Search JIM Advanced Search

Journal of Integrative Medicine ›› 2015, Vol. 13 ›› Issue (6): 400-411.doi: 10.1016/S2095-4964(15)60201-1

• Research Article • Previous Articles     Next Articles

Ultra-highly diluted plant extracts of Hydrastis canadensis and Marsdenia condurango induce epigenetic modifications and alter gene expression profiles in HeLa cells in vitro

Santu Kumar Sahaa, Sourav Royb, Anisur Rahman Khuda-Bukhsha   

  1. a Cytogenetics and Molecular Biology Laboratory, Department of Zoology, University of Kalyani, Kalyani-741235, West Bengal, India
    b Department of Entomology and Institute for Integrative Genome Biology, University of California Riverside, CA 92521, USA
  • Received:2015-02-17 Accepted:2015-07-09 Online:2015-11-10 Published:2015-11-15


Methylation-specific epigenetic process and gene expression profiles of HeLa cells treated with ultra-high dilutions (HDs) of two plant extracts, Hydrastis canadensis (HC-30) and Marsdenia condurango (Condu-30), diluted 1060 times, were analyzed against placebo 30C (Pl-30) for alterations in gene profiles linked to epigenetic modifications. 


Separate groups of cells were subjected to treatment of Condu-30, HC-30, and Pl-30 prepared by serial dilutions and succussions. Global microarray data recorded on Affymetrix platform, using 25-mer probes were provided by iLifeDiscoveries, India. Slides were scanned with 3000 7G microarray scanner and raw data sets were extracted from Cel (raw intensity) files. Analyses of global microarray data profile, differential gene expression, fold change and clusters were made using GeneSpring GX12.5 software and standard normalization procedure. Before microarray study, concentration of RNA (ng/μL), RIN value and rRNA ratio for all the samples were analysed by Agilant Bioanalyzer 2100. Reverse transcriptase polymerase chain reaction (RT-PCR) and quantitative RT-PCR were done for analyzing SMAD-4 expression. Fluorescence-activated cell sorting study was further made to elucidate fate of cells at divisional stages. Methylation-specific restriction enzyme assay was conducted for ascertaining methylation status of DNA at specific sites. 


HDs of HC-30 and Condu-30 differentially altered methylation in specific regions of DNA and expression profiles of certain genes linked to carcinogenesis, as compared to Pl-30. Two separate cut sites were found in genomic DNA of untreated and placebo-treated HeLa cells when digested with McrBC, compared to a single cut observed in Condu-30-treated genomic DNA. SMAD-4 gene expression validated the expression pattern observed in microarray profile. Methylation-specific restriction enzyme assay elucidated differential epigenetic modifications in drug-treated and control cells.

HDs triggered epigenetic modifications and alterations in microarray gene expression profiles of many genes associated with carcinogenesis in HeLa cells in vitro.

Key words: Plant extracts, Homeopathy, Reactive oxygen species, Apoptosis, Gene expression, Epigenesis, Genetic, Smad4 protein

[1] Kola I, Landis J . Can the pharmaceutical industry reduce attrition rates? Nat Rev Drug Discov. 2004; 3(8):711-715.
doi: 10.1038/nrd1470
[2] Bensoussan M, Jovenin N, Garcia B, Vandromme L, Jolly D, Bouché O, Thiéfin G, Cadiot G . Complementary and alternative medicine use by patients with inflammatory bowel disease: results from a postal survey. Gastroenterol Clin Biol, 2006,30(1):14-23
doi: 10.1016/S0399-8320(06)73072-X
[3] Ernst E . The role of complementary and alternative medicine in cancer. Lancet Oncol, 2000,1: 176-180
doi: 10.1016/S1470-2045(00)00031-0
[4] U.S. Food and Drug Administration. CPG Sec. 400.400 Conditions under which homeopathic drugs may be marketed. (1995-03)[ 2012-03-22]. .
[5] 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
doi: 10.1021/la303477s
[6] Stovbun SV, Kiselev AV, Zanin AM, Kalinina TS, Voronina TA, Mikhailov AI, Berlin AA . Effects of physicochemical forms of phenazepam and Panavir on their action at ultra-low doses. Bull Exp Biol Med, 2012,153(4):455-458
doi: 10.1007/s10517-012-1739-z pmid: 22977843
[7] Saha SK, Roy S, Khuda-Bukhsh AR . Evidence in support of gene regulatory hypojournal: Gene expression profiling manifests homeopathy effect as more than placebo. Int. High Dilution Res. 2013; 12(45):162-167
[8] Bishayee K, Sikdar S, Khuda-Bukhsh AR . Evidence of an epigenetic modification in cell-cycle arrest caused by the use of ultra-highly-diluted Gonolobus condurango extract. J Pharmacopuncture, 2013,16(4):7-13
[9] Sikdar S, Saha SK, Khuda-Bukhsh AR . Relative apoptosis-inducing potential of homeopa thic Condurango 6C and 30C in H460 lung cancer cells in vitro. J Pharmacopuncture, 2014,17(1):59-69
doi: 10.3831/KPI.2014.17.008
[10] Witt CM, Bluth M, Albrecht H, Weisshuhn TE, Baumgartner S, Willich SN . The in vitro evidence for an effect of high homeopathic potencies — a systematic review of the literature. Complement Ther Med, 2007,15(2):128-138
doi: 10.1016/j.ctim.2007.01.011
[11] Khuda-Bukhsh AR . Potentized homoeopathic drugs act through regulation of gene-expression: a hypothesis to explain their mechanism and pathways of action in vitro. Complement Ther Med, 1997,5(1):43-46
doi: 10.1016/S0965-2299(97)80090-8
[12] De A, Das D, Dutta S, Chakraborty D, Boujedaini N, Khuda-Bukhsh AR . Potentiated homeopathic drug Arsenicum Album 30C inhibits intracellular reactive oxygen species generation and up-regulates expression of arsenic resistance gene in arsenite-exposed bacteria Escherichia coli. J Chin Integr Med, 2012,10(2):210-227
doi: 10.3736/jcim201202
[13] Das S, Saha SK, De A, Das D, Khuda-Bukhsh AR . Potential of the homeopathic remedy, Arnica Montana 30C, to reduce DNA damage in Escherichia coli exposed to ultraviolet irradiation through up-regulation of nucleotide excision repair genes. J Chin Integr Med, 2012,10(3):337-346
doi: 10.3736/jcim
[14] Saha SK, Das S, Khuda-Bukhsh AR . Phenotypic evidence of ultra-highly diluted homeopathic remedies acting at gene expression level: a novel probe on experimental phage infectivity in bacteria. J Chin Integr Med, 2012,10(4):462-470
[15] Bell IR, Koithan M . A model for homeopathic remedy effects: low dose nanoparticles, allostatic cross-adaptation, time-dependent sensitization in a complex adaptive system. BMC Complement Altern Med, 2012,12: 191
doi: 10.1186/1472-6882-12-S1-P191
[16] Betti L, Trebbi G, Zurla M, Nani D, Peruzzi M, Brizzi M . A review of three simple plant models and corresponding statistical tools for basic research in homeopathy. ScientificWorldJournal, 2010,10: 2330-2347
doi: 10.1100/tsw.2010.224
[17] Sharma SV, Haber DA, Settleman J . Cell line-based platforms to evaluate the therapeutic efficacy of candidate anticancer agents. Nat Rev Cancer, 2010,10(4):241-253
doi: 10.1038/nrc2820 pmid: 2030010520300105
[18] Fernandez AF, Rosales C, Lopez-Nieva P, Graa O, Ballestar E, Ropero S, Espada J, Melo SA, Lujambio A, Fraga MF, Pino I, Javierre B, Carmona FJ, Acquadro F, Steenbergen RD, Snijders PJ, Meijer CJ, Pineau P, Dejean A, Lloveras B, Capella G, Quer J, Buti M, Esteban JI, Allende H, Rodriguez-Frias F, Castellsague X, Minarovits J, Ponce J, Capello D, Gaidano G, Cigudosa JC, Gomez-Lopez G, Pisano DG, Valencia A, Piris MA, Bosch FX, Cahir-McFarland E, Kieff E, Esteller M . The dynamic DNA methylomes of double-stranded DNA viruses associated with human cancer. Genome Res, 2009,19(3):438-451
doi: 10.1101/gr.083550.108 pmid: 19208682
[19] Paschos K, Allday MJ . Epigenetic reprogramming of host genes in viral and microbial pathogenesis. Trends Microbiol, 2010,18(10):439-447
doi: 10.1016/j.tim.2010.07.003 pmid: 3089700
[20] Davenas E, Beauvais F, Amara J, Oberbaum M, Robinzon B, Miadonna A, Tedeschi A, Pomeranz B, Fortner P, Belon P, Sainte-Laudy J, Poitevin B, Benveniste J . Human basophil degranulation triggered by very dilute antiserum against IgE. Nature, 1988,333(6176):816-818
doi: 10.1038/333816a0 pmid: 2455231
[21] Belon P, Cumps J, Ennis M, Mannaioni PF, Roberfroid M, Sainte-Laudy J, Wiegant FA . Histamine dilutions modulate basophil activation. Inflamm Res, 2004,53(5):181-188
doi: 10.1007/s00011-003-1242-0 pmid: 15105967
[22] Van Wijk R, Wiegant FAC. Homoeopathy and the mammalian cell: programmed cell recovery and new therapeutic strategies. In: Ernst E, Hahn EG.Homeopathy — A critical appraisal[M]. Woburn: Butterworth-Heinemann, Reed Educational and Professional Publishers, 1998: 180-186
[23] Nascarella MA, Calabrese EJ . A method to evaluate hormesis in nanoparticle dose-responses. Dose Response, 2012,10(3):344-354
doi: 10.2203/dose-response.10-025.Nascarella pmid: 3430396
[24] Demangeat JL . Nanosized solvent superstructures in ultramolecular aqueous dilutions: twenty years’ research using water proton NMR relaxation. Homeopathy. 2013. 102(2):87-105.
doi: 10.1016/j.homp.2013.01.001
[25] Montagnier L, Aissa J, Lavallee C, Mbamy M, Varon J, Chenal H . Electromagnetic detection of HIV DNA in the blood of AIDS patients treated by antiretroviral therapy. Interdiscip Sci, 2009,1(2):81-90
doi: 10.1007/s12539-009-0036-7
[26] Rey L . Thermoluminescence of ultra-high dilutions of lithium chloride and sodium chloride. Physica A, 2003,323: 67-74
doi: 10.1016/S0378-4371(03)00047-5
[27] Khuda-Bukhsh AR . Towards understanding molecular mechanisms of action of homeopathic drugs: an overview. Mol Cell Biochem, 2003,253(1-2):339-345
doi: 10.1023/A:1026048907739 pmid: 14619985
[28] Saha SK, Khuda-Bukhsh AR . Berberine alters epigenetic modifications, disrupts microtubule network, and modulates HPV-18 E6-E7 oncoproteins by targeting p53 in cervical cancer cell HeLa: a mechanistic study including molecular docking. Eur J Pharmacol, 2014,744: 132-146
doi: 10.1016/j.ejphar.2014.09.048
[29] Bellavite P, Marzotto M, Debora O, Christofoleti M, Brizzi M, Tononi P . Cellular and transcriptional responses of neurocytes following in vitro exposure to Gelsemium sempervirens. Int J High Dilution Res, 2012,11(40):113-114
[30] Bigagli E, Luceri C, Bernardini S, Dei A, Filippini A, Dolara P . Exploring the effects of homeopathic Apis mellifica preparations on human gene expression profiles. Homeopathy, 2014,103(2):127-132
doi: 10.1016/j.homp.2014.01.003
[31] Marotti I, Betti L, Bregola V, Bosi S, Trebbi G, Borghini G, Nani D, Dinelli G . Transcriptome profiling of wheat seedlings following treatment with ultrahigh diluted arsenic trioxide. Evid Based Complement Alternat Med, 2014,2014: 851263
[32] Khuda-Bukhsh AR . Current trends in high dilution research with particular reference to gene regulatory hypothesis. Nucleus, 2014,57(1):3-17
doi: 10.1007/s13237-014-0105-0
[33] Sambrook J, Russell DW. Molecular cloning.3rd ed[M]. New York: Cold Spring Harbor Laboratory Press,2001:6.28- 6.30
[34] Das D, De A, Dutta S, Biswas R, Boujedaini N, Khuda-Bukhsh AR . Potentized homeopathic drug Arsenicum Album 30C positively modulates protein biomarkers and gene expressions in Saccharomyces cerevisae exposed to arsenate. J Chin Integr Med, 2011,9(7):752-760
doi: 10.3736/jcim
[1] Cai-lian Fana, Wan-jun Caib, Meng-nan Ye, Miao Chen, Yi Dai. Qili Qiangxin, a compound herbal medicine formula, alleviates hypoxia-reoxygenation-induced apoptotic and autophagic cell death via suppression of ROS/AMPK/mTOR pathway in vitro. Journal of Integrative Medicine, 2022, 20(4): 365-375.
[2] 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.
[3] Hong-xiao Li, Ling Shi, Shang-jie Liang Chen-chen Fang, Qian-qian Xu, Ge Lu, Qian Wang, Jie Cheng, Jie Shen, Mei-hong Shen. Moxibustion alleviates decreased ovarian reserve in rats by restoring the PI3K/AKT signaling pathway. Journal of Integrative Medicine, 2022, 20(2): 163-172.
[4] Mohamed S. Othman, Sofian T. Obeidat, Amal H. Al-Bagawi, Mohamed A. Fareid, Alaa Fehaid, Ahmed E. Abdel Moneim. Green-synthetized selenium nanoparticles using berberine as a promising anticancer agent#br#
. Journal of Integrative Medicine, 2022, 20(1): 65-72.
[5] Hyongjun Jeon, Hee-Young Kim, Chang-Hwan Bae, Yukyung Lee, Sungtae Koo, Seungtae Kim. Korean red ginseng decreases 1-methyl-4-phenylpyridinium-induced mitophagy in SH-SY5Y cells. Journal of Integrative Medicine, 2021, 19(6): 537-544.
[6] Mu-yan Kong, Le-yan Li, Yan-mei Lou, Hong-yu Chi, Jin-jun Wu. Chinese herbal medicines for prevention and treatment of colorectal cancer: from molecular mechanisms to potential clinical applications. Journal of Integrative Medicine, 2020, 18(5): 369-384.
[7] Porwornwisit Tritripmongkol, Tullayakorn Plengsuriyakarn, Mayuri Tarasuk, Kesara Na-Bangchang. In vitro cytotoxic and toxicological activities of ethanolic extract of Kaempferia galanga Linn. and its active component, ethyl-p-methoxycinnamate, against cholangiocarcinoma. Journal of Integrative Medicine, 2020, 18(4): 326-333.
[8] 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.
[9] Maged Mohamed Maher Abou-Hashem, Dina Mohamed Abo-elmatty, Noha Mostafa Mesbah, Ahmed Mohamed Abd EL-Mawgoud. Induction of sub-G0 arrest and apoptosis by seed extract of Moringa peregrina (Forssk.) Fiori in cervical and prostate cancer cell lines. Journal of Integrative Medicine, 2019, 17(6): 410-422.
[10] Supakanya Kumkarnjana, Rutt Suttisri, Ubonthip Nimmannit, Apirada Sucontphunt, Mattaka Khongkow, Thongchai Koobkokkruad, Nontima Vardhanabhuti. Flavonoids kaempferide and 4,2′-dihydroxy-4′,5′,6′-trimethoxychalcone inhibit mitotic clonal expansion and induce apoptosis during the early phase of adipogenesis in 3T3-L1 cells. Journal of Integrative Medicine, 2019, 17(4): 288-295.
[11] 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.
[12] Shriniwas S. Basaiyye, Sanjay Kashyap, Kannan Krishnamurthi, Saravanadevi Sivanesan. Induction of apoptosis in leukemic cells by the alkaloid extract of garden cress (Lepidium sativum L.). Journal of Integrative Medicine, 2019, 17(3): 221-228.
[13] Carlos R. Oliveira, Daniel G. Spindola, Daniel M. Garcia, Adolfo Erustes, Alexandre Bechara, Caroline Palmeira-dos-Santos, Soraya S. Smaili, Gustavo J.S. Pereira, André Hinsberger, Ezequiel P. Viriato, Maria Cristina Marcucci, Alexandra C.H.F. Sawaya, Samantha L. Tomaz, Elaine G. Rodrigues, Claudia Bincoletto. Medicinal properties of Angelica archangelica root extract: Cytotoxicity in breast cancer cells and its protective effects against in vivo tumor development. Journal of Integrative Medicine, 2019, 17(2): 132-140.
[14] Mohammad Ansaripour, Mohsen Naseri, Mohammad Mahdi Esfahani, Iraj Nabipour, Fatemeh Rakhshani, Arman Zargaran, Roya Kelishadi. Periconceptional care and offspring health at birth and long term, from the perspective of Avicenna. Journal of Integrative Medicine, 2019, 17(2): 80-86.
[15] Bruno José Martins Da Silva, Sandro Wilson Gomes Pereira, Ana Paula Drummond Rodrigues, José Luiz Martins Do Nascimento, Edilene Oliveira Silva. In vitro antileishmanial effects of Physalis angulata root extract on Leishmania infantum. Journal of Integrative Medicine, 2018, 16(6): 404-410.
Full text



[1] Jin-zhou Tian, Jing Shi, Xin-qing Zhang, Qi Bi, Xin Ma, Zhi-liang Wang, Xiao-bin Li, Shu-li Shen, Lin Li, Zhen-yun Wu, Li-yan Fang, Xiao-dong Zhao, Ying-chun Miao, Peng-wen Wang, Ying Ren, Jun-xiang Yin, Yong-yan Wang, Beijing United Study Group on MCI of the Capital Foundation of Medical Developments. An explanation on "guiding principles of clinical research on mild cognitive impairment (protocol)". Journal of Chinese Integrative Medicine, 2008, 6(1): 15-21
[2] Yi-ting He, Qing-lin Zha, Jian-ping Yu, Yong Tan, Cheng Lu, Ai-ping Lv. Principal factor analysis of symptoms of rheumatoid arthritis and their correlations with efficacy of traditional Chinese medicine and Western medicine. Journal of Chinese Integrative Medicine, 2008, 6(1): 32-36
[3] Jun Cai, Hua Wang, Sheng Zhou, Bin Wu, Hua-rong Song, Zheng-rong Xuan. Effect of Sijunzi Decoction and enteral nutrition on T-cell subsets and nutritional status in patients with gastric cancer after operation: A randomized controlled trial. Journal of Chinese Integrative Medicine, 2008, 6(1): 37-40
[4] Wei Zhang, Xiang-feng Lu, Xiao-mei Zhang, Jian-jun Wu, Liang-duo Jiang. A rat model of pulmonary fibrosis induced by infusing bleomycin quickly through tracheal intubation. Journal of Chinese Integrative Medicine, 2008, 6(1): 60-67
[5] A-gao Zhou, Yong Zhang, Gang Kui, De-Yun Kong, Hai-liang Ge, Qiu-hua Ren, Jia-rong Dong, Sheng Hong, Xu-ming Mao, Yin Wang, Hui-zheng Zhang, Shu-jun Wang. Influence of traditional Chinese compound recipes with different efficacy on body weight, tumor weight and immune function in H22 cancer-bearing mice. Journal of Chinese Integrative Medicine, 2008, 6(1): 77-82
[6] Guo-hong Yuan, Xiao-jing Pang, He-chao Ma. Synergic effects of Danggui Buxue Decoction in reducing toxicity of cytoxan in tumor-bearing mice. Journal of Chinese Integrative Medicine, 2008, 6(1): 83-88
[7] Li Zhou, Hong-xing Zhang, Ling-guang Liu, Wen-jun Wan. Effect of electro-acupuncture at Fenglong (GV 16) on nitric oxide and endothelin in rats with hyperlipidemia. Journal of Chinese Integrative Medicine, 2008, 6(1): 89-92
[8] Jin-zhou Tian, Jing Shi, Xin-qing Zhang, Qi Bi, Xin Ma, Zhi-liang Wang, Xiao-bin Li, Shu-li Shen, Lin Li, Zhen-yun Wu, Li-yan Fang, Xiao-dong Zhao, Ying-chun Miao, Peng-wen Wang, Ying Ren, Jun-xiang Yin, Yong-yan Wang, Beijing United Study Group on MCI of the Capital Foundation of Medical Developments. Guiding principles of clinical research on mild cognitive impairment (protocol). Journal of Chinese Integrative Medicine, 2008, 6(1): 9-14
[9] Xin-jun Wang, Ling-ling Wang . A mechanism of endogenous opioid peptides for rapid onset of acupuncture effect in treatment of depression. Journal of Chinese Integrative Medicine, 2010, 8(11): 1014-1017
[10] Bo Wang , Wei Yan , Li-hui Hou, Xiao-ke Wu. Disorder of Tiangui (kidney essence) and reproductive dysfunction in patients with polycystic ovary syndrome. Journal of Chinese Integrative Medicine, 2010, 8(11): 1018-1022