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Volume 39 Issue 4
Jul.  2021
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REN Dan, QI Fangyuan, HUANG Ziyan, QIN Luping, ZHU Bo. Determination of harpagide and harpagoside contents in Scrophularia ningpoensis from 15 origins[J]. Journal of Pharmaceutical Practice and Service, 2021, 39(4): 313-316, 330. doi: 10.12206/j.issn.1006-0111.202008102
Citation: REN Dan, QI Fangyuan, HUANG Ziyan, QIN Luping, ZHU Bo. Determination of harpagide and harpagoside contents in Scrophularia ningpoensis from 15 origins[J]. Journal of Pharmaceutical Practice and Service, 2021, 39(4): 313-316, 330. doi: 10.12206/j.issn.1006-0111.202008102

Determination of harpagide and harpagoside contents in Scrophularia ningpoensis from 15 origins

doi: 10.12206/j.issn.1006-0111.202008102
  • Received Date: 2020-08-26
  • Rev Recd Date: 2021-05-16
  • Available Online: 2021-07-21
  • Publish Date: 2021-07-25
  •   Objective  To establish an HPLC method for the simultaneous determination of harpagide and harpagoside content in Scrophularia ningpoensis (SN).  Methods  An Eclipse C18 column was used for determination of methanol extract of S. ningpoensis with a HPLC-PDA method and mobile phase of acetonitrile-0.03% phosphate solution in a gradient elution manner. The flow rate of mobile phase was 1.0 ml/min, and the detection wavelengths were 210 nm and 280 nm.  Results  Harpagide and harpagoside contents in SN showed good linear relationships within 0.1020-0.5100 mg/ml (r=0.9999) and 0.0340-0.1700 mg/ml (r=0.9999). Their average recovery rates were 97.44% and 97.08%.The RSDs were 0.93% and 1.24%.. There were significant differences in the contents of harpagide and harpagoside in SN from 15 origins (P<0.01). The content of harpagoside in Sichuan Long-dong, Zhejiang Lin-an, Zhejiang Pan-an and Henan Nan-feng is higher.  Conclusion  This method is stable, accurate and reproducible and can be used for the quality control of SN.
  • [1] 胡瑛瑛, 黄真. 玄参的化学成分及药理作用研究进展[J]. 浙江中医药大学学报, 2008, 32(2):268-270. doi:  10.3969/j.issn.1005-5509.2008.02.076
    [2] 薛刚强, 杜婧, 潘新艳, 等. 玄参化学成分研究[J]. 中药材, 2014, 37(9):1597-1599.
    [3] 刘树民, 张宁, 于卉, 等. 玄参性味的本草考证[J]. 中华中医药杂志, 2017, 32(2):429-431.
    [4] 李杰, 詹琤琤, 段时振, 等. 中药玄参研究概况[J]. 湖北中医杂志, 2013, 35(8):73-75. doi:  10.3969/j.issn.1000-0704.2013.08.055
    [5] SHEN X, EICHHORN T, GRETEN H J, et al. Effects of Scrophularia ningpoensis hemsl. on inhibition of proliferation, apoptosis induction and NF-κB signaling of immortalized and cancer cell lines[J]. Pharmaceuticals (Basel),2012,5(2):189-208. doi:  10.3390/ph5020189
    [6] CHEN B, LIU Y, LIU H W, et al. Iridoid and aromatic glycosides from Scrophularia ningpoensis Hemsl. and their inhibition of [Ca2+](i) increase induced by KCl[J]. Chem Biodivers,2008,5(9):1723-1735. doi:  10.1002/cbdv.200890161
    [7] HUANG T H, TRAN V H, DUKE R K, et al. Harpagoside suppresses lipopolysaccharide-induced iNOS and COX-2 expression through inhibition of NF-kappa B activation[J]. J Ethnopharmacol,2006,104(1-2):149-155. doi:  10.1016/j.jep.2005.08.055
    [8] XUE H Y, GAO G Z, LIN Q Y, et al. Protective effects of aucubin on H2O2-induced apoptosis in PC12 cells[J]. Phytother Res,2012,26(3):369-374. doi:  10.1002/ptr.3562
    [9] HUA J, QI J, YU B Y. Iridoid and phenylpropanoid glycosides from Scrophularia ningpoensis Hemsl. and their α-glucosidase inhibitory activities[J]. Fitoterapia,2014,93:67-73. doi:  10.1016/j.fitote.2013.11.011
    [10] 刘青坡, 甄攀, 冯洁. 玄参的化学成分和生物活性研究[J]. 海峡药学, 2018, 30(10):47-50. doi:  10.3969/j.issn.1006-3765.2018.10.015
    [11] 刘宾, 梁晨, 徐思思, 等. 不同产地玄参饮片哈巴苷与哈巴俄苷含量比较研究[J]. 时珍国医国药, 2014, 25(3):555-556.
    [12] JEONG E J, LEE K Y, KIM S H, et al. Cognitive-enhancing and antioxidant activities of iridoid glycosides from Scrophularia buergeriana in scopolamine-treated mice[J]. Eur J Pharmacol,2008,588(1):78-84. doi:  10.1016/j.ejphar.2008.04.015
    [13] 崔忠生, 邸科前, 马焕云. 哈巴苷及哈巴俄苷对过氧化氢损伤血管内皮细胞的保护作用[J]. 医学研究与教育, 2009, 26(2):11-12. doi:  10.3969/j.issn.1674-490X.2009.02.004
    [14] 丁逸雪, 徐继校, 吴威, 等. HPLC法同时测定白术中4种倍半萜[J]. 中成药, 2020, 42(4):927-931. doi:  10.3969/j.issn.1001-1528.2020.04.020
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Determination of harpagide and harpagoside contents in Scrophularia ningpoensis from 15 origins

doi: 10.12206/j.issn.1006-0111.202008102

Abstract:   Objective  To establish an HPLC method for the simultaneous determination of harpagide and harpagoside content in Scrophularia ningpoensis (SN).  Methods  An Eclipse C18 column was used for determination of methanol extract of S. ningpoensis with a HPLC-PDA method and mobile phase of acetonitrile-0.03% phosphate solution in a gradient elution manner. The flow rate of mobile phase was 1.0 ml/min, and the detection wavelengths were 210 nm and 280 nm.  Results  Harpagide and harpagoside contents in SN showed good linear relationships within 0.1020-0.5100 mg/ml (r=0.9999) and 0.0340-0.1700 mg/ml (r=0.9999). Their average recovery rates were 97.44% and 97.08%.The RSDs were 0.93% and 1.24%.. There were significant differences in the contents of harpagide and harpagoside in SN from 15 origins (P<0.01). The content of harpagoside in Sichuan Long-dong, Zhejiang Lin-an, Zhejiang Pan-an and Henan Nan-feng is higher.  Conclusion  This method is stable, accurate and reproducible and can be used for the quality control of SN.

REN Dan, QI Fangyuan, HUANG Ziyan, QIN Luping, ZHU Bo. Determination of harpagide and harpagoside contents in Scrophularia ningpoensis from 15 origins[J]. Journal of Pharmaceutical Practice and Service, 2021, 39(4): 313-316, 330. doi: 10.12206/j.issn.1006-0111.202008102
Citation: REN Dan, QI Fangyuan, HUANG Ziyan, QIN Luping, ZHU Bo. Determination of harpagide and harpagoside contents in Scrophularia ningpoensis from 15 origins[J]. Journal of Pharmaceutical Practice and Service, 2021, 39(4): 313-316, 330. doi: 10.12206/j.issn.1006-0111.202008102
  • 玄参别名元参、黑参、浙玄参等,为我国传统大宗药材,主产地为浙江、安徽、四川、贵州等地[1-2]。玄参,始载于《神农本草经》,被列为中品,味甘、苦、咸,性微寒[3];归肺、胃、肾经,具有清热凉血,滋阴降火,解毒散结的功效,常用于热病伤阴,舌绛烦渴,津伤便秘、目赤、咽痛等症[4-5]。环烯醚萜苷类是玄参的主要有效成分,包括哈巴苷、哈巴俄苷等,临床上主要用于脑缺血、高血压、糖尿病等疾病,具有抗炎、保护心血管、抗肿瘤、免疫调节等作用[6-9]

    关于不同产地玄参有效成分含量测定的报道较少,仅有的报道也只集中在少数产地[10-11]。玄参药材原植物生长环境差别大,不同的生境因子下药材质量参差不齐,且在栽培过程中,栽培品系繁多混杂,严重阻碍了玄参产业的可持续发展。2020版《中国药典》规定将哈巴苷、哈巴俄苷作为玄参药材的质控成分,现代药理学研究也表明哈巴苷、哈巴俄苷具有保护心血管、保护神经的药理作用[12-13]。因此,课题组以哈巴苷和哈巴俄苷成分为指标性成分,收集全国玄参15个主产区玄参种质资源,建立HPLC法对其进行含量测定,分析不同产地玄参环烯醚萜苷类成分之间的差异性,以期为玄参药材质量控制提供理论依据。

  • 2019年11月至2019年12月,收集浙江磐安、安徽谯城区、贵州阳溪、河南朱庄、四川陇东等15个产地新鲜玄参种质资源,经浙江中医药大学药学教研室秦路平教授鉴定为玄参科植物玄参(Scrophularia ningpoensis Hemsl.)的干燥根,信息见表1

    编号产地经度(°)纬度(°)海拔(l/m)降雨量(l/mm)年平均温度(t/℃)
    S1浙江磐安120.4329.05 1020 1440.1 17.4
    S2浙江临安119.7230.2310501463.316.2
    S3杭州植物园120.1330.27251400.316.1
    S4安徽涡阳116.2233.5240809.815.1
    S5安徽谯城区115.7733.8835805.014.5
    S6安徽利辛116.2033.1522823.914.8
    S7河南太丘116.1333.9332740.514.3
    S8河南南丰115.2033.6534738.614.6
    S9河南朱庄114.5133.0133740.514.3
    S10四川陇东102.7130.4814281000.012.6
    S11四川永富102.6530.551456984.613.4
    S12四川五龙102.7730.401410993.412.5
    S13贵州洛龙107.7129.068321170.422.1
    S14贵州阳溪107.5729.0713001100.316.3
    S15贵州玉溪107.3629.0513241070.315.6
  • Waters Alliance e2695型高效液相色谱仪(美国Waters公司);ZORBAX Eclipse C18色谱柱(250 mm×4.6 mm, 5 μm);XS105型电子分析天平(梅特勒-多利多公司);超纯水仪(美国Millipore公司);DXF–60D型中药打粉机(广州市大祥电子机械设备有限公司);KQ–300DV型超声波(广州颖汉科技有限公司);DHG–9031A型电热恒温鼓风干燥箱(上海森信试验仪器有限公司)。哈巴苷(B20481)、哈巴俄苷(B20480)对照品均购于上海源叶生物科技有限公司。乙腈(色谱级,美国天地公司),磷酸(色谱级,上海阿拉丁公司),其余试剂均为分析纯。

  • ZORBAX Eclipse Plus C18色谱柱(250 mm×4.6 mm,5 μm);流动相:乙腈(A)–0.03%磷酸水溶液(B),梯度洗脱(1~10 min,3%~10% A;10~20 min,10%~33% A;20~25 min,33%~50% A;25~30 min,50%~80% A;30~35 min,80% A;35~37 min,80%~3% A;37~42 min,3% A);体积流量1.0 ml/min,柱温30 ℃,进样量为10 μl;检测波长为210、280 nm。色谱图见图1

  • 精密称取哈巴苷、哈巴俄苷对照品适量,用30%甲醇溶液制备成终质量浓度为每1 ml含哈巴苷0.6 mg、哈巴俄苷0.2 mg的混合对照品溶液,于4 ℃冰箱中保存备用。分别精密吸取哈巴苷、哈巴俄苷混合对照品溶液0.34、0.67、1.00、1.40、1.70 ml置于2 ml容量瓶中,30%甲醇溶液定容至2 ml,吸取上述溶液适量,0.45 μm微孔滤膜过滤,按“1.3.1”项下色谱条件依次进样。哈巴苷的检测波长为210 nm,哈巴俄苷的检测波长为280 nm。以对照品溶液质量浓度为横坐标(X),峰面积为纵坐标(Y)进行回归,结果见表2,可知哈巴苷与哈巴俄苷在各自范围内线性关系良好。

    成分回归方程线性范围r
    哈巴苷Y=2964.3X+5156.40.1020~0.5100 mg/ml0.9999
    哈巴俄苷Y=13919X+366.10.0340~0.1700 mg/ml0.9999
  • 将洗净后的玄参根切片,置于60 ℃烘箱中烘至恒定质量,粉碎后过3号筛。精密称取玄参根样品粉末0.25 g,置于25 ml具塞锥形瓶中,精密加入50%甲醇溶液25 ml,密塞,称定重量,浸泡1 h,超声处理45 min(300 W, 40 kHz),放冷,再称定重量,用50%甲醇溶液补足损失的重量,摇匀,用0.45 μm微孔滤膜过滤,取续滤液,即得。按“1.3.1”项下色谱条件平行测定3次。

  • 精密吸取哈巴苷、哈巴俄苷对照品溶液适量,按“1.3.1”项下色谱条件依次进样6次,测得哈巴苷、哈巴俄苷峰面积RSD分别为0.11%、0.09%,表明仪器精密度良好。

  • 取浙江磐安样品,按“1.3.3”项下方法制备供试品溶液,分别于0、2、4、8、12、18、24 h,按“1.3.1”项下色谱条件进样,测得哈巴苷、哈巴俄苷峰面积RSD分别为0.24%、0.17%,表明供试品溶液在24 h内稳定性良好。

  • 取浙江磐安样品6份,按“1.3.3”项下方法制备供试品溶液,按“1.3.1”项下色谱条件进样,测得哈巴苷、哈巴俄苷含量的RSD分别为0.59%、0.50%,表明该方法重复性良好。

  • 取含量已知的浙江磐安样品,精密称定0.125 g, 加入哈巴苷、哈巴俄苷对照品适量,按“1.3.3”项下方法制备成供试品溶液,按“1.3.1”项下色谱条件进样,计算回收率,结果见表3

    成分称样量(m/g)原有量(m /mg)加入量(m /mg)测得量(m /mg)回收率(%)平均回收率(RSD)(%)
    哈巴苷0.12510.07480.07480.14780.975997.44(0.93)
    0.12500.07480.07480.14760.9733
    0.12510.07480.07480.14860.9866
    0.12540.07500.07500.14820.9760
    0.12530.07490.07490.14800.9760
    0.12510.07480.07480.14650.9586
    哈巴俄苷0.12500.10650.10650.20930.965397.08(1.24)
    0.12540.10680.10680.21260.9906
    0.12520.10670.10680.20890.9569
    0.12510.10660.10680.21090.9766
    0.12530.10680.10680.20960.9625
    0.12510.10660.10680.21050.9728
  • 应用SPSS 19.0软件处理数据。方差分析采用单因素ANOVA分析,聚类分析采用组间连接法[14]

  • 15个产地间玄参根中哈巴苷、哈巴俄苷的含量存在统计学差异(P<0.01),结果见表4。哈巴苷、哈巴俄苷含量以及两者总量分别为0.791、0.228、1.265 mg/g。四川陇东产哈巴苷含量最高、安徽谯城区含量最低;浙江磐安产哈巴俄苷含量最高,四川永富含量最低;四川陇东产哈巴苷与哈巴俄苷总量最高,杭州植物园最低,含量范围在0.752~1.529 mg/g之间,均符合《中国药典》2020版质量标准。

    样品哈巴苷含量哈巴俄苷含量哈巴苷与哈巴俄苷总量
    S10.598±0.0030.852±0.0031.450±0.006
    S20.979±0.0020.520±0.0041.499±0.006
    S30.600±0.0070.152±0.0090.752±0.013
    S40.625±0.0030.542±0.0031.167±0.004
    S50.489±0.0020.831±0.0081.320±0.009
    S60.558±0.0030.615±0.0061.173±0.009
    S70.730±0.0210.626±0.0171.355±0.038
    S80.921±0.0070.563±0.0061.484±0.002
    S90.642±0.0040.655±0.0041.297±0.008
    S101.147±0.0060.382±0.0021.529±0.008
    S110.992±0.0160.036±0.0021.029±0.017
    S120.560±0.0020.422±0.0010.983±0.002
    S130.759±0.0060.385±0.0051.144±0.009
    S141.120±0.0090.234±0.0151.355±0.024
    S151.140±0.0070.297±0.2941.434±0.010
    均值0.791**0.228**1.265**
    **P<0.01,表示不同产地的哈巴苷或哈巴俄苷含量存在极显著差异。
  • 对不同产地之间中的有效成分与生境因子进行相关性分析,结果如表5所示,哈巴苷与海拔、降雨量、年平均温度呈正相关关系,与海拔正相关系数最高为(0.440);与经度、纬度之间呈负相关关系,与经度负相关系数最高为(–0.419)。哈巴俄苷与经度、纬度呈正相关,与海拔、降雨量、年平均温度呈负相关,与海拔负相关系数最高为(–0.360)。

    项目哈巴苷哈巴
    俄苷
    经度纬度海拔年降
    雨量
    年平均
    温度
    哈巴苷1.000
    哈巴俄苷−0.5531.000
    经度−0.4190.5721.000
    纬度−0.3410.5060.3701.000
    海拔0.440−0.360−0.620*−0.768**1.000
    年降雨量0.098−0.2430.184−0.795**0.5081.000
    年平均温度0.079−0.237−0.005−0.5070.0090.3631.000
    *P<0.05,表示显著相关;**P<0.01,表示极显著相关。
  • 以哈巴苷、哈巴俄苷含量以及两者总量为参考指标,对15个产地玄参进行聚类分析,根据欧式距离D2=19.98将不同产地划分为3个类群,如图2所示。类群I共5个产地,分别为浙江临安,河南南丰,贵州阳溪、玉溪,四川陇东,该类玄参中哈巴苷含量、哈巴苷和哈巴俄苷总量最高;类群II有8个产地、分别为贵州洛龙,四川五龙,安徽利辛、谯城区、涡阳,浙江磐安,河南太丘、朱庄,这些产地哈巴俄苷含量次之;类群III共有2个产地,分别是四川永富和杭州植物园,这两个产地的哈巴俄苷含量、哈巴苷和哈巴俄苷总量最低。

  • 本试验采用高效液相法测定玄参哈巴苷与哈巴俄苷含量,经PDA检测器对哈巴苷和哈巴俄苷标准品溶液进行波长扫描,结果显示,哈巴苷的最大检测波长是210 nm,与溶剂甲醇的吸收波长一致,此处会有溶剂峰的出现,而哈巴俄苷的最大检测波长是280 nm,此时基线平稳,未与溶剂峰相重叠,因此,确定检测波长为210 nm和280 nm;采用2020版《中国药典》中哈巴苷与哈巴俄苷的含量测定的提取方法及流动相,结果显示,提取、分离效果良好,哈巴苷与哈巴俄苷在甲醇中超声处理45 min即可提取完全,各峰之间的分离度>1.5,理论塔板数>5000,哈巴苷与哈巴俄苷的保留时间分别是10.107、24.116 min。

    药用植物的生长环境对次生代谢产物的积累起着非常关键的作用,包括经纬度、海拔、降雨量等[14]。本实验结果显示,15个产地玄参根中哈巴苷、哈巴俄苷的含量存在统计学差异(P<0.01),其中四川陇东、河南南丰、浙江临安、磐安含量最高;海拔、降雨量、年平均温度与哈巴苷之间呈正相关,与哈巴俄苷呈负相关。课题组发现,浙江临安、磐安的海拔分别是1050 m、1020 m,远高于杭州植物园的25 m,因此,临安、磐安哈巴苷的含量要高于植物园,这与实验结果一致;经纬度与哈巴俄苷呈正相关,且相关系数较大,经纬度越高,哈巴俄苷的含量就越大,四川陇东、永富、五龙的经纬度低于安徽利辛、涡阳、谯城区,因此,前者的哈巴俄苷的含量低于后者。结果显示,经纬度越高越有利于哈巴俄苷的积累,降雨量越大,海拔越高,越有利于哈巴苷的积累,故可优先在拥有上述环境条件的种质中选择。本实验只对玄参中哈巴苷、哈巴俄苷的含量进行测定,下一步将继续增加其他活性成分的含量测定,为玄参的质量控制提供参考依据。

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