广地龙配方颗粒质量标准规范化研究及地龙凝胶剂的研制
|
摘要
1.目的
为了更好地控制广地龙配方颗粒的质量,保证临床疗效,本研究在中医药理论和《中药配方颗粒质量标准研究的技术要求》指导下,对广地龙饮片入选标准、广地龙配方颗粒质量标准进行系统研究,对指标成分次黄嘌呤和琥珀酸进行药效学验证。同时研制地龙凝胶剂并进行初步的药效学评价。
2.方法
研究内容主要包括:广地龙饮片的入选标准,广地龙配方颗粒的质量标准及稳定性研究,指标成分次黄嘌呤和琥珀酸止咳平喘的药效学验证,地龙凝胶剂的制备及治疗烫伤的药效学初探等。
广地龙饮片入选标准研究中,采用薄层色谱法鉴别氨基酸、琥珀酸及氯仿提取物,建立高效液相色谱法及高效毛细管电泳法分别测定次黄嘌呤和琥珀酸含量的方法。高效液相法测定次黄嘌呤含量的色谱条件:色谱柱为ODS-EP 5μm ,4.6 mm×150 mm,流动相为水-甲醇-四氢呋喃(100﹕0.5﹕0.5),流速为1.0 ml/ min ,检测波长λ=254 nm,柱温为室温,理论板数按次黄嘌呤峰计算不低于3888。高效毛细管电泳测定琥珀酸含量的条件:以未涂层融硅毛细管(50 cm×75μm)为分离柱,1mmol/L Tris-10 mmol/L H3BO3-0.5%四乙烯五胺-0.025 mmol/L CTAB (pH=11.45)体系为电泳介质,分离电压为-10KV。还对广地龙饮片进行杂质、水分及浸出物等项目的检查。
配方颗粒采用传统汤剂的制备方法,并通过离心除杂、喷雾干燥及制粒获得。以次黄嘌呤的含量为指标,用正交设计法优选有效成分的最佳提取工艺。
配方颗粒的薄层鉴别和含量测定与饮片类似,并按照2005版中国药典一部规定的颗粒剂项下对广地龙配方颗粒装量差异、溶化性和含水量进行检查。
初步稳定性实验采用实验室留样观察三个月。指标成分的药效学验证分别以组胺和乙酰胆碱混合溶液引喘法及雾化氨水引咳法来评价次黄嘌呤和琥珀酸的止咳平喘作用。
地龙凝胶剂的制备选用卡波姆为凝胶基质,并含有甘油和丙二醇等附加成分。药效学实验则分别以烫伤后面积,含水量,羟脯氨酸含量变化以及肉眼观察和病理切片来评价。
3.结果
广地龙饮片入选标准研究中改进了2005版中国药典一部地龙项下氨基酸薄层鉴别方法,增加丙氨酸的斑点,并增加了琥珀酸的斑点鉴别;HPLC测得不同产地的广地龙次黄嘌呤含量为0.54～1.25 mg/g;HPEC测得药材中琥珀酸(产地:广西)含量为0.8 mg/g。
广地龙配方颗粒制备研究中,确定最佳提取工艺条件为:取广地龙30g,剪碎,加入12倍量的水,浸泡1小时,煎煮3次,过滤,合并煎煮液,浓缩。
广地龙配方颗粒质量标准研究中,配方颗粒的次黄嘌呤含量测定方法色谱条件与饮片一致,不同批号的配方颗粒次黄嘌呤含量为1.5～3.3mg/袋。稳定性实验:实验室留样观察三个月,按质量标准项目检查,包括外观、鉴别、检查、含量测定、卫生学,均符合规定。
小鼠的哮喘和咳嗽实验证明次黄嘌呤、琥珀酸有止咳平喘疗效。选择这两个成分作为指标成分是合理的。
地龙凝胶剂治疗烫伤实验中受伤面积数据表明广地龙无糖凝胶和广地龙含糖凝胶对烫伤有治疗作用。含水量实验数据表明广地龙无糖凝胶和广地龙含糖凝胶可减少水肿。羟脯氨酸含量测定数据表明广地龙含糖凝胶使烫伤部位恢复较快。
4.结论
本研究建立的广地龙饮片和配方颗粒质量控制方法规范,科学,通过药效学的验证,说明以次黄嘌呤和琥珀酸含量作为控制广地龙饮片及配方颗粒质量的合理性,为广地龙及其产品的质量控制提供科学依据。另外本课题对广地龙促进伤口的愈合作用的制剂及药效学的进行初步研究,为今后广地龙外用制剂的开发奠定了一定的基础。
Objective: In order to control the quality of Phertima dispensing granule and assure its clinical results, System study on quality standard of Phertima material and its dispensing granule was carried out according to the theories of Traditional Chinese Medicine and”the Technical Requirement of Research on the Quality Standard of Chinese Medicine Granule”and the validity of hypoxanthine and succinic acid as the active components was testified, In addition, earthworm gel preparation was researched, and its therapeutic efficacy of scald was evaluated.
Methods: This research includes quality standard of Phertima and its dispensing granule, preparation technology, pharmacodynamics evaluation of hypoxanthine and succinic acid, stability, earthworm gel preparation, pharmacodynamic action of scald. In the quality research of the raw materials, thin layer chromatography (TLC) was used to identify amino acids, succinic acid and extractive of trichloromethane. High performace liquid chromatography (HPLC) method used for determing the content of hypoxanthine using chromatographic column of Inertsil ODS-EP, mobile phase of H2O-CH3OH-C4H8O (100﹕0.5﹕0.5), flow rate of 1.0mL/min and detection wavelength of 254 nm. Another component, succinic acid, was determined by High Performance Capillary Electrophoresis (HPCE). The succinic acid could be separated and detected by buffer (0.36 mmol/L Tris-1.25 mmol/L H3BO3-0.5% 4-ethylene -5-amino-0.025 mmol/L CTAB adjusted to pH = 11.45 ) at -10kV of constant voltage. Other determination indexes, for example, dopant, moisture content, extractive assaying, also were determined.
The dispensing granules were preparated according to traditional decoction, removing impurity by centrifuge, drying by spray and granulation and the extraction technics of active components were optimized by orthogonal design using the content of hypoxanthine as the valuation index. Same methods including TLC, HPLC which were used in the raw materials were applied to control quality of Phertima dispensing granule, other indexes were ascertained according to requirement of granules in appendix of Pharmacopoeia and preliminary experiments on stability were carried out.
Asthmatic model of mice induced by histamine and acetylcholine and cough model of mice induced by ammonia water, were used to evaluate the pharmacodynamics of extract, dispensing granules, hypoxanthine, succinic acid and so on.
Earthworm gel is composed of earthworm extract, carbomer, glycerine, propanediol and so on and its scald pharmacodynamics evaluated by scald area, vary of hydroxyproline and pathology slice.
Results: In the quality research of the raw materials, TLC in China Pharmacopeia (version 2005) was improved, 2-aminopropionic acid and succinic acid, were added as the marker substance. The content range of hypoxanthine in different herbal Pheretima aspergillum were 0.54 mg/g～1.25 mg/g and 1.6 mg/bag~3.3 mg/bag in is dispensing granule by HPLC. The content of succinic acid was 0.8 mg/g in herbal Pheretima aspergillum (region:GuangXi).
The optimum extract processing of active components was: 30 gram Phertima was weighed and soaked 1hour, then bored 1h and extracted with water third, used 12 volumes of water, afterward, filtrated, concentrated, dried and granulated. Dispensing granules were taken stability examination in the laboratory for three months including the outward appearance, the discrimination, the inspection, the assaying and the hygieology and all above were conformed to the requirement.
Hypoxanthine and succinic acid have effects on cough-remedy, asthma-smooth. It can be concluded that the two ingredients as the standard components are reasonable and reliable. In the scald-remedy experiment, the data indicated that both earthworm syrup gel and earthworm non-syrup gel have therapeutics effects.
Conclusions: The all methods to control the quality both the raw materials and the granules were standard and scientific and hypoxanthine and succinic acid as the standard components was reasonable. This thesis provided scientific and reliable basis for controlling the quality of Phertima dispensing granules. In addition, the earthworm gel preparation in agglutinating and the vertification of pharmacodynamics will be established an experimental foundation in Phertima comprehend research and development.
为了更好地控制广地龙配方颗粒的质量,保证临床疗效,本研究在中医药理论和《中药配方颗粒质量标准研究的技术要求》指导下,对广地龙饮片入选标准、广地龙配方颗粒质量标准进行系统研究,对指标成分次黄嘌呤和琥珀酸进行药效学验证。同时研制地龙凝胶剂并进行初步的药效学评价。
2.方法
研究内容主要包括:广地龙饮片的入选标准,广地龙配方颗粒的质量标准及稳定性研究,指标成分次黄嘌呤和琥珀酸止咳平喘的药效学验证,地龙凝胶剂的制备及治疗烫伤的药效学初探等。
广地龙饮片入选标准研究中,采用薄层色谱法鉴别氨基酸、琥珀酸及氯仿提取物,建立高效液相色谱法及高效毛细管电泳法分别测定次黄嘌呤和琥珀酸含量的方法。高效液相法测定次黄嘌呤含量的色谱条件:色谱柱为ODS-EP 5μm ,4.6 mm×150 mm,流动相为水-甲醇-四氢呋喃(100﹕0.5﹕0.5),流速为1.0 ml/ min ,检测波长λ=254 nm,柱温为室温,理论板数按次黄嘌呤峰计算不低于3888。高效毛细管电泳测定琥珀酸含量的条件:以未涂层融硅毛细管(50 cm×75μm)为分离柱,1mmol/L Tris-10 mmol/L H3BO3-0.5%四乙烯五胺-0.025 mmol/L CTAB (pH=11.45)体系为电泳介质,分离电压为-10KV。还对广地龙饮片进行杂质、水分及浸出物等项目的检查。
配方颗粒采用传统汤剂的制备方法,并通过离心除杂、喷雾干燥及制粒获得。以次黄嘌呤的含量为指标,用正交设计法优选有效成分的最佳提取工艺。
配方颗粒的薄层鉴别和含量测定与饮片类似,并按照2005版中国药典一部规定的颗粒剂项下对广地龙配方颗粒装量差异、溶化性和含水量进行检查。
初步稳定性实验采用实验室留样观察三个月。指标成分的药效学验证分别以组胺和乙酰胆碱混合溶液引喘法及雾化氨水引咳法来评价次黄嘌呤和琥珀酸的止咳平喘作用。
地龙凝胶剂的制备选用卡波姆为凝胶基质,并含有甘油和丙二醇等附加成分。药效学实验则分别以烫伤后面积,含水量,羟脯氨酸含量变化以及肉眼观察和病理切片来评价。
3.结果
广地龙饮片入选标准研究中改进了2005版中国药典一部地龙项下氨基酸薄层鉴别方法,增加丙氨酸的斑点,并增加了琥珀酸的斑点鉴别;HPLC测得不同产地的广地龙次黄嘌呤含量为0.54～1.25 mg/g;HPEC测得药材中琥珀酸(产地:广西)含量为0.8 mg/g。
广地龙配方颗粒制备研究中,确定最佳提取工艺条件为:取广地龙30g,剪碎,加入12倍量的水,浸泡1小时,煎煮3次,过滤,合并煎煮液,浓缩。
广地龙配方颗粒质量标准研究中,配方颗粒的次黄嘌呤含量测定方法色谱条件与饮片一致,不同批号的配方颗粒次黄嘌呤含量为1.5～3.3mg/袋。稳定性实验:实验室留样观察三个月,按质量标准项目检查,包括外观、鉴别、检查、含量测定、卫生学,均符合规定。
小鼠的哮喘和咳嗽实验证明次黄嘌呤、琥珀酸有止咳平喘疗效。选择这两个成分作为指标成分是合理的。
地龙凝胶剂治疗烫伤实验中受伤面积数据表明广地龙无糖凝胶和广地龙含糖凝胶对烫伤有治疗作用。含水量实验数据表明广地龙无糖凝胶和广地龙含糖凝胶可减少水肿。羟脯氨酸含量测定数据表明广地龙含糖凝胶使烫伤部位恢复较快。
4.结论
本研究建立的广地龙饮片和配方颗粒质量控制方法规范,科学,通过药效学的验证,说明以次黄嘌呤和琥珀酸含量作为控制广地龙饮片及配方颗粒质量的合理性,为广地龙及其产品的质量控制提供科学依据。另外本课题对广地龙促进伤口的愈合作用的制剂及药效学的进行初步研究,为今后广地龙外用制剂的开发奠定了一定的基础。
Objective: In order to control the quality of Phertima dispensing granule and assure its clinical results, System study on quality standard of Phertima material and its dispensing granule was carried out according to the theories of Traditional Chinese Medicine and”the Technical Requirement of Research on the Quality Standard of Chinese Medicine Granule”and the validity of hypoxanthine and succinic acid as the active components was testified, In addition, earthworm gel preparation was researched, and its therapeutic efficacy of scald was evaluated.
Methods: This research includes quality standard of Phertima and its dispensing granule, preparation technology, pharmacodynamics evaluation of hypoxanthine and succinic acid, stability, earthworm gel preparation, pharmacodynamic action of scald. In the quality research of the raw materials, thin layer chromatography (TLC) was used to identify amino acids, succinic acid and extractive of trichloromethane. High performace liquid chromatography (HPLC) method used for determing the content of hypoxanthine using chromatographic column of Inertsil ODS-EP, mobile phase of H2O-CH3OH-C4H8O (100﹕0.5﹕0.5), flow rate of 1.0mL/min and detection wavelength of 254 nm. Another component, succinic acid, was determined by High Performance Capillary Electrophoresis (HPCE). The succinic acid could be separated and detected by buffer (0.36 mmol/L Tris-1.25 mmol/L H3BO3-0.5% 4-ethylene -5-amino-0.025 mmol/L CTAB adjusted to pH = 11.45 ) at -10kV of constant voltage. Other determination indexes, for example, dopant, moisture content, extractive assaying, also were determined.
The dispensing granules were preparated according to traditional decoction, removing impurity by centrifuge, drying by spray and granulation and the extraction technics of active components were optimized by orthogonal design using the content of hypoxanthine as the valuation index. Same methods including TLC, HPLC which were used in the raw materials were applied to control quality of Phertima dispensing granule, other indexes were ascertained according to requirement of granules in appendix of Pharmacopoeia and preliminary experiments on stability were carried out.
Asthmatic model of mice induced by histamine and acetylcholine and cough model of mice induced by ammonia water, were used to evaluate the pharmacodynamics of extract, dispensing granules, hypoxanthine, succinic acid and so on.
Earthworm gel is composed of earthworm extract, carbomer, glycerine, propanediol and so on and its scald pharmacodynamics evaluated by scald area, vary of hydroxyproline and pathology slice.
Results: In the quality research of the raw materials, TLC in China Pharmacopeia (version 2005) was improved, 2-aminopropionic acid and succinic acid, were added as the marker substance. The content range of hypoxanthine in different herbal Pheretima aspergillum were 0.54 mg/g～1.25 mg/g and 1.6 mg/bag~3.3 mg/bag in is dispensing granule by HPLC. The content of succinic acid was 0.8 mg/g in herbal Pheretima aspergillum (region:GuangXi).
The optimum extract processing of active components was: 30 gram Phertima was weighed and soaked 1hour, then bored 1h and extracted with water third, used 12 volumes of water, afterward, filtrated, concentrated, dried and granulated. Dispensing granules were taken stability examination in the laboratory for three months including the outward appearance, the discrimination, the inspection, the assaying and the hygieology and all above were conformed to the requirement.
Hypoxanthine and succinic acid have effects on cough-remedy, asthma-smooth. It can be concluded that the two ingredients as the standard components are reasonable and reliable. In the scald-remedy experiment, the data indicated that both earthworm syrup gel and earthworm non-syrup gel have therapeutics effects.
Conclusions: The all methods to control the quality both the raw materials and the granules were standard and scientific and hypoxanthine and succinic acid as the standard components was reasonable. This thesis provided scientific and reliable basis for controlling the quality of Phertima dispensing granules. In addition, the earthworm gel preparation in agglutinating and the vertification of pharmacodynamics will be established an experimental foundation in Phertima comprehend research and development.
引文
[1] 雷载权 张延模. 《中华临床中药学》[M].下册 人民卫生出版社. 1459
[2] 《中华本草》[M].下册 上海科学技术出版社.2344
[3] 《中国药典》2005 版[S].80
[4] 于德江.《中药材广地龙》[J]. 中国药学杂志,1993;28(10):594～596
[5] 陈平等.《中药广地龙的药源调查与商品鉴定》[J] .中草药, 1997;28(8):492～495
[6] 宋立人等.《现代中药学大辞典》[M].上册 人民卫生出版社. 758
[7] 王光忠等.广地龙类药材化学成分分析[J]. 中药材,1998; 21(3):133～135
[8] 杨得坡等.《参环毛蚓脂类挥发性成分分析》[J]. 中药材,2000;23(1):31～33
[9] 郑虎占等. 《中药现代研究与应用》[M]. 第 5 卷 4879
[10] 崔建等.《广地龙的药理作用研究进展》[J].长春中医学院学报, 1996; 12(57):61～62
[11] 贺石林等.《广地龙提取液的抗凝作用与毒性》[J].湖南医科大学学报, 1990;15(2):107～110
[12] 郭怀芳等.《蚓激酶的临床应用进展》[J].药物生物技术, 2001;9(1):57～60
[13] 李勇文等.《广地龙的研究进展》[J].广西医学, 2004; 26(5):699～701
[14] 汪珊等.《中药广地龙药理与哮喘气道重建相关性研究》[J]. 广东药学院学报, 2004;60(2):60～62
[15] 张凤春等.《广地龙对巨噬细胞免疫活性的增强作用》[J]. 中国药学杂志,1998; 33 (9):532～535
[16] 张凤春等.《广地龙促进大白兔背部创伤伤口收缩的实验研究》[J]. 中国中药杂志,1998;23 (9):560～561
[17] 章梅等.《广地龙提取物对大鼠深Ⅱ度烫伤创面愈合的影响》[J]. 中药材, 2004;27(10):744～747
[18] 雷载权 张延模. 《中华临床中药学》[M].下册 人民卫生出版社. 1463
[19] 隋海明等.《血管吻合术后中药抗凝作用的临床观察》[J].中医正骨, 2005; 17(6):6
[20] 汪建华等.《疏血通治疗急性脑梗塞 56 例》[J].陕西中医, 2004;25(7): 607～608
[21] 李亚君等.《普恩复治疗脑梗塞疗效观察》[J]. 黑龙江医学, 1999;4:40
[22] 张洁等.《复方广地龙胶囊对脑梗死患者的临床疗效及血液流变学影响》.江西 中医药,2003;34(4):17～18
[23] 李启荣. 《广地龙注射液治疗脑梗塞的初步临床观察》[J].广西中医学院学报, 2001;4(2):52～53
[24] 陈梅晞.《广地龙提取液对哮喘患者肺功能的影响》[J]. 右江民族医学院学报, 1997; 19(4) 606～607
[25] 傅洁. 《广地龙液即刻平喘的临床和实验研究》[J]. 上海中医药杂志,1991;2:24-26
[26] 周嘉莅. 《咳喘平煎剂治疗小儿肺炎 60 例》[J]. 陕西中医, 2003;(24):6 484～485
[27] 曾凡举等.《广地龙浸出液治疗烧伤168 例疗效观察》[J]. 国医论坛,2001; 16(1):36
[28] 孙海燕等.《鲜广地龙外敷治疗创伤性溃疡临床观察》[J].河北医学,1998;12(4):50
[29] 安静等.《鲜广地龙外敷治疗感染性褥疮的观察及护理》[J]. 黑龙江护理杂志,1998;4(10):5
[30] 张文斌等.《地龙不同炮制品中琥珀酸含量的比较》[J]. 中国药事, 2001;15(3):216
[31] 李坚等. 《HPLC 测定不同产地广地龙次黄嘌呤含量》[J].中药材,2006;29(5):448～449
[32] 黄庆华等.《广地龙中琥珀酸的毛细管电泳电导法测定》[J]. 中国现代应用药学杂志,2006;23(5):484～486
[33] 王光忠.《薄层扫描法测定广地龙类药材中琥珀酸的含量》[J]. 药物分析杂志,1997;17(4):274～275
[34] 李仪奎. 中药药理实验方法学[M]. 上海:上海科技卫生出版社,1991: 424～425
[35] 李仪奎.中药药理实验方法学[M]. 上海:上海科技卫生出版社,1991: 443～444
[36] 陈云峰等.《广地龙促进创面愈合的实验研究》[J].中华整形外科杂志, 2000; 16 (3)
[37] 王素香. 《广地龙白糖浸出液治疗烧伤18例》[J].荷泽医专学报, 1997;9(3): 14
[38] 曾凡举等. 《广地龙浸出液治疗烧伤168例疗效观察》[J].国医论坛, 2001;16(1):36
[39] 黄淑华等. 《广地龙浸液治疗烫火伤》[J].中国民间疗法, 2001;9(2):64
[40] 苗明三. 《实验动物和动物实验技术》[M].中国中医药出版社 1997.252
[41] 章梅等. 《广地龙提取物对大鼠深Ⅱ度烫伤创面愈合的影响》[J]. 中药材, 2004;27(10)
[42] 许志勤等. 《组织羟脯氨酸测定方法的改进》[J].解放军预防医学杂志, 1990; 8(1): 40～42 [ 43 ] Berry B.W.Smith G.C. and Carpenter Z.L.,Relationship of certain muscle,cartilage and bone traits to tenderness of the beef longissimus[J]. J.Food Sci., 1974, 39:819
[44] 高文铸. 《华佗遗书》[M]. 华夏出版社, 614
[2] 《中华本草》[M].下册 上海科学技术出版社.2344
[3] 《中国药典》2005 版[S].80
[4] 于德江.《中药材广地龙》[J]. 中国药学杂志,1993;28(10):594～596
[5] 陈平等.《中药广地龙的药源调查与商品鉴定》[J] .中草药, 1997;28(8):492～495
[6] 宋立人等.《现代中药学大辞典》[M].上册 人民卫生出版社. 758
[7] 王光忠等.广地龙类药材化学成分分析[J]. 中药材,1998; 21(3):133～135
[8] 杨得坡等.《参环毛蚓脂类挥发性成分分析》[J]. 中药材,2000;23(1):31～33
[9] 郑虎占等. 《中药现代研究与应用》[M]. 第 5 卷 4879
[10] 崔建等.《广地龙的药理作用研究进展》[J].长春中医学院学报, 1996; 12(57):61～62
[11] 贺石林等.《广地龙提取液的抗凝作用与毒性》[J].湖南医科大学学报, 1990;15(2):107～110
[12] 郭怀芳等.《蚓激酶的临床应用进展》[J].药物生物技术, 2001;9(1):57～60
[13] 李勇文等.《广地龙的研究进展》[J].广西医学, 2004; 26(5):699～701
[14] 汪珊等.《中药广地龙药理与哮喘气道重建相关性研究》[J]. 广东药学院学报, 2004;60(2):60～62
[15] 张凤春等.《广地龙对巨噬细胞免疫活性的增强作用》[J]. 中国药学杂志,1998; 33 (9):532～535
[16] 张凤春等.《广地龙促进大白兔背部创伤伤口收缩的实验研究》[J]. 中国中药杂志,1998;23 (9):560～561
[17] 章梅等.《广地龙提取物对大鼠深Ⅱ度烫伤创面愈合的影响》[J]. 中药材, 2004;27(10):744～747
[18] 雷载权 张延模. 《中华临床中药学》[M].下册 人民卫生出版社. 1463
[19] 隋海明等.《血管吻合术后中药抗凝作用的临床观察》[J].中医正骨, 2005; 17(6):6
[20] 汪建华等.《疏血通治疗急性脑梗塞 56 例》[J].陕西中医, 2004;25(7): 607～608
[21] 李亚君等.《普恩复治疗脑梗塞疗效观察》[J]. 黑龙江医学, 1999;4:40
[22] 张洁等.《复方广地龙胶囊对脑梗死患者的临床疗效及血液流变学影响》.江西 中医药,2003;34(4):17～18
[23] 李启荣. 《广地龙注射液治疗脑梗塞的初步临床观察》[J].广西中医学院学报, 2001;4(2):52～53
[24] 陈梅晞.《广地龙提取液对哮喘患者肺功能的影响》[J]. 右江民族医学院学报, 1997; 19(4) 606～607
[25] 傅洁. 《广地龙液即刻平喘的临床和实验研究》[J]. 上海中医药杂志,1991;2:24-26
[26] 周嘉莅. 《咳喘平煎剂治疗小儿肺炎 60 例》[J]. 陕西中医, 2003;(24):6 484～485
[27] 曾凡举等.《广地龙浸出液治疗烧伤168 例疗效观察》[J]. 国医论坛,2001; 16(1):36
[28] 孙海燕等.《鲜广地龙外敷治疗创伤性溃疡临床观察》[J].河北医学,1998;12(4):50
[29] 安静等.《鲜广地龙外敷治疗感染性褥疮的观察及护理》[J]. 黑龙江护理杂志,1998;4(10):5
[30] 张文斌等.《地龙不同炮制品中琥珀酸含量的比较》[J]. 中国药事, 2001;15(3):216
[31] 李坚等. 《HPLC 测定不同产地广地龙次黄嘌呤含量》[J].中药材,2006;29(5):448～449
[32] 黄庆华等.《广地龙中琥珀酸的毛细管电泳电导法测定》[J]. 中国现代应用药学杂志,2006;23(5):484～486
[33] 王光忠.《薄层扫描法测定广地龙类药材中琥珀酸的含量》[J]. 药物分析杂志,1997;17(4):274～275
[34] 李仪奎. 中药药理实验方法学[M]. 上海:上海科技卫生出版社,1991: 424～425
[35] 李仪奎.中药药理实验方法学[M]. 上海:上海科技卫生出版社,1991: 443～444
[36] 陈云峰等.《广地龙促进创面愈合的实验研究》[J].中华整形外科杂志, 2000; 16 (3)
[37] 王素香. 《广地龙白糖浸出液治疗烧伤18例》[J].荷泽医专学报, 1997;9(3): 14
[38] 曾凡举等. 《广地龙浸出液治疗烧伤168例疗效观察》[J].国医论坛, 2001;16(1):36
[39] 黄淑华等. 《广地龙浸液治疗烫火伤》[J].中国民间疗法, 2001;9(2):64
[40] 苗明三. 《实验动物和动物实验技术》[M].中国中医药出版社 1997.252
[41] 章梅等. 《广地龙提取物对大鼠深Ⅱ度烫伤创面愈合的影响》[J]. 中药材, 2004;27(10)
[42] 许志勤等. 《组织羟脯氨酸测定方法的改进》[J].解放军预防医学杂志, 1990; 8(1): 40～42 [ 43 ] Berry B.W.Smith G.C. and Carpenter Z.L.,Relationship of certain muscle,cartilage and bone traits to tenderness of the beef longissimus[J]. J.Food Sci., 1974, 39:819
[44] 高文铸. 《华佗遗书》[M]. 华夏出版社, 614