氯霉素—羟丙基-β-环糊精滴眼液稳定性及抑菌作用的研究
|
摘要
氯霉素(chloramphenicol)是由委内瑞拉链霉菌产生的抗生素,其化学结构含对硝基苯基、丙二醇和二氯乙酰胺三个部分,其抗菌活性主要与丙二醇有关。氯霉素是白色至微黄色细针状或片状结晶,无臭,味极苦。在甲醇、乙醇、丙二醇、丙酮中易溶,在干燥时稳定,在中性和弱酸性溶液中比较稳定,遇碱类则易失效。氯霉素对革兰阳性、阴性细菌均有抑制作用,且对后者的作用较强。其抗菌机制是能够与核蛋白体50 S亚基结合,抑制肽酰基转移酶,从而抑制了蛋白质的合成。
氯霉素广泛地被用于治疗眼部感染,是治疗敏感细菌所引起的结膜炎、角膜炎、沙眼等外眼感染的首选药物。氯霉素滴眼液对光和热敏感,光照和温度升高能够引起氯霉素加速水解生成氯霉素的二醇物。在炎热季节里的运输和销售等环节,由于短时间或局部温度过高会造成氯霉素含量迅速下降、氯霉素的二醇物明显增加,因此增强氯霉素滴眼液的稳定性成了亟待解决的难题。
羟丙基-p-环糊精(hydroxypropyl-β-cyclodextrin,HP-β-CD)是由β-环糊精和1,2-环氧丙烷缩合成的亲水性衍生物。HP-p-CD是非结晶性粉末,在水中其溶解度大于50%,并能够溶于醇的水溶液：HP-p-CD肾毒性比较低,可以通过非肠道给药途径给药；HP-β-CD不能被胃酸和a-淀粉酶水解,几乎不参加任何生物体内代谢,也不蓄积,口服给药后基本上以完整形态排出体外；HP-β-CD在与药物形成复合物后对药物具有促释作用,能促进药物在生物体内迅速释放；HP-β-CD表面活性低,基本没有刺激性和溶血性。鉴于上述优点,HP-β-CD被作为一种安全、稳定的辅料,广泛应用为眼科药物的增溶剂。用HP-β-CD制成的滴眼剂黏度低、无刺激性,还可提高角膜对药物的生物利用度。
1.氯霉素-羟丙基-β-环糊精滴眼液稳定性试验研究
根据氯霉素滴眼液目前所存在的问题,筛选处方,加入了合适的稳定剂,增加了药物的稳定性。通过改变药物剂型,利用氯霉素的性质,采用固-液,非水相-水相两个独立的包装,保证了氯霉素滴眼液在有效期内合格。
我们通过对本品进行处方研究、辅料筛选确定了抑制氯霉素水解的优选处方,并进行了相关的质量研究和稳定性考察,最后结果表明本品变更了辅料、工艺后符合现行质量标准规定,并且在贮存期内氯霉素二醇物含量明显降低。
2.氯霉素-羟丙基-β-环糊精滴眼液抑菌试验的作用
本试验采用抑菌环测量法比较氯霉素-羟丙基-β-环糊精滴眼液、氯霉素-玻璃酸钠滴眼液、氯霉素滴眼液对金黄色葡萄球菌、大肠埃希菌抑菌作用的差别。结果表明经过HP-β-CD包裹的氯霉素滴眼液无论是对革兰阳性菌金黄色葡萄球菌还是对革兰阴性菌大肠埃希菌均有良好的抑菌效果。经统计学分析与玻璃酸钠-氯霉素滴眼液和氯霉素滴眼液的抑菌能力相比无明显差异。
本课题就氯霉素-羟丙基-β-环糊精滴眼液进行了研究,主要研究结论如下：
(1)拟订了几个比较稳定的制剂处方,能够改善目前存在的稳定性问题。
(2)加入HP-β-CD不但提高了氯霉素滴眼液的稳定性且保证了其抑菌能力,为临床上预防和治疗金黄色葡萄菌和大肠埃希菌等提供了可靠的实验依据,并为新药开发带来广阔的前景。
Chloramphenicol is produced by the Streptomyces venezuelae, its chemical structure contains nitrophenyl, propylene glycol and dichloro-acetamide. The antimicrobial activity is mainly related with propylene glycol. Chloramphenicol is white or slight yellow-green needle-like, long flaky crystal or crystalline powder with bitter taste. It is soluble in methanol, ethanol, propylene glycol and acetone. It is stable at dry condition and fairly stable in weak acid and neutral solutions. It could easily become invalid when it meet with alkali. Chloramphenicol inhibites Gram-positive and Gram-negative bacteria, and the latter is stronger. Antibacterial mechanism is that chloramphenicol combines with 50 S ribosomal subunit, inhibites peptide acyltransferase, thereby inhibites protein synthesis.
Chloramphenicol is widely used to treat eye infections, is the first choice to cure conjunctivitis, keratitis, trachoma and so on. Chloramphenicol eye drops are sensitive for heat and light. Heat and light will accelerate the hydrolysis of chloramphenicol. During the sale and the transportation in hot seasons, local temperature is too high will result in rapid hydrolysis of chloramphenicol, meanwhile chloramphenicol diol materials increase significantly, thus enhancing the stability of chloramphenicol eye drops has become urgent.
Hydroxypropyl-β-cyclodextrin(HP-β-CD) is hydrophilic derivatives condensed byβ-cyclodextrin((3-CD) and 1,2-propylene oxide. HP-β-CD is amorphous powder, more than 50% soluble in water, soluble in alcohol aqueous solution. HP-β-CD is relatively low renal toxicity, then it can be used for parenteral route.HP-β-CD is not hydrolyzed by acid andα-amylase,almost not appears in vivo metabolism, not accumulates, largely excreted in full form. It complexing with drugs promotes the release of the drugs in vivo. The surface activity of HP-β-CD is low, and HP-β-CD is little or no hemolytic and irritant. As a safe, stable accessories, HP-β-CD are widely used as ophthalmic drug solubilizer. Eye drops made of HP-β-CD is low viscosity, non-irritating, enhancing the corneal drug bioavailability.
1. Stability test of chloramphenicol eye drops with hydroxypropyl-β-cyclodextrin
According to the current problems of chloramphenicol eye drops, prescriptions were screened and suitable stabilizing agent was added to increase the stability of drug. Changing drug dosage form, taking advantage of the nature of chloramphenicol, and using independent package of solid-liquid, non-aqueous phase-aqueous phase ensure the qualification in the validity term.
The preferred prescription by the screening of pharmaceutic adjuvants was identified, and the quality and stability of chloramphenicol eye drops were researched. The results showed that the products complied with the provisions of the existing quality standards after changing adjuvants and process, and chloramphenicol diol significantly reduced during the product storage.
2. Study on bacteriostatic action of chloramphenicol eye drops with hydroxypropy1-β-cyclodextrin
The bacterial inhibition ring test was used to compare the bacteriostatic action of chloramphenicol-hydroxypropyl-β-cyclodextrin eye drops, chloramphenicol-sodium hyaluronate eye drops and chloramphenicol eye drops on Staphylococcus aureus and Escherichia coli. The results showed that chloramphenicol eye drops with HP-β-CD have good bacteriostatic action on Staphylococcus aureus and Escherichia coli, and by the statistics analysis there was no significant difference in bacteriostatic action of three different eye drops.
Achievements of this research:
(1) Several relatively stable prescriptions to improve the stability of chloramphenicol eye drops were developed.
(2) The adding of HP-(3-CD not only improves the stability of chloramphenicol eye drops but also ensures its bacteriostatic action. It provides a reliable experimental basis for clinical prevention and treatment of Staphylococcus aureus and Escherichia coli, and brings a broad prospect for the development of new drugs.
氯霉素广泛地被用于治疗眼部感染,是治疗敏感细菌所引起的结膜炎、角膜炎、沙眼等外眼感染的首选药物。氯霉素滴眼液对光和热敏感,光照和温度升高能够引起氯霉素加速水解生成氯霉素的二醇物。在炎热季节里的运输和销售等环节,由于短时间或局部温度过高会造成氯霉素含量迅速下降、氯霉素的二醇物明显增加,因此增强氯霉素滴眼液的稳定性成了亟待解决的难题。
羟丙基-p-环糊精(hydroxypropyl-β-cyclodextrin,HP-β-CD)是由β-环糊精和1,2-环氧丙烷缩合成的亲水性衍生物。HP-p-CD是非结晶性粉末,在水中其溶解度大于50%,并能够溶于醇的水溶液：HP-p-CD肾毒性比较低,可以通过非肠道给药途径给药；HP-β-CD不能被胃酸和a-淀粉酶水解,几乎不参加任何生物体内代谢,也不蓄积,口服给药后基本上以完整形态排出体外；HP-β-CD在与药物形成复合物后对药物具有促释作用,能促进药物在生物体内迅速释放；HP-β-CD表面活性低,基本没有刺激性和溶血性。鉴于上述优点,HP-β-CD被作为一种安全、稳定的辅料,广泛应用为眼科药物的增溶剂。用HP-β-CD制成的滴眼剂黏度低、无刺激性,还可提高角膜对药物的生物利用度。
1.氯霉素-羟丙基-β-环糊精滴眼液稳定性试验研究
根据氯霉素滴眼液目前所存在的问题,筛选处方,加入了合适的稳定剂,增加了药物的稳定性。通过改变药物剂型,利用氯霉素的性质,采用固-液,非水相-水相两个独立的包装,保证了氯霉素滴眼液在有效期内合格。
我们通过对本品进行处方研究、辅料筛选确定了抑制氯霉素水解的优选处方,并进行了相关的质量研究和稳定性考察,最后结果表明本品变更了辅料、工艺后符合现行质量标准规定,并且在贮存期内氯霉素二醇物含量明显降低。
2.氯霉素-羟丙基-β-环糊精滴眼液抑菌试验的作用
本试验采用抑菌环测量法比较氯霉素-羟丙基-β-环糊精滴眼液、氯霉素-玻璃酸钠滴眼液、氯霉素滴眼液对金黄色葡萄球菌、大肠埃希菌抑菌作用的差别。结果表明经过HP-β-CD包裹的氯霉素滴眼液无论是对革兰阳性菌金黄色葡萄球菌还是对革兰阴性菌大肠埃希菌均有良好的抑菌效果。经统计学分析与玻璃酸钠-氯霉素滴眼液和氯霉素滴眼液的抑菌能力相比无明显差异。
本课题就氯霉素-羟丙基-β-环糊精滴眼液进行了研究,主要研究结论如下：
(1)拟订了几个比较稳定的制剂处方,能够改善目前存在的稳定性问题。
(2)加入HP-β-CD不但提高了氯霉素滴眼液的稳定性且保证了其抑菌能力,为临床上预防和治疗金黄色葡萄菌和大肠埃希菌等提供了可靠的实验依据,并为新药开发带来广阔的前景。
Chloramphenicol is produced by the Streptomyces venezuelae, its chemical structure contains nitrophenyl, propylene glycol and dichloro-acetamide. The antimicrobial activity is mainly related with propylene glycol. Chloramphenicol is white or slight yellow-green needle-like, long flaky crystal or crystalline powder with bitter taste. It is soluble in methanol, ethanol, propylene glycol and acetone. It is stable at dry condition and fairly stable in weak acid and neutral solutions. It could easily become invalid when it meet with alkali. Chloramphenicol inhibites Gram-positive and Gram-negative bacteria, and the latter is stronger. Antibacterial mechanism is that chloramphenicol combines with 50 S ribosomal subunit, inhibites peptide acyltransferase, thereby inhibites protein synthesis.
Chloramphenicol is widely used to treat eye infections, is the first choice to cure conjunctivitis, keratitis, trachoma and so on. Chloramphenicol eye drops are sensitive for heat and light. Heat and light will accelerate the hydrolysis of chloramphenicol. During the sale and the transportation in hot seasons, local temperature is too high will result in rapid hydrolysis of chloramphenicol, meanwhile chloramphenicol diol materials increase significantly, thus enhancing the stability of chloramphenicol eye drops has become urgent.
Hydroxypropyl-β-cyclodextrin(HP-β-CD) is hydrophilic derivatives condensed byβ-cyclodextrin((3-CD) and 1,2-propylene oxide. HP-β-CD is amorphous powder, more than 50% soluble in water, soluble in alcohol aqueous solution. HP-β-CD is relatively low renal toxicity, then it can be used for parenteral route.HP-β-CD is not hydrolyzed by acid andα-amylase,almost not appears in vivo metabolism, not accumulates, largely excreted in full form. It complexing with drugs promotes the release of the drugs in vivo. The surface activity of HP-β-CD is low, and HP-β-CD is little or no hemolytic and irritant. As a safe, stable accessories, HP-β-CD are widely used as ophthalmic drug solubilizer. Eye drops made of HP-β-CD is low viscosity, non-irritating, enhancing the corneal drug bioavailability.
1. Stability test of chloramphenicol eye drops with hydroxypropyl-β-cyclodextrin
According to the current problems of chloramphenicol eye drops, prescriptions were screened and suitable stabilizing agent was added to increase the stability of drug. Changing drug dosage form, taking advantage of the nature of chloramphenicol, and using independent package of solid-liquid, non-aqueous phase-aqueous phase ensure the qualification in the validity term.
The preferred prescription by the screening of pharmaceutic adjuvants was identified, and the quality and stability of chloramphenicol eye drops were researched. The results showed that the products complied with the provisions of the existing quality standards after changing adjuvants and process, and chloramphenicol diol significantly reduced during the product storage.
2. Study on bacteriostatic action of chloramphenicol eye drops with hydroxypropy1-β-cyclodextrin
The bacterial inhibition ring test was used to compare the bacteriostatic action of chloramphenicol-hydroxypropyl-β-cyclodextrin eye drops, chloramphenicol-sodium hyaluronate eye drops and chloramphenicol eye drops on Staphylococcus aureus and Escherichia coli. The results showed that chloramphenicol eye drops with HP-β-CD have good bacteriostatic action on Staphylococcus aureus and Escherichia coli, and by the statistics analysis there was no significant difference in bacteriostatic action of three different eye drops.
Achievements of this research:
(1) Several relatively stable prescriptions to improve the stability of chloramphenicol eye drops were developed.
(2) The adding of HP-(3-CD not only improves the stability of chloramphenicol eye drops but also ensures its bacteriostatic action. It provides a reliable experimental basis for clinical prevention and treatment of Staphylococcus aureus and Escherichia coli, and brings a broad prospect for the development of new drugs.
引文
[1]Ehrlich J, Bartz QR, Smith RM, et al. Chloranmycetin, a antibiotic from a soil actinomycete[J]. Science.1947,106:417.
[2]赵永福,刘春泉,冯敏等.γ辐照降解氯霉素的研究[J].江苏农业学报.2006,22(3)：289-292.
[3]中华人民共和国药典2010年版二部[S],2010：1029.
[4]吴晓平,杨鹭华.氯霉素残留的危害及其检测方法[J].动物医学进展.2004,25(3)：41-43.
[5]凌沛学.眼科药物与制剂学[M].北京：中国轻工业出版社.2010：16
[6]顾学裘.药物制剂注解[M].北京：人民卫生出版社,1983：90-94,891-892.
[7]杨家海,颜耀洞,吕士杰,等.正交设计法优选氯霉素眼液处方[J].空军医高专学报.1996,18(2)：173-178.
[8]四川医学院附属医院药房.制剂操作规程[M].1973：148.
[9]袁素华,凌沛学.玻璃酸钠在氯霉素滴眼液中的作用[J].中国生化药物杂志.2007,28(3)：201-202.
[10]周维书,陆丽珠译.药物的化学稳定性(第一版)[M].北京：人民卫生出版社.1983：173-178
[11]Meakin BJ, Davies DJG., Richardson NE., et al. The quality control of chloramphenicol in pharmaceutical preparations [J]. Act@ Pham Tech.1979, 25(1):29-48.
[12]吴听.临床应用氯霉素的不良反应[J].临床合理用药,2008,1(1)：36-37.
[13]陈岳敏.氯霉素滴眼液的不良反应[J].抗感染药学,2005,2(1)：44-46.
[14]王凤山,凌沛学.生物化学与生化技术[M].北京：人民卫生出版社,2007,2：195.
[15]Meyer K, Palmer JW. The polysaccharide of the vitreous humor[J]. J Biol Chem, 1934,107:629-634.
[16]Rapport MM, Weissman B, Linker A, et al. Isolation of a crystalline disaccharide, hyalobiuronic acid[J]. Nature.1951,168:996-997.
[17]Laurent TC. Biochemistry of hyaluronan[J]. Acta otolaryngol(Stockh),1987 (suppl),442:7-24.
[18]黎洪珊,王培玉.β-环糊精衍生物的研究进展及在药剂学上的应用[J].中国药学杂志,1999,34(4)：220-223.
[19]张秀荣,林天慕,牛松青.环糊精衍生物的非肠道应用[J].中国药学杂志,2000,35(10)：649-652.
[20]谢伯泰,杨国武,谢薇梅.羟丙基-β-环糊精特性及其在医药领域中的应用与安全性[J],国外医药—合成药、生化药、制剂分册,2002,23(5)：302.
[21]Lrie T, Uekama K.Parmaceutical applications of cyclodextrins.Ⅲ.toxicological issues and safety evaluation[J]J Pharm Sci,1997;86(2):147-162.
[22]蔡双霜,黄华,陈莉.羟丙基-β-环糊精的应用发展[J].中国药业,2008,17(10)：79.
[23]王兰,张芳侠,薛显周.羟丙基-β-环糊精在中药制剂学中的应用概况[J].陕西中医,2005,26(2)：172.
[24]胡士高,孙备,杨士友.羟丙基-β-环糊精在注射剂中的应用研究进展[J].药学专论,2010,19(13)：1-2.
[25]陆斌.药物新剂型与新技术[M]北京：人民卫生出版社,1998：40-46.
[26]郑俊民主译.药用辅料手册[M]北京：化学工业出版社,2004：218-222.
[27]伊曲康唑注射液说明书.
[28]Jozsef Szejtli.Past, present, and future of cyclodextrin research [J] Pure Appl Chem,2004,76(10):1825-1845.
[29]Florence Maurin, Vailhauques, Bernard Pages,Montbazin,Glaude Coquelet,Saint Gely du Fesc,et al. Ready-to-use eye lotion containing indomethacin. US 5744154[P].1998
[30]张颖,高永良.环糊精及其衍生物在眼用制剂中的应用[J].国外医学药学分册,2006,33(2)：143-147.
[31]齐红艺,李莉,吴纯洁,等.羟丙基-β-环糊精在眼用制剂中的应用进展[J].中国新药杂志,2006,15(18)：1539-1551.
[32]黄秀凤.氯霉素滴眼液质量稳定性的考察[J].中国实用医药,2009,4(30)：62-63.
[33]陈国广,陈振华,李学明,等.氯霉素滴眼液的稳定性及其处方的工艺改进[J].华西药学杂志,2006,21(5)：443-445.
[34]凌沛学.眼科药物与制剂学[M].北京：中国轻工业出版社,2010：16-17,194-195.
[35]郭维儿.β-环糊精增强氯霉素滴眼液稳定性研究[J].西北药学杂志,1998,13(3)：114.
[36]中华人民共和国卫生部卫生法制与监督司.消毒技术规范[M].2002：48-131
[37]王桂亭,宋艳艳,王志波,等.大蒜液体外抑菌作用的实验研究[J].预防医学论坛,2006,12(2)：131-133.
[38]王桂亭,宋艳艳,王志玉,等.双氧胍复合消毒湿巾抑菌效果的实验研究[].中国消毒学杂志,2005,22(2)：189-190.
[39]王桂亭,王皋,宋艳艳,等.金银花复方洗手液体外抑菌研究[J].中国消毒学杂志,2006,23(1)：18-19.
[2]赵永福,刘春泉,冯敏等.γ辐照降解氯霉素的研究[J].江苏农业学报.2006,22(3)：289-292.
[3]中华人民共和国药典2010年版二部[S],2010：1029.
[4]吴晓平,杨鹭华.氯霉素残留的危害及其检测方法[J].动物医学进展.2004,25(3)：41-43.
[5]凌沛学.眼科药物与制剂学[M].北京：中国轻工业出版社.2010：16
[6]顾学裘.药物制剂注解[M].北京：人民卫生出版社,1983：90-94,891-892.
[7]杨家海,颜耀洞,吕士杰,等.正交设计法优选氯霉素眼液处方[J].空军医高专学报.1996,18(2)：173-178.
[8]四川医学院附属医院药房.制剂操作规程[M].1973：148.
[9]袁素华,凌沛学.玻璃酸钠在氯霉素滴眼液中的作用[J].中国生化药物杂志.2007,28(3)：201-202.
[10]周维书,陆丽珠译.药物的化学稳定性(第一版)[M].北京：人民卫生出版社.1983：173-178
[11]Meakin BJ, Davies DJG., Richardson NE., et al. The quality control of chloramphenicol in pharmaceutical preparations [J]. Act@ Pham Tech.1979, 25(1):29-48.
[12]吴听.临床应用氯霉素的不良反应[J].临床合理用药,2008,1(1)：36-37.
[13]陈岳敏.氯霉素滴眼液的不良反应[J].抗感染药学,2005,2(1)：44-46.
[14]王凤山,凌沛学.生物化学与生化技术[M].北京：人民卫生出版社,2007,2：195.
[15]Meyer K, Palmer JW. The polysaccharide of the vitreous humor[J]. J Biol Chem, 1934,107:629-634.
[16]Rapport MM, Weissman B, Linker A, et al. Isolation of a crystalline disaccharide, hyalobiuronic acid[J]. Nature.1951,168:996-997.
[17]Laurent TC. Biochemistry of hyaluronan[J]. Acta otolaryngol(Stockh),1987 (suppl),442:7-24.
[18]黎洪珊,王培玉.β-环糊精衍生物的研究进展及在药剂学上的应用[J].中国药学杂志,1999,34(4)：220-223.
[19]张秀荣,林天慕,牛松青.环糊精衍生物的非肠道应用[J].中国药学杂志,2000,35(10)：649-652.
[20]谢伯泰,杨国武,谢薇梅.羟丙基-β-环糊精特性及其在医药领域中的应用与安全性[J],国外医药—合成药、生化药、制剂分册,2002,23(5)：302.
[21]Lrie T, Uekama K.Parmaceutical applications of cyclodextrins.Ⅲ.toxicological issues and safety evaluation[J]J Pharm Sci,1997;86(2):147-162.
[22]蔡双霜,黄华,陈莉.羟丙基-β-环糊精的应用发展[J].中国药业,2008,17(10)：79.
[23]王兰,张芳侠,薛显周.羟丙基-β-环糊精在中药制剂学中的应用概况[J].陕西中医,2005,26(2)：172.
[24]胡士高,孙备,杨士友.羟丙基-β-环糊精在注射剂中的应用研究进展[J].药学专论,2010,19(13)：1-2.
[25]陆斌.药物新剂型与新技术[M]北京：人民卫生出版社,1998：40-46.
[26]郑俊民主译.药用辅料手册[M]北京：化学工业出版社,2004：218-222.
[27]伊曲康唑注射液说明书.
[28]Jozsef Szejtli.Past, present, and future of cyclodextrin research [J] Pure Appl Chem,2004,76(10):1825-1845.
[29]Florence Maurin, Vailhauques, Bernard Pages,Montbazin,Glaude Coquelet,Saint Gely du Fesc,et al. Ready-to-use eye lotion containing indomethacin. US 5744154[P].1998
[30]张颖,高永良.环糊精及其衍生物在眼用制剂中的应用[J].国外医学药学分册,2006,33(2)：143-147.
[31]齐红艺,李莉,吴纯洁,等.羟丙基-β-环糊精在眼用制剂中的应用进展[J].中国新药杂志,2006,15(18)：1539-1551.
[32]黄秀凤.氯霉素滴眼液质量稳定性的考察[J].中国实用医药,2009,4(30)：62-63.
[33]陈国广,陈振华,李学明,等.氯霉素滴眼液的稳定性及其处方的工艺改进[J].华西药学杂志,2006,21(5)：443-445.
[34]凌沛学.眼科药物与制剂学[M].北京：中国轻工业出版社,2010：16-17,194-195.
[35]郭维儿.β-环糊精增强氯霉素滴眼液稳定性研究[J].西北药学杂志,1998,13(3)：114.
[36]中华人民共和国卫生部卫生法制与监督司.消毒技术规范[M].2002：48-131
[37]王桂亭,宋艳艳,王志波,等.大蒜液体外抑菌作用的实验研究[J].预防医学论坛,2006,12(2)：131-133.
[38]王桂亭,宋艳艳,王志玉,等.双氧胍复合消毒湿巾抑菌效果的实验研究[].中国消毒学杂志,2005,22(2)：189-190.
[39]王桂亭,王皋,宋艳艳,等.金银花复方洗手液体外抑菌研究[J].中国消毒学杂志,2006,23(1)：18-19.