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Volume 41 Issue 6
Jun.  2023
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JIANG Yuetong, GAO Congcong, ZHANG Yujia, OU Yangyu, ZHOU Tingting. Comparison of contents variation of six isoflavones in black beans, semifinished and finished Sojae Semen Praeparatum[J]. Journal of Pharmaceutical Practice and Service, 2023, 41(6): 372-376. doi: 10.12206/j.issn.2097-2024.202209085
Citation: JIANG Yuetong, GAO Congcong, ZHANG Yujia, OU Yangyu, ZHOU Tingting. Comparison of contents variation of six isoflavones in black beans, semifinished and finished Sojae Semen Praeparatum[J]. Journal of Pharmaceutical Practice and Service, 2023, 41(6): 372-376. doi: 10.12206/j.issn.2097-2024.202209085

Comparison of contents variation of six isoflavones in black beans, semifinished and finished Sojae Semen Praeparatum

doi: 10.12206/j.issn.2097-2024.202209085
  • Received Date: 2022-09-30
  • Rev Recd Date: 2022-11-08
  • Available Online: 2023-07-14
  • Publish Date: 2023-06-25
  •   Objective  To compare the contents variation of six flavonoids includingdaidzin, glycitin, genistin, daidzein, glycitein and genisteinin black beans, semifinished and finished Sojae Semen Praeparatum.Methods The contents of flavonoids were determined by HPLC, the condition were Diamonsil C18 column (4.6×250 mm, 5 μm) , column temperature 30 ℃, detection wavelength 260 nm, mobile phase 0.2% acetic acid water (A) - methanol (B), gradient elution, flow rate 1.0 ml/min.  Results  The linearity of this method to determine 6 isoflavones was good (r≥0.9993) within the determination range, and the recovery rate met the requirements. The RSD of precision, repeatability and stability experiment was less than 4%, 3%and 3%. The results of HPLC showed that the contents of six flavonoidsin Sojae Semen Praeparatum increased significantly compared with black beans. And, the contents of six flavonoids in finished Sojae Semen Praeparatum were slightly more than those in semifinished Sojae Semen Praeparatum.   Conclusion  The HPLC method established in this study could accurately determine the content of 6 isoflavones in Sojae Semen Praeparatum. The content of six isoflavones in black beans could be increased by the fermentation, and the combined isoflavones were transformed into free isoflavones during the fermentation process.
  • [1] 国家药典委员会. 中华人民共和国药典(一部)2020 年版 [S]. 北京: 中国医药科技出版社, 2020: 350.
    [2] 陈丽艳, 官雪莲, 张蕾, 等. 淡豆豉对人体肠道六种常住菌的调节作用[J]. 中国微生态学杂志, 2017, 29(10):1122-1126.
    [3] 胡斌, 王秋红, 姜海, 等. 淡豆豉抗菌活性及化学成分分析[J]. 中国实验方剂学杂志, 2019, 25(6):163-167.
    [4] 曹冬英, 李鸷, 许文, 等. 4种市售黑豆及成品淡豆豉中异黄酮含量分析[J]. 药学研究, 2020, 39(10):581-584.
    [5] 李鸷, 曹冬英, 许文, 等. 基于发酵过程的淡豆豉6种黄酮类成分质量控制研究[J]. 药学研究, 2019, 38(10):563-566,573.
    [6] 田赛赛, 何金城, 韩燕, 等. 大豆及其发酵品的活性成分研究进展[J]. 药学服务与研究, 2016, 16(1):15-18.
    [7] GUO H, ZHANG Z, YAO Y, et al. A new strategy for statistical analysis-based fingerprint establishment: application to quality assessment of Semen sojae praeparatum[J]. Food Chem,2018,258:189-198. doi:  10.1016/j.foodchem.2018.03.067
    [8] YAOY, MAX, LI T, et al. Quantification of isoflavone glycosides and aglycones in rat plasma by LC-MS/MS: Troubleshooting of interference from food and its application to pharmacokinetic study of Semen Sojae Praeparatum extract[J]. J Pharm Biomed Anal,2018,161:444-454. doi:  10.1016/j.jpba.2018.09.011
    [9] 张景, 冯亭亭, 张明柱. 淡豆豉中异黄酮含量测定及其抑制乙酰胆碱酯酶活性研究[J]. 天然产物研究与开发, 2017, 29(2):310-315.
    [10] 廖丽娜, 张明敏, 曹尉尉, 等. 淡豆豉药材的高效液相指纹图谱研究[J]. 药学实践杂志, 2012, 30(5):351-352,386.
    [11] QULP, FANG R, PENG J Y, et al. Isolation of six isoflavones from semen soja epraeparatum by preparative HPLC[J]. Fitoterapia,2007,78(3):200-204. doi:  10.1016/j.fitote.2006.11.002
    [12] 国家药典委员会. 中华人民共和国药典 (四部)2020 年版 [S]. 北京: 中国医药科技出版社, 2020: 480.
    [13] CHANGRR, LIUJ L, LUO Y S, et al. Isoflavones' effects on pharmacokinetic profiles of main iridoids from Gardeniae Fructus in rats[J]. J Pharm Anal,2020,10(6):571-580. doi:  10.1016/j.jpha.2019.11.004
    [14] 董雨薇, 刘学, 薛晓欣, 等. 浅谈黑豆萌发过程中营养成分的变化[J]. 广东蚕业, 2019, 53(06):25-26.
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Comparison of contents variation of six isoflavones in black beans, semifinished and finished Sojae Semen Praeparatum

doi: 10.12206/j.issn.2097-2024.202209085

Abstract:   Objective  To compare the contents variation of six flavonoids includingdaidzin, glycitin, genistin, daidzein, glycitein and genisteinin black beans, semifinished and finished Sojae Semen Praeparatum.Methods The contents of flavonoids were determined by HPLC, the condition were Diamonsil C18 column (4.6×250 mm, 5 μm) , column temperature 30 ℃, detection wavelength 260 nm, mobile phase 0.2% acetic acid water (A) - methanol (B), gradient elution, flow rate 1.0 ml/min.  Results  The linearity of this method to determine 6 isoflavones was good (r≥0.9993) within the determination range, and the recovery rate met the requirements. The RSD of precision, repeatability and stability experiment was less than 4%, 3%and 3%. The results of HPLC showed that the contents of six flavonoidsin Sojae Semen Praeparatum increased significantly compared with black beans. And, the contents of six flavonoids in finished Sojae Semen Praeparatum were slightly more than those in semifinished Sojae Semen Praeparatum.   Conclusion  The HPLC method established in this study could accurately determine the content of 6 isoflavones in Sojae Semen Praeparatum. The content of six isoflavones in black beans could be increased by the fermentation, and the combined isoflavones were transformed into free isoflavones during the fermentation process.

JIANG Yuetong, GAO Congcong, ZHANG Yujia, OU Yangyu, ZHOU Tingting. Comparison of contents variation of six isoflavones in black beans, semifinished and finished Sojae Semen Praeparatum[J]. Journal of Pharmaceutical Practice and Service, 2023, 41(6): 372-376. doi: 10.12206/j.issn.2097-2024.202209085
Citation: JIANG Yuetong, GAO Congcong, ZHANG Yujia, OU Yangyu, ZHOU Tingting. Comparison of contents variation of six isoflavones in black beans, semifinished and finished Sojae Semen Praeparatum[J]. Journal of Pharmaceutical Practice and Service, 2023, 41(6): 372-376. doi: 10.12206/j.issn.2097-2024.202209085
  • 黑豆是由豆科植物大豆[Glycine max (Linn.) Merr.]的成熟种子,淡豆豉是由黑豆经发酵后加工而成[1]。淡豆豉性味苦且辛凉,具有解表、除烦和清解郁热的功效,可用于治疗头痛感冒、胸闷烦躁、失眠等症状。淡豆豉中含有异黄酮、多糖、氨基酸、蛋白、皂苷、纤溶酶等成分,其中异黄酮类被广泛认为是主要活性成分之一。研究发现,淡豆豉还具有抗细菌、降低血糖、调节肠道菌群及抗癌等药理活性[2-6]

    本课题组按照前期优化的发酵工艺自制淡豆豉,并建立通过高效液相色谱对淡豆豉中6种异黄酮进行含量测定的方法[7-8]。依据建立的分析方法对发酵前的黑豆、发酵后的半成品和成品淡豆豉中6种异黄酮的含量进行检测,比较黑豆发酵前后和不同程度发酵条件下淡豆豉中6种异黄酮的含量变化,为进一步完善淡豆豉的发酵工艺和深入研究淡豆豉中异黄酮类化合物的药理活性奠定研究基础。

    • SHIMAZULC-20ADXR高效液相色谱仪(日本岛津制作所);KQ-800DE数控超声波清洗器(昆山市超声仪器有限公司);Sartorius SQP型电子天平(北京赛多利斯科学仪器有限公司);YF103-200G高速中药粉碎机(瑞安市永历制药机械有限公司);DHG9420电热鼓风干燥箱(上海一恒科学仪器有限公司);AnkeTDL-2B台式低速离心机(济南存昌生物技术有限公司);Hitech Smart-S15纯水仪(上海和泰仪器有限公司)。

    • 大豆苷对照品(批号AZ21091701)、黄豆黄苷对照品(批号AZ22052701) 、染料木苷对照品(批号AZ21090901)、大豆苷元对照品(批号AZ21102201)、黄豆黄素对照品(批号AZ22061011) 、染料木素对照(批号AZ21101501),均购于成都埃法生物科技有限公司;甲醇和冰醋酸为色谱纯(默克股份两合公司,德国);黑豆购买于安徽亳州神农谷药材商品交易中心,经海军军医大学药学系辛海量教授鉴定为豆科植物大豆的成熟种子;淡豆豉为本实验室发酵而成。

    • 根据《中国药典》中淡豆豉的制作标准[1],按质量比1∶1∶10称量青蒿、桑叶和黑豆,用量筒量取青蒿桑叶总质量的8倍水,将青蒿、桑叶倒入圆底烧瓶中,加入量好的水,冷凝回流煎煮1 h,将煎煮好的中药用尼龙纱布过滤,药渣放入托盘中,滤液倒入烧杯中。将处理后的黑豆放入滤液中,浸泡16 h。取出黑豆,放入蒸煮锅蒸煮1 h。晾约1 h至黑豆表面基本干燥,之后放入烧杯中,将一定量的半干半湿的青蒿桑叶置于黑豆上层,覆盖1.5 cm,置于35 ℃、湿度50%的恒温培养箱中发酵,6天后取出一半,做为半成品淡豆豉样品。剩余一半清洗稍晾后继续放入烧杯中,上层覆盖干燥的混匀后的青蒿、桑叶,置于35 ℃、湿度50%的恒温培养箱继续发酵15天,即为成品淡豆豉样品。

    • 精密称取对照品大豆苷10.93 mg、黄豆黄苷10.18 mg、染料木苷10.84 mg、大豆苷元5.09 mg、黄豆黄素5.19 mg和染料木素8.95 mg,分别加入甲醇超声溶解,于10 ml容量瓶内定容,制成浓度分别为1093 μg/ml、1018 μg/ml、1084 μg/ml、509 μg/ml、519 μg/ml和895 μg/ml的单一对照品储备液,再分别精密量取6种异黄酮对照品储备液适量,用甲醇稀释,配制成一系列浓度的混合对照品溶液[9-10]

    • 将黑豆及自制淡豆豉样品60 ℃干燥,粉碎,过四号筛。取样品粉末约0.5 g,精密称定,置具塞锥形瓶中,精密加入10 ml色谱甲醇,混匀,超声处理2 h,放冷,再次称重,甲醇补足减失重,摇匀,进样前用 0.45 μm 滤膜滤过,取续滤液,得供试品溶液。

    • 色谱柱:DiamonsilC18(4.6×250 mm,5 μm);流动相为0.2%醋酸水(A)-甲醇(B),梯度洗脱(0~6 min,20%B;6~7 min,20%~40%B;7~12 min,40%B;12~22 min,40%~60%B;22~27 min,60%B;27~35 min,60%~90%B;35~40 min,90%B;40~41 min,90%~20%B;41~45 min,20%B),流速:1.0 ml/min;柱温:30 ℃;检测波长:260 nm;进样量:10 μl。空白溶剂、对照品、黑豆和淡豆豉样品色谱峰如图1所示。大豆苷、黄豆黄苷、染料木苷、大豆苷元、黄豆黄素和染料木素的保留时间依次为16.72 min、17.56 min、20.63 min、27.40 min、28.03 min和30.95 min。

    • 分别精密量取“2.1”项下对照品储备液溶液适量,置同一10ml容量瓶中,加50%甲醇至刻度,即得大豆苷54.65 μg/ml、黄豆黄苷50.90 μg/ml、染料木苷54.20 μg/ml、大豆苷元50.90 μg/ml、黄豆黄素51.90 μg/ml和染料木素44.75 μg/ml混合对照品溶液。取混合对照品溶液加50%甲醇进行逐级稀释,制成系列浓度的混合对照品溶液,进样分析。将信噪比大于且接近于10的对照品溶液浓度作为定量限(LOQ),对于高于定量限的系列浓度的混合对照品溶液,以峰面积(Y)为纵坐标、浓度(X)为横坐标,进行线性回归,得到大豆苷、黄豆黄苷、染料木苷、大豆苷元、黄豆黄素和染料木素的标准曲线,结果见表1。结果显示 6 种化学成分在相应浓度范围内呈现良好线性关系。

      成分回归方程线性范围
      (μg/ml)
      rLOQ
      (μg/ml)
      大豆苷Y=36742X-109991.093~54.650.99990.1093
      黄豆黄苷Y=46941X-147431.018~50.900.99990.1018
      染料木苷Y=49764X-189481.084~54.200.99990.1084
      大豆苷元Y=49161X-146141.018~50.900.99990.1018
      黄豆黄素Y=32743X-8632.11.038~51.900.99930.1038
      染料木素Y=80493X-272000.895~44.750.99990.0895
    • 取浓度分别为低、中、高的对照品溶液,在同一天内连续重复进样3次,以峰面积计算3次结果的RSD值。低浓度对照品中大豆苷、黄豆黄苷、染料木苷、大豆苷元、黄豆黄素和染料木素峰面积结果的RSD值分别为1.83%、0.39%、1.56%、1.45%、2.89%和0.59%;中浓度对照品中6种异黄酮成分峰面积结果的RSD值分别为0.08%、0.05%、0.05%、0.01%、0.41%和0.07%;高浓度对照品中6种异黄酮成分面积结果的RSD值分别为0.48%、0.42%、0.38%、0.44%、1.10%和0.59%。结果表明,此方法的日内精密度良好。

      取浓度分别为低、中、高的对照品溶液,连续3天进样检测,以峰面积计算3次结果的RSD值。低浓度对照品中6种异黄酮成分峰面积结果的RSD值分别为0.13%、3.72%、3.52%、2.33%、2.15%和1.18%;中浓度对照品中6种异黄酮成分峰面积结果的RSD值分别为2.46%、2.61%、2.58%、2.28%、1.06%和1.81%;高浓度对照品中6种异黄酮成分面积结果的RSD值分别为3.44%、3.62%、3.70%、3.37%、3.08%和2.98%。结果表明,此方法的日间精密度良好。

    • 取同一批自制淡豆豉按上述条件制备 6 份供试品溶液,按“2.3”项下色谱条件进样测定, 6种异黄酮大豆苷、黄豆黄苷、染料木苷、大豆苷元、黄豆黄素和染料木素的平均含量分别为124.2 μg/g、37.11 μg/g、252.5 μg/g、273.4 μg/g、58.65 μg/g、283.3 μg/g,并计算含量的RSD值分别为1.89%、1.79%、2.50%、2.04%、2.51%、2.67%,结果表明该方法的重复性良好。

    • 取同一批自制淡豆豉的供试品溶液,按“2.3”项下色谱条件,在制备后0 h、2 h、4 h、6 h、8 h进样测定。测定6种异黄酮的峰面积,计算6种异黄酮峰面积的RSD 值,结果分别为1.93%、2.43%、0.94%、0.88%、1.01%和0.89%,结果表明供试品溶液在室温下放置8h的稳定性良好。

    • 取重复性试验同批次淡豆豉粉末约0.5 g,精密称定,平行6份,分别置于具塞锥形瓶中,每份分别精密加入近似等量的 6种对照品溶液,精密加入色谱甲醇至10 ml,混匀,超声处理2h,放冷,再次称重,甲醇补足减失重,摇匀,进样前用0.45 μm滤膜滤过,取续滤液,得加样回收供试品溶液。按上述色谱条件测定各成分的峰面积,计算各个化合物的回收率和回收率的RSD值,结果见表2。结果表明该方法回收率良好。

      成分药材粉末称量(g)原有量(μg)加入量(μg)测得量(μg)回收率(%)平均回收率(%)RSD(%)
      大豆苷0.4923
      0.4956
      0.5001
      0.4927
      0.5004
      0.5012
      63.16
      63.58
      64.16
      63.21
      64.20
      64.30
      64.78
      64.78
      64.78
      64.78
      64.78
      64.78
      121.5
      123.7
      123.2
      121.5
      123.9
      124.4
      90.11
      92.79
      91.20
      90.05
      92.21
      92.70
      91.51


      1.36


      黄豆黄苷0.4923
      0.4956
      0.5001
      0.4927
      0.5004
      0.5012
      18.02
      18.14
      18.31
      18.04
      18.32
      18.35
      18.48
      18.48
      18.48
      18.48
      18.48
      18.48
      37.69
      37.42
      37.81
      37.18
      37.90
      37.82
      106.4
      104.3
      105.5
      103.5
      106.0
      105.3
      105.21.02
      染料木苷0.4923
      0.4956
      0.5001
      0.4927
      0.5004
      0.5012
      110.6
      111.4
      112.4
      110.7
      112.4
      112.6
      113.4
      113.4
      113.4
      113.4
      113.4
      113.4
      215.8
      213.8
      216.4
      213.4
      217.2
      217.2
      92.73
      90.32
      91.64
      90.49
      92.29
      92.22
      91.611.09
      大豆苷元0.4923
      0.4956
      0.5001
      0.4927
      0.5004
      0.5012
      126.0
      126.8
      128.0
      126.1
      128.1
      128.3
      129.2
      129.2
      129.2
      129.2
      129.2
      129.2
      248.7
      247.6
      250.1
      246.7
      250.8
      251.2
      94.94
      93.44
      94.49
      93.31
      95.01
      95.10
      94.380.86
      黄豆黄素0.4923
      0.4956
      0.5001
      0.4927
      0.5004
      0.5012
      29.77
      29.97
      30.24
      29.79
      30.26
      30.31
      30.53
      30.53
      30.53
      30.53
      30.53
      30.53
      58.40
      57.78
      58.05
      57.37
      58.22
      58.44
      93.77
      91.08
      91.10
      90.34
      91.59
      92.16
      91.671.30
      染料木素0.4923
      0.4956
      0.5001
      0.4927
      0.5004
      0.5012
      127.2
      128.0
      129.2
      127.2
      129.2
      129.4
      130.4
      130.4
      130.4
      130.4
      130.4
      130.4
      254.0
      253.8
      256.9
      253.4
      257.9
      257.8
      97.25
      96.43
      97.98
      96.75
      98.69
      98.40
      97.580.94
    • 用上述建立的方法对发酵前的黑豆、发酵中的半成品淡豆豉和发酵后的成品淡豆豉中6种异黄酮的含量进行检测,每个样品平行测定3次,计算3次检测结果的平均值,并对检测结果比较。结果见表3

      含量(μg/g) 大豆苷黄豆黄苷染料木苷大豆苷元黄豆黄素染料木素
      黑豆73.4420.58128.285.9930.4592.48
      半成品淡豆豉88.7825.29211.6213.9249.10239.7
      成品淡豆豉123.644.02217.0239.356.75245.4

      将上述结果用柱形图表示,如图2

    • 本实验建立了一种通过高效液相色谱法对黑豆及发酵淡豆豉中的6种异黄酮成分进行含量检测的分析方法,并对建立的分析方法进行了系统性的方法学验证。从图1中可以看出,该方法能够检测出黑豆及淡豆豉中的大豆苷、黄豆黄苷、染料木苷、大豆苷元、黄豆黄素和染料木素这6种异黄酮成分,且空白溶剂对检测无影响。说明该高效液相色谱分析方法专属性良好,适用于本实验发酵工艺下的淡豆豉中异黄酮的含量测定。经线性、精密度、重复性、稳定性和加样回收率试验考查,该分析方法检测6种异黄酮的含量线性关系良好,较为精密和稳定,回收率符合《中国药典》规定标准[11-12]。其中,大豆苷、染料木苷和黄豆黄素的回收率偏低,可能是由于这3种化合物的稳定性较差,易分解或转化,也可能是在浓缩等预处理过程中由于溶解度偏低的原因导致化合物一定程度上的损失。

      通过上述分析方法对发酵前的黑豆和课题组自制的半成品淡豆豉及成品淡豆豉进行含量测定,并对检测出的含量进行比较。根据图2所示,6种异黄酮在发酵后淡豆豉中的含量相较于在未发酵的黑豆中,均有显著性提升。其中,相较于大豆苷、黄豆黄苷、染料木苷等结合型糖苷,大豆苷元、黄豆黄素和染料木素等游离型苷元的提升更加明显。此外,与半成品淡豆豉相比较,成品淡豆豉中的6种异黄酮的含量均较高,其中大豆苷、黄豆黄苷、大豆苷元这3种异黄酮的含量增多较为明显。从整体趋势来看,在黑豆发酵成半成品淡豆豉再至成品淡豆豉的过程中,这6种异黄酮的含量逐渐增多,在黑豆发酵为淡豆豉后,其中的异黄酮含量显著升高,但随着发酵时间的延长,淡豆豉中异黄酮含量的增速变缓。

      大豆异黄酮多以结合型糖苷的形式存在,一般认为结合型的糖苷是无活性的,大量研究表明,异黄酮苷元具有较强的生物活性[13]。所以,上述结果可以表明,黑豆经过发酵成淡豆豉后,其生物活性有明显提升。另外,在黑豆发酵过程中,6种异黄酮的含量持续提升,但随着发酵时间的推移,异黄酮含量提升趋势变缓。这种趋势的原因可能是,在黑豆发酵过程中,随着呼吸作用的增强,异黄酮的生物合成的关键酶——苯丙氨酸氨基裂解酶(PAL)含量也随之提高,使得发酵后的淡豆豉中的异黄酮的含量随之提高,但随着发酵时间的延长,呼吸作用的增强变缓[14]

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