CN1117525A - 新疆紫草细胞固体两步培养生产工艺 - Google Patents
新疆紫草细胞固体两步培养生产工艺 Download PDFInfo
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Abstract
采用新疆紫草幼苗的不同器官作外植体诱导愈伤组织并获细胞系;建立了细胞固体二步培养生产工艺流程,包括AG-7生长培养基与AP-5生产培养基。试验证明用本方法其细胞培养物中紫草宁衍生物的含量较原植物中高4-8倍。具抗菌、抗肿瘤活性的乙酰紫草素的含量比原植物高6-10倍,蜡质含量仅为天然提取物的1/16。
Description
本发明属于植物细胞工程技术领域,具体地说它是一种新疆紫草细胞固体培养的方法。
新疆紫草(Arnebia euchroma)为我国传统中草药,其有效成分紫草宁及其衍生物为萘醌类化合物。主要用于治疗皮肤烧伤、冻伤、烫伤、溃疡久治不愈及妇科炎症等。临床还用于各种皮肤疾症,如湿疹、丘疹、粉刺、角皮症、妇女色斑及头部浅在性白藓等。尤其是紫草宁衍生物中的乙酰紫草素具有抗肿瘤活性。此外紫草宁及其衍生物还是极好的天然色素,色价高,理化性能稳定。可用于化妆品,食品和染料等。但是由于新疆紫草生长在海拔3000米左右的高山地带,故引种栽培至今尚未成功,目煎仍果用果挖野生紫草提取紫草宁及其衍生物的方法。该技术的缺点在于:
①新疆紫草为多年生植物,紫草宁及其衍生物只存在于天然植栋的根部,故采集根部无疑是“杀鸡取卵”,对野生资源具有严重的破坏性。为此,新疆紫草早已被林业部列为国家级保护植物。
②紫草野生植株生长在不同的生长地带,其根中有效成份的含量不稳定,而且含量较低,通常为干重的1-4%。
③从天然植株根中提取的紫草宁及其衍生物中含有15-30%的蜡质。
除从野生新疆紫草根中提取紫草宁及其衍生物外,近年来我国东北地区,已将不同属的辽宁紫草(Lithospermum erythorhizon)引种栽培成功。此种生产紫草宁及其衍生物的途径也存在如下缺点:
①我国临床实践证明,新疆紫草临床药效远超过辽宁紫草。
②新疆紫草根中紫草宁及其衍生物的含量亦比辽宁紫草根中更高。
③人工栽培要占据大量农田,在可耕面积日渐缩小的形势下,这一举措应视为下策。
④人工栽培不能摆脱自然环境的影响,如不良气候条件,病虫害等,故有效成份的含量和质量将不稳定。
本发明的目的在于针对上述问题和存在缺陷,提出一种新疆紫草细胞固体两步培养生产方法。该方法采用植物细胞大量培养技术,实现了工业化生产紫草宁及其衍生物。
以下是本发明的主要内容:
1.新疆紫草细胞系的制取:
A.果用新疆紫草种子苗的不同器官(根、胚轴、子叶及真叶等)作外植体诱导愈伤组织。具体操作如下:将采自新疆地区的新疆紫草种子,经8%的次氯酸钠浸泡12分钟以进行表面消毒,播于经高压灭菌而浸有无菌水的滤纸上,在温度为25±1℃下暗培养,5天后开始发芽。待幼苗真叶形成,根和胚轴生长到0.8--1.2cm,将幼苗分成根、胚轴、子叶和真叶。把这四部分外植体分别接种在附加KT 0.5mg和2,4-D1mg/l的LS培养基上,放置于25±1℃暗室培养。经6周培养,即可在外植体的切口部位形成愈伤组织。
B.愈伤组织在生长培养基(AG—7)上继代培养(25±1℃暗培养),每25天转代一次,经过10-30代的继代培养可获得A-1细胞系。
2.新疆紫草细胞固体二步培养法中培养基组成:
A.生长培养基 B.生产培养基AG-7生长培养基组成成分 AP-5生产培养基组成成分
(单位:mg/L) (单位:mg/L)KNO3 1,900.0 KNO3 30.0(NH4)2SO4 463.0 Ca(NO3)·4B2O 694.0KH2PO4 170.0 MgSO4·7H2O 750.0MgSO4·7H2O 370.0 NaH2PO4·2H2O 19.0CaCl2·2H2O 440.0 KCl 65.0B3BO3 6.2 Na2SO4 1480.0KI 0.83MnSO4·4H2O 22.3 FeSO4·7H2O 27.8ZnSO4·7H2O 8.6 Na2EDTA 37.3Na2MoO4·2H2O 0.25 ZnSO4·7H2O 3.0CuSO4·5H2O 0.025 H3BO3 4.5CoCl·6H2O 0.025 CuSO4·5H2O 0.025FeSO4·7H2O 27.8 KT 1.0NNa2EDTA 37.3 Suerosse 20,000.0—50,000.0Inosiiol 100.0 Agar 7,000.0—10,000.0VitamineBl-HCl 0.1 *在高压消毒之前pH至4.8—6.2IAA 0.2KT 1.0Sucrosse 20,000.0—50,000.0Ager 7,000.0—10,000.0*在高压消毒之前pH调至4.8—6.2
3.新疆紫草细胞固体二步培养生产工艺流程
(A)按本发明AG-7生长培养基的配方组成,配制生长培养基。用pH计测定pH值,并用1N的HCl及1N的KOH调节培养基pH值在4.8—6.2范围内。加入0.7-1%琼脂,煮沸后分装至5号中方瓶(北京玻璃5厂出品)内,每瓶20—60毫升。用封口膜封口后,进行高压消毒,压力为1.0-1.2kg/cm2,时间为15—25分钟。
(B)将经过继代培养获得的A-1细胞系,在超净工作台中接种于上述生长培养基中,接种量为1.5-5.0%(w/v,g.fw/ML)。将接种后的培养瓶放入暗培养室中培养18—28天,温度为15—30℃。
(c)按(A)项所述的程序,按本专利申请AP-5生产培养基的配方组成,配制生产培养基。将(B)项在生长培养基上生长18—28天的愈伤组织,在超净工作台中转接至生产培养基中,接种量为3.0-7.0克鲜重/瓶,培养室条件同(B)项。培养20—25天后,用不锈钢勺将培养物全部取出,放入塘瓷盘内,送入50℃的烘箱中烘干,进行下述第4项的紫草宁衍生物的分离提取。
图1为新疆紫草细胞固体二步培养生产工艺流程示意图。
4.紫草宁衍生物的分离提取工艺:
取紫草细胞培养物于空气循环干燥器60℃烘干后,以粉碎机粉碎成20目的粗粉,装于组合式连续提取器中,用30-60沸程的石油醚做浸出溶剂进行循环浸出。浸出液的出液系数控制在5以下。浸出液经过滤后再用旋转式蒸发器减压回收石油醚,将浸出液浓缩成膏状,再于60℃干燥得紫草宁衍生物(AC-90)产品。
试验证明在固体培养下,用本发明提供的培养基,其细胞生长速率可迭10.0克·干重/升/月,紫草宁衍生物的含量最高可达培养物干重的7.0%;液体悬浮培养,细胞生长速率可达22.0克·干重/升/月,紫草宁衍生物的含量可达17.0%;应用生物反应器培养(10—30升)细胞生长速率可达18.0干重/升/月,紫草宁衍生物的含量最高可达培养物干重的12.0%。
图2为紫草宁衍生物提取工艺流程图。
本发明较已有技术相比具有如下优点及积极效果:
(1).新疆紫草为国家保护植物,利用植物细胞大量培养生产紫草宁衍生物,走工业化生产的途径,可有效地保护野生资源,保护生态环境。
(2).应用细胞大量培养生物合成紫草宁衍生物,可获得稳定的质量和产量,不受自然条件的限制和影响。
(3).不需要占用农田,只需要占用有限的厂房和培养室,高度集约化。
(4).通过人工调节与控制细胞的生长和次生产物的合成,可极大地提高紫草宁衍生物的产量与含量(见表1),尤其是提高紫草宁衍生物中具抗菌(见表2)、抗肿瘤活性的乙酰紫草素(Acetylsbikonin)的含量比例(见表3)。比原植株根中的紫草宁衍生物含量高4-8倍,乙酰紫草素的含量高6-10倍,蜡质含量仅为天然提取物的1/16。
(5).该项技术,不使用生物反应器进行工业化生产,故可免去购置生物反应器的昂贵投资。一次性投资量小,产量与含量稳定,污染率极低。
以下叙述本发明的实施例:实施例1
按本发明AG—7生长培养基的配方组成,配制生长培养基,蔗糖浓度为30克/升,调pH值为5.8,琼脂浓度为0.7%。用5号中方瓶,每瓶装培养液40毫升。高压消毒,压力为1.0Kg/CM2,2分钟。接入A—1细胞系,接种量为3.0%,效入暗培养室培养,温度为24°±1℃。培养23天后每瓶鲜重可达15.0克。将其转接至AP—5生产培养基中(配制程序同生长培养基),接种量为5.0克鲜重/瓶,培养室条件同生长阶段。培养23天后收获,每瓶可获培养物0.68克干重,细胞培养物经分离提取后,用高压液相色谱分析,并与天然植株中提取的紫草宁衍生物进行对照结果如下:表1 新疆紫草细胞培养物与天然植栋紫草宁衍生物含量及特性的比较
*.A.去氧紫草素, B.β,β—二甲基丙烯酰紫草素C.乙酰紫草素, 0.2,3—二甲基戊烯酰紫草素E.β—羟基异戊酰紫草素
总灰分(%) | 含蜡量(%) | 纯化合物含量比例(%)* | 色价E10%1CM(520nm) | |||||
A | B | C | D | E | ||||
天然植株细胞培养物 | 0.120.23 | 15.60.62 | 1.87.3 | 11.621.6 | 6.020.6 | 5.215.0 | 0.24.2 | ≥138≥167 |
实施例2
按实施例1通过培养物提取分离获得的紫草宁衍生物其抗菌活性超过天然植株提取的紫草宁衍生物。试验结果如下表。表中BA—I代表β,β—二甲基丙烯酰紫草素,BA—II代表乙酰紫草素,BA—P代表从植物中提取的总紫草宁化合物,BA—C代表从细胞培养物中提取的总紫草宁化合物。
表2对15种细菌的抗菌作用
试验细菌 | MIC(μg/Ml) | |||
BA—I | BA—II | BA—P | BA—C | |
金葡菌209p金葡菌15表葡菌26069八叠球菌枯草杆菌6638大肠杆菌22绿脓杆菌11绿脓杆菌17变形杆菌9肺炎杆菌14福氏痢疾杆菌宗氏痢疾杆菌伤寒杆菌鼠伤寒沙门氏菌粘质沙雷氏菌 | 0.390.39501.5650>10010010010010010050100100100 | 0.190.39>500.7825>50>50>5050>50>5050>50>50>50 | 0.783.125>1006.2525>100>100>100>100>100>100>100>100>100>100 | 0.190.78>1000.7812.5>100>100>100>100>100>10050>100>100>100 |
实施例3
按实施例1通过培养物提取分离获得的紫草宁衍生物其抗肿瘤活性超过天然植株提取的紫草宁衍生物。试验结果如下表。表中BA—P代表从植物中提取的总紫草宁化合物,BA—C代表从细胞培养物中提取的总紫草宁化合物,BA—II代表乙酰紫草素。
表3紫草宁衍生物抗艾氏腹水癌(EC)的活性比较
样 品 | 剂 量(Mg/Kg/日) | 小鼠生命延长率% |
BA—PBA—CBA—II | 555 | 2210769 |
Claims (3)
1.一种紫草细胞系,其特征在于:A.将新疆紫草幼苗的根或胚轴或子叶或真叶外植体接种于附加KT 0.5mg/l和2,4-D 1mg/l的LS培养基上,放置于25±1℃暗室培养,6周后,即可获得愈伤组织;
B.愈伤组织在下列培养基(AG—7)上继代培养,每25天转代一次,经过10-30代的继代培养可获得A-1细胞系。
AG-7生长培养基组成成分
(单位:mg/L)
KNO3 1,900.0
(NH4)2SO4 463.0
KB2PO4 170.0
MgSO4·7H20 370.0
CaCl2·2H2O 440.0
B3BO3 6.2
KI 0.83
MnS04·4H2O 22.3
ZnSO4·7H2O 3.6
Na2MoO4·2H2O 0.25
CuSO4·5H2O 0.025
CoCl·6H2O 0.025
FeSO4·7H2O 27.8
Na2EDFA 37.3
Inositol 100.0
VitamineB1-HC1 0.1
IAA 0.2
KT 1.0
Sucrosse 20,000.0—50,000.0
Agar 7,000.0—10,000.0
2.一种紫草细胞系的生产方法,其特征在于:A.将新疆紫草幼苗的根或胚轴或子叶或真叶外植体分别接种在附加KT 0.5mg/1和2,4-D1mg/l的LS培养基上,放置于25±1℃暗室培养,6周后,即可获得愈伤组织;
B.愈伤组织在下列培养基(AG—7)上继代培养,每25天转代一次,经过10-30代的继代培养可获得A-1细胞系。
AG-7生长培养基组成成分
(单位:mg/L)
KNO3 1,900.0
(KN4)2SO4 463.0
KH2PO4 170.0
MgSO4·7H2O 370.0
CaCl2·2H2O 440.0
H3BO3 6.2
KI 0.83
MnSO4·4H2O 22.3
ZnSO4·7H2O 8.6
Na2MoO4·2H2O 0.25
CuSO4·5H2O 0.025
CoCl·6H2O 0.025
FeSO4·7H2O 27.8
Na2EDTA 37.3
Inositol 100.0
VitamineBl-HCl 0.1
IAA 0.2
KT 1.0
Sucrosse 20,000.0—50,000.0
Agar 7,000.—10,000.0
3.一种利用新疆紫草细胞固体二步培养生产紫草宁衍生物的方法,其特征在于:
(A)将权利要求1所述的A-1细胞系接种于下述AG-7生长培养基中,接种量为1.5-5.0%(w/v,g.fw/ML);接种后放入暗培养室中培养18—28天,温度为15—30℃;
(B)将(A)中得到的细胞系转接至下述AP-5生产培养基中,接种量为3.0-7.0克鲜重/瓶,培养室条件同(A)项;培养20—28天后,获得新疆紫草培养物;
(C)将(B)中得到的培养物送入60℃的烘箱中烘干,用粉碎机粉碎成20目的粗粉,装于组合式连续提取器中,用石油醚浸提浸出液过滤、浓缩、干燥得紫草宁衍生物(AC-90)产品。
A. 生长培养基 B.生产培养基AG-7生长培养基组成成分 AP-5生产培养基组成成分
(单位:mg/L) (单位:mg/L)KNO3 1,900.0 KNO3 30.0(KH4)2SO4 463.0 Ca(NO3)·4H2O 694.0KH2PO4 170.0 MgSO4·TH2O 750.0MgSO4·7H2O 370.0 NaH2PO4·2H2O 19.0CaCl2·2H2O 440.0 KCl 65.0H3BO3 6.2 Na2SO4 1480.0KI 0.83MnSO4·4H2O 22.3 FeSO4·7H2O 27.8ZnSO4·7H2O 8.6 Na2EDTA 37.3Na2MoO4·2H2O 0.25 ZnSO4·7H2O 3.0CuSO4·5H2O 0.025 H3BO3 4.5CoCl·6H2O 0.025 CuSO4·5H2O 0.025FeSO4·7H2O 27.8 KT 1.0Na2EDTA 37.3 Sucrosse 20,000.0—50,000.0Inositol 100.0 Agar 7,000.0—10,000.0VitamineBl-HCl 0.1IAA 0.2KT 1.0Sucrosse 20,000.0—50,000.0Agar 7,000.0—10,000.0
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WO2004073699A1 (en) * | 2003-02-21 | 2004-09-02 | Beijing Jbc Chinese Traditional Medicine Science And Technology Develop Co. Ltd. | Pharmaceuticals comprising shikonins as active constituent |
EP1649743A1 (en) * | 2004-10-21 | 2006-04-26 | Guru Jambheshwar University | Method of direct regeneration, Shikonin induction in callus and Agrobacterium rhizogenes-mediated genetic transformation of Arnebia hispidissima |
CN1304565C (zh) * | 2003-12-26 | 2007-03-14 | 中国科学院过程工程研究所 | 一种用稀土元素促进新疆紫草愈伤组织生长的方法 |
CN101869591A (zh) * | 2010-06-03 | 2010-10-27 | 新疆农业大学 | 利用新疆紫草毛状根生产紫草素的方法 |
CN111194692A (zh) * | 2018-11-19 | 2020-05-26 | 四川农业大学 | 一种新疆紫草组培快繁方法和新疆紫草组培快繁培养基 |
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CN100391334C (zh) * | 2004-12-13 | 2008-06-04 | 中国科学院植物研究所 | 新疆紫草的一种繁殖方法 |
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CN1099051A (zh) * | 1993-08-17 | 1995-02-22 | 徐宝明 | 提取天然植物色素的原料和方法 |
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EP1649743A1 (en) * | 2004-10-21 | 2006-04-26 | Guru Jambheshwar University | Method of direct regeneration, Shikonin induction in callus and Agrobacterium rhizogenes-mediated genetic transformation of Arnebia hispidissima |
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