半枝莲总黄酮中7种成分的含量测定及抗肿瘤活性

Determination of Seven Compounds and Antitumor Activity of Total Flavonoids from Scutellariae Barbatae Herba

目的:建立半枝莲总黄酮中7种成分的含量测定方法,并对半枝莲总黄酮及7种成分进行体外抗肿瘤活性的评价。方法:采用HPLC方法,测定半枝莲总黄酮中7种成分野黄芩苷,芹菜素-5-O-β-D-吡喃葡萄糖苷,野黄芩素,异高山黄芩素-8-O-β-D-葡萄糖醛酸苷,木犀草素,4'-羟基汉黄芩素及芹菜素的含量。采用噻唑蓝(MTT)法评价半枝莲总黄酮及7种化学成分分别对人肝癌细胞(Hep3B),人胃癌细胞(AGS),人肺癌细胞(A549),小鼠黑色素瘤细胞(B16),小鼠乳腺癌细胞(4T1)5种癌细胞存活率的影响。结果:建立了测定半枝莲总黄酮中7种成分野黄芩苷,芹菜素-5-O-β-D-吡喃葡萄糖苷,野黄芩素,异高山黄芩素-8-O-β-D-葡萄糖醛酸苷,木犀草素,4'-羟基汉黄芩素及芹菜素含量测定方法,线性范围分别为9.98~249.60,1.06~26.50,2.16~54.00,2.56~64.05,1.16~29.10,0.52~13.00,1.18~29.50 mg·L^(-1);平均加样回收率分别为103.47%,96.15%,98.66%,98.91%,98.25%,102.67%,102.32%,RSD分别为2.1%,1.1%,1.7%,3.2%,2.1%,1.8%,1.9%。半枝莲总黄酮(300 mg·L^(-1))对Hep3B,AGS,A549及B16共4种肿瘤细胞的增殖抑制率均>50%。木犀草素和4'-羟基汉黄芩素抑制肿瘤细胞生长的活性较显著,木犀草素的半数抑制浓度(IC50)为26.64~36.97 mmol·L^(-1),4'-羟基汉黄芩素的IC50为20.14~37.91 mmol·L^(-1)。结论:该方法专属性强,准确度高,重复性好,可用于半枝莲总黄酮中多种有效成分的含量测定。半枝莲总黄酮对Hep3B,AGS,A549,B16及4T1肿瘤细胞的增殖均有一定的抑制作用,木犀草素和4'-羟基汉黄芩素对该5种细胞增殖抑制活性显著,推测半枝莲总黄酮是半枝莲抗肿瘤的主要活性部位,而木犀草素和4'-羟基汉黄芩素是其主要活性成分。

Objective： To establish an HPLC method for detecting the contents of seven compounds in total flavonoids from Scutellariae Barbatae Herba,and further study the antitumor activity of total flavonoids and seven compounds from Scutellariae Barbatae Herba. Method： The contents of scutellarin,apigenin-5-O-β-Dglucopyranoside,suctellarein, isoscutellarein 8-glucuronide, luteolin,4＇-hydroxywogonin, and apigenin were detected by HPLC. The antitumor activity of total flavonoids and the seven compounds were assayed in human hepatocellular carcinoma cells（Hep3B）,human gastric cancer cells（AGS）,human lung cancer cells（A549）,mouse melanoma cells（B16） and mouse breast cancer cells（4T1） by using MTT assat. Result： The HPLC method was established to detect the contents of above seven compounds. The linear range was 9. 98-249. 60,1. 06-26. 50,2. 16-54. 00,2. 56-64. 05,1. 16-29. 10,0. 52-13. 00 and 1. 18-29. 50 mg·L^-1respectively for the above 7 components,with an average recovery rate of 103. 47%,96. 15%,98. 66%,98. 91%,98. 25%,102. 67% and 102. 32% respectively,and RSD of 2. 1%,1. 1%,1. 7%,3. 2%,2. 1%,1. 8%,and 1. 9%.The total flavonoids（300 mg·L^-1） showed obvious inhibitory effect on the proliferation of Hep3 B,AGS,A549 and B16 cells（the inhibitory rate was 50% for the 4 kinds of cells）. Luteolin and 4＇-hydroxywogonin showed significant anti-tumor activity（IC5026. 64-36. 97 mmol·L^-1and 20. 14-36. 97 mmol·L^-1,respectively）.Conclusion： The method is simple,rapid,accurate and is suitable for the determination of effective compounds in total flavonoids from Scutellariae Barbatae Herba. The total flavonoids have certain inhibitory effect,while luteolin and 4＇-hydroxywogonin have significant inhibitory effect on the proliferation of Hep3 B,AGSA549,B16 and 4T1 cells. All these results indicated that the total flavonoids are the main active fraction in Scutellariae Barbatae Herba for anti-tumor effect,and luteolin and 4＇-hydroxywogonin are the main active ingredient.