SCIENCE CHINA Life Sciences, Volume 60, Issue 2: 215-224(2017) https://doi.org/10.1007/s11427-015-4911-7

In vitro and in vivo evaluation of cucurbitacin E on rat hepatic CYP2C11 expression and activity using LC-MS/MS

More info
  • ReceivedJul 3, 2015
  • AcceptedJul 27, 2015
  • PublishedSep 9, 2015


This study explored the effects of cucurbitacin E (CuE), a bioactive compound from Cucurbitaceae, on the metabolism/ pharmacokinetic of tolbutamide, a model CYP2C9/11 probe substrate, and hepatic CYP2C11 expression in rats. Liquid chromatography-(tandem) mass spectrometry (LC-MS/MS) assay was used to detect tolbutamide as well as 4-hydroxytolbutamide, and then successfully applied to the pharmacokinetic study of tolbutamide in rats. The effect of CuE on CYP2C11 expression was determined by western blot. CuE (1.25–100 μmol L-1) competitively inhibited tolbutamide 4-hydroxylation (CYP2C11) activity only in concentration-dependent manner with a Ki value of 55.5 μmol L-1in vitro. In whole animal studies, no significant difference in metabolism/pharmacokinetic of tolbutamide was found for the single pretreatment groups. In contrast, multiple pretreatments of CuE (200 µg kg-1 d-1, 3 d, i.p.) significantly decreased tolbutamide clearance (CL) by 25% and prolonged plasma half-time (T1/2) by 37%. Moreover, CuE treatment (50–200 µg kg-1 d-1, i.p.) for 3 d did not affect CYP2C11 expression. These findings demonstrated that CuE competitively inhibited the metabolism of CYP2C11 substrates but had no effect on rat CYP2C11 expression. This study may provide a useful reference for the reasonable and safe use of herbal or natural products containing CuE to avoid unnecessary drug-drug interactions.

Funded by

National Natural Science Foundation of China(81301908)

Science and Technology Commission of Shanghai Municipality(13ZR1412600,14DZ2270100)


Acknowledgements This work was supported by the National Natural Science Foundation of China (81301908), and the Science and Technology Commission of Shanghai Municipality (13ZR1412600, 14DZ2270100).

Interest statement

Compliance and ethics The authors declare that they have no conflict of interest.


[1] Abbas S., Vincourt J.B., Habib L., Netter P., Greige-Gerges H., Magdalou J.. The cucurbitacins E, D and I: Investigation of their cytotoxicity toward human chondrosarcoma SW 1353 cell line and their biotransformation in man liver. Toxicol Lett, 2013, 216: 189-199 CrossRef PubMed Google Scholar

[2] Abdelwahab, S.I., Hassan, L.E., Sirat, H.M., Yagi, S.M., Koko, W.S., Mohan, S., Taha, M.M., Ahmad, S., Chuen, C.S., Narrima, P., Rais, M.M., and Hadi, A.H. (2011). Anti-inflammatory activities of cucurbitacin E isolated from Citrullus lanatus var. citroides: role of reactive nitrogen species and cyclooxygenase enzyme inhibition. Fitoterapia 82, 1190–1197. Google Scholar

[3] Attard E., Brincat M.P., Cuschieri A.. Immunomodulatory activity of cucurbitacin E isolated from Ecballium elaterium. Fitoterapia, 2005, 76: 439-441 CrossRef PubMed Google Scholar

[4] Bhandari P., Kumar N., Singh B., Kaul V.K.. Cucurbitacins from Bacopa monnieri☆. Phytochemistry, 2007, 68: 1248-1254 CrossRef PubMed Google Scholar

[5] Brown R.E., Jarvis K.L., Hyland K.J.. Protein measurement using bicinchoninic acid: elimination of interfering substances. Anal Biochem, 1989, 180: 136-139 CrossRef Google Scholar

[6] Chen A., Qin X., Tang Y., Liu M., Wang X.. Evaluation of enzyme inhibition kinetics in drug–drug interactions. Chemico-Biol Interactions, 2014, 222: 133-134 CrossRef PubMed Google Scholar

[7] China Pharmacopoeia Commission. (2010). Pharmacopoeia of the People’s Republic of China (Part One). (Beijing: China Medical Science Press). Google Scholar

[8] Ding T., Zhang Y., Chen A., Tang Y., Liu M., Wang X.. Effects of Cucurbitacin E, a Tetracyclic Triterpene Compound fromCucurbitaceae , on the Pharmacokinetics and Pharmacodynamics of Warfarin in Rats. Basic Clin Pharmacol Toxicol, 2015, 116: 385-389 CrossRef PubMed Google Scholar

[9] Dong Y., Lu B., Zhang X., Zhang J., Lai L., Li D., Wu Y., Song Y., Luo J., Pang X., Yi Z., Liu M.. Cucurbitacin E, a tetracyclic triterpenes compound from Chinese medicine, inhibits tumor angiogenesis through VEGFR2-mediated Jak2-STAT3 signaling pathway. Carcinogenesis, 2010, 31: 2097-2104 CrossRef PubMed Google Scholar

[10] Guengerich, F.P. (2004). Cytochrome P450: what have we learned and what are the future issues? Drug Metab Rev 36, 159–197. Google Scholar

[11] Jensen B.P., Chin P.K.L., Roberts R.L., Begg E.J.. Influence of adult age on the total and free clearance and protein binding of (R)- and (S)-warfarin. British J Clinical Pharmacology, 2012, 74: 797-805 CrossRef PubMed Google Scholar

[12] Kakkar, T., Boxenbaum, H., and Mayersohn, M. 1999E. stimation of Ki in a competitive enzyme-inhibition model: comparisons among three methods of data analysis. Drug Metab Dispos 27, 756–762. Google Scholar

[13] Kaminsky L.S., Zhang Z.Y.. Human P450 metabolism of warfarin. Pharmacology Therapeutics, 1997, 73: 67-74 CrossRef Google Scholar

[14] Lan, T., Wang, L., Xu, Q., Liu, W., Jin, H., Mao, W., and Wang, X. (2013). Growth inhibitory effect of Cucurbitacin E on breast cancer cells. Int J Clin Exp Pathol 6, 1799–1805. Google Scholar

[15] Li Y., Wang R., Ma E., Deng Y., Wang X., Xiao J., Jing Y.. The induction of G2/M cell-cycle arrest and apoptosis by cucurbitacin E is associated with increased phosphorylation of eIF2α in leukemia cells. Anti-Cancer Drugs, 2010, 21: 389-400 CrossRef PubMed Google Scholar

[16] Ouellet D., Bramson C., Roman D., Remmers A.E., Randinitis E., Milton A., Gardner M.. Effects of three cytochrome P450 inhibitors, ketoconazole, fluconazole, and paroxetine, on the pharmacokinetics of lasofoxifene. Br J Clin Pharmacol, 2007, 63: 59-66 CrossRef PubMed Google Scholar

[17] Qiao J., Xu L., He J., Ouyang D., He X.. Cucurbitacin E exhibits anti-inflammatory effect in RAW 264.7 cells via suppression of NF-κB nuclear translocation. Inflamm Res, 2013, 62: 461-469 CrossRef PubMed Google Scholar

[18] Rettie A.E., Jones J.P.. CLINICAL AND TOXICOLOGICAL RELEVANCE OF CYP2C9: Drug-Drug Interactions and Pharmacogenetics. Annu Rev Pharmacol Toxicol, 2005, 45: 477-494 CrossRef Google Scholar

[19] Schenkman, J.B, and Jansson, I. (1998) Spectral analysis of cytochrome P450. In Methods in Molecular Biology, vol. 107: Cytochrome P450 Protocols, I.R. Phillips, and E.A. Stephard, ed. (Totowa: Human Press). Google Scholar

[20] Sun C., Zhang M., Shan X., Zhou X., Yang J., Wang Y., Li-Ling J., Deng Y.. Inhibitory effect of cucurbitacin E on pancreatic cancer cells growth via STAT3 signaling. J Cancer Res Clin Oncol, 2010, 136: 603-610 CrossRef PubMed Google Scholar

[21] Sun, M., Ding, T.G., Tang, Y., Liu, M.Y., and Wang, X. (2013). Cucurbitacin E exhibits inhibitory effects on CYP2C, 3A activities in rat and human liver in vitro. Acta Pharmacol Sin 34, 29–29. Google Scholar

[22] Sun M., Tang Y., Ding T., Liu M., Wang X.. Inhibitory effects of celastrol on rat liver cytochrome P450 1A2, 2C11, 2D6, 2E1 and 3A2 activity. Fitoterapia, 2014, 92: 1-8 CrossRef PubMed Google Scholar

[23] Tateishi T., Asoh M., Nakura H., Watanabe M., Tanaka M., Kumai T., Kobayashi S.. Carbamazepine induces multiple cytochrome P450 subfamilies in rats. Chemico-Biol Interactions, 1999, 117: 257-268 CrossRef Google Scholar

[24] Wang, X. (2014). Evaluation of Cytochrome P450 enzymes in drug interactions. In Cytochrome P450 Enzymes: Biochemistry, Pharmacology and Health Implications, J. Wu, ed. (New York: Nova Science Publishers). Google Scholar

[25] Wang X., Lee W.Y.W., Or P.M.Y., Yeung J.H.K.. Effects of major tanshinones isolated from Danshen (Salvia miltiorrhiza) on rat CYP1A2 expression and metabolism of model CYP1A2 probe substrates. Phytomedicine, 2009, 16: 712-725 CrossRef PubMed Google Scholar

[26] Wang X., Lee W.Y.W., Or P.M.Y., Yeung J.H.K.. Pharmacokinetic interaction studies of tanshinones with tolbutamide, a model CYP2C11 probe substrate, using liver microsomes, primary hepatocytes and in vivo in the rat. Phytomedicine, 2010, 17: 203-211 CrossRef PubMed Google Scholar

[27] Wang X., Yeung J.H.K.. Effects of the aqueous extract fromSalvia miltiorrhiza Bunge on caffeine pharmacokinetics and liver microsomal CYP1A2 activity in humans and rats. J Pharmacy Pharmacology, 2010, 62: 1077-1083 CrossRef PubMed Google Scholar

[28] Wu W.W.P., Yeung J.H.K.. Inhibition of warfarin hydroxylation by major tanshinones of Danshen (Salvia miltiorrhiza) in the rat in vitro and in vivo. Phytomedicine, 2010, 17: 219-226 CrossRef PubMed Google Scholar

[29] Yeung J.H.K., Or P.M.Y.. Polysaccharide peptides from Coriolus versicolor competitively inhibit tolbutamide 4-hydroxylation in specific human CYP2C9 isoform and pooled human liver microsomes. Phytomedicine, 2011, 18: 1170-1175 CrossRef PubMed Google Scholar

[30] Zhang T., Li J., Dong Y., Zhai D., Lai L., Dai F., Deng H., Chen Y., Liu M., Yi Z.. Cucurbitacin E inhibits breast tumor metastasis by suppressing cell migration and invasion. Breast Cancer Res Treat, 2012, 135: 445-458 CrossRef PubMed Google Scholar

[31] Zhou S., Gao Y., Jiang W., Huang M., Xu A., Paxton J.W.. Interactions of Herbs with Cytochrome P450. Drug Metabolism Rev, 2003, 35: 35-98 CrossRef PubMed Google Scholar

  • Figure 1

    Chemical structure of cucurbitacin E.

  • Figure 2

    Representative chromatograms after multiple reaction monitoring (MRM) of tolbutamide and 4-hydroxytolbutamide in rat plasma. A, A blank plasma sample. B, A blank plasma spiked with tolbutamide, 4-hydroxytolbutamide and IS at lower limit of quantification (LLOQ) sample. C, A plasma sample collected at 60 min after caudal intravenous administration of 10 mg kg-1 tolbutamide in rats.

  • Figure 3

    (Color online) Inhibition of CYP2C11 activity by CuE in rat liver microsomes, presented as 4-hydroxytolbutamide/tolbutamide ratios. Results were x¯ ±SE of six rats. Statistical significance was determined by analysis of variance. **, P<0.01; ***, P<0.001.

  • Figure 4

    (Color online) Primary Lineweaver-Burk plot (A) and the secondary plot for Ki (B) in the inhibition of CYP2C11-mediated tolbutamide 4-hydroxylase by various concentrations of CuE (5–50 μmol L-1) in rat liver microsomes. Tolbutamide was used at concentrations of 25, 50, 75 and 100 μmol L-1. Each data point represents the mean of six rats.

  • Figure 5

    (Color online) Concentration-time profiles of (A) tolbutamide and (B) 4-hydroxytolbutamide after a single administration of CuE (50–200 μg kg-1, i.p.) after a standard dose of tolbutamide (10 mg kg-1, i.v.). Results were x ¯±SE of 5–8 rats. *, P<0.05; **, P<0.01; ***, P<0.001.

  • Figure 6

    (Color online) Concentration-time profiles of (A) tolbutamide and (B) 4-hydroxytolbutamide after a 3 d administration of CuE (50–200 μg kg-1 day-1, i.p.) after a standard dose of tolbutamide (10 mg kg-1, i.v.). Results were x ¯±SE of 5–8 rats. *, P<0.05; **, P<0.01; ***, P<0.001.

  • Figure 7

    Effects of CuE on CYP2C11 expression in the rat. Rats received 3 d pretreatment according to the following groups. A, Control. B, Carbamazepine (60 mg kg-1 d-1, i.p.). C, CuE (50 µg kg-1 d-1, i.p.). D, CuE (100 µg kg-1 d-1, i.p.). E, CuE (200 µg kg-1 d-1, i.p.). Data were expressed as the x¯±SE of six rats. *, P<0.05.

  • Table 1   Pharmacokinetics of tolbutamide (10 mg kg, i.v.) in rats after a single administration of CuE (50–200 μg kg, i.p.)




    60 mg kg-1

    CuE treatment

    50 μg kg-1

    100 μg kg-1

    200 μg kg-1

    T1/2 (min)






    Cinitial (μg mL-1)






    AUC 0-T (min μg mL-1)






    AUC 0-∞ (min μg mL-1)






    Vd (mL kg-1)






    CL (mL min-1 kg-1)






    MRT (min)






    Fluconazole (60 mg kg-1, i.p.) was used as a positive control for enzyme inhibition studies. Results were x ¯±SE of 5–8 animals. **, P<0.01; ***, P<0.001.

  • Table 2   Pharmacokinetics of tolbutamide (10 mg kg, i.v.) in rats after multiple pretreatments with CuE (50–200 μg kg d, i.p.) for 3 d




    60 mg kg-1 d-1, 3 d

    CuE treatment

    50 μg kg-1 d-1,

    3 d

    100 μg kg-1 d-1,

    3 d

    200 μg kg-1 d-1,

    3 d

    T1/2 (min)











    Cinitial (μg mL-1)











    AUC 0-T (min μg mL-1)











    AUC 0- (min μg mL-1)











    Vd (mL kg-1)











    CL (mL min kg-1)











    MRT (min)











    Carbamazepine (60 mg kg-1 d-1, i.p., 3 d) was used as a positive control for enzyme induction studies. Results were x ¯±SE of 5–8 animals. *, P<0.05; **, P<0.01.

Copyright 2019 Science China Press Co., Ltd. 《中国科学》杂志社有限责任公司 版权所有