Search JIM Advanced Search

Journal of Integrative Medicine ›› 2014, Vol. 12 ›› Issue (5): 425-438.doi: 10.1016/S2095-4964(14)60045-5

• Research Article • Previous Articles     Next Articles

Low doses of ethanolic extract of Boldo (Peumus boldus) can ameliorate toxicity generated by cisplatin in normal liver cells of mice in vivo and in WRL-68 cells in vitro, but not in cancer cells in vivo or in vitro

Jesmin Mondala, Kausik Bishayeea, Ashis Kumar Panigrahib, Anisur Rahman Khuda-Bukhsha   

  1. Cytogenetics and Molecular Biology Laboratory, Department of Zoology, University of Kalyani, Kalyani-741235, India
    Fisheries and Aquaculture Laboratory, Department of Zoology, University of Kalyani, Kalyani-741235, India
  • Received:2014-03-23 Accepted:2014-07-08 Online:2014-09-10 Published:2014-09-15


Use of cisplatin, a conventional anticancer drug, is restricted because it generates strong hepatotoxicity by accumulating in liver. Therefore its anticancer potential can only be fully exploited if its own toxicity is considerably reduced. Towards this goal, ethanolic extract of the plant, Boldo (Peumus boldus), known for its antihepatotoxic effects, was used simultaneously with cisplatin, to test its ability to reduce cisplatin’s cytotoxicity without affecting its anticancer potential. 


The cytotoxicity of Boldo extract (BE) and cisplatin, administered alone and in combination, was determined in three cancer cell lines (A549, HeLa, and HepG2) and in normal liver cells (WRL-68). Drug-DNA interaction, DNA damage, cell cycle, apoptosis, reactive oxygen species (ROS) and mitochondrial membrane potential (MMP, ΔΨ) were also studied. Hepatotoxicity and antioxidant activity levels were determined by alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase and glutathione assays in mice. The cytotoxicity of related proteins was tested by Western blotting. 


Co-administration of BE and cisplatin increased viability of normal cells, but had no effect on the viability of cancer cells. Boldo protected liver from damage and normalized different antioxidant enzyme levels in vivo and also reduced ROS and re-polarized MMP in vitro. Bax and cytochrome c translocation was reduced with caspase 3 down-regulation. Further, a drug-DNA interaction study revealed that BE reduced cisplatin’s DNA-binding capacity, resulting in a reduction in DNA damage. 


Results indicated that a low dose of BE could be used beneficially in combination with cisplatin to reduce its toxicity without hampering cisplatin’s anticancer effect. These findings signify a potential future use of BE in cancer therapy.

Key words: Drug-related toxicity, Peumus boldus, Plant ethanolic extract, Cytotoxicity, Liver, Anti-oxidant

Figure 1

Cell viability assay by MTTCell viability was checked by MTT with different doses of cisplatin and BE on different cell lines. A and B: Cisplatin (A) and BE (B) treatment on WRL-68 and HepG2 cell lines. C: Co-treatment of cisplatin and BE on WRL-68, HepG2, HeLa and A549 cells. Data are represented as percentage of control and are presented as mean±standard error of mean. **P<0.01 (for WRL-68) versus untreated control and △△P<0.01 (for HepG2) versus untreated group was considered statistically significant. MTT: thiazolyl blue tetrazolium bromide."

Figure 2

Drug-DNA interaction studyCD spectral analysis of DNA-binding ability of cisplatin and BE alone and co-administration. CD: circular dichroism; BE: Boldo extract."

Figure 3

Morphological analysis (A) The microscopic study of different cancer cells under binocular phase contrast microscope (Leica, Germany, ×40) revealed that cisplatin could induce cellular structural distortion. Co-treatment of BE and cisplatin did not show any cellular structural reformation. Cisplatin also induced damage to the normal liver cells (WRL-68). BE co-treatment with cisplatin could prevent the damage caused by cisplatin (CisP (cisplatin), 20 μmol/L; BE1, 48 μg/mL; and BE2, 64 μg/mL for in vitro). (B) 4′,6-Diamidino-2-phenylindole staining indicates that there was damage in DNA when cisplatin was administered on both HepG2 and WRL-68 cells. Co-administration of BE reduced the damage induced by cisplatin in WRL-68 cells but not in HepG2 cells."

Figure 4

DNA damage and apoptosis analysis DNA-gel assay of normal cells (A, WRL-68) and cancer cells (C, HepG2) reveals that cisplatin treatment in both normal and cancer cells induced DNA damage. Co-administration of Boldo extract (BE) reduced damage in DNA of normal cells but not in cancer cells. Annexin V/PI assay indicates apoptosis induction when cisplatin was administered on both WRL-68 cells (B) and HepG2 cells (D). Co-administration with BE reduced apoptosis induced by cisplatin in normal cells but there was no visible effect in cancer cells. In B and D, X-axis denotes annexin V-fluorescein isothiocyanate and Y-axis denotes propidium iodide (PI). LN1=untreated, LN2=64 μg/mL BE, LN3=20 μmol/L cisplatin, LN4=20 μmol/L cisplatin+64 μg/mL BE. The quadrants of lower left, lower right, upper right and upper left show the percentage of live (annexin-ve; PI-ve), early apoptotic (annexin+ve; PI-ve), late apoptotic (annexin+ve; PI+ve) and necrotic cells (PI +ve) respectively."

Figure 5

Cell cycle analysis (A) Cell cycle analysis indicates that increase in number of sub-G cells with reduction in S-phase population, while cisplatin was administered. But BE treatment along with cisplatin reduced DNA damage and increased DNA synthesis in normal WRL-68 cells [M1=Sub-G, M2=G0/G1, M3=S, M4=G2/M]. Y axis denotes counts of cells. (B) GSH depletion in WRL-68 cells (LN1=control, LN2=BE2, LN3=cisplatin, LN4=cisplatin+BE1, LN5=cisplatin+BE2). Data are represented as percentage of control and are presented as mean ± standard error of mean. Statistical significance was considered as **P<0.01, vs untreated control. (C) Protein expression study by Western blot: cytosolic and mitochondrial Bax and cytochrome c activity were analyzed, along with caspase 3 activity. GAPDH and VDAC1 served as loading control for cytosolic and mitochondrial fraction, respectively. GAPDH: glyceraldehyde 3-phosphate dehydrogenase; VDAC1: voltage-dependent anion-selective channel protein 1."

Figure 6

Microscopical and flow-cytometrical examining for ROS estimation in both HepG2 and WRL-68 cells In normal cell line, cisplatin treatment increased ROS generation compared to the control, but co-administration of BE with cisplatin reduced this ROS generation, whereas in cancer cell lines treatment with cisplatin alone as well as cisplatin plus BE increased ROS generation compared to control. ROS: reactive oxygen species; BE: Boldo extract."

Figure 7

Microscopical and flow-cytometrical checking for MMP depolarization in both HepG2 and WRL-68 cells MMP: mitochondrial membrane potential, BE: Boldo extract."

Figure 8

Effects on the survival time, GSH and pathologic change (A) Survivability curve of cancerous mice receiving drug treatment as compared to the control. Carcinogen-treated mice receiving no treatment survived less than those which received drug treatment. This plot shows the survivability of mice in last three months after cancer induction. (B) Cisplatin could introduce damage to the DNA of the cancerous liver tissue. BE co-treatment did not show any ameliorative effect on histological features of cancerous liver tissue. In the normal mice, when cisplatin was injected, it induced damage to the liver cells, but when BE was co-treated with cisplatin, the damage was minimum (CisP (cisplatin), 10 mg/kg bw; BE1, 20 mg/kg bw; and BE2, 40 mg/kg bw). (C) GSH depletion in mice liver. LN1=control, LN2=BE2 (40 mg/kg bw), LN3=cisplatin (10 mg/kg bw), LN4=cisplatin+BE1 [cisplatin (10 mg/kg bw) + BE1 (20 mg/kg bw)], LN5=cisplatin+BE2 [cisplatin (10 mg/kg bw)+BE2 (40 mg/kg bw)]. Data are represented as percentage of control and are presented as mean±standard error of mean. Statistical significance was considered as **P<0.01 versus untreated control."


Figure 9

Schematic representation of action of Boldo extract in reducing cytotoxicity generated by cisplatin in liver cells in vitro and in vivo ALT: alanine aminotrasferase; AST: aspartate aminotrasferase; GSH: redused glutathione; LDH: lactate dehydrogenase; MMP: mitochondrial membrance protential; ROS: reactive oxygen species."

[1] Gregg RW, Molepo JM, Monpetit VJ, Mikael NZ, Redmond D, Gadia M, Stewart DJ . Cisplatin neurotoxicity: the relationship between dosage, time, and platinum concentration in neurologic tissues, and morphologic evidence of toxicity[J]. J Clin Oncol, 1992,10(5):795-803
doi: 10.1200/JCO.1992.10.5.795
[2] Lu Y, Cederbaum AI . Cisplatin-induced hepatotoxicity is enhanced by elevated expression of cytochrome P450 2E1[J]. Toxicol Sci, 2006,89(2):515-523
doi: 10.1093/toxsci/kfj031
[3] Rabik CA, Dolan ME . Molecular mechanisms of resistance and toxicity associated with platinating agents[J]. Cancer Treat Rev, 2007,33(1):9-23
doi: 10.1016/j.ctrv.2006.09.006
[4] Anderson ME, Naganuma A, Meister A . Protection against cisplatin toxicity by administration of glutathione ester[J]. FASEB J, 1990,4:3251-3255
doi: 10.1096/fasebj.4.14.2227215
[5] Brady HR, Kone BC, Stromski ME, Zeidel ML, Giebisch G, Gullans SR . Mitochondrial injury: an early event in cisplatin toxicity to renal proximal tubules[J]. Am J Physiol, 1990,258(5 Pt 2):F1181-F1187
[6] Sugihara K, Gemba M . Modification of cisplatin toxicity by antioxidants[J]. Jpn J Pharmacol, 1986,40:353-355
doi: 10.1254/jjp.40.353
[7] Shellard SA, Fichtinger-Schepman AM, Lazo JS, Hill BT . Evidence of differential cisplatin-DNA adduct formation, removal and tolerance of DNA damage in three human lung carcinoma cell lines[J]. Anticancer Drugs, 1993,4(4):491-500
doi: 10.1097/00001813-199308000-00011
[8] Hurley LH . DNA and its associated processes as targets for cancer therapy[J]. Nat Rev Cancer, 2002,2(3):188-200
doi: 10.1038/nrc749
[9] Chevallier A. Encyclopaedia of herbal medicine. 2nd ed. New York: Dorling Kindersley Publishing Inc. 2001.
[10] Committee on Herbal Medicinal Products ( HMPC). Assessment report on Peumus boldus molina, folium. European Medicines Agency: Evaluation of Medicines for Human Use. London. Assessment report on Peumus boldus molina, folium. European Medicines Agency: Evaluation of Medicines for Human Use. London. (2009-01-14) [2014-2-12]. .
[11] Petigny L, Périno-Issartier S, Wajsman J, Chemat F . Batch and continuous ultrasound assisted extraction of Boldo leaves(Peumus boldus Mol.)[J]. Int J Mol Sci, 2013,14:5750-5764
doi: 10.3390/ijms14035750
[12] Passone MA , Etcheverry M. Antifungal impact of volatile fractions of Peumus boldus and Lippia turbinata on Aspergillus section Flavi and residual levels of these oils in irradiated peanut. Int J Food Microbiol. 2014; 168- 169:17-23.
[13] Boerick W . Pocket manual of homeopathic materia medica & repertory, Reprint Edn[M]. New Delhi, India: B. Jain Publishers (P) Ltd, 2004: 468-469
[14] Mosmann T . Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays[J]. J Immunol Methods, 1983,65(1-2):55-63
doi: 10.1016/0022-1759(83)90303-4
[15] Bishayee K, Ghosh S, Mukherjee A, Sadhukhan R, Mondal J, Khuda-Bukhsh AR . Quercetin induces cytochrome-c release and ROS accumulation to promote apoptosis and arrest the cell cycle in G2/M, in cervical carcinoma: signal cascade and drug-DNA interaction[J]. Cell Prolif, 2013,46(2):153-163
doi: 10.1111/cpr.2013.46.issue-2
[16] Matassov D, Kagan T, Leblanc J, Sikorska M, Zakeri Z . Measurement of apoptosis by DNA fragmentation[J]. Methods Mol Biol, 2004,282:1-17
[17] Mukherjee A, Sikdar S, Bishayee K, Boujedaini N, Khuda-Bukhsh AR . Flavonol isolated from ethanolic leaf extract of Thuja occidentalis arrests the cell cycle at G2-M and induces ROS-independent apoptosis in A549 cells, targeting nuclear DNA[J]. Cell Prolif, 2014,47(1):56-71
doi: 10.1111/cpr.12079
[18] Hissin PJ, Hilf R . A fluorometric method for determination of oxidized and reduced glutathione in tissues[J]. Anal Biochem, 1976,74:214-226
doi: 10.1016/0003-2697(76)90326-2
[19] Paul S, Bhattacharyya SS, Samaddar A, Boujedaini N, Khuda-Bukhsh AR . Anticancer potentials of root extract of Polygala senega against benzo[a]pyrene-induced lung cancer in mice[J]. J Chin Integr Med, 2011,9(3):320-327
doi: 10.3736/jcim201103
[20] Dominguez MF, Macias RI , Izco-Basurko I, de La Fuente A, Pascual MJ, Criado JM, Monte MJ, Yajeya J, Marin JJ.Low in vivo toxicity of a novel cisplatin-ursodeoxycholic derivative(Bamet-UD2) with enhanced cytostatic activity versus liver tumors[J]. J Pharmacol Exp Ther, 2001,297:1106-1112
[21] Andrade RJ, Robles M , Fernández-Casta?er A, López-Ortega S, López-Vega MC, Lucena MI.Assessment of drug-induced hepatotoxicity in clinical practice: a challenge for gastroenterologists[J]. World J Gastroenterol, 2007,13(3):329-340
doi: 10.3748/wjg.v13.i3.329
[22] Hou XM, Zhang XH, Wei KJ, Ji C, Dou SX, Wang WC, Li M, Wang PY . Cisplatin induces loop structures and condensation of single DNA molecules[J]. Nucleic Acids Res, 2009,37(5):1400-1410
doi: 10.1093/nar/gkn933
[23] Neidle S, Waring M. Molecular aspects of anticancer drug-DNA interactions.Vol 2[M]. Boca Raton: CRC Press, 1994: 162-168
[24] Schuliga M, Chouchane S, Snow ET . Upregulation of glutathione-related genes and enzyme activities in cultured human cells by sublethal concentrations of inorganic arsenic[J]. Toxicol Sci, 2002,70(2):183-192
doi: 10.1093/toxsci/70.2.183
[25] Battin EE, Brumaghim JL . Antioxidant activity of sulfur and selenium: a review of reactive oxygen species scavenging, glutathione peroxidase, and metal-binding antioxidant mechanisms[J]. Cell Biochem Biophys, 2009,55(1):1-23
doi: 10.1007/s12013-009-9054-7
[26] Sharma SS, Dietz KJ . The relationship between metal toxicity and cellular redox imbalance[J]. Trends Plant Sci, 2009,14(1):43-50
[27] Sheehan D, Meade G, Foley VM, Dowd CA . Structure, function and evolution of glutathione transferases: implications for classification of non-mammalian members of an ancient enzyme superfamily[J]. Biochem J, 2001,360(Pt 1):1-16
doi: 10.1042/bj3600001
[28] Snoke JE, Bloch K . Studies on the mechanism of action of glutathione synthetase[J]. J Biol Chem, 1955,213:825-835
[29] Rowe LA, Degtyareva N, Doetsch PW . DNA damage-induced reactive oxygen species(ROS) stress response in Saccharomyces cerevisiae[J]. Free Radic Biol Med, 2008,45:1167-1177
doi: 10.1016/j.freeradbiomed.2008.07.018
[30] Kroemer G, Galluzzi L, Brenner C . Mitochondrial membrane permeabilization in cell death[J]. Physiol Rev, 2007,87(1):99-163
doi: 10.1152/physrev.00013.2006
[31] Haupt S, Berger M, Goldberg Z, Haupt Y . Apoptosis — the p53 network[J]. J Cell Sci, 2003,116:4077-4085
doi: 10.1242/jcs.00739
[32] Gowda S, Desai PB, Hull VV, Math AA, Vernekar SN, Kulkarni SS . A review on laboratory liver function tests[J]. Pan Afr Med J, 2009,3:17
[33] Fan TJ, Han LH, Cong RS, Liang J . Caspase family proteases and apoptosis[J]. Acta Biochim Biophys Sin (Shanghai), 2005,37(11):719-727
doi: 10.1111/abbs.2005.37.issue-11
[1] Meng-ya Shan, Ying Dai, Xiao-dan Ren, Jing Zheng, Ke-bin Zhang, Bin Chen, Jun Yan, Zi-hui Xu. Berberine mitigates nonalcoholic hepatic steatosis by downregulating SIRT1-FoxO1-SREBP2 pathway for cholesterol synthesis. Journal of Integrative Medicine, 2021, 19(6): 545-554.
[2] Gai Ran, Xi-lin Feng, Yi-lin Xie, Qing-yun Zheng, Peng-peng Guo, Ming Yang, Ying-lu Feng, Chen Ling, Li-qing Zhu, Chen Zhong. The use of miR122 and its target sequence in adeno-associated virus-mediated trichosanthin gene therapy. Journal of Integrative Medicine, 2021, 19(6): 515-525.
[3] Mao-xing Pan, Chui-yang Zheng, Yuan-jun Deng, Kai-rui Tang, Huan Nie, Ji-qian Xie, Dong-dong Liu, Gui-fang Tu, Qin-he Yang, Yu-pei Zhang. Hepatic protective effects of Shenling Baizhu powder, a herbal compound, against inflammatory damage via TLR4/NLRP3 signalling pathway in rats with nonalcoholic fatty liver disease . Journal of Integrative Medicine, 2021, 19(5): 428-438.
[4] Hao Gou, Ruo-chen Huang, Yong-hua Su, Wei Li. Design of dual targeting immunomicelles loaded with bufalin and study of their anti-tumor effect on liver cancer. Journal of Integrative Medicine, 2021, 19(5): 408-417.
[5] Liang Ding, Xin-you Zhang, Di-yao Wu, Meng-ling Liu. Application of an extreme learning machine network with particle swarm optimization in syndrome classification of primary liver cancer. Journal of Integrative Medicine, 2021, 19(5): 395-407.
[6] Varuni Colamba Pathiranage, Ira Thabrew, Sameera R. Samarakoon, Kamani H. Tennekoon, Umapriyatharshini Rajagopalan, Meran K. Ediriweera. Evaluation of anticancer effects of a pharmaceutically viable extract of a traditional polyherbal mixture against non-small-cell lung cancer cells. Journal of Integrative Medicine, 2020, 18(3): 242-252.
[7] Kayode Ezekiel Adewole. Nigerian antimalarial plants and their anticancer potential: A review. Journal of Integrative Medicine, 2020, 18(2): 92-113.
[8] Yogesh Subhash Biradar, Swathi Bodupally, Harish Padh. Evaluation of antiplasmodial properties in 15 selected traditional medicinal plants from India. Journal of Integrative Medicine, 2020, 18(1): 80-85.
[9] Stefania Lamponi, Anna Maria Aloisi, Claudia Bonechi, Marco Consumi, Alessandro Donati, Gemma Leone, Claudio Rossi, Gabriella Tamasi, Luana Ghiandai, Ersilia Ferrini, Paolo Fiorenzani, Ilaria Ceccarelli, Agnese Magnani. Evaluation of in vitro cell and blood compatibility and in vivo analgesic activity of plant-derived dietary supplements. Journal of Integrative Medicine, 2019, 17(3): 213-220.
[10] Chang-quan Ling, Jia Fan, Hong-sheng Lin, Feng Shen, Zhen-ye Xu, Li-zhu Lin, Shu-kui Qin, Wei-ping Zhou, Xiao-feng Zhai, Bai Li, Qing-hui Zhou, on behalf of the Chinese Integrative Therapy of Primary Liver Cancer Working Group. Clinical practice guidelines for the treatment of primary liver cancer with integrative traditional Chinese and Western medicine. Journal of Integrative Medicine, 2018, 16(4): 236-248.
[11] Akintayo Lanre Ogundajo, Lateef Apollo Adeniran, Anofi Omotayo Ashafa. Medicinal properties of Ocotea bullata stem bark extracts: Phytochemical constituents, antioxidant and anti-inflammatory activity, cytotoxicity and inhibition of carbohydrate-metabolizing enzymes. Journal of Integrative Medicine, 2018, 16(2): 132-140.
[12] Pathom Somwong, Rutt Suttisri. Cytotoxic activity of the chemical constituents of Clerodendrum indicum and Clerodendrum villosum roots. Journal of Integrative Medicine, 2018, 16(1): 57-61.
[13] Khanh Nguyen, Jean Sparks, Felix O. Omoruyi. Investigation of the cytotoxicity, antioxidative and immune-modulatory effects of Ligusticum porteri (Osha) root extract on human peripheral blood lymphocytes. Journal of Integrative Medicine, 2016, 14(6): 465-472.
[14] Matthias Kröz, Marcus Reif, Danilo Pranga, Roland Zerm, Friedemann Schad, Erik Wim Baars, Matthias Girke. The questionnaire on autonomic regulation: A useful concept for integrative medicine?. Journal of Integrative Medicine, 2016, 14(5): 315-321.
[15] Boris V.Dons'koi, Viktor P.Chernyshov, Dariia V.Osypchuk, Sergiy M.Baksheev. Repeated cupping manipulation temporary decreases natural killer lymphocyte frequency, activity and cytotoxicity. Journal of Integrative Medicine, 2016, 14(3): 197-202.
Full text



[1] Wei-xiong Liang. Problems-solving strategies in clinical treatment guideline for traditional Chinese medicine and integrative medicine. Journal of Chinese Integrative Medicine, 2008, 6(1): 1-4
[2] Zhao-guo Li. Discussion on English translation of commonly used sentences in traditional Chinese medicine: part one. Journal of Chinese Integrative Medicine, 2008, 6(1): 107-110
[3] Jun Hu, Jian-ping Liu. Non-invasive physical treatments for chronic/recurrent headache. Journal of Chinese Integrative Medicine, 2008, 6(1): 31
[4] Xue-mei Liu, Qi-fu Huang, Yun-ling Zhang, Jin-li Lou, Hong-sheng Liu, Hong Zheng. Effects of Tribulus terrestris L. saponion on apoptosis of cortical neurons induced by hypoxia-reoxygenation in rats. Journal of Chinese Integrative Medicine, 2008, 6(1): 45-50
[5] . Uniform requirements for manuscripts submitted to biomedical journals: Writing and editing for biomedical publication (Chinese version, part two). Journal of Chinese Integrative Medicine, 2010, 8(11): 1001-1005
[6] Daniel Weber, Janelle M Wheat, Geoffrey M Currie. Inflammation and cancer: Tumor initiation, progression and metastasis,and Chinese botanical medicines. Journal of Chinese Integrative Medicine, 2010, 8(11): 1006-1013
[7] Hong Liu , Guo-liang Zhang, Li Shen , Zhen Zeng, Bao-luo Zhou, Cheng-hai Liu, Guang Nie . Application and evaluation of a pseudotyped virus assay for screening herbs for anti-H5Nl avian influenza virus. Journal of Chinese Integrative Medicine, 2010, 8(11): 1036-1040
[8] Zhao-guo Li . A discussion of English translation of 1995 and 1997 Chinese National Standards of Traditional Chinese Medical Terminologies for Clinical Diagnosis and Treatment. Journal of Chinese Integrative Medicine, 2010, 8(11): 1090-1096
[9] Rui Jin, Bing Zhang. A complexity analysis of Chinese herbal property theory: the multiple formations of herbal property (Part 1). Journal of Chinese Integrative Medicine, 2012, 10(11): 1198-1205
[10] Hui-min Liu, Xian-bo Wang, Yu-juan Chang, Li-li Gu. Systematic review and meta-analysis of randomized controlled trials of integrative medicine therapy for treatment of chronic severe hepatitis. Journal of Chinese Integrative Medicine, 2012, 10(11): 1211-1228