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Establishment of HPLC Fingerprint and Content Determination of 5 Kinds of Isoflavone Components in
Sojae Semen Nigrum
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GUO Qianxiang ，LIANG Youling ，SHI Xuhua ，BAI Junqi ，HUANG Juan ，HUANG Zhihai ，QIU Xiaohui （1.
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Guangdong Province Hospital of TCM & The Second College of Clinical Medicine，Guangzhou University of
TCM，Guangzhou 510120，China；2. Guangdong Provincial Key Laboratory of Clinical Research of TCM
Syndrome，Guangzhou 510006，China）
ABSTRACT OBJECTIVE：To establish the fingerprint of Sojae Semen Nigrum and content determination method of 5 kinds of
isoflavones，so as to provide reference for controlling its quality better. METHODS：HPLC method was adopted to establish the
fingerprint and detect the contents of 5 kinds of isoflavones. The determination was performed on Phenomenex C18 column with
mobile phase consisted of acetonitrile-0.12％ formic acid solution（gradient elution）at the flow rate of 1 mL/min. The detection
wavelength was set at 260 nm；the column temperature was 30 ℃ and sample size was 10 μL. Using daidzin as reference，HPLC
fingerprints of 12 batches of samples were determined. The similarity of 12 batches of samples was evaluated by TCM
Chromatographic Fingerprint Similarity Evaluation System （2012A） to confirm common peak. Cluster analysis and principal
component analysis were performed by using SPSS 20.0 software and SIMCA 13.0 software. RESULTS：There were 19 common
peaks in HPLC fingerprints of 12 batches of samples，the similarity of which was higher than 0.94. Totally 5 components were
identified，such as daidzin，glycitin，genistin，daidzein，genistein. Cluster analysis showed that 12 batches of Sojae Semen Nigrum
were clustered into 2 categories，i.e. S1-S3 clustered into one category，and S4-S12 clustered into the other category. By principal
component analysis，the contribution rates of two principle components were 53.261％ and 40.715％；accumulative contribution
rate was 93.976％. The linear range of above 5 components were 5.97-191.00 µg/mL（r＝0.999 9），1.05-33.46 µg/mL（r＝0.999 9），
8.93-285.61 µg/mL（r＝0.999 5），0.82-26.33 µg/mL（r＝0.999 9），0.93-29.64 µg/mL（r＝0.999 7），respectively. The limits of
quantitation were 0.881 1，0.611 6，0.078 6，0.243 3，0.511 6 μg/mL，respectively. The limits of detection were 0.264 3，0.244 7，
0.021 4，0.124 8，0.106 7 μg/mL，respectively. RSDs of precision，stability，reproducibility and durability tests were all lower
than 5％ . Recoveries were 95.15％ -96.56％（RSD＝0.51％ ，n＝6），98.52％ -103.45％（RSD＝1.88％ ，n＝6），95.37％ -97.91％
（RSD＝0.95％，n＝6），99.75％-102.00％（RSD＝0.78％，n＝6），100.26％-103.65％（RSD＝1.21％，n＝6）. Among 12 batches
of Sojae Semen Nigrum，the contents of above 5 components were 0.178 3-0.265 9，0.021 7-0.096 2，0.288 5-0.597 2，0.014 1-
0.058 8，0.012 9-0.082 9 mg/g. CONCLUSIONS：Established HPLC fingerprint and content determination method of 5 kinds of
isoflavones can be used for quality control of Sojea Semen Nigrum. The Isoflavone components are similar，but the contents are
different among Sojae Semen Nigrum from different producing areas.
KEYWORDS Sojae Semen Nigrum；Isoflavones；HPLC；Fingerprint；Cluster analysis；Principal component analysis；Content
determination

黑豆为豆科植物大豆[Glycine max（L.）Merr.]的干燥 豆为2010年版《中国药典》（一部）新收录的品种，现行标
[1]
成熟种子 。《本草纲目》中记载，大豆有黑、白、黄、褐、 准收载于2015年版《中国药典》（一部），包括性状鉴别、
[1]
[2]
青、斑数色，黑者入药 。中医认为，五色入五脏，黑色属 显微鉴别和薄层色谱鉴别等 ，尚无含量测定项。虽然，
[7]
[3]
水，水走肾，黑豆专入肾经，故被认定为“补肾”之品 。 陆泉 采用反相高效液相色谱法测定黑豆中大豆苷的含
2015 年版《中国药典》（一部）收录黑豆，载其有益精明 量；刘洋等 研究发现，不同产地黑豆黄酮提取物的抗氧
[8]
目、养血祛风、利水、解毒等功效 。现代药理研究表明， 化还原能力不同，其中产地影响较大，因此认为不同产
[1]
黑豆具有抗氧化、调节激素水平、预防动脉血管硬化、延 地黑豆的药效也存在一定差异。但这些研究的指标单
年益智、防癌抗癌等作用 。该药的作用机制与其所含 一，存在一定的局限性，且关于黑豆的指纹图谱、不同产
[4]
多种生物活性成分有关，尤以异黄酮最为突出。该类成 地质量差异的研究较少。本课题组通过前期化学成分
分除具有缓解更年期综合征、防治骨质疏松及心血管疾 和药理研究证实，黑豆中含有大豆苷、黄豆黄苷、染料木
病等作用外，还具有显著的防癌抗癌作用，故备受学者 苷、大豆苷元和染料木素等5种异黄酮成分，这5种成分
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关注 。 也是其发挥药理作用的活性成分 。为更加全面地评价
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化学成分是中药药效发挥作用的物质基础，中药质 黑豆质量，本研究采用高效液相色谱法（HPLC）建立了
量与其复杂的有效成分密切相关，因此采用指纹图谱结 12 批黑豆药材样品的 HPLC 指纹图谱，测定了上述 5 种
合多指标成分的含量测定可更好地控制中药质量 。黑 异黄酮类成分的含量，同时结合聚类分析和主成分分析
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中国药房 2020年第31卷第4期 China Pharmacy 2020 Vol. 31 No. 4 ·429 ·