多硫酸化硫酸软骨素的制备、性质及其口服制剂对兔膝骨关节炎的关节保护作用
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摘要
随着世界人口进入老龄化,骨关节炎(Osteoarthritis,OA)的患病率不断上升,患病人群日益增多。OA会导致疼痛、残疾,严重影响患者的劳动能力。OA发生时,首先受到损伤的是关节软骨。关节软骨变薄、变性、破坏,软骨下骨硬化、关节间隙狭窄。对OA的治疗目前常用非甾体类抗炎药和多糖类软骨保护剂。多糖类软骨保护剂一般都是肝素类似物,如多硫酸氨基聚糖、多硫酸戊糖钠、多硫酸糖胺聚糖等。它们的作用是维持关节软骨的蛋白多糖含量,刺激软骨细胞合成Ⅱ型胶原及蛋白多糖,从而有助于维持关节软骨完整性。
硫酸软骨素(Chondroitin sulfate,CS)是关节软骨的重要成分,是由重复双糖→4)-β-D-GlcUA-(1→3)-β-D-GalNAc-(1→构成的糖胺聚糖。OA发生时,软骨受到损害,CS含量下降。补充CS能有效缓解症状,减轻炎症区域的肿胀度、下调基质金属蛋白酶-9,减轻OA对软骨的损害;还可能降低发病率、关节温度、痛感。临床上已有CS的复方口服药物或膳食补充剂,用于骨关节炎患者的治疗和保健。而深入研究发现,CS由于糖链上硫酸基位置的不同具有多种异构体。目前使用的CS药物多从动物软骨组织提取,其成分多是4-硫酸软骨素(CSA)和6-硫酸软骨素(CSC)。而在人关节软骨中主要含有的是4,6-二硫酸软骨素(CSE)。体外实验发现,当模拟OA发生时,培养的软骨组织中4,6-双硫酸软骨素含量下降,而有针对性的补充4,6-双硫酸软骨素在低浓度即可刺激软骨基质合成,并抑制蛋白酶合成,具有显著的软骨保护效应。提示硫酸根和其负电荷可能对OA的发病和治疗都非常重要。
为开发针对OA发病机制的特异性的软骨保护剂,本课题采用氯磺酸-甲酰胺磺化法对4-硫酸软骨素进行多硫酸化衍生,制备了含有4,6-双硫酸软骨素结构的多硫酸化硫酸软骨素(Polysulfated chondroitin sulfate,PSCS),使硫酸软骨素带上更多的负电荷,改变了其构象。由于PSCS是带有大量负电荷的大分子,口服不易吸收,要开发成口服治疗OA的药物,需要制备成带负电荷少、容易吸收的口服制剂。因此我们研究了PSCS的制备、鉴定、剂型及对兔膝OA的药理活性。
1 PSCS的制备、纯化及性质
用氯磺酸-甲酰胺磺化法对CS进行多硫酸化,制备PSCS。通过SO_4~(2-)含量测定和SO_4~(2-)/COO~-比值测定验证磺化结果,SO_4~(2-)含量从12.76%上升到20.62%,增加了7.76%;SO_4~(2-)/COO~-比值从1.35上升到3.35。产率达78.9%。对产物PSCS进行毛细管电泳分析和高效液相色谱分析,均只出现一个对称尖峰,证明所得产品较纯。并测定了其理化性质,包括糖醛酸含量、氨基己糖含量、绝对分子量、比旋度等。同时测定了PSCS的抗凝效价为40usp/mg。说明PSCS具有一定的抗凝活性。
2 PSCS的结构研究
用UV、IR、~1H-NMR和~(13)C-NMR分析了PSCS的结构。UV显示,样品不含共轭双键,也不含有蛋白质和核酸等杂质。经与CS红外光谱对比,证实了PSCS具有CS基本母链结构,即含有葡萄糖醛酸与半乳糖胺双糖单位,吡喃糖为β构型。并且在PSCS的IR上出现了硫酸基加在了半乳糖胺6位碳上的特征吸收峰818cm~(-1),可以判断半乳糖胺6位碳上羟基被硫酸基取代。经过~1H-NMR和~(13)C-NMR进一步解析,葡萄糖醛酸的C-2、C-3位都连接上了硫酸基。PSCS主要双糖结构应该为由2,3位硫酸化的葡萄糖醛酸和4,6位O硫酸化的半乳糖胺组成→4)-β-D-GlcUA(2S,3S)-(1→3)-β-D-GalNAc(4S,6S)-(1→。
3 PSCS口服制剂的制备
筛选了PSCS-壳聚糖纳米粒剂型的制备条件和方法。其优化条件为:壳聚糖的分子量为5kDa,其浓度为2mg╱ml,溶液的pH6.0,加入的PSCS浓度为1mg/ml,体积为壳聚糖溶液的四分之一,滴加速度为2秒/滴,滴加时壳聚糖反应液涡旋转速为600r/min。所得PSCS-壳聚糖纳米粒径在200nm左右,所带电荷为+46mV左右。具有较好的均一性、圆整度和稳定性。
4 PSCS对兔膝骨关节炎的软骨保护作用
用兔膝关节腔内注射Ⅱ型胶原酶建立了骨关节炎早期模型。
每日灌胃给PSCS-壳聚糖纳米粒,持续4w。行为学、影像学、病理学检测证实了口服PSCS-壳聚糖纳米粒对兔膝骨关节炎具有一定的疗效。行为学观察可见口服给药PSCS,对骨关节炎的膝疼痛有一定缓解作用。X光片显示,模型组关节面粗糙,关节间隙变窄,有疑似骨赘。而用药组关节面较光滑,关节间隙只是略有变窄,无骨赘。剥离软骨,观察软骨表面:用药组关节软骨表面与模型组相比,软化灶更少,光泽度更好。观察软骨HE染色病理切片,显示PSCS用药组能有效保护软骨表面完整性和深层细胞及结构;经Markin评分,结果显示,模型组为5.76±3.23,PSCS低剂量组为3.98±1.88,PSCS高剂量组为3.32±2.02。PSCS高、低剂量组与模型组相比都具有统计学差异,说明低剂量PSCS即具有一定的缓解软骨退行性变的作用。可以认为PSCS具有一定的软骨保护功能。
检测了兔血清中TNF-α和IL-1β的含量。PSCS低剂量组TNF-α含量为8.98±1.98pg/ml,与模型组18.76±1.23pg/ml相比具有显著性差异,说明PSCS能有效抑制骨关节炎模型兔血清中TNF-α的增高,且与阳性药物组9.92±1.16pg/ml的效果无统计学差异。IL-1β的含量PSCS组与模型组对比无显著性差异。
本课题取得的主要成果:
■成功的制备了纯度较高的多硫酸化硫酸软骨素PSCS,并确认了它含有4,6-双硫酸软骨素结构。
■成功的制备了PSCS-壳聚糖纳米粒,并筛选了PSCS-壳聚糖纳米粒制备的最优条件。
■将PSCS-壳聚糖纳米粒用于兔膝骨关节炎治疗,证实了PSCS对早期OA具有明显的软骨保护作用。同时也验证了PSCS-壳聚糖纳米粒被机体有效吸收。
The Osteoarthritis(OA)morbidity is rising as the aging of population in the world.OA causes great pain and even disability,and affects people' work ability seriously.Articular cartilage is injured first when OA happens.It becomes thinner, denatured and destroyed,and joint space becomes narrower.Non-steroidal anti-inflammatory drugs and chondroprotective agents are now used in the therapy of OA.Chondroprotective agents are often polysaccharide,such as polysulfated aminoglycans,polysulfated Fondaparinux sodium and polysulfated glycosaminoglycans.Their effects are to maintain the concentration of proteoglycans in cartilage and stimulate the synthesis of typeⅡcollagen by chondrocyte,thus maintaining the integrity of articular cartilage.
Chondroitin sulfate(CS),the main component of articular cartilage,is a glycosaminoglycan with a disaccharide unit of→4)-β-D-GlcUA-(1→3)-β-D-GalNAc-(1→. The content of CS decreases when OA happens and articular cartilage injured.Administration of CS can down-regulate matrix metalloproteinases-9,ease symptoms of OA,and may also decrease incidence of the disease.CS compound recipe has been used clinically for therapy of OA.Now CS is often extracted from animals,the main components of which are 4-sulfate CS(CSA)and 6-sulfate CS(CSC).The main component in human articular cartilage is 4,6-disulfate CS(CSE). In vitro experiments show that the content of 4,6-disulfate CS decreases in cultured articular cartilage when simulating OA,while the addition of 4,6-disulfate CS can stimulate the synthesis of cartilage,suggesting that sulfate group may be import for the treatment of OA.
In order to develop chondroprotective agent,PSCS containing 4,6-disulfate structure were prepared by the sulfation of 4-sulfate CS with chlorosulfonic acid-formamide methods.As PSCS is a macromolecule with large amounts of anions, it is difficult to be absorpted after oral administration.So it is necessary to prepare suitable dosages in which the PSCS with easy absorption after oral taken.Therefore, we investigated the PSCS preparation,structural determination,and chondroprotective effect on the OA of rabbit knee.
1.Preparation,purification and the properties of PSCS.
We prepared the polysulfated CS derivatives with chlorosulfonic acid-formamide methods,and test the results by the determination of the content of SO_4~(2-)and SO_4~(2-)/COO~- ratio.SO_4~(2-)was increased from 12.76%to 20.62%;SO_4~(2-)/COO~- ratio was from 1.35 to 3.35.The yield of the reaction was 78.9%.The capillary electrophoresis and HPLC results of PSCS showed a symmetrical sharp peak, suggesting the high purity of PSCS.The physico-chemical properties were determined such as the content of glycuronic acid,aminohexose,molecular weight and specific optical rotation.And its anticoagulant activity is 40usp/mg,suggesting that PSCS has anticoagulant activities.
2 Structure of PSCS
The structure of PSCS was analysized by the spectra of UV,IR,~1H-NMR and ~(13)C-NMR.UV shows that there is no conjugated double band in its structure,and no protein or nucleic acid in the sample.By comparising its IR with that of CS,it is shown that PSCS has the same major structure as CS,that is the disaccharide unit of→4)-β-D-GlcUA-(1→3)-β-D-GalNAc-(1→,and the absorption at 818cm~(-1)suggests that 6-hydroxy of galactosamine is substituted by sulfate radical.~1H-NMR and ~(13)C-NMR spectropy shows that C-2 and C-3 of glucuronic acid are sulfated.The major disaccharide unit of PSCS could be→4)-β-D-GlcUA(2S,3S)-(1→3)-β-D-GalNAc(4S, 6S)-(1→.
3.Preparation of oral dosage form
The conditions and method of preparaing PSCS-chitosan nanoparticle were screened.The molecular weight of chitosan is 5kDa,the concentration is 2mg/ml,and pH at 6.0.A quarter of the volume of 1mg/ml PSCS was titrated at a rate of 2s/drop, with the stirring rate of 600 r/min.The diameters and the charges of the nanoparticles are 200 nm and+46mV respectively,with good homogeneity,spherical degree,and good stability.
4.The chondroprotective effect of PSCS on rabbit Knee Osteoarthritis
The animal knee OA model was made by intraarticular injection of typeⅡcollagenase to rabbits.
PSCS-chitonan nanoparticules were administered by gastric perfusion daily for 4 weeks.The ethology,iconography and pathology studies showed that PSCS-chitosan nanoparticules therapeutic effects for rabbit knee OA.Ethology observation showed that the administration of PSCS can ease the pain of knee OA.X-ray photography showed that the rough articular facet,narrower joint space and doubtful splints exist in the articulations of model group,while the articulations in the PSCS group have smooth articular facet,less narrower joint space and no splints.The pathological sections were assessed with Markin method.The results showed scores of model group,PSCS low dosage group and the PSCS high dosage group are 3.32±2.02, 3.98±1.88,5.76±3.23,respectively.Statistics difference exist between the model group and PSCS low dosage group,suggesting that PSCS has chondroprotective effect even at a low dosage.
The concentrations of TNF-αand IL-1βin the serum of the rabbits were detected. The concentration of TNF-αin PSCS low dosage group and model group are 18.76±1.23pg/ml and 8.98±1.98pg/ml,respectively,with significant difference, suggesting that PSCS can decrease the concentration of TNF-αin OA rabbit serum, and has no significant difference with positive control group.The concentration of IL-1βin the PSCS group and control group has no significant difference.
The accomplishments of the subject:
■Successfully prepared highly purified PSCS,containing the structure of 4,6-bisulfate CS.
■Successfully prepared the PSCS-chitinan nanoparticles and screened the conditions.
■The PSCS-chitinan nanoparticles were administrated orally for treatment of rabbit knee osteoarthritis and confirmed the protective effects.
硫酸软骨素(Chondroitin sulfate,CS)是关节软骨的重要成分,是由重复双糖→4)-β-D-GlcUA-(1→3)-β-D-GalNAc-(1→构成的糖胺聚糖。OA发生时,软骨受到损害,CS含量下降。补充CS能有效缓解症状,减轻炎症区域的肿胀度、下调基质金属蛋白酶-9,减轻OA对软骨的损害;还可能降低发病率、关节温度、痛感。临床上已有CS的复方口服药物或膳食补充剂,用于骨关节炎患者的治疗和保健。而深入研究发现,CS由于糖链上硫酸基位置的不同具有多种异构体。目前使用的CS药物多从动物软骨组织提取,其成分多是4-硫酸软骨素(CSA)和6-硫酸软骨素(CSC)。而在人关节软骨中主要含有的是4,6-二硫酸软骨素(CSE)。体外实验发现,当模拟OA发生时,培养的软骨组织中4,6-双硫酸软骨素含量下降,而有针对性的补充4,6-双硫酸软骨素在低浓度即可刺激软骨基质合成,并抑制蛋白酶合成,具有显著的软骨保护效应。提示硫酸根和其负电荷可能对OA的发病和治疗都非常重要。
为开发针对OA发病机制的特异性的软骨保护剂,本课题采用氯磺酸-甲酰胺磺化法对4-硫酸软骨素进行多硫酸化衍生,制备了含有4,6-双硫酸软骨素结构的多硫酸化硫酸软骨素(Polysulfated chondroitin sulfate,PSCS),使硫酸软骨素带上更多的负电荷,改变了其构象。由于PSCS是带有大量负电荷的大分子,口服不易吸收,要开发成口服治疗OA的药物,需要制备成带负电荷少、容易吸收的口服制剂。因此我们研究了PSCS的制备、鉴定、剂型及对兔膝OA的药理活性。
1 PSCS的制备、纯化及性质
用氯磺酸-甲酰胺磺化法对CS进行多硫酸化,制备PSCS。通过SO_4~(2-)含量测定和SO_4~(2-)/COO~-比值测定验证磺化结果,SO_4~(2-)含量从12.76%上升到20.62%,增加了7.76%;SO_4~(2-)/COO~-比值从1.35上升到3.35。产率达78.9%。对产物PSCS进行毛细管电泳分析和高效液相色谱分析,均只出现一个对称尖峰,证明所得产品较纯。并测定了其理化性质,包括糖醛酸含量、氨基己糖含量、绝对分子量、比旋度等。同时测定了PSCS的抗凝效价为40usp/mg。说明PSCS具有一定的抗凝活性。
2 PSCS的结构研究
用UV、IR、~1H-NMR和~(13)C-NMR分析了PSCS的结构。UV显示,样品不含共轭双键,也不含有蛋白质和核酸等杂质。经与CS红外光谱对比,证实了PSCS具有CS基本母链结构,即含有葡萄糖醛酸与半乳糖胺双糖单位,吡喃糖为β构型。并且在PSCS的IR上出现了硫酸基加在了半乳糖胺6位碳上的特征吸收峰818cm~(-1),可以判断半乳糖胺6位碳上羟基被硫酸基取代。经过~1H-NMR和~(13)C-NMR进一步解析,葡萄糖醛酸的C-2、C-3位都连接上了硫酸基。PSCS主要双糖结构应该为由2,3位硫酸化的葡萄糖醛酸和4,6位O硫酸化的半乳糖胺组成→4)-β-D-GlcUA(2S,3S)-(1→3)-β-D-GalNAc(4S,6S)-(1→。
3 PSCS口服制剂的制备
筛选了PSCS-壳聚糖纳米粒剂型的制备条件和方法。其优化条件为:壳聚糖的分子量为5kDa,其浓度为2mg╱ml,溶液的pH6.0,加入的PSCS浓度为1mg/ml,体积为壳聚糖溶液的四分之一,滴加速度为2秒/滴,滴加时壳聚糖反应液涡旋转速为600r/min。所得PSCS-壳聚糖纳米粒径在200nm左右,所带电荷为+46mV左右。具有较好的均一性、圆整度和稳定性。
4 PSCS对兔膝骨关节炎的软骨保护作用
用兔膝关节腔内注射Ⅱ型胶原酶建立了骨关节炎早期模型。
每日灌胃给PSCS-壳聚糖纳米粒,持续4w。行为学、影像学、病理学检测证实了口服PSCS-壳聚糖纳米粒对兔膝骨关节炎具有一定的疗效。行为学观察可见口服给药PSCS,对骨关节炎的膝疼痛有一定缓解作用。X光片显示,模型组关节面粗糙,关节间隙变窄,有疑似骨赘。而用药组关节面较光滑,关节间隙只是略有变窄,无骨赘。剥离软骨,观察软骨表面:用药组关节软骨表面与模型组相比,软化灶更少,光泽度更好。观察软骨HE染色病理切片,显示PSCS用药组能有效保护软骨表面完整性和深层细胞及结构;经Markin评分,结果显示,模型组为5.76±3.23,PSCS低剂量组为3.98±1.88,PSCS高剂量组为3.32±2.02。PSCS高、低剂量组与模型组相比都具有统计学差异,说明低剂量PSCS即具有一定的缓解软骨退行性变的作用。可以认为PSCS具有一定的软骨保护功能。
检测了兔血清中TNF-α和IL-1β的含量。PSCS低剂量组TNF-α含量为8.98±1.98pg/ml,与模型组18.76±1.23pg/ml相比具有显著性差异,说明PSCS能有效抑制骨关节炎模型兔血清中TNF-α的增高,且与阳性药物组9.92±1.16pg/ml的效果无统计学差异。IL-1β的含量PSCS组与模型组对比无显著性差异。
本课题取得的主要成果:
■成功的制备了纯度较高的多硫酸化硫酸软骨素PSCS,并确认了它含有4,6-双硫酸软骨素结构。
■成功的制备了PSCS-壳聚糖纳米粒,并筛选了PSCS-壳聚糖纳米粒制备的最优条件。
■将PSCS-壳聚糖纳米粒用于兔膝骨关节炎治疗,证实了PSCS对早期OA具有明显的软骨保护作用。同时也验证了PSCS-壳聚糖纳米粒被机体有效吸收。
The Osteoarthritis(OA)morbidity is rising as the aging of population in the world.OA causes great pain and even disability,and affects people' work ability seriously.Articular cartilage is injured first when OA happens.It becomes thinner, denatured and destroyed,and joint space becomes narrower.Non-steroidal anti-inflammatory drugs and chondroprotective agents are now used in the therapy of OA.Chondroprotective agents are often polysaccharide,such as polysulfated aminoglycans,polysulfated Fondaparinux sodium and polysulfated glycosaminoglycans.Their effects are to maintain the concentration of proteoglycans in cartilage and stimulate the synthesis of typeⅡcollagen by chondrocyte,thus maintaining the integrity of articular cartilage.
Chondroitin sulfate(CS),the main component of articular cartilage,is a glycosaminoglycan with a disaccharide unit of→4)-β-D-GlcUA-(1→3)-β-D-GalNAc-(1→. The content of CS decreases when OA happens and articular cartilage injured.Administration of CS can down-regulate matrix metalloproteinases-9,ease symptoms of OA,and may also decrease incidence of the disease.CS compound recipe has been used clinically for therapy of OA.Now CS is often extracted from animals,the main components of which are 4-sulfate CS(CSA)and 6-sulfate CS(CSC).The main component in human articular cartilage is 4,6-disulfate CS(CSE). In vitro experiments show that the content of 4,6-disulfate CS decreases in cultured articular cartilage when simulating OA,while the addition of 4,6-disulfate CS can stimulate the synthesis of cartilage,suggesting that sulfate group may be import for the treatment of OA.
In order to develop chondroprotective agent,PSCS containing 4,6-disulfate structure were prepared by the sulfation of 4-sulfate CS with chlorosulfonic acid-formamide methods.As PSCS is a macromolecule with large amounts of anions, it is difficult to be absorpted after oral administration.So it is necessary to prepare suitable dosages in which the PSCS with easy absorption after oral taken.Therefore, we investigated the PSCS preparation,structural determination,and chondroprotective effect on the OA of rabbit knee.
1.Preparation,purification and the properties of PSCS.
We prepared the polysulfated CS derivatives with chlorosulfonic acid-formamide methods,and test the results by the determination of the content of SO_4~(2-)and SO_4~(2-)/COO~- ratio.SO_4~(2-)was increased from 12.76%to 20.62%;SO_4~(2-)/COO~- ratio was from 1.35 to 3.35.The yield of the reaction was 78.9%.The capillary electrophoresis and HPLC results of PSCS showed a symmetrical sharp peak, suggesting the high purity of PSCS.The physico-chemical properties were determined such as the content of glycuronic acid,aminohexose,molecular weight and specific optical rotation.And its anticoagulant activity is 40usp/mg,suggesting that PSCS has anticoagulant activities.
2 Structure of PSCS
The structure of PSCS was analysized by the spectra of UV,IR,~1H-NMR and ~(13)C-NMR.UV shows that there is no conjugated double band in its structure,and no protein or nucleic acid in the sample.By comparising its IR with that of CS,it is shown that PSCS has the same major structure as CS,that is the disaccharide unit of→4)-β-D-GlcUA-(1→3)-β-D-GalNAc-(1→,and the absorption at 818cm~(-1)suggests that 6-hydroxy of galactosamine is substituted by sulfate radical.~1H-NMR and ~(13)C-NMR spectropy shows that C-2 and C-3 of glucuronic acid are sulfated.The major disaccharide unit of PSCS could be→4)-β-D-GlcUA(2S,3S)-(1→3)-β-D-GalNAc(4S, 6S)-(1→.
3.Preparation of oral dosage form
The conditions and method of preparaing PSCS-chitosan nanoparticle were screened.The molecular weight of chitosan is 5kDa,the concentration is 2mg/ml,and pH at 6.0.A quarter of the volume of 1mg/ml PSCS was titrated at a rate of 2s/drop, with the stirring rate of 600 r/min.The diameters and the charges of the nanoparticles are 200 nm and+46mV respectively,with good homogeneity,spherical degree,and good stability.
4.The chondroprotective effect of PSCS on rabbit Knee Osteoarthritis
The animal knee OA model was made by intraarticular injection of typeⅡcollagenase to rabbits.
PSCS-chitonan nanoparticules were administered by gastric perfusion daily for 4 weeks.The ethology,iconography and pathology studies showed that PSCS-chitosan nanoparticules therapeutic effects for rabbit knee OA.Ethology observation showed that the administration of PSCS can ease the pain of knee OA.X-ray photography showed that the rough articular facet,narrower joint space and doubtful splints exist in the articulations of model group,while the articulations in the PSCS group have smooth articular facet,less narrower joint space and no splints.The pathological sections were assessed with Markin method.The results showed scores of model group,PSCS low dosage group and the PSCS high dosage group are 3.32±2.02, 3.98±1.88,5.76±3.23,respectively.Statistics difference exist between the model group and PSCS low dosage group,suggesting that PSCS has chondroprotective effect even at a low dosage.
The concentrations of TNF-αand IL-1βin the serum of the rabbits were detected. The concentration of TNF-αin PSCS low dosage group and model group are 18.76±1.23pg/ml and 8.98±1.98pg/ml,respectively,with significant difference, suggesting that PSCS can decrease the concentration of TNF-αin OA rabbit serum, and has no significant difference with positive control group.The concentration of IL-1βin the PSCS group and control group has no significant difference.
The accomplishments of the subject:
■Successfully prepared highly purified PSCS,containing the structure of 4,6-bisulfate CS.
■Successfully prepared the PSCS-chitinan nanoparticles and screened the conditions.
■The PSCS-chitinan nanoparticles were administrated orally for treatment of rabbit knee osteoarthritis and confirmed the protective effects.
引文
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[25]管剑龙,韩星海.中国骨关节炎十年[M].上海:第二军医大学出版社 2006,1:139-57
[26]Das A Jr,Hammad TA.Efficacy of a combination of FCHG49 glucosamine hydrochloride,TRH122 low molecular weight sodium chondroitin sulfate and manganese ascorbate in the management of knee osteoarthritis[J].Osteoarthritis Cartilage 2000,8(5):343-50
[27]卢京光,姬胜利,张尊建.硫酸软骨素及其衍生物的研究进展[J].中国海洋药物杂志 2006,25(1):59-63
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