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Journal of Chinese Integrative Medicine Free Full Text
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Original Experimental Research
Journal of Chinese Integrative Medicine: Volume 6   January, 2008   Number 1

DOI: 10.3736/jcim20080111
Antitumor activities of kushen flavonoids in vivo and in vitro
1. Ming-yu SUN (Institute of Liver Diseases, Shuguang Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China E-mail: mysun248@hotmail.com)
2. Jian ZUO (Department of Pharmacology, Hutchison Medipharma Ltd, Zhangjiang Hi-Technology Park, Shanghai 201203, China )
3. Ji-feng DUAN (Department of Pharmacology, Hutchison Medipharma Ltd, Zhangjiang Hi-Technology Park, Shanghai 201203, China )
4. Jun HAN (Department of Pharmacognosy, School of Pharmacy, Second Military Medical University, Shanghai 200433, China )
5. Shi-ming FAN (Department of Pharmacology, Hutchison Medipharma Ltd, Zhangjiang Hi-Technology Park, Shanghai 201203, China )
6. Wei ZHANG (Department of Pharmacology, Hutchison Medipharma Ltd, Zhangjiang Hi-Technology Park, Shanghai 201203, China )
7. Li-fang ZHU (Department of Pharmacology, Hutchison Medipharma Ltd, Zhangjiang Hi-Technology Park, Shanghai 201203, China )
8. Ming-hui YAO (Department of Pharmacology, Shanghai Medical College, Fudan University, Shanghai 200032, China )

Objective: To explore the antitumor activities of kushen (Sophora flavescens) flavonoids (KS-Fs) in vivo and in vitro.

Methods: Cell proliferation was assayed by using methyl thiazolyl tetrazolium (MTT) method. H22 hepatocellular carcinoma and S180 sarcoma were induced in ICR mice. Lewis lung carcinoma was induced in C57BL/6 mice. H460 and Eca-109 tumor were induced in Balb/c nude mice by injecting 5×105 or 5×106 tumor cells in the right flank, respectively.

Results: KS-Fs could inhibit the growth of a variety of human tumor cell lines (A549, SPC-A-1, NCI-H460, etc.) in vitro. The antitumor efficacies were confirmed in the mice models of H22, S180 and Lewis lung tumors and the nude mice models of human H460 and Eca-109 xenografted tumors. The oral or intravenous maximum tolerated dose of KS-Fs was more than 2.8 g/kg or 750 mg/kg respectively, far more than the oral medial lethal dose of kushen alkaloids (≤1.18 g/kg). No adverse reactions were observed.

Conclusion: These results suggest that KS-Fs or kurarinone may be developed as a novel antitumor agent.

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Sun MY, Zuo J, Duan JF, Han J, Fan SM, Zhang W, Zhu LF, Yao MH. J Chin Integr Med/Zhong Xi Yi Jie He Xue Bao. 2008; 6(1): 51-59. Received March 5, 2007; published online January 15, 2008. Free full text (PDF) is available at www.jcimjournal.com. Indexed/abstracted in and full text link-out at PubMed. Forward linking and reference linking via CrossRef. DOI: 10.3736/jcim20080111

 

Correspondence: Ming-yu SUN, MD, Associate Professor; Tel: 021-51328888-8112; Email: mysun248@hotmail.com

 

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      苦参(Sophora flavescens Aiton)为豆科槐属植物,是中国传统常用中药之一,始载于《神农本草经》,列为中品,常用于治疗急、慢性炎症和癌症等疾病。苦参主要含有生物碱和黄酮两大类化学成分11992年以苦参碱和氧化苦参碱为主的苦参总碱注射液吗特灵[(92)卫药试字X-102)号]已作为抗肿瘤药批准上市。相对于苦参碱,苦参黄酮的研究较迟。现已从苦参根中分离出56种黄酮类化合物,其中大部分化合物的A环上存在异戊烯基侧链2-14。研究表明异戊烯基侧链黄酮有抗病原微生物、抗炎、抗肿瘤(体外)等生物活性2-14

      一系列从苦参中分离出的含异戊烯基侧链的苦参黄酮在体外对一些人肿瘤细胞株有细胞毒活性,例如人白血病HL-602K562,小鼠白血病L121010,人乳腺癌MCF-711,非小细胞肺腺癌A549,卵巢癌SKOV-3121A99,皮肤黑素瘤SK-MEL-2,中枢神经系统肿瘤XF498,肠癌HCT12,子宫颈癌HeLa10,鼻咽上皮癌KBKB-Vindrug-resistant variant KB)等细胞株10。这些黄酮还可以抑制一些酶的活性,例如phospholipase Cγ17diacylglycerol acyltransferase913tyrosinase514testosterone 5α-reductase6cGMP phosphodiesterase 58。但苦参黄酮在体内是否有抑制肿瘤作用,目前国内外均没有相关报道。

      本研究提取分离苦参总黄酮(kushen flavonoids, KS-Fs)和苦参酮(kurarinone, Kur),检测KS-FsKur对肿瘤细胞增殖的影响,并比较KS-Fs与市售苦参碱(kushen alkaloids, KS-As)制剂(主要成分为苦参生物碱或氧化苦参碱)的细胞毒作用。考察KS-FsKur对小鼠S180肉瘤、H22肝癌和Lewis肺癌及裸小鼠人移植肿瘤非小细胞肺癌H460和食管癌Eca-109的影响。

 
   

1   材料和方法
1.1  
材料
1.1.1  
药物   苦参饮片,雷允上医药有限公司,经鉴定为豆科槐属植物苦参的根,苦参碱含量大于2%,符合《中华人民共和国药典》1995年版规定。苦参素(kushenin),宁夏制药厂;苦参碱注射液,广州明兴制药厂。体外实验:将KS-FsKur用二甲亚砜(dimethyl sulfoxide, DMSO)配制成20 mg/ml储备液,-20 ℃贮存,临用时用相应细胞培养液稀释到所需终浓度。体内药效实验:将KS-FsKur5% DMSO9% Cremophor EL助溶后,加85%蒸馏水配成溶液。急性毒性实验注射配方:用10% DMSO18% Cremophor EL溶解到最大浓度,加蒸馏水配成终浓度为50 mg/ml溶液。急性毒性实验口服配方:20%聚乙二醇(PEG 400),18% Cremophor溶解到最大浓度,加蒸馏水配制成混悬液,终浓度为70 mg/ml。注射用环磷酰胺(cytoxan, CTX),江苏恒瑞医药股份有限公司,批号为04061821;注射用顺铂(cisplatin),齐鲁制药有限公司,批号为04051241;临用时用生理盐水稀释。
1.1.2   细胞株   肺癌A549SPC-A-1NCI-H460H460)、H446,人乳腺癌MDA-MB-231MCF-7,子宫颈癌HeLa,肠癌HCT-116HT-29SW-620CaCo-2细胞株,均购自美国菌种细胞保藏数据库(American Type Culture Collection, ATCC);白血病HL-60、食管癌Eca-109、胃癌BGC-823和肝癌SMMC-7721细胞株以及小鼠S180肉瘤、H22肝癌和Lewis肺癌细胞,均购自中国科学院细胞生物研究所。
1.1.3   动物   Balb/c-nu/nu裸小鼠,雄性,68周龄,1822 g,上海市肿瘤研究所,合格证号:2002-0001。雌性ICRC57BL/6小鼠,2022 g;昆明鼠,雌雄各半,2022 g。均购自斯莱克动物中心,合格证号:2003-0003
1.1.4   试剂   伊思考夫改良杜尔贝可培养基(Iscove's modified Dulbecco's medium, IMDM)、达尔伯克改良伊格尔培养基(Dulbecco's modified Eagle's medium, DMEM)、1640培养基、胎牛血清和0.05%胰酶-乙二胺四乙酸,来自GIBCO公司;青霉素、链霉素、DMSO、甲基噻唑基四唑(methyl thiazolyl tetrazolium, MTT)和Cremophor EL,为Sigma公司产品。
1.2   方法
1.2.1   KS-FsKur
的分离提取   由和记黄埔医药有限公司提取鉴定。将苦参饮片粉碎成粗粉,10倍量95%乙醇渗漉,渗漉液经减压浓缩,加水以石油醚萃取,回收后得到KS-Fs粗提物。乙醇重溶解,过大孔树脂得到KS-Fs,紫外分光光度计检测(ultraviolet rays, UV)方法检测黄酮含量和纯度。KS-Fs经过聚酰胺和硅胶柱联用层析分离提取得到Kur

      5 kg苦参用乙醇渗漉、溶剂萃取得到KS-Fs粗提物126 g,得率为2.52%,黄酮含量超过55%。乙醇重溶解,过大孔树脂得到KS-FsUV方法检测黄酮含量,纯度超过80%KS-Fs经过聚酰胺和硅胶柱联用层析分离提取得到KurKS-Fs主要含有Kur,降苦参酮(norkurarinone, NKur)和二甲氧基苦参酮(2'-methoxy-kurarinone, MKur)及其他黄酮,其中KurNKurMKur的含量分别是29.5%4.6%1.4%。见图1

1   KS-Fs的高效液相图谱和主要化合物结构  

Figure 1   High-performance liquid chromatograms and chemical structures of compounds in KS-Fs
    
1.2.2   KS-Fs
Kur在体外对肿瘤细胞株的抑制作用   将(0.51×105个生长至对数生长期的细胞接种于96孔板,细胞在37 ℃5% CO2培养箱中培养24 h后,更换成含不同浓度药物的培养基,其中KS-FsKur和其他待测药物按梯度浓度加入,共设8个浓度,每孔中药物溶剂(DMSO或水)的体积不超过1%总体积。每个浓度设3个复孔,继续培养48 hMTT法检测15。以溶剂孔为对照,数据用XLfit Wizard软件分析,计算各药物对细胞生长抑制的半数抑制浓度(median inhibitory concentration, IC50)。实验重复3次。
1.2.3   KS-FsKur体内抗肿瘤作用
1.2.3.1  
小鼠肿瘤模型的建立   小鼠S180肉瘤、H22肝癌和Lewis肺癌细胞,采用中国科学院上海细胞研究所推荐的培养基和血清浓度培养在2575 ml培养瓶中,置于37 ℃5% CO2培养箱中静止培养。

      分别收集对数生长期S180H22细胞,调整细胞数为5×106/mlICR小鼠腹腔注射细胞悬液,每只0.2 ml。体内腹水传两代后,无菌条件下抽取生长良好的S180肉瘤或肝癌H22小鼠腹水,用生理盐水稀释至5×106/ml,充分混匀种植于小鼠右前腋下,每只0.2 ml

      收集对数生长期Lewis肺癌细胞,以5×106/ml种植于C57BL/6小鼠右前腋下,每只0.2 ml。动物种植肿瘤细胞后,每天观察小鼠情况,待肿瘤长出后,每隔23 d测量肿瘤直径,并估计肿瘤质量。体内传两代后,待Lewis肺癌瘤块长到直径大约1 cm左右时,选取肿瘤生长旺盛,无溃破的肿瘤,在无菌条件下剥离肿瘤,去除坏死部分,剪成约1.52.0 mm3大小组织块,皮下接种于68周龄健康的雌性C57BL/6小鼠右侧腋下。
1.2.3.2   裸鼠肿瘤模型的建立   收集对数生长期的H460Eca-109细胞,调整细胞数为5×105/ml5×106/ml,无菌注射于Balb/c-nu/nu裸鼠右侧背部近腋窝处,每只0.2 ml。裸鼠在标准无菌环境下饲养,每天观察并测量肿瘤直径,待肿瘤直径大约为1 cm左右时,保种传代。肿瘤体积计算公式:V=W2×L/2W代表肿瘤的宽度,L代表肿瘤的长度16。在无菌条件下自保种传代的H460Eca-109裸鼠中剥离肿瘤,剪成约1.52.0 mm3大小组织块,分别皮下接种于Balb/c-nu/nu裸鼠右侧腋下,肿瘤块体积大致相等。
1.2.3.3   分组、给药、测量和取材   小鼠接种H22肝癌第2天,将小鼠随机分组,设KS-Fs溶剂对照组,KS-Fs高(500 mg/kg)、中(100 mg/kg)、低(20 mg/kg)剂量组,CTX30 mg/kg)阳性对照组。小鼠接种S180肉瘤第2天,Lewis肺癌小鼠肿瘤长至150 mm3左右时(接种后第8天),将小鼠随机分组,设KS-Fs溶剂对照组,KS-Fs高(200 mg/kg)、低(60 mg/kg)剂量组,CTX30 mg/kg)阳性对照组。各组小鼠均腹腔注射给药,1/d,连续7 d,给药体积为10 ml/kg体质量。每天观察小鼠的一般情况,体质量变化,每周测量2次小鼠体质量。小鼠接种S180肉瘤、H22肝癌肿瘤后10 d及接种Lewis肺癌后18 d结束实验。脱颈椎处死小鼠,剥离肿瘤,称瘤质量,计算瘤质量抑瘤率,肿瘤质量抑制率(%=(对照组平均瘤质量-给药组平均瘤质量)/对照组平均瘤质量×10017

      裸小鼠肿瘤长到100 mm3时,将裸鼠随机分组并开始给药。设溶媒对照组,只给予相应溶剂;阳性药对照组,静脉注射顺铂(2 mg/kg1/d,连续给药7 d);KS-Fs组,静脉注射KS-Fs 200 mg/kg1/d,连续给药21 d。人移植肿瘤Eca-109裸小鼠加设Kur静脉注射高(200 mg/kg)、低(50 mg/kg)剂量组,1/d,连续给药21 d。人非小细胞肺癌H460和食管癌Eca-109肿瘤移植裸小鼠分别于给药后第25天和第22天结束实验。脱颈椎处死裸小鼠,剥离肿瘤,称取瘤质量,计算瘤质量抑瘤率。

      每天观察裸鼠的一般情况,体质量变化,计算相对体质量(relative body weight, RBW),每只裸鼠的RBW是测量时的裸鼠体质量与实验初始状态时裸鼠体质量的比;每周测量2次移植瘤体积,计算相对瘤体积(relative tumor volume, RTV),每个肿瘤的RTV是测量时的肿瘤体积与实验初始状态时肿瘤体积的比18)。描绘肿瘤生长曲线,并计算肿瘤的生长抑制率。生长抑制率(%=(溶媒组RTV-治疗组RTV/溶媒组RTV×100
1.2.4   急性毒性试验   昆明鼠随机分为4组:口服溶媒对照组,口服KS-Fs组,静脉注射溶媒对照组,静脉注射KS-Fs组,每组各10只,雌雄各半。实验开始前,小鼠禁食16 h,口服组单次给予最大给药体积0.04 ml/gKS-Fs给药剂量2.80 g/kg;静脉注射组单次给予最大给药体积0.015 ml/gKS-Fs给药剂量750 mg/kg,连续观察14 d
1.2.5   统计学方法   实验数据均以`x±s`x表示,采用SPSS 12.0统计软件,组间比较采用方差分析和q检验。

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2     
2.1   KS-FsKur体外对肿瘤细胞株的抑制作用   首先考察了KS-Fs对多种人肿瘤细胞增殖的影响,结果表明KS-Fs可抑制H460A549BGC-823SMMC-7721HT-29SW-620细胞的增殖,IC50分别为12.48.912.118.0621.9518.26 μg/mlKS-Fs与市售苦参碱抑制肿瘤细胞增殖作用的比较实验结果表明KS-FsSPC-A-1Eca-109细胞的IC50分别为(15.3±0.5μg/ml和(12.4±0.4μg/ml,均远低于市售苦参素和苦参碱(IC50>200 μg/ml)。见图2

 

 

2   KS-Fs与市售苦参碱制剂的体外细胞毒性

Figure 2   Cytotoxicity of marketed preparation of kushen alkaloids and KS-Fs in vitro    

**P<0.01, vs KS-Fs-treated group.

 

      在多个细胞株上验证了KS-Fs抑制肿瘤增殖作用。结果证明KS-Fs对小细胞肺癌细胞株H446,非小细胞肺癌细胞株H460A549SPC-A-1,结直肠癌细胞株CaCo-2HCT-116HT-29SW-620,乳腺癌细胞MCF-7MDA-MB-231,食管癌细胞Eca-109,肝癌SMMC-7721等均有细胞毒作用,IC504.9~29.4 μg/ml,其中对H446HeLaA549IC50均小于10 μg/ml,对乳腺癌细胞MDA-MB-231IC50则小于5 μg/mlKur细胞毒作用强于KS-FsIC502.013.1 μg/ml
2.2   KS-FsKur体内抗肿瘤作用及毒性实验
2.2.1   KS-FsKurH22肝癌、S180肉瘤和Lewis肺癌小鼠的影响   为考察KS-Fs是否在体内有直接抗肿瘤作用,首先利用H22肝癌模型小鼠评价KS-Fs的剂量效应关系。溶媒组小鼠在接种H22肝癌第10天肿瘤质量达到2.47 g,体积超过800 mm320100500 mg/kg KS-Fs腹腔注射,对小鼠肝癌H22肿瘤的生长呈明显的剂量依赖抑制作用(P<0.01),瘤质量抑瘤率分别为43.40%66.45%78.98%。见表1和图3。低、中、高剂量KS-Fs对小鼠的体质量均没有显著影响,小鼠一般状态良好。见表1

 

1   KS-FsH22肝癌小鼠肿瘤和体质量的影响

Table 1   Effects of KS-Fs on tumor and body weights in H22 hepatoma mice

                                                                                                                                                                     (`x±s`x )  

Group

n

Body weight after tumor excision (g)

Tumor weight (g)

Inhibition rate (%)

Vehicle control

19

32.04±0.12

2.47±0.03

CTX-treated

10

32.23±0.23

0.47±0.02**

81.00

20 mg/kg KS-Fs-treated

10

32.88±0.22

1.40±0.08**

43.40

100 mg/kg KS-Fs-treated

10

32.69±0.18

0.83±0.05**

66.45

500 mg/kg KS-Fs-treated

10

33.77±0.10

0.52±0.02**

78.98

**P<0.01, vs vehicle control group.
    

3   KS-FsH22肝癌和S180肉瘤小鼠肿瘤的影响

Figure 3   Effects of different doses of KS-Fs on the tumor of H22 hepatoma and S180 sarcoma in mice

A: H22 hepatoma; B: S180 sarcoma.
    
      接下来,为进一步确证KS-Fs的体内抗肿瘤作用,我们设计了S180Lewis肺癌小鼠肿瘤实验。溶媒组小鼠在接种S180肉瘤第10天肿瘤质量达到2.91 g,体积超过1 000 mm360200 mg/kg KS-Fs腹腔注射,对小鼠S180肉瘤的生长有明显的抑制作用(P<0.01),瘤质量抑瘤率分别为72.52%82.14%。见表2和图3。溶媒组小鼠在接种Lewis肺癌第18天肿瘤质量达到2.57 g,腹腔注射60200 mg/kg KS-Fs,对小鼠Lewis肺癌的生长有明显抑制作用(P<0.01),瘤质量抑瘤率分别为40.26%59.74%。与溶媒组相比,CTX组动物体质量显著下降(P<0.05),而KS-Fs高、低剂量组小鼠的体质量均没有明显下降。见表3

2   KS-FsS180肉瘤小鼠肿瘤和体质量的影响

Table 2   Effects of KS-Fs on tumor and body weights in S180 sarcoma mice

                                                                                                                                                                (`x±s`x )     

Group

n

Body weight after tumor excision (g)

Tumor weight (g)

Inhibition rate (%)

Vehicle control

10

29.65±0.51

2.91±0.11

CTX-treated

10

27.51±0.14

0.43±0.02**

85.11

60 mg/kg KS-Fs-treated

10

31.73±0.42

0.80±0.08**

72.52

200 mg/kg KS-Fs-treated

10

34.41±0.29*

0.52±0.05**

82.14

*P<0.05, **P<0.01 vs vehicle control group.
    

3   KS-FsLewis肺癌小鼠肿瘤和体质量的影响

Table 3   Effects of KS-Fs on tumor and body weights in Lewis mice with lung carcinoma

                                                                                                                                                         (`x±s`x )  

Group

n

Body weight after tumor excision (g)

Tumor weight (g)

Inhibition rate (%)

Vehicle control

6

19.58±0.19

2.57±0.09

CTX-treated

6

18.38±0.07*

0.28±0.01**

88.96

60 mg/kg KS-Fs-treated

6

19.23±0.13

1.53±0.05**

40.26

200 mg/kg KS-Fs-treated

6

19.70±0.15

1.03±0.06**

59.74

*P<0.05, **P<0.01, vs vehicle control group.

      为评价KS-Fs的安全性,我们进行了小鼠急性毒性试验,结果口服及静脉注射KS-Fs的半数致死量(lethal dose 50%, LD50)都未测出。口服最大耐受剂量大于2.8 g/kg;静脉注射最大耐受剂量大于750 mg/kg。动物无任何异常表现。
2.2.2   KS-Fs和(或)Kur对人非小细胞肺癌H460和人Eca-109裸鼠移植模型的影响   为验证Ks-Fs在体内对人移植肿瘤裸小鼠的抗肿瘤作用,考察了KS-Fs和(或)Kur对人非小细胞肺癌H460和人Eca-109裸鼠移植模型的影响。接种H460肿瘤的裸鼠,溶媒对照组的肿瘤RTV在治疗开始第4天达到2.41,至第25天,RTV达到了36.04,肿瘤质量达到3.19 g;静脉注射200 mg/kg KS-Fs,第4天至第25天均能显著地抑制肿瘤生长(P<0.05P<0.01),第25天肿瘤质量降至1.70 g,瘤质量抑瘤率(46.8%)与顺铂组相当(47.1%)。顺铂组裸小鼠体质量在治疗第4天开始显著下降(P<0.01),在整个治疗过程中,体质量与溶媒组的差异有统计学意义(P<0.01),最低体质量下降至治疗前体质量的75%P<0.05P<0.01)。KS-Fs组的相对体质量在472125 d与溶媒组的差异有统计学意义(P<0.05);在47111518 d,明显高于顺铂组(P<0.05)。见表4和图4

     

4   25天时KS-Fs对人H460非小细胞肺癌裸小鼠移植肿瘤模型的影响

Table 4   Effects of KS-Fs on the tumor weight of H460 in xenografted nude mice on the 25th day
                                                                                                                                     (`x±s`x )   

Group

n

RTV

Tumor weight (g)

Inhibition rate (%)

Vehicle control

8

36.04±4.59

3.19±0.18

Cisplatin-treated

5

16.78±1.94**

1.69±0.06*

47.10

200 mg/kg KS-Fs-treated

8

19.24±1.03**

1.70±0.21*

46.80

*P<0.05, **P<0.01, vs vehicle control group.
 

   

4   KS-FsKur对人H460非小细胞肺癌和Eca-109食管癌裸小鼠移植肿瘤模型肿瘤体积和体质量的影响

Figure 4   Effects of KS-Fs or Kur on the relative tumor volume and relative body weight of H460 and Eca-109 xenografted tumors in nude mice 

*P<0.05, **P<0.01, vs vehicle control group; P<0.05, △△P<0.01, vs cisplatin-treated group.

 

      接种Eca-109肿瘤的裸鼠,对照组的肿瘤RTV在治疗开始第4天达到1.8,至第22RTV达到了9.1,而肿瘤质量达到1.1 g;静脉注射200 mg/kg KS-FsKur,从第4天开始至第22天均能显著抑制肿瘤生长(P<0.05),肿瘤质量分别为0.30 g0.31 g,瘤体积抑瘤率分别为47.2%42.7%,与顺铂(50.1%)组相当,KS-Fs组和Kur组的相对体质量与模型组差异无统计学意义(P>0.05);KS-Fs组在48111518 d,明显高于顺铂组(P<0.05)。见表5和图4
        

5   22天时KS-FsKur对人Eca-109食管癌裸小鼠移植肿瘤模型的影响

Table 5   Effects of KS-Fs or Kur on the tumor weight of Eca-109 in xenografted nude mice on the 22nd day    

                                                                                                                                                              (`x±s`x )  

Group

n

RTV

Tumor weight (g)

Inhibition rate (%)

Vehicle control

6

9.08±1.06

0.53±0.04

Cisplatin-treated

6

5.25±0.63**

0.27±0.02**

49.81

200 mg/kg Kur-treated

6

5.21±0.63*

0.31±0.02**

41.29

50 mg/kg Kur-treated

6

6.59±0.60

0.50±0.08

4.92

200 mg/kg KS-Fs-treated

6

4.80±0.79*

0.30±0.02*

42.71

*P<0.05, **P<0.01, vs vehicle control group.


      综上所述,体内药效试验表明KS-Fs和(或)Kur对小鼠H22肝癌、S180肉瘤和Lewis肺癌和人移植肿瘤食管癌Eca-109和非小细胞肺癌H460裸小鼠模型均有较好的抗肿瘤作用。20500 mg/kg KS-Fs对小鼠S180H22Lewis肺癌的抑制率均大于40%200 mg/kg KS-Fs腹腔注射对S180肿瘤的抑制率超过80%,对Lewis肺癌达到59.7%500 mg/kg KS-FsH22肿瘤瘤质量抑制率达到79%200 mg/kg KS-Fs静脉注射对人移植肿瘤H460Eca-109裸小鼠的肿瘤瘤质量抑制率也大于40%200 mg/kg Kur静脉注射对Eca-109的抑制肿瘤作用与KS-Fs相当。

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3   讨   论
      文献报道苦参黄酮对多种细胞株有细胞毒作用,其中
KurKS-Fs中最主要的抑制肿瘤细胞增殖的黄酮之一。Kur对人白血病HL-60、肝癌HepG2、肺癌A549、卵巢癌SKOV-3、黑色素皮肤癌SK-MEL-2、中枢神经系统肿瘤XF498、结肠癌HCT15、结肠癌HCT15/CL02、神经胶质瘤SF295IC50分别为18.536.29.07.296.45.97.3714.347.38 μg/ml781319KS-Fs中另外两个主要成分NKurMKur也对肿瘤细胞表现出一定的细胞毒作用,NKurSF295SKOV-3HCT15HCT15/CL02A549 SKMEL-2IC50分别为4.255.635.4612.1415.412.9 μg/ml1319MKurHL-60HepG2IC50分别是13.721.1 μg/ml713。我们的研究结果揭示,KS-FsKur对多个肿瘤细胞生长呈剂量依赖抑制作用,KS-FsIC504.929.4 μg/mlKur作为KS-Fs的主要成分,细胞毒作用强于KS-FsIC50范围为2.013.1 μg/ml

      文献记载苦参碱或氧化苦参碱对白血病K562、乳腺癌MCF-7、卵巢癌SKOV-3和肝癌HepG2 等肿瘤细胞株的IC50均在200 μg/ml以上,一般多为5001 000 μg/ml20-22。我们的实验结果证明KS-Fs对非小细胞肺癌SPC-A-1和食管癌Eca-109细胞的细胞毒作用远强于已经上市的苦参碱(氧化苦参碱)制剂——苦参素(宁夏)和苦参碱(广州)。可见KS-FsKur都比苦参碱(氧化苦参碱)有更好的细胞毒作用,有进一步开发研究的价值。

      目前国内外均没有文献报道苦参黄酮的体内抑制肿瘤作用。我们首次证明KS-Fs能抑制小鼠H22肝癌和S180肉瘤,而且对小鼠Lewis肺癌及人食管癌Eca-109和非小细胞肺癌H460肿瘤移植裸小鼠模型均有较好的肿瘤抑制作用。200 mg/kg KS-Fs连续腹腔注射7 d对小鼠H22S180肿瘤的瘤质量抑制率均大于80%,作用强于苦参碱和氧化苦参碱(40%60%1。而Kur作为KS-Fs的主要组成成分,对食管癌Eca-109裸鼠移植肿瘤的瘤质量抑制率与KS-Fs作用相近,大于40%;对胃癌BGC-823和喉癌Hep-2裸小鼠移植肿瘤的瘤质量抑制率分别为39%31%Kur基本能够代表KS-Fs抗肿瘤作用的主成分。苦参总碱小鼠口服的LD501.18 g/kg23

      苦参碱和氧化苦参碱腹腔注射,小鼠的LD50分别是150 mg/kg750 mg/kg24KS-Fs口服和静脉注射的LD50均没有测出,口服最大耐受量大于2.8 g/kg,静脉注射最大耐受剂量则大于750 mg/kg,动物均无异常表现。治疗剂量连续静脉注射两周,对动物体质量和血象、骨髓均无影响。可见KS-Fs比苦参碱毒性更小,更为安全。

      本实验的研究结果证明KS-FsKur是安全有效的中药抗肿瘤有效部位(单体),极有可能像苦参碱一样开发成新的抗肿瘤植物药。

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References
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