Arnica,Homeopathy,Bias,Statistics,Zene expression,Tamm-Horsfall protein,Cell line," /> Arnica,Homeopathy,Bias,Statistics,Zene expression,Tamm-Horsfall protein,Cell line,"/> Homeopathic potencies of <em>Arnica montana</em> L. change gene expression in a Tamm-Horsfall protein-1 cell line <em>in vitro</em> model: The role of ethanol as a possible confounder and statistical bias
Search JIM Advanced Search

Journal of Integrative Medicine ›› 2017, Vol. 15 ›› Issue (4): 255-264.doi: 10.1016/S2095-4964(17)60346-7

Special Issue: Natural Drug

• Commentary •     Next Articles

Homeopathic potencies of Arnica montana L. change gene expression in a Tamm-Horsfall protein-1 cell line in vitro model: The role of ethanol as a possible confounder and statistical bias

Salvatore Chirumboloa, Geir Bjørklundb   

  1. a Department of Neurological and Movement Sciences, University of Verona, 37134 Verona, Italy 
    b Council for Nutritional and Environmental Medicine, 8610 Mo i Rana, Norway
  • Received:2017-01-10 Accepted:2017-04-06 Online:2017-07-15 Published:2017-07-15
  • Contact: Salvatore Chirumbolo, PhD; E-mail:

Marzotto et al. showed that homeopathic preparations of Arnica montana L. acted directly on gene expression of Tamm-Horsfall protein-1 (THP-1) monocyte/macrophage cell lines activated with phorbol12-myristate13-acetate and interleukin-4 (IL-4). A. montana homeopathic dilutions are used in complementary and alternative medicine to treat inflammation disorders and post-traumatic events as well as for wound repair. The French Pharmacopoeia of these remedies uses 0.3% ethanol in each centesimal dilution. In this paper, we discuss how ethanol-containing A. montana homeopathic centesimal dilutions can change gene expression in IL-4-treated monocyte/macrophage THP-1. We assessed the role of ethanol in the Arnica homeopathic dilutions containing this alcohol by investigating its action on gene expression of THP-1 cell. Evidence would strongly suggest that the presence of ethanol in these remedies might play a fundamental role in the dilutions ability to affect gene expression, particularly for doses from 5c to 15c. Where, rather than playing a major role in the mesoscopic structure of water, the ethanol might have a chemical-physical role in the induction of THP-1 gene expression, apoptosis, and deoxyribonucleic acid function. This evidence generates a debate about the suggestion that the use of a binary-mixed solvent in homeopathic chemistry, used by Hahnemann since 1810, may be fundamental to explain the activity of homeopathy on cell models.

Key words: Arnica, Homeopathy, Bias, Statistics, Zene expression, Tamm-Horsfall protein, Cell line

1 Marzotto M, Bonafini C, Olioso D, Baruzzi A, Bettinetti L, Di Leva F, Galbiati E, Bellavite P.Arnica montana stimulates extracellular matrix gene expression in a macrophage cell line differentiated to wound-healing phenotype. PLoS One. 2016; 11(11): e0166340.
2 Endrullat C, Glökler J, Franke P, Frohme M.Standardization and quality management in next-generation sequencing.Appl Transl Genom. 2016; 10: 2-9.
3 Staneva J, Denkova P, Todorova M, Evstatieva L.Quantitative analysis of sesquiterpene lactones in extract ofArnica montana L. by 1H NMR spectroscopy. J Pharm Biomed Anal. 2011; 54(1): 94-99.
4 Merfort I.Perspectives on sesquiterpene lactones in inflammation and cancer.Curr Drug Targets. 2011; 12(11): 1560-1573.
5 Mary-Huard T, Daudin JJ, Baccini M, Biggeri A, Bar- Hen A.Biases induced by pooling samples in microarray experiments.Bioinformatics. 2007; 23(13): i313-i318.
6 Fasold M, Binder H.Variation of RNA quality and quantity are major sources of batch effects in microarray expression data.Microarrays (Basel). 2014; 3(4): 322-339.
7 Zimmerman DW, Zumbo BD.Relative power of the Wilcoxon test, the Friedman test, and repeated-measures ANOVA on ranks.J Exper Educ. 1993; 62(1): 75-86.
8 Bürkner PC, Doebler P, Holling H.Optimal design of the Wilcoxon-Mann-Whitney test.Biom J. 2017; 59(1): 25-40.
9 Zimmerman DW, Zumbo BD.The relative power of the Wilcoxon-Mann-Whitney test and Student’st test under simple bounded transformations. J Gen Psychol. 1990; 117(4): 425-436.
10 Olioso D, Marzotto M, Bonafini C, Brizzi M, Bellavite P.Arnica montana effects on gene expression in a human macrophage cell line. Evaluation by quantitative real-time PCR. Homeopathy. 2016; 105(2): 131-147.
11 Chirumbolo S, Bjørklund G.Commentary:Arnica montana effects on gene expression in a human macrophage cell line: evaluation by quantitative real-time PCR. Front Immunol. 2016; 7: 280.
12 Olioso D, Marzotto M, Bonafini C, Brizzi M, Bellavite P.Response to commentary:Arnica montana effects on gene expression in a human macrophage cell line. Evaluation by quantitative real-time PCR. Front Immunol. 2016; 7: 320.
13 Parekh S, Ziegenhain C, Vieth B, Enard W, Hellmann I.The impact of amplification on differential expression analyses by RNA-seq.Sci Rep. 2016; 6: 25533.
14 Rajkumar AP, Qvist P, Lazarus R, Lescai F, Ju J, Nyegaard M, Mors O, Børglum AD, Li Q, Christensen JH.Experimental validation of methods for differential gene expression analysis and sample pooling in RNA-seq.BMC Genomics. 2015; 16: 548.
15 Wang C, Gong B, Bushel PR, Thierry-Mieg J, Thierry- Mieg D, Xu J, Fang H, Hong H, Shen J, Su Z, Meehan J, Li X, Yang L, Li H, Łabaj PP, Kreil DP, Megherbi D, Gaj S, Caiment F, van Delft J, Kleinjans J, Scherer A, Devanarayan V, Wang J, Yang Y, Qian HR, Lancashire LJ, Bessarabova M, Nikolsky Y, Furlanello C, Chierici M, Albanese D, Jurman G, Riccadonna S, Filosi M, Visintainer R, Zhang KK, Li J, Hsieh JH, Svoboda DL, Fuscoe JC, Deng Y, Shi L, Paules RS, Auerbach SS, Tong W. The concordance between RNA-seq and microarray data depends on chemical treatment and transcript abundance.Nat Biotechnol. 2014; 32(9): 926-932.
16 Yuan F, Lei Y, Wang Q, Esberg LB, Huang Z, Scott GI, Li X, Ren J.Moderate ethanol administration accentuates cardiomyocyte contractile dysfunction and mitochondrial injury in high-fat diet-induced obesity.Toxicol Lett. 2015; 233(3): 267-277.
17 Cahill A, Stabley GJ, Wang X, Hoek JB.Chronic ethanol consumption causes alterations in the structural integrity of mitochondrial DNA in aged rats.Hepatology. 1999; 30(4): 881-888.
18 Bolnick JM, Karana R, Chiang PJ, Kilburn BA, Romero R, Diamond MP, Smith SM, Armant DR.Apoptosis of alcohol-exposed human placental cytotrophoblast cells is downstream of intracellular calcium signaling.Alcohol Clin Exp Res. 2014; 38(6): 1646-1653.
19 Guan Z, Lui CY, Morkin E, Bahl JJ.Oxidative stress and apoptosis in cardiomyocyte induced by high-dose alcohol.J Cardiovasc Pharmacol. 2004; 44(6): 696-702.
20 Zhang J, He S, Zhou W, Yuan B.Ethanol induces oxidative stress and apoptosis in human umbilical vein endothelial cells.Int J Clin Exp Med. 2016; 9(2): 4125-4130.
21 Rehman S, Chandel N, Salhan D, Rai P, Sharma B, Singh T, Husain M, Malhotra A, Singhal PC.Ethanol and vitamin D receptor in T cell apoptosis.J Neuroimmune Pharmacol. 2013; 8(1): 251-261.
22 Pourhoseingholi MA, Baghestani AR, Vahedi M.How to control confounding effects by statistical analysis.Gastroenterol Hepatol Bed Bench. 2012; 5(2): 79-83.
23 Chiba Y.The sign of the unmeasured confounding bias under various standard populations.Biom J. 2009; 51(4): 670-676.
24 Süt N.Study designs in medicine.Balkan Med J. 2014; 31(4): 273-277.
25 Ishida N, Inoue T, Miyahara M, Higashitani K.Nanbubbles in a hydrophobic surface in water observed by tappingmode atomic force microscopy.Langmuir. 2000; 16(16): 6377-6380.
26 Ruckenstein E.Nanodispersions of bubbles and oil drops in water.Colloids Surf A Physicochem Eng Asp. 2013; 423: 112-114.
27 Thorpe SA, Stubbs AR, Hall AJ, Turner J.Wave-produced bubbles observed by side-scan sonar.Nature. 1982; 296: 636-638.
28 Jin F, Ye J, Hong L, Lam H, Wu C.Slow relaxation mode in mixtures of water and organic molecules: supramolecular structures or nanobubbles?J Phys Chem B. 2007; 111(9): 2255-2261.
29 Häbich A, Ducker W, Dunstan DE, Zhang X.Do stable nanobubbles exist in mixtures of organic solvents and water?J Phys Chem B. 2010; 114(20): 6962-6967.
30 An H, Liu G, Atkin R, Craig VSJ.Surface nanobubbles in nonaqueous media: looking for nanobubbles in DMSO, formamide, propylene carbonate, ethylammniumnitrate and propylammonium nitrate.ACS Nano. 2015; 9: 7596-7607.
31 Uchida T, Oshita S, Ohmori M, Tsuno T, Soejima K, Shinozaki S, Take Y, Mitsuda K.Transmission electron microscopic observations of nanobubbles and their capture of impurities in wastewater.Nanoscale Res Lett. 2011; 6(1): 295.
32 Craig VSJ.Viewpoint: surface nanobubbles or Knudsen bubbles?Physics. 2011; 4: 70.
33 Liu S, Oshita S, Makino Y, Wang Q, Kawagoe Y, Uchida T.Oxidative capacity of nanobubbles and its effect on seed germination.ACS Sustainable Chem Eng. 2016; 4(3): 1347-1353.
34 Uchida T, Liu S, Enari M, Oshita S, Yamazaki K, Gohara K.Effect of NaCl on the lifetime of micro- and nano-bubbles.Nanomaterials. 2016; 6: 31.
35 Attard P.The stability of nanobubbles.Eur Phys J Special Topics. 2013; 2013: 1-22.
36 Chan CU, Ohl CD.Total internal reflection fluorescence microscopy for the study of nanobubble dynamics.Phys Rev Lett. 2012; 109: 174501.
37 Sedlák M, Rak D.Large-scale inhomogeneities in solutions of low molar mass compounds and mixtures of liquids: supramolecular structures or nanobubbles?J Phys Chem B. 2013; 117(8): 2495-2504.
38 Simonsen AC, Hansen PL, Klösgen B.Nanobubbles give evidence of incomplete wetting at a hydrophobic interface.J Colloid Interface Sci. 2004; 273(1): 291-299.
39 Yang S, Dammer SM, Bremond N, Zandvliet HJ, Kooij ES, Lohse D.Characterization of nanobubbles on hydrophobic surfaces in water.Langmuir. 2007; 23(13): 7072-7077.
40 Sedlák M, Rak D.On the origin of mesoscale structures in aqueous solutions of tertiary butyl alcohol: the mystery resolved.J Phys Chem B. 2014; 118(10): 2726-2737.
41 Chikramane PS, Kalita D, Suresh AK, Kane SG, Bellare JR.Why extreme dilutions reach non-zero asymptotes: a nanoparticulate hypothesis based on froth flotation.Langmuir. 2012; 28(45): 15864-15875.
42 Chirumbolo S.Bias in homeopathy: technical note.Malays J Med Biol Res. 2015; 2(3): 191-199.
43 Pawitan Y, Murthy KR, Michiels S, Ploner A.Bias in the estimation of false discovery rate in microarray studies.Bioinformatics. 2005; 21(20): 3865-3872.
44 Liao JG, Lin Y, Selvanayagam ZE, Shih WJ.A mixture model for estimating the local false discovery rate in DNA microarray analysis.Bioinformatics. 2004; 20(16): 2694-2701.
45 Mayo MS, Gajewski BJ, Morris JS.Some statistical issues in microarray gene expression data.Radiat Res. 2006; 165(6): 745-748.
[1] Ubiratan Cardinalli Adler, Maristela Schiabel Adler, Ana Elisa Madureira Padula, Livia Mitchiguian Hotta, Amarilysde Toledo Cesar, José Nelson Martins Diniz, Helen de Freitas Santos, Edson Zangiacomi Martinez. Homeopathy for COVID-19 in primary care: A randomized, double-blind, placebo-controlled trial (COVID-Simile study). Journal of Integrative Medicine, 2022, 20(3): 221-229.
[2] Stergios Tsiormpatzis. Is the N-of-1 method applicable in bodywork research? Lessons learned using a trial as a methodological pilot. Journal of Integrative Medicine, 2021, 19(3): 203-210.
[3] Mikhail Teppone. Medicine has always been “Modern” and “Scientific” from ancient times to the present day. Journal of Integrative Medicine, 2019, 17(4): 229-237.
[4] Ubiratan Cardinalli Adler, Ana Carolina Acorinte, Fernando Oliveira Calzavara, Adriano Andréda Silva, Amarilys de Toledo Cesar, Maristela Schiabel Adler, Edson Zangiacomi Martinez, José Carlos Fernandes Galduróz. Double-blind evaluation of homeopathy on cocaine craving: A randomized controlled pilot study. Journal of Integrative Medicine, 2018, 16(3): 178-184.
[5] Shubha Singhal, Vandana Roy. Awareness, practice and views about integrating AYUSH in allopathic curriculum of allopathic doctors and interns in a tertiary care teaching hospital in New Delhi, India. Journal of Integrative Medicine, 2018, 16(2): 113-119.
[6] Bellavite Paolo, Marzotto Marta, Bonafini Clara. Arnica montana experimental studies: Confounders and biases?. Journal of Integrative Medicine, 2018, 16(2): 72-76.
[7] Saeed Ahmad, Tayyeba Rehman, Waheed Mumtaz Abbasi. Effects of homoeopathic ultrahigh dilutions of Aconitum napellus on Baker's yeast-induced fever in rabbits. Journal of Integrative Medicine, 2017, 15(3): 209-213.
[8] Jesmin Mondal, Asmita Samadder, Anisur Rahman Khuda-Bukhsh. Psorinum 6× triggers apoptosis signals in human lung cancer cells. Journal of Integrative Medicine, 2016, 14(2): 143-153.
[9] Santu Kumar Saha, Sourav Roy, Anisur Rahman Khuda-Bukhsh. Ultra-highly diluted plant extracts of Hydrastis canadensis and Marsdenia condurango induce epigenetic modifications and alter gene expression profiles in HeLa cells in vitro. Journal of Integrative Medicine, 2015, 13(6): 400-411.
[10] Arthur Yin Fan. The methodology flaws in Hinman’s acupuncture clinical trial, Part II: Zelen design and effectiveness dilutions. Journal of Integrative Medicine, 2015, 13(3): 136-139.
[11] Arthur Yin Fan. The methodology flaws in Hinman’s acupuncture clinical trial, Part I: Design and results interpretation. Journal of Integrative Medicine, 2015, 13(2): 65-68.
[12] Subhranil Saha​, Munmun Koley, Jogendra Singh Arya, Gurudev Choubey, Shubhamoy Ghosh​, Subhasish Ganguly​, Aloke Ghosh​, Sangita Saha​, Malay Mundle​. Medicine prescription practices of homeopathic undergraduate students in West Bengal, India. Journal of Integrative Medicine, 2014, 12(1): 7-12.
[13] Subhranil Saha​, Munmun Koley​, Subhasish Ganguly​, Prasanta Rath​, Pulak Roy Chowdhury, Seikh Intaj Hossain. Developing the criteria for evaluating quality of individualization in homeopathic clinical trialreporting: A preliminary study. Journal of Integrative Medicine, 2014, 12(1): 13-19.
[14] Munmun Koley, Subhranil Saha, Jogendra Singh Arya, Gurudev Choubey, Shubhamoy Ghosh, Rajib Purkait, Ramkumar Mondal, Bapi Kundu, Rajarshi Mukherjee. A study on drug utilization and prescription habits of physicians in a government homeopathic hospital in West Bengal, India. Journal of Chinese Integrative Medicine, 2013, 11(5): 305-313.
[15] Farha A. Kabeer, Geetha B. Sreedevi, Mangalam S. Nair, Dhanya S. Rajalekshmi, Latha P. Gopalakrishnan, Sujathan Kunjuraman, Remani Prathapan. Antineoplastic effects of deoxyelephantopin, a sesquiterpene lactone from Elephantopus scaber, on lung adenocarcinoma (A549) cells. Journal of Chinese Integrative Medicine, 2013, 11(4): 269-277.
Full text



[1] Dong Yang, Yong-ping Du, Qing Shen, Wei Chen, Yan Yu, Guang-lei Chen. Expression of alpha-smooth muscle actin in renal tubulointerstitium in patients with kidney collateral stasis. Journal of Chinese Integrative Medicine, 2008, 6(1): 41-44
[2] Hai-feng Wei, Bai-liu Ya, Ling Zhao, Cui-fei Ye, Li Zhang, Lin Li. Evaluation of tongue manifestation of blood stasis syndrome and its relationship with blood rheological disorder in a rat model of transient brain ischemia. Journal of Chinese Integrative Medicine, 2008, 6(1): 73-76
[3] Xi Lin, Jian-ping Liu. Herbal medicines for viral myocarditis. Journal of Chinese Integrative Medicine, 2008, 6(1): 76
[4] Xi Lin, Jian-ping Liu. Tai chi for treating rheumatoid arthritis. Journal of Chinese Integrative Medicine, 2008, 6(1): 82
[5] Liang-ping Hu, Hui Gao. Discrimination of errors in statistical analysis of medical papers published in the first issue of 2006 in Journal of Chinese Integrative Medicine. Journal of Chinese Integrative Medicine, 2008, 6(1): 98-106
[6] Yan-bo Zhu , Qi Wang, Cheng-yu Wu, Guo-ming Pang, Jian-xiong Zhao, Shi-lin Shen, Zhong-yuan Xia , Xue Yan . Logistic regression analysis on relationships between traditional Chinese medicine constitutional types and overweight or obesity. Journal of Chinese Integrative Medicine, 2010, 8(11): 1023-1035
[7] Wei Xu, Meng Shi, Jian-gang Liu, Cheng-long Wang . Collagen protein expressions in ischemic myocardium of rats with acute myocardial infarction and effects of qi-tonifying, yin-tonifying and blood-activating herbs and detoxifying and blood-activating herbs. Journal of Chinese Integrative Medicine, 2010, 8(11): 1041-1047
[8] Tao Wang , Feng Qin. Effects of Chinese herbal medicine Xiaoyao Powder on monoamine neurotransmitters in hippocampus of rats with postpartum depression. Journal of Chinese Integrative Medicine, 2010, 8(11): 1075-1079
[9] Ying Xu , Chang-chun Zeng , Xiu-yu Cai , Rong-ping Guo , Guang Nie , Ying Jin. Chromaticity and optical spectrum colorimetry of the tongue color in different syndromes of primary hepatic carcinoma. Journal of Chinese Integrative Medicine, 2012, 10(11): 1263-1271
[10] Xiang-ying Mao , Qin Bian , Zi-yin Shen. Analysis of the osteogenetic effects exerted on mesenchymal stem cell strain C3H10T1/2 by icariin via MAPK signaling pathway in vitro. Journal of Chinese Integrative Medicine, 2012, 10(11): 1272-1278