several.
  • R is an alkyl group having 1 to 6 carbon atoms.
  • the chlorogenic acid acylate of such a structure can improve bioavailability and enhance drug efficacy.
  • the medicament is an oral preparation prepared by adding chlorogenic acid acylate as an active ingredient and adding one or more pharmaceutically acceptable pharmaceutical excipients.
  • the oral preparation is a tablet, a capsule, a granule, a powder, a pill, or an oral solution.
  • the unit preparation of the oral preparation contains 1-10 mg of chlorogenic acid acylate, and the unit preparation contains 10-200 mg of chlorogenic acid acylate in an optimized scheme.
  • the preparation method of the chlorogenic acid acylate in the present invention is as follows:
  • the chlorogenic acid and the solvent are stirred and mixed in a vessel, and the catalyst is added dropwise, and then an acetylating agent is added to react, dried, dissolved in a solvent, washed with water to remove other impurities, and dried again to obtain a chlorogenic acid acylate.
  • dimethylformamide (DMF) is stirred magnetically in a container, chlorogenic acid is added, triethylamine is added dropwise, then acetic anhydride is added, dried in vacuum to dryness, dissolved in ethanol, and poured into high-speed stirred water to precipitate. After filtration and drying, the acylated chlorogenic acid, ie, chlorogenic acid acylate is obtained; or DMF is electromagnetically stirred, chlorogenic acid is added, pyridine is added dropwise, and then an appropriate amount of acetic anhydride is added to react, and the mixture is dried to dryness in ethyl acetate. After dissolving, it was washed three times with distilled water, and the ethyl acetate was separated and dried to obtain chlorogenic acid in which the phenolic hydroxyl group was acetylated.
  • DMF dimethylformamide
  • chlorogenic acid commercially available; chlorogenic acid acylate: R 1 , R 2 and R 3 are H + ions, wherein R 4 and R 5 are C 2 H 3 O + , labeled as sample 1; R 1 , R 2 and R 3 are C 3 H 5 O + , wherein R 4 and R 5 are H + ions, which are labeled as sample 2; R 1 , R 4 and R 5 are H + , wherein R 2 and R 3 are C 7 H 5 O + ion, labeled as sample 3.
  • Sample 1 can be prepared by: 100 ml DMF electromagnetic stirring, adding 50 g of chlorogenic acid, constant temperature 10 ° C, adding 0.1 ml of pyridine, adding 10 ml of acetic anhydride for 24 hours, drying at 80 ° C for 12 hours to dry, in 100 ml of acetic acid After dissolving in the ethyl ester (room temperature), it was washed three times with 20 ml of distilled water, and the ethyl acetate was separated and dried to obtain chlorogenic acid in which the phenolic hydroxyl group was acetylated at the 4th and 5th positions.
  • Samples 2 and 3 may be a similar or close preparation method as in Sample 1.
  • Preparation of the reference solution Take the appropriate amount of chlorogenic acid reference substance, accurately weighed, dissolved in methanol-0.2% phosphoric acid (20:80) solution and make a solution containing about 8mg per 1ml as a reference solution for chlorogenic acid. .
  • the refrigerator is refrigerated (the reserve is stable for 1 month) and diluted before use.
  • Preparation of the internal standard reference solution Take the appropriate amount of puerarin reference substance, accurately weigh it, dissolve it with methanol and make a solution containing about 84 ⁇ g per 1 ml, as an internal standard solution, and refrigerate in the refrigerator.
  • test solution Take 100 ⁇ l of serum, add 100 ⁇ l of methanol-0.2% phosphoric acid (80:20) solution, internal standard solution and methanol, vortex for 3 min, let stand, centrifuge (12000 rpm) for 10 min, remove the supernatant, As a serum test solution.
  • the content of chlorogenic acid in serum was calculated by the internal standard method using the internal standard method.
  • chlorogenic acid acylate can significantly increase the concentration of drugs in the blood and increase green by intragastric administration.
  • mice BALB/C-nu mice, weighing 18-24 g, were provided by the Experimental Animal Center of Sichuan University.
  • R 1 , R 2 and R 3 are H + ions, wherein R 4 and R 5 are C 2 H 3 O + , labeled as sample 1; 1 , R 2 and R 3 are C 3 H 5 O + , wherein R 4 and R 5 are H + ions, which are labeled as sample 2; R 1 , R 2 , R 4 and R 5 are H + ions, wherein R 3 For C 7 H 5 O + , labeled as sample 3.
  • Cell culture SPCA-1 cells are human lung adenocarcinoma cell lines. The cells are routinely resuscitated and passaged. The second-generation mouse tumors are taken and washed with Hank's solution for 3 times to remove blood stains, fat and necrotic tissue, and the tumor is cut. Cut into 1mm ⁇ 1mm ⁇ 1mm pieces, Hank's solution was washed twice, proportionally added physiological saline (1g: 3ml), and then ground in a glass homogenizer, filtered through 80-100 mesh screen to make single cells The suspension was counted by trypan blue staining.
  • the average tumor of the negative group was greater than 1.0 g (the tumor volume was about 0.5 cm 3 )
  • the experiment was stopped, the mice were sacrificed by cervical dislocation and weighed, and the tumor was removed.
  • Tumor inhibition rate% [1-(mean tumor weight of the administration group/average tumor weight of the negative group)] ⁇ 100%;
  • Chlorogenic acid acylate sample 1 Chlorogenic acid acylate sample 2 Chlorogenic acid acylate sample 3 Chlorogenic acid control group Negative control group 6d 0.272 ⁇ 0.306*# 0.190 ⁇ 0.051*# 0.259 ⁇ 0.117*# 0.451 ⁇ 0.420 ⁇ 0.679 ⁇ 0.243 8d 0.353 ⁇ 0.377*# 0.210 ⁇ 0.058*# 0.353 ⁇ 0.194*# 0.598 ⁇ 0.571 ⁇ 0.744 ⁇ 0.439 10d 0.595 ⁇ 0.503*# 0.533 ⁇ 0.219*# 0.643 ⁇ 0.307*# 0.860 ⁇ 0.372 ⁇ 1.002 ⁇ 0.405 12d 0.782 ⁇ 0.727*# 0.704 ⁇ 0.276*# 0.897 ⁇ 0.416*# 1.224 ⁇ 1.009 ⁇ 1.405 ⁇ 0.300 14d 0.857 ⁇ 0.783*# 0.791 ⁇ 0.340*# 1.012 ⁇ 0.560*# 1.252 ⁇ 1.172 ⁇ 1.499 ⁇ 0.795 16d 1.006 ⁇ 0.912*# 1.054 ⁇ 0.471*# 1.128
  • chlorogenic acid significantly inhibited the growth curve of SPLA-1 lung cancer BALB/C-nu mouse xenografts from day 6 to day 14 after administration (p ⁇ 0.05). The inhibitory effect was not significant after the 14th day after administration.
  • chlorogenic acid acylate sample 1, sample 2 and sample 3 On the 6th day after the administration of chlorogenic acid acylate sample 1, sample 2 and sample 3, the growth curve of SPLA-1 lung cancer BALB/C-nu mice had a significant inhibitory effect, and its inhibitory effect was observed. It is significantly higher than the chlorogenic acid group; it shows that chlorogenic acid acylate has better anti-tumor effect than chlorogenic acid by intragastric administration.
  • Chlorogenic acid acylate sample 1, sample 2 and sample 3 have higher inhibition rate of SPCA-1 lung cancer BALB/C-nu mouse xenografts, and have significant advantages compared with the chlorogenic acid control group, further illustrating the green Acid acylate can enhance the anti-tumor effect.
  • Chlorogenic acid acylate 1 R 1 , R 2 and R 3 are H + ions, wherein R 4 and R 5 are C 2 H 3 O +
  • a chlorogenic acid acylate tablet is prepared by a wet granule tableting method.
  • (1) Prepare an aqueous solution of hypromellose according to the prescription; (2) mix the prescribed amount of chlorogenic acid acylate, starch and lactose, add hypromellose aqueous solution, stir well and make it.
  • (3) The prepared soft material is subjected to conventional wet granulation operation procedure, sieved, dried and granulated to obtain granules of suitable size; (4) the prepared granules are mixed with magnesium stearate. After uniform tableting, a total of 1000 tablets were prepared, each containing 100 mg of chlorogenic acid acylate.
  • Chlorogenic acid acylate 2 R 4 and R 5 are H + ions, wherein R 1 , R 2 and R 3 are C 2 H 3 O +
  • each capsule contains chlorogenic acid 50mg.
  • Chlorogenic acid acylate 3 R 1 , R 2 , R 4 , R 5 are H + ions, wherein R 3 is C 7 H 5 O +
  • Povidone K30 was taken and made into a solution with water. After the prescription amount of chlorogenic acid acylate, mannitol and sucrose were uniformly mixed, the povidone K30 solution was added to prepare a soft material. According to the conventional preparation process of granules, the soft materials are sieved, dried and finished. After the granules, granules are obtained. The granules were dispensed under aseptic conditions to prepare 400 bags of granules each containing 500 mg of chlorogenic acid acylate.
  • Chlorogenic acid acylate 4 R 1 , R 2 , R 4 , R 5 are H + ions, wherein R 3 is C 2 H 3 O +
  • Chlorogenic acid acylate 5 R 1 , R 4 , and R 5 are H + ions, wherein R 2 and R 3 are C 2 H 3 O +
  • Chlorogenic acid acylate 6 R 3 , R 4 , R 5 are H + ions, wherein R 1 and R 2 are C 4 H 7 O +
  • each oral liquid is 10 mL containing 200 mg of chlorogenic acid acylate.
  • the invention provides a chlorogenic acid acylate, which can increase the blood concentration of chlorogenic acid in blood after oral administration, suggesting that the chlorogenic acid acylate can improve the bioavailability of chlorogenic acid in vivo.
  • the chlorogenic acid acylate can significantly improve the antitumor effect by oral administration, indicating that the chlorogenic acid acylate has a synergistic effect in the field of treating tumors.

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Abstract

本发明提供一种绿原酸酰化物及其应用。具有本发明结构式的绿原酸酰化物可提高绿原酸的生物利用度，可用于制备治疗肿瘤的药物。

Description

提高绿原酸生物利用度的绿原酸酰化物及应用 技术领域
本发明属于生物医药领域，尤其涉及提高绿原酸生物利用度的绿原酸酰化物及应用。
背景技术
绿原酸(英语：Chlorogenic acid)亦称咖啡鞣酸。即3-咖啡酰奎宁酸，为多酚类化合物，广泛存在于高等双子叶植物和蕨类植物中，主要存在于忍冬科忍冬属(Lonicera)、菊科蒿属(Artemisia)植物中，其中含量较高的植物主要为杜仲、金银花、向日葵、继木、咖啡、可可树等。
绿原酸具有消炎、抗病毒、抗肿瘤、提高免疫等作用，在酸、碱、光照和高温条件下不稳定，周宏灏、Olthof MR和Gonthier MP等文献报道表明，绿原酸属不稳定的多酚类化合物，且易受到消化道菌群等因素的影响，口服给药的生物利用度较低。Margreet等在大鼠回肠造口术试验中进一步证实了，经口服给药的大鼠，在小肠吸收进入血液循环的只有给药剂量的三分之一，这一实验现象与肠道菌群的代谢有密切关系；因此绿原酸通过口服给药的方式，很难充分发挥其药效作用。
发明内容
为了解决上述问题，本发明人提供提高绿原酸生物利用度的绿原酸酰化物及应用，提高口服给药在抗肿瘤时的生物利用度和抑瘤效果。
Figure PCTCN2016076820-appb-000001
解决以上技术问题的一种提高绿原酸生物利用度的绿原酸酰化物，其特征在于：具有以下结构式：
其中：R1、R2、R3、R4、R5为RCO+功能团或H+离子，RCO+功能团在R1、R2、R3、R4、R5位置当中任意一个或几个，所述RCO+功能团中R为含1-6个碳原子的烷基。
所述R可为甲基，乙基，丙基、丁基、戊基、己基、异丙基和异丁基等。
所述结构式中R1、R2及R3为H+离子，其中R4、R5为C2H3O+。
这样结构的绿原酸酰化物能提高生物利用度、增强药效。
所述药物是由绿原酸酰化物为有效成分，加入一种或多种药学上可接受的药用赋形剂制备而成的口服制剂。
其中，所述口服制剂是片剂、胶囊剂、颗粒剂、散剂、丸剂、口服液。
所述口服制剂的单位制剂含绿原酸酰化物1-1000mg，优化方案中单位制剂含绿原酸酰化物10-200mg。
本发明中绿原酸酰化物可用制备方法如下：
将绿原酸与溶剂在容器中搅拌混合溶解，滴加催化剂，再加入乙酰化剂反应，干燥，再加入溶剂溶解后再用水洗涤去除其它杂质，再次干燥后得到绿原酸酰化物。
比如将二甲基甲酰胺(DMF)在容器中电磁搅拌，加入绿原酸，滴加三乙胺，再加入乙酸酐反应，真空干燥至干，于乙醇中溶解后倒入高速搅拌的水中沉淀，过滤干燥后得到被酰化的绿原酸，即绿原酸酰化物；或DMF电磁搅拌，加入绿原酸，滴加吡啶，再加入适量乙酸酐反应，真空干燥至干，于乙酸乙酯中溶解后分别加入蒸馏水洗涤三次，分离乙酸乙酯旋干后得到酚羟基被乙酰化的绿原酸。
具体步骤可如下：
将100ml DMF在容器中电磁搅拌，加入50g绿原酸，滴加0.5ml三乙胺，再加入50ml乙酸酐反应36小时，80℃真空干燥12小时至干，于50ml乙醇，温度60℃中溶解后倒入2000ml高速搅拌的水中沉淀，过滤干燥后得到5个羟基被酰化的绿原酸，即绿原酸酰化物。
或100ml DMF电磁搅拌，加入50g绿原酸，恒温10℃，滴加0.1ml吡啶，再加入10ml乙酸酐反应24小时，80℃真空干燥12小时至干，于100ml乙酸乙酯中溶解后(室温)分别加入20ml蒸馏水洗涤三次，分离乙酸乙酯旋干后得到4、5位酚羟基被乙酰化的绿原酸。
本发明中的绿原酸酰化物经口服给药后能够提高绿原酸在血液中血药浓度，即能够提高绿原酸在体内的生物利用度，解决了口服给药时绿原酸受到肠道菌群的影响而使吸收率偏低的问题；同时显著地提高了绿原酸对肿瘤的抑制效果。
附图说明
图1为本发明中犬血清中绿原酸血药浓度-曲线图
图2为本发明中肿瘤生长曲线图
具体实施方式
实施例1
一种提高绿原酸生物利用度的绿原酸酰化物，其特征在于：具有以下结构式：
Figure PCTCN2016076820-appb-000002
其中：R1、R2、R3、R4、R5为RCO+功能团或H+离子，RCO+功能团在R1、R2、R3、R4、R5位置当中任意一个或几个。
所述RCO+功能团中R为含1-6个碳原子的烷基。
这样结构的绿原酸酰化物能提高生物利用度，增强药效。
所述药物是由绿原酸酰化物为有效成分，加入一种或多种药学上可接受的药用赋形剂制备而成的口服制剂。
其中，所述口服制剂是片剂、胶囊剂、颗粒剂、散剂、丸剂、口服液。
所述口服制剂的单位制剂含绿原酸酰化物1-1000mg，优化方案中单位制剂含绿原酸酰化物10-200mg。
本发明中绿原酸酰化物可用制备方法如下：
将绿原酸与溶剂在容器中搅拌混合溶解，滴加催化剂，再加入乙酰化剂反应，干燥，再加入溶剂溶解后再用水洗涤去除其它杂质，再次干燥后得到绿原酸酰化物。
比如将二甲基甲酰胺(DMF)在容器中电磁搅拌，加入绿原酸，滴加三乙胺，再加入乙酸酐反应，真空干燥至干，于乙醇中溶解后倒入高速搅拌的水中沉淀，过滤干燥后得到被酰化的绿原酸，即绿原酸酰化物；或DMF电磁搅拌，加入绿原酸，滴加吡啶，再加入适量乙酸酐反应，真空干燥至干，于乙酸乙酯中溶解后分别加入蒸馏水洗涤三次，分离乙酸乙酯旋干后得到酚羟基被乙酰化的绿原酸。
将100ml DMF在容器中电磁搅拌，加入50g绿原酸，滴加0.5ml三乙胺，再加入 50ml乙酸酐反应36小时，80℃真空干燥12小时至干，于50ml乙醇，温度60℃中溶解后倒入2000ml高速搅拌的水中沉淀，过滤干燥后得到5个羟基被酰化的绿原酸，即绿原酸酰化物。
实施例2
(1)绿原酸，市购；绿原酸酰化物：R1、R2及R3为H+离子，其中R4、R5为C2H3O+，标记为样品1；R1、R2及R3为C3H5O+，其中R4、R5为H+离子，标记为样品2；R1、R4及R5为H+，其中R2、R3为C7H5O+离子，标记为样品3。
样品1的制备方法可为：100ml DMF电磁搅拌，加入50g绿原酸，恒温10℃，滴加0.1ml吡啶，再加入10ml乙酸酐反应24小时，80℃真空干燥12小时至干，于100ml乙酸乙酯中溶解后(室温)分别加入20ml蒸馏水洗涤三次，分离乙酸乙酯旋干后得到4、5位酚羟基被乙酰化的绿原酸。
样品2和3的制备方法可为如样品1中类似或接近的制备方法。
(2)受试动物Beagle犬，12只，体重8～10kg，雌雄各半。
(3)给药途径和方式单剂量灌胃(剂量：100mg/kg)
(4)血样采集将12只Beagle犬分4组，每组3只，其中一组作为绿原酸给药组，其余三组为绿原酸酰化物给药组；给药前禁食12小时，于给药前5分钟抽取静脉血样5ml，制备空白血清；再分别于给药后5、10、20、30、45、60、90、120、150和180分钟时，抽取静脉血样5ml，室温静置30分钟后，离心(3000rpm，15min)，分取血清(约2ml)冻藏(-20℃)，待测。
(5)溶液制备
对照品溶液的制备：取绿原酸对照品适量，精密称定，用甲醇-0.2％磷酸(20:80)溶液溶解并制成每1ml约含8mg的溶液，作为绿原酸对照品储备液。冰箱冷藏(储备液稳定期为1个月)，临用前稀释。
内标对照品溶液的制备：取葛根素对照品适量，精密称定，用甲醇溶解并制成每1ml约含84μg的溶液，作为内标溶液，冰箱冷藏。
供试品溶液的制备：取血清100μl，加入甲醇-0.2％磷酸(80:20)溶液、内标溶液和甲醇各100μl，漩涡振荡3min，静置，离心(12000rpm)10min，取出上清液，作为血清供试品溶液。
(6)血清中绿原酸含量测定
采用十八烷基硅烷键合硅胶柱(150mm×4.6mm，5μm)及保护柱；以流动相A(甲醇)与流动相 B(0.2％磷酸)，按表1的程序进行梯度洗脱；检测波长为325nm；流速为1ml/min；柱温40℃。
精密量取血清供试品溶液20μl，注入液相色谱仪，记录色谱图。
(7)计算方法
采用内标法，以随行标准曲线计算血清中绿原酸的含量。
表1 梯度程序洗脱表
Figure PCTCN2016076820-appb-000003
(6)试验结果
表2 各实验组不同时间的犬血清中平均血药浓度(n＝3)
Figure PCTCN2016076820-appb-000004
(7)数据分析
(1)由表2的数据分析，灌胃给药45min后，绿原酸酰化物组的血药浓度均值分别为139.85±15.23、114.70±8.44和118.19±13.27μg/ml，与绿原酸组的42.45±6.93μg/ml比较，具有明显的差异性。
(8)结论
实验结果显示，通过灌胃给药方式，绿原酸酰化物能够明显提高血液中药物的浓度，提高绿 原酸的生物利用度。
实施例3
(1)动物 BALB/C-nu小鼠，体重18～24g，由四川大学实验动物中心提供。
(2)药物 绿原酸，市购；绿原酸酰化物：R1、R2及R3为H+离子，其中R4、R5为C2H3O+，标记为样品1；R1、R2及R3为C3H5O+，其中R4、R5为H+离子，标记为样品2；R1、R2、R4及R5为H+离子，其中R3为C7H5O+，标记为样品3。
(3)细胞培养 SPCA-1细胞为人肺腺癌细胞株，细胞经常规复苏，传代，取二代种鼠肿瘤，用Hank′s液冲洗3次，清除血污、脂肪及坏死组织，将肿瘤剪切成1mm×1mm×1mm碎块，Hank′s液冲洗2次，按比例加入生理盐水(1g∶3ml)，再在玻璃匀浆器内研磨，经80-100目筛网过滤制成单细胞悬液，用台盼蓝染色法计数活细胞数。
(4)体内实验 将制备好的细胞悬液按0.2ml·只-1(约含细胞数1×106个)，接种于BALB/C-nu小鼠左前腋窝下，并按体重随机分为5组，每组5只，分别为绿原酸酰化物样品1组、绿原酸酰化物样品2组、绿原酸酰化物样品3组、绿原酸对照组、阴性对照组(N.S生理盐水)。于接种后的第二日灌胃给药，给药剂量为50mg/kg，每日一次，连续给药12次。于接种第6日开始间日测量肿瘤体积(V＝0.52×移植瘤长径L×移植瘤最短径W2)，并每周称小鼠重量两次。待阴性组平均瘤重大于1.0g时(瘤体积约为0.5cm3)停止实验，脱颈椎处死小鼠并称重，剥取肿瘤，计算：
抑瘤率％＝[1-(给药组平均瘤重/阴性组平均瘤重)]×100％；
(5)实验结果
(1)绿原酸酰化物对SPCA-1肺腺癌BALB/C-nu小鼠移植瘤生长体积的影响
表3 绿原酸酰化物对SPCA-1肺腺癌BALB/C-nu小鼠移植瘤生长体积的影响
Figure PCTCN2016076820-appb-000005
接种后 绿原酸酰化物样品1 绿原酸酰化物样品2 绿原酸酰化物样品3 绿原酸对照组 阴性对照组
6d 0.272±0.306*# 0.190±0.051*# 0.259±0.117*# 0.451±0.420△ 0.679±0.243
8d 0.353±0.377*# 0.210±0.058*# 0.353±0.194*# 0.598±0.571△ 0.744±0.439
10d 0.595±0.503*# 0.533±0.219*# 0.643±0.307*# 0.860±0.372△ 1.002±0.405
12d 0.782±0.727*# 0.704±0.276*# 0.897±0.416*# 1.224±1.009△ 1.405±0.300
14d 0.857±0.783*# 0.791±0.340*# 1.012±0.560*# 1.252±1.172△ 1.499±0.795
16d 1.006±0.912*# 1.054±0.471*# 1.128±0.769*# 1.443±1.367 1.636±0.932
18d 1.259±0.995*# 1.243±0.537*# 1.159±0.703*# 1.583±1.254 1.771±1.115
20d 1.315±0.555*# 1.449±0.585*# 1.327±0.709*# 2.004±1.611 2.126±1.308
与阴性对照组比较△p＜0.05，*p＜0.01；与绿原酸对照组比较#p＜0.01
(2)绿原酸酰化物对SPCA-1肺腺癌BALB/C-nu小鼠移植瘤重量和抑瘤率的影响
表4 绿原酸酰化物对SPCA-1肺腺癌BALB/C-nu小鼠移植瘤重量和抑瘤率的影响
Figure PCTCN2016076820-appb-000006
Figure PCTCN2016076820-appb-000007
与阴性组比较△p＜0.05，*p＜0.01；与绿原酸对照组比较#p＜0.01
(6)数据分析
由表3的移植瘤生长体积结果可以分析出，在接种后的第6天起，绿原酸酰化物样品1、样品2及样品3组与阴性对照组对比，具有显著差异性(p＜0.01)；与绿原酸对照组比较具有显著差异性(p＜0.01)。
由表4的移植瘤重量和抑瘤率结果可以分析出，绿原酸酰化物样品1、样品2及样品3组与阴性对照组对比，具有显著差异性(p＜0.01)；与绿原酸对照组比较具有显著差异性(p＜0.01)，抑瘤率比绿原酸对照组高出22.19％至28.13％。
(7)结论
通过灌胃给药方式，绿原酸在给药后第6天至第14天对SPCA-1肺癌BALB/C-nu小鼠移植瘤的生长曲线均有明显的抑制效果(p＜0.05)，给药后第14天后抑制效果不明显。而绿原酸酰化物样品1、样品2及样品3给药后第6天直至实验结束对SPCA-1肺癌BALB/C-nu小鼠移植瘤的生长曲线始终有显著的抑制作用，其抑制效果明显高于绿原酸组；说明了通过灌胃给药方式，绿原酸酰化物相比绿原酸有更好的抗肿瘤效果。
绿原酸酰化物样品1、样品2及样品3对SPCA-1肺癌BALB/C-nu小鼠移植瘤抑瘤率较高，与绿原酸对照组相比具有显著优势，进一步说明了绿原酸酰化物能够提高抗肿瘤效果。
实施例4：片剂
1.绿原酸酰化物1
绿原酸酰化物1：R1、R2及R3为H+离子，其中R4、R5为C2H3O+
2.处方：
绿原酸酰化物1 200g
淀粉 100g
乳糖 200g
羟丙甲纤维素 50g
硬脂酸镁 5g
3.制法：
本实施例采用制湿颗粒压片法制备绿原酸酰化物片剂。(1)按处方量取羟丙甲纤维素制成水溶液；(2)取处方量的绿原酸酰化物、淀粉和乳糖混合均匀后，加入羟丙甲纤维素水溶液，充分搅拌均匀后制成软材；(3)将制备好的软材按常规的湿法制粒的操作规程，过筛、干燥和整粒后得到大小合适的颗粒；(4)将制得的颗粒与硬脂酸镁混合均匀后压片，共制成1000片剂，每片含绿原酸酰化物100mg。
实施例5：胶囊剂
1.绿原酸酰化物2
绿原酸酰化物2：R4、R5为H+离子，其中R1、R2及R3为C2H3O+
2.处方：
绿原酸酰化物2 100g
淀粉 200g
3.制法：
取处方量的绿原酸酰化物和淀粉，混合均匀，加入80％乙醇溶液制成软材，干燥，整粒后按照胶囊剂的常规制备工艺制备2000粒胶囊，每粒胶囊含绿原酸酰化物50mg。
实施例6：颗粒剂
1.绿原酸酰化物3
绿原酸酰化物3：R1、R2、R4、R5为H+离子，其中R3为C7H5O+
2.处方：
绿原酸酰化物3 200g
甘露醇 100g
蔗糖 400g
聚维酮K30 10
3.制法：
取聚维酮K30，用水制成溶液。取处方量的绿原酸酰化物、甘露醇和蔗糖混合均匀之后，加入聚维酮K30溶液，制成软材。按照颗粒剂的常规制备工艺，对软材进行过筛、干燥和整 粒之后，得到颗粒剂。在无菌条件下分装颗粒剂，制备400袋颗粒剂，每袋颗粒剂含绿原酸酰化物500mg。
实施例7：散剂
1.绿原酸酰化物4
绿原酸酰化物4：R1、R2、R4、R5为H+离子，其中R3为C2H3O+
2.处方
绿原酸酰化物4   1000g
3.制法
取处方量绿原酸酰化物过筛后，按照散剂的常规制备工艺，无菌分装成含1000瓶/袋散剂，每瓶/袋散剂含绿原酸酰化物1000mg。
实施例8：丸剂
1.绿原酸酰化物5
绿原酸酰化物5：R1、R4、R5为H+离子，其中及R2、R3为C2H3O+
2.处方
绿原酸酰化物5 1g
淀粉 50g
聚维酮K30 适量
   
3.制法：
取适量的聚维酮K30，用水配制成溶液，再取处方量的绿原酸酰化物和淀粉，采用等量稀释法混合均匀之后，加入聚维酮K30溶液，充分搅拌后制得软材，采用搓丸法制得绿原酸酰化物丸剂1000粒，每粒丸剂含绿原酸酰化物1mg。
实施例9：口服液
1.绿原酸酰化物6
绿原酸酰化物6：R3、R4、R5为H+离子，其中R1、R2为C4H7O+
2.处方
Figure PCTCN2016076820-appb-000008
3.制法
取处方量的绿原酸酰化物、磷脂、胆固醇，溶解于乙醇，搅拌均匀后浓缩回收乙醇，再加入到10L水中超声充分分散后，无菌灌装成1000支口服液，每支口服液为10mL，含绿原酸酰化物200mg。
本发明提供一种绿原酸酰化物，该酰化物经口服给药后能够提高绿原酸在血液中血药浓度，提示着该绿原酸酰化物能够提高绿原酸在体内的生物利用度；同时通过与绿原酸抑瘤效果对比试验发现，通过口服给药方式该绿原酸酰化物能够明显提高抑瘤效果，说明该绿原酸酰化物在治疗肿瘤领域中具有增效的作用。

Claims (7)

  1. 一种提高绿原酸生物利用度的绿原酸酰化物，其特征在于：具有以下结构式：
    Figure PCTCN2016076820-appb-100001
    其中：R1、R2、R3、R4、R5为RCO+功能团或H+离子，RCO+功能团在R1、R2、R3、R4、R5位置当中任意一个或几个，所述RCO+功能团中R为含1-6个碳原子的烷基。
  2. 根据权利要求1中所述一种提高绿原酸生物利用度的绿原酸酰化物，其特征在于：所述结构式中R1、R2及R3为H+离子，其中R4、R5为C2H3O+。
  3. 根据权利要求1或2中所述一种提高绿原酸生物利用度的绿原酸酰化物的应用，其特征在于：所述绿原酸酰化物应用于制备治疗肿瘤的药物。
  4. 根据权利要求3中所述一种提高绿原酸生物利用度的绿原酸酰化物的应用，其特征在于：所述药物是由绿原酸酰化物为有效成分，加入一种或多种药学上可接受的药用赋形剂制备而成的口服制剂。
  5. 根据权利要求4中所述一种提高绿原酸生物利用度的绿原酸酰化物的应用，其特征在于：所述口服制剂是片剂、胶囊剂、颗粒剂、散剂、丸剂、口服液。
  6. 根据权利要求4所述一种提高绿原酸生物利用度的绿原酸酰化物的应用，其特征在于：所述口服制剂的单位制剂含绿原酸酰化物1-1000mg。
  7. 根据权利要求6所述一种提高绿原酸生物利用度的绿原酸酰化物的应用，其特征在于：所述口服制剂的单位制剂含绿原酸酰化物10-200mg。
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