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YI Bowen, LIU Huining, ZHENG Rui, REN Jiawei, LIU Yang. Determination and effect of five alkaloids from extracts of Piper longum on rats with experimental myocardial ischemia induced by injection of pituitrin[J]. Journal of Pharmaceutical Practice and Service. doi: 10.12206/j.issn.2097-2024.202112011
Citation: YI Bowen, LIU Huining, ZHENG Rui, REN Jiawei, LIU Yang. Determination and effect of five alkaloids from extracts of Piper longum on rats with experimental myocardial ischemia induced by injection of pituitrin[J]. Journal of Pharmaceutical Practice and Service. doi: 10.12206/j.issn.2097-2024.202112011

Determination and effect of five alkaloids from extracts of Piper longum on rats with experimental myocardial ischemia induced by injection of pituitrin

doi: 10.12206/j.issn.2097-2024.202112011
  • Received Date: 2021-12-05
  • Rev Recd Date: 2022-05-29
  •   Objective  To determine the content of five alkaloids from extracts of piper longum and test the pharmacodynamic effect of them on rats with experimental myocardial ischemia induced by injection of pituitrin.   Methods  The content of five alkaloids was determined simultaneously by HPLC. The experimental myocardial ischemia in rats was induced by injection of pituitrin, and the absolute value of T wave change and change of heart rate before and after model establishment were chosen to be the observation index. The effects of large, medium and small dose groups were evaluated.   Results  Three batches of samples were analyzed, with the contents of piperine for 56.1%, 49.7%, 51.6%; N-isobutyl-(2E,4E)octadecatrienamide for 4.5%, 4.2%, 4.3%; guineensine for 0.46%, 0.38%, 0.40%; piplartine for 1.73%, 1.67%, 1.70% and piperamide for 0.55%, 0.46%, 0.49%, respectively. All dose groups from extracts of piper longum had significantly reduced the absolute value of T wave and almost have no effect on the change of heart rate, except the high dose group showed the effect of reducing heart rate at some time .  Conclusion  The HPLC method was suitable for the simultaneous determination of five alkaloids from extracts of piper longum. It was shown that extracts of piper longum had good bioactivity in anti-myocardial ischemia.
  • [1] 王睿, 曲惠翀, 李丹, 等. 药食两用中药荜茇研究进展[J]. 食品与药品, 2020, 22(5):442-446. doi:  10.3969/j.issn.1672-979X.2020.05.021
    [2] 王如梦, 杨宏新. 荜茇及其有效成分胡椒碱的研究进展[J]. 中国药事, 2021, 35(3):350-356. doi:  10.16153/j.1002-7777.2021.03.015
    [3] 惠昱昱, 陈镝, 杨秀芳. 荜茇根化学成分及其抗肿瘤活性[J]. 中成药, 2021, 43(1):98-102. doi:  10.3969/j.issn.1001-1528.2021.01.019
    [4] 章圣朋, 张超, 李丽华, 等. 基于网络药理学预测荜茇抗肝纤维化作用及验证研究[J]. 中国中药杂志, 2021, 46(4):845-854.
    [5] LI D, WANG R, CHENG X H, et al. Chemical constituents from the fruits of Piper longum L. and their vascular relaxation effect on rat mesenteric arteries[J]. Nat Prod Res,2022,36(2):674-679. doi:  10.1080/14786419.2020.1797726
    [6] 肖娟, 张华果, 陈洁, 等. 荜茇酰胺逆转人小细胞肺癌H446/DDP细胞耐药性及机制的研究[J]. 中国免疫学杂志, 2020, 36(2):193-197. doi:  10.3969/j.issn.1000-484X.2020.02.012
    [7] LIM H J, BAK S G, PARK E J, et al. Retrofractamide C derived from Piper longum alleviates xylene-induced mouse ear edema and inhibits phosphorylation of ERK and NF-κB in LPS-induced J774A. 1[J]. Molecules,2020,25(18):4058. doi:  10.3390/molecules25184058
    [8] 唐诗, 孙伟, 孔祥清, 等. 荜茇明碱抑制BMP2/pSmad1/5信号减轻高钙高磷诱导的主动脉瓣膜间质细胞钙化[J]. 南京医科大学学报(自然科学版), 2020, 40(4):509-514,569. doi:  10.7655/NYDXBNS20200409
    [9] 谭丽娟, 刘雅倩, 闫洁, 等. 荜茇酰胺的合成、抗肿瘤机制及结构修饰的研究进展[J]. 中国药房, 2021, 32(4):508-512. doi:  10.6039/j.issn.1001-0408.2021.04.20
    [10] 李颖, 黎美欢, 陈铭泰, 等. 基于数据挖掘探究名老中医治疗冠心病用药规律[J]. 辽宁中医药大学学报, 2020, 22(8):150-154.
    [11] 蒋云聪, 李杨, 张晶晶, 等. 胡椒加工过程中胡椒碱稳定性研究[J]. 中国食品添加剂, 2021, 32(4):12-15.
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Determination and effect of five alkaloids from extracts of Piper longum on rats with experimental myocardial ischemia induced by injection of pituitrin

doi: 10.12206/j.issn.2097-2024.202112011

Abstract:   Objective  To determine the content of five alkaloids from extracts of piper longum and test the pharmacodynamic effect of them on rats with experimental myocardial ischemia induced by injection of pituitrin.   Methods  The content of five alkaloids was determined simultaneously by HPLC. The experimental myocardial ischemia in rats was induced by injection of pituitrin, and the absolute value of T wave change and change of heart rate before and after model establishment were chosen to be the observation index. The effects of large, medium and small dose groups were evaluated.   Results  Three batches of samples were analyzed, with the contents of piperine for 56.1%, 49.7%, 51.6%; N-isobutyl-(2E,4E)octadecatrienamide for 4.5%, 4.2%, 4.3%; guineensine for 0.46%, 0.38%, 0.40%; piplartine for 1.73%, 1.67%, 1.70% and piperamide for 0.55%, 0.46%, 0.49%, respectively. All dose groups from extracts of piper longum had significantly reduced the absolute value of T wave and almost have no effect on the change of heart rate, except the high dose group showed the effect of reducing heart rate at some time .  Conclusion  The HPLC method was suitable for the simultaneous determination of five alkaloids from extracts of piper longum. It was shown that extracts of piper longum had good bioactivity in anti-myocardial ischemia.

YI Bowen, LIU Huining, ZHENG Rui, REN Jiawei, LIU Yang. Determination and effect of five alkaloids from extracts of Piper longum on rats with experimental myocardial ischemia induced by injection of pituitrin[J]. Journal of Pharmaceutical Practice and Service. doi: 10.12206/j.issn.2097-2024.202112011
Citation: YI Bowen, LIU Huining, ZHENG Rui, REN Jiawei, LIU Yang. Determination and effect of five alkaloids from extracts of Piper longum on rats with experimental myocardial ischemia induced by injection of pituitrin[J]. Journal of Pharmaceutical Practice and Service. doi: 10.12206/j.issn.2097-2024.202112011
  • 荜茇为胡椒科植物荜茇(Piper longum L.)的干燥未成熟果穗,为常见的药食同源中药。果穗变黑时采收,除去杂质,晒干后可入药。荜茇主要产于印度、菲律宾和越南,我国云南、广东和广西等多地也有栽培[1]。荜茇具有多种生物活性,药理实验表明其具有抗癌、抗氧化、保肝和神经系统保护等作用[1,2]。荜茇含有生物碱、黄酮、木脂素、挥发油和脂肪酸等多种化学成分, 其中最重要的化学成分为酰胺类生物碱成分[1,3-5], 而胡椒碱是其中代表性的生物碱类化合物[2,5]。研究表明,荜茇中生物碱类成分具有较强的药理活性,如抗肿瘤作用[6-8];抑制高钙高磷导致的主动脉瓣膜间质细胞钙化作用[9];大鼠肠系膜动脉血管舒张作用等[5]。同时也有研究表明,荜茇在传统用药中常用于冠心病的治疗[10]。本实验采用HPLC法对荜茇提取物中5个生物碱类成分(胡椒碱、N-异丁基-2E,4E-十八烷二烯酰胺、几内亚胡椒碱、荜茇明碱和胡椒酰胺)的含量进行同时测定,并观察了荜茇提取物灌胃给药对垂体后叶素所致大鼠实验性心肌缺血的影响,为荜茇用于治疗冠心病提供实验依据。

    • LC-10AT高效液相色谱仪(日本岛津公司);UV-2501PC型紫外分光光度计(日本岛津公司);CPA225D型电子分析天平(德国SARTROIUS公司);SB-5200DT型超声波清洗仪器(宁波新芝生物科技股份有限公司);BIOPAC系统16道生理记录仪(BIOPAC Systems, Inc.);冷光单孔手术灯(上海医疗器械五厂生产)。

      甲醇(色谱纯,Fisher公司);乙腈(色谱纯,Fisher公司);水为重蒸水;其他试剂均为分析纯;胡椒碱对照品(自制,纯度99.5%,批号:BB-01-0105);几内亚胡椒碱对照品(自制,纯度99.0%,批号:BB-02-0108);胡椒酰胺对照品(自制,纯度98.3%,批号:BB-03-0115);荜茇明碱对照品(自制,纯度98.8%,批号:BB-04-0118);N-异丁基-2E,4E-十八烷二烯酰胺对照品(自制,纯度97.3%,批号:BB-05-0123);荜茇提取物(自制,批号081001、081002和081003);盐酸地尔硫卓片(天津田边制药有限公司,批号170604);氯化钠注射((四川科伦药业股份有限公司,批号:150910223);垂体后叶注射液(宁波第二激素厂,批号:150624);氨基甲酸乙酯(北京化学试剂公司,批号:20161101);Wistar大鼠(雄性,体重220~240 g,斯贝福(北京)实验动物技术有限公司,许可证号:SCXK(京)2011-0004)。

    • 色谱柱为RP-C18柱(4.6 mm×250 mm,5 μm),流动相为乙腈(A)-水(B)梯度洗脱:0~30 min,50%~100% A;30~50 min,100% A;50~55 min,100%~50% A。检测波长260 nm和343 nm,流速1.0 ml/min,柱温30 ℃。

    • 对照品溶液制备:精密称取5个生物碱对照品各10 mg,加入甲醇溶解并定容于50 ml棕色容量瓶中,摇匀,即得浓度为0.2 mg/ml的对照品储备液,备用。取胡椒碱溶液(浓度208.90 μg/ml), N-异丁基-2E,4E-十八烷二烯酰胺溶液(稀释5倍,浓度39.59 μg/ml),几内亚胡椒碱溶液(稀释110倍,浓度1.84 μg/ml),荜茇明碱溶液(稀释9倍,浓度21.84 μg/ml)和胡椒酰胺溶液(稀释50倍,浓度4.17 μg/ml)作为定位溶液和线性溶液1。精密量取胡椒碱溶液(208.90 μg/ml), N-异丁基-2E,4E-十八烷二烯酰胺(197.96 μg/ml),荜茇明碱(196.52 μg/ml)和胡椒酰胺(208.47 μg/ml)对照品储备液各5.0、1.0、0.55、0.1 ml和几内亚胡椒碱储备液稀释20倍后溶液(10.13 μg/ml)0.9 ml置于10 ml棕色容量瓶中,加甲醇定容,得线性溶液2。再将线性溶液2按2、4、8、16倍逐级稀释,摇匀,即得线性溶液3~6。

      供试品溶液制备:取荜茇提取物约0.025 g,精密称定,置50 ml棕色容量瓶中,甲醇超声5 min使溶解(250 W,33 KHz),放至室温,用甲醇定容至刻度,摇匀。精密移取10 ml置25 ml棕色容量瓶中,加甲醇稀释至刻度,摇匀,用0.22 μm滤膜过滤,取续滤液即得。

      空白样品溶液:将甲醇用0.22 μm滤膜过滤,取续滤液即得。

    • 分别吸取上述对照品线性溶液1~6各10 μl依次注入液相色谱仪,按“2.1.1”项下色谱条件测定,以对照品浓度为横坐标(X),峰面积为纵坐标(Y),绘制标准曲线,结果见表1。在线性范围之内,浓度和峰面积的线性关系良好。高效液相色谱图见图1

      化合物线性回归方程r线性范围(μg/ml)
      胡椒碱Y=60744550.07X+
      72196.06
      0.9995 6.53~209
      N-异丁基-2E,4E-
      十八烷二烯酰胺
      Y=30260.88X+4536.60.9998 1.24~39.6
      几内亚胡椒碱Y=72122.11X+70.350.99980.058~1.84
      荜茇明碱Y=61103.03X+6392.770.99990.676~21.8
      胡椒酰胺Y=71662.81X+2764.790.99980.130~4.16
    • 精密吸取10 μl线性溶液2,按“2.1.1”项下色谱条件连续进样6次。胡椒碱RSD为0.37%,N-异丁基-2E,4E-十八烷二烯酰胺RSD为0.62%,几内亚胡椒碱RSD为0.59%,荜茇明碱RSD为0.88%,胡椒酰胺RSD为1.2%,表明仪器精密度良好。

    • 精密取同一供试品(批号081001)溶液,放置在室温避光条件下,分别于0、1、2、4、8、24 h测定峰面积。结果显示:胡椒碱、N-异丁基-2E,4E-十八烷二烯酰胺、几内亚胡椒碱、荜茇明碱和胡椒酰胺峰面积RSD分别为1.1%、1.2%、0.51%、0.83% 和0.46%,表明本品在室温避光条件下24 h内稳定性良好。

    • 取同一批荜茇提取物(批号081001)6份,按供试品溶液制备方法制成样品溶液,按“2.1.1”项下色谱条件测定。测得胡椒碱平均含量为56.1%,RSD为0.64%;N-异丁基-2E,4E-十八烷二烯酰胺平均含量为4.48%,RSD为1.32%;几内亚胡椒碱平均含量为0.461%,RSD为1.03%;荜茇明碱平均含量为1.73%,RSD为0.591%;胡椒酰胺平均含量为0.554%,RSD为0.814%,表明本法重复性良好。

    • 取6份同一供试品粉末(批号081001)约0.0125 g,精密称定,置50 ml棕色容量瓶中,甲醇超声5 min,放至室温,用甲醇定容至刻度,摇匀。精密移取4.0 ml置25 ml棕色容量瓶中,并加入一定量的对照品溶液(按1∶1比例),制成样品溶液,精密吸取该样品溶液10 μl,按“2.1.1”项下色谱条件测定,计算回收率。结果见表2,表明回收率良好。

      化合物原有量
      (m/μg)
      加入量
      (m/μg)
      测得量
      (m/μg)
      回收率
      (%)
      RSD
      (%)
      胡椒碱540.0500.0103098.120.85
      N-异丁基-2E,4E-
      十八烷二烯酰胺
      43.7443.5086.95 99.332.11
      几内亚胡椒碱4.5084.525 9.078101.011.86
      荜茇明碱16.3316.5032.54 98.191.31
      胡椒酰胺5.3085.56010.86299.890.96
    • 取3批荜茇提取物,每一批样品平行制备3份,并按“2.1.1”项下色谱条件测定,计算供试品中5个生物碱的含量。3批样品测定结果见表3

      成分批号平均值RSD(%)
      081001081002081003
      胡椒碱56.149.751.652.56.27
      N-异丁基-2E,4E-
      十八烷二烯酰胺
      4.484.214.284.323.20
      几内亚胡椒碱0.4610.3780.3960.41210.6
      荜茇明碱1.731.671.701.701.87
      胡椒酰胺0.5540.4610.4930.5009.46
    • 将Wistar大鼠60只,随机分为6组,每组10只,分别为:空白对照组、模型组、盐酸地尔硫卓组(6 mg/kg)、荜茇提取物小剂量组(10 mg/kg)、荜茇提取物中剂量组(30 mg/kg)、荜茇提取物大剂量组(60 mg/kg)。

    • 大鼠腹腔注射氨基甲酸乙酯(1000 mg/kg)麻醉,然后按2.0 IU/kg经舌下静脉注射垂体后叶素造成急性心肌缺血模型,以造模前后T波变化绝对值、心率及其变化百分率为观测指标。

    • 荜茇提取物3个剂量组和盐酸地尔硫卓组均按1 ml/100 g连续灌胃给药大鼠7天,空白对照组和模型组给予同体积生理盐水,荜茇提取物三个剂量组和盐酸地尔硫卓组于第7天灌胃给药后1 h造模,比较各剂量组造模后T波的变化、心率及其变化百分率。

    • 实验数据以均数±标准差($ \overline{\text{x}}\text{±}\text{s} $)表示,其他各组与模型组比较,采用F检验和t检验进行统计分析,P<0.05表示差异有显著统计学意义。

    • 与空白对照组相比,模型组T波变化绝对值增高(表4),心率变化百分率加大(表5),差异具有显著性,表明实验造模成功;与模型组相比,盐酸地尔硫卓组(即阳性药组)T波变化绝对值降低,差异具有显著性,表明实验评价方法可行。荜茇提取物灌胃给药对垂体后叶素所致实验性心肌缺血大鼠T波变化绝对值的影响结果见表4,对心率变化百分率的影响结果见表5

      组别造模后时间
      1 min5 min15 min
      空白对照组0.008±0.0030.007±0.0040.007±0.003
      模型组0.033±0.018△△0.051±0.023△△0.053±0.022△△
      盐酸地尔硫卓组0.024±0.0220.018±0.014**0.020±0.016**
      小剂量组0.031±0.0310.042±0.040.016±0.013**
      中剂量组0.018±0.011*0.021±0.012**0.020±0.015**
      大剂量组0.023±0.022*0.018±0.012**0.022±0.016**
      组别造模后时间
      1 min5 min15 min
      空白对照组0.06±0.020.05±0.050.06±0.03
      模型组0.53±0.15△△△0.43±0.05△△△0.30±0.07△△△
      盐酸地尔硫卓组0.58±0.130.41±0.040.34±0.05
      小剂量组0.52±0.160.42±0.150.35±0.15
      中剂量组0.57±0.180.43±0.130.34±0.09
      大剂量组0.73±0.11*0.51±0.090.38±0.11
      注:与空白对照组比较,△△P<0.01,△△△P<0.001;与模型组比较*P<0.05,**P<0.01

      舌下静脉注射垂体后叶素后,荜茇提取物小、中、大剂量组均有降低T波变化绝对值的作用;大剂量组除在个别时间点有降低心率的作用,其他各组在各时间点对心率变化百分率没有明显影响。表明荜茇提取物有良好的抗心肌缺血活性作用。

    • 荜茇属于生物碱含量较高的药食同源中药材,我们研究所用药材中胡椒碱含量约为3.0%。采用大孔吸附树脂制备的荜茇提取物中胡椒碱含量高达50%左右,5个生物碱含量之和约为60%。

      荜茇提取物中的5个生物碱成分主要是两种化学母核结构组成:二烯酰胺类(N-异丁基-2E,4E-十八烷二烯酰胺、几内亚胡椒碱和胡椒酰胺)和苯并二烯酰胺类(胡椒碱和荜茇明碱)。二烯酰胺类成分紫外最大吸收波长约在260 nm, 苯并二烯酰胺类最大吸收波长约在340 nm。因此,为了提高检测灵敏度,我们建立高效液相色谱分析方法时采用双波长同时检测这两类生物碱成分。荜茇中生物碱类成分因为含有共轭二烯结构导致对光稳定性很差,我们研究中发现胡椒碱对照品溶液在自然光下照射20 h,浓度下降约50%。文献报道胡椒碱固体光照含量也会有所下降,但较液体稳定性好[11]。因此,在测定荜茇中生物类成分时,供试品和对照品溶液均需要放置在棕色量瓶中避光保存。在大鼠抗心肌缺血试验中,与空白对照组相比,模型组T波变化绝对值增高,心率减慢,差异具有显著性,说明本次实验造模成功;与模型组相比,阳性药组(盐酸地尔硫卓组)T波变化绝对值降低,差异具有显著性,说明实验评价方法可行。与模型组比较,舌下静脉注射垂体后叶素后,荜茇提取物小、中和大剂量组均能够显著降低T波变化绝对值,经统计学处理差异具有显著性;除大剂量组在1 min时可降低心率之外,荜茇提取物各组在各时间点对心率变化百分率基本没有明显影响。结果表明,荜茇提取物具有良好的抗心肌缺血活性。实验中我们也考查了胡椒碱单体抗心肌缺血的生物活性,结果显示,其也具有一定的抗心肌缺血活性,但生物活性弱于荜茇提取物。

      本研究建立了荜茇提物中5个生物碱类成分含量同时测定的分析方法,同时也对高生物碱含量的荜茇提取物灌胃给药对垂体后叶素所致实验性心肌缺血大鼠的影响开展初步的药效试验。结果表明,所建立的含量测定方法可以用来有效控制荜茇提取物的质量;药效试验证明荜茇提取物具有较好的抗心肌缺血活性,其作用机制有待后续进一步研究。

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