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Journal of Chinese Integrative Medicine ›› 2012, Vol. 10 ›› Issue (4): 462-470.doi: 10.3736/jcim20120416

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Phenotypic evidence of ultra-highly diluted homeopathic remedies acting at gene expression level: A novel probe on experimental phage infectivity in bacteria

Santu Kumar Saha, Sreemanti Das, Anisur Rahman Khuda-Bukhsh()   

  1. Cytogenetics and Molecular Biology Laboratory, Department of Zoology, University of Kalyani, Kalyani 741235, India
  • Received:2011-11-11 Accepted:2011-12-21 Online:2012-04-20 Published:2018-04-15

Objective: To explore if some ultra-highly diluted homeopathic remedies claimed to have antiviral effects can demonstrate any discernible action in the bacteriaEscherichia coli through modulating infectivity potentials of the bacteriophage ФX174 DNA.
Methods: ФX174 was selected because of its known host specificity to E. coli and its constitutive expression of lytic gene E when inside the bacterial host. We deployed the “bacteriophage assay system” by “top layer agar plating” method of plaque-counting for evaluation of efficacy of the homeopathic remedies in rendering the bacteria’s protective ability against the attack of ФX174. The plaque number in the agar-plated Petri dishes, either containing the phage-bacteria mixture subjected to one of the diluted homeopathic drugs under test (1% volume ratio; Belladonna 30C, Rhus Tox 30C, Arnica 30C) or the succussed 1% “alcoholic vehicle” of the drug was recorded. The plaques represented the bacterial colony actually infected and lysed by ФX174. Conversely, we subjected ФX174 to the homeopathic drug treatment before allowing them to interact with the bacteria to ascertain if the drug itself had any direct effect on the infective potential of the phage DNA entering into the bacterial cell.
Results: Each homeopathic remedy showed a significant decrease in plaque number on pretreated bacteria (1 h prior to infection) with respect to untreated and placebo-treated controls; there was only an insignificant change in the plaque number when ФX174 was pretreated with the drugs. As ФX174 starts lytic cycle when inside the bacterial cell, the loss of plaque number would mean that either the lytic gene E in many was repressed or the entire phage DNA was annihilated by the bacterial gene product (restriction enzymes) known to be regulated by a cluster of genes.
Conclusion: This provides phenotypic evidence for the ability of ultra-highly diluted homeopathic remedies to regulate expression of certain gene(s) depending on need of the organism.

Key words: bacteriophage phi X 174, Escherichia coli, homeopathy, gene expression regulation, plaque assay

Figure 1

Representation of phage infectivity on drug-pretreated bacteria A: Control group without any treatment; B: Placebo treated with 1% of alcohol; C: Bacteria treated with 1% Belladona 30C prior to infection; D: Bacteria treated with 1% Rhus Tox 30C prior to infection; E: Bacteria treated with 1% Arnica 30C prior to infection."

Figure 2

Plaque-forming units in drug-treated bacteria prior to phage infection Data are expressed as mean±standard error of mean of plaque-forming unit. n=5; *P<0.05, vs control group; △P<0.05, vs placebo group."

Figure 3

Representation of phage infectivity on drug-pretreated phage A: Control group without any treatment; B: Placebo treated with 1% of alcohol; C: Phage treated with 1% Belladona 30C prior to infection; D: Phage treated with 1% Rhus Tox 30C prior to infection; E: Phage treated with 1% Arnica 30C prior to infection."

Figure 4

Plaque-forming units in drug-treated phage prior to infection Data are expressed as mean±standard error of mean of plaque-forming unit. n=5."

[1] Sinsheimer RL, Starman B, Nagler C, Guthrie S . The process of infection with bacteriophage <pX174. I . Evidence for a “ replicative form”.J Mol Biol. 1962 : 142-160.
[2] Arai K, Low R, Kobori J, Shlomai J, Kornberg A . Mechanism of dnaB protein action. V . Association of dnaB protein, protein n’,and other repriming proteins in the primosome of DNA replication.J Biol Chem. 1981; 256(10) : 5273-5280.
[3] Goulian M, Kornberg A, Sinsheimer RL . Enzymatic synthesis of DNA, XXN • Synthesis of infectious phage 9-X174 DNA. Proc Natl Acad Sci USA. 1967; 58(6):2321-2328.
doi: 10.1073/pnas.58.6.2321
[4] Kornberg A . Ten commandments : lessons from the enzymology of DNA replication.J Bacteriol. 2000 ; 182(13) : 3613-3618.
doi: 10.1128/JB.182.13.3613-3618.2000
[5] Sanger F, Coulson AR, Friedmann T, Air GM, Barrell BG, Brown NL, Fiddes JC , Hutchison CA 3rd, Slocombe PM, Smith M. The nucleotide sequence of bacteriophage (pX174.J Mol Biol. 1978; 125(2):225-246.
doi: 10.1016/0022-2836(78)90346-7
[6] Smith HO , Hutchison CA 3rd, Pfannkoch C, Venter JC. Generating a synthetic genome by whole genome assembly: <pX174 bacteriophage from synthetic oligonucleotides. Proc Natl Acad Sci USA. 2003; 100(26):15440-15445.
doi: 10.1073/pnas.2237126100
[7] Witte A, Lubitz W . Biochemical characterization of (pX174-protein-E-mediated lysis of Escherichia coli. Eur J Biochem. 1989; 180(2):393-398.
doi: 10.1111/ejb.1989.180.issue-2
[8] Eigner J , Stouthamer AH, van der Sluys I, Cohen JA. A study of the 70 s component of bacteriophage <pX174.J Mol Biol. 1963; 6(1) : 61-84.
doi: 10.1016/S0022-2836(63)80082-0
[9] Markert A, Zillig W . Studies on the lysis of Escherichia coli C by bacteriophage <pX174. Virology. 1965; 25:88-97.
doi: 10.1016/0042-6822(65)90256-4
[10] Cao Y, Lu HM, Liang J . Stochastic probability landscape model for switching efficiency, robustness, and differential threshold for induction of genetic circuit in phage X. Conf Proc IEEE Eng Med Biol Soc. 2008; 2008:611-614.
[11] Khan NC, Poddar RK . Photodynamic inactivation of antigenic determinants of single-stranded DNA bacteriophage (pX174.J Virol. 1974; 13(5):997-1000.
[12] de Mik G, de Groot I . Mechanisms of inactivation of bacteriophage <pX174 and its DNA in aerosols by ozone and ozonized cyclohexene. J Hyg (Lond). 1977 ; 78(2):199-211.
doi: 10.1017/S0022172400056096
[13] Hsu WT, Lin EJ, Harvey RG, Weiss SB . Mechanism of phage <pX174 DNA inactivation by benzo(a)pyrene-7, 8-dihydrodiol-9,10-epoxide. Proc Natl Acad Sci USA. 1977; 74(8) : 3335-3339.
doi: 10.1073/pnas.74.8.3335
[14] Rueckert RR, Zillig W . Biosynjournal of virus protein in Escherichia coli C in vivo following infection with bacteriophage <pXl74.J Mol Biol. 1962; 5:1-9.
doi: 10.1016/S0022-2836(62)80056-4
[15] Hayashi M, Hayashi MN, Spiegelman S . Restriction of in vivo genetic transcription to one of the complementary strands of DNA. Proc Natl Acad Sci USA. 1963; 50:664-672.
doi: 10.1073/pnas.50.4.664
[16] 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
[17] Dulbecco R . Experiments on photoreactivation of bacteriophages inactivated with ultraviolet radiation.J Bacteriol. 1950; 59(3) : 329-347.
[18] Denhardt DT, Sinsheimer RL . The process of infection with bacteriophage <pX174. 3. Phage maturation and lysis after synchronized infection.J Mol Biol. 1965; 12(3):641-646.
doi: 10.1016/S0022-2836(65)80318-7
[19] Saha S, Jana B, Basu T . The two inducible responses, SOS and heat-shock, in Escherichia coli act synergistically during Weigle reactivation of the bacteriophage <pX174. Int J Radiat Biol. 2007; 83(7):463-469.
doi: 10.1080/09553000701371389
[20] Goossens M, Laekeman G, Aertgeerts B, Buntinx F; ARCH study group. Evaluation of the quality of life after individualized homeopathic treatment for seasonal allergic rhinitis. A prospective, open, non-comparative study. Homeopathy. 2009; 98(1):11-16.
doi: 10.1016/j.homp.2008.11.008
[21] Kirkby R, Herscu P . Homeopathic trial design in influenza treatment. Homeopathy. 2010 ; 99(1) : 69-75.
doi: 10.1016/j.homp.2009.09.001
[22] Lockie A . The family guide to homeopathy: the safe form of medicine for the future. London: Elm Tree Books. 1989.
[23] Horvath P, Barrangou R . CRISPR/Cas, the immune system of bacteria and archaea. Science. 2010 ; 327(5962):167-170.
doi: 10.1126/science.1179555
[24] Makarova KS, Wolf YI, Snir S, Koonin EV . Defense islands in bacterial and archaeal genomes and prediction of novel defense systems.J Bacteriol. 2011 ; 193(21):6039-6056.
doi: 10.1128/JB.05535-11
[25] Khuda-Bukhsh AR, De A, Das D, Dutta S, Boujedaini N . Analysis of the capability of ultra-highly diluted glucose to increase glucose uptake in arsenite-stressed bacteria Escherichia coli. J Chin Integr Med. 2011; 9 (8) : 901-912. English with abstract in Chinese.
Khuda-Bukhsh AR, De A, Das D, Dutta S, Boujedaini N. 高度稀释的葡萄糖溶液提高含亚砷酸盐培养基中大肠埃希氏杆菌的葡萄糖摄取.中西医结合学报. 2011; 9(8) : 901-912.
[26] De A, Das D, Dutta S, Chakraborty D, Boujedaini N, Khuda-Bukhsh AR . Potentized homeopathic drug Arsenicum Album 30C inhibits intracellular reactive oxygen species generation and up-regulates expression of arsenic resistance gene in arsenic-exposed bacteria Escherichia coli. J Chin Integr Med. 2012; 10(2): 210-227. English with abstract in Chinese.
De A, Das D, Dutta S, Chakraborty D, Boujedaini N, Khuda-Bukhsh AR. 顺势疗法药物白砷剂抑制暴露于三氧化二砷的大肠杆菌细胞内活性氧的产生并上调其抗三氧化二砷基因的表达.中西医结合学报. 2012; 10(2):210-227.
[27] Das S, Saha SK, De A, Das D, Khuda-Bukhsh AR . A study of the 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. English with abstract in Chinese.
Das S, Saha SK, De A, Das D, Khuda-Bukhsh AR. 顺势疗法药物山金车30C通过上调核苷酸切除修复基因的表达减少紫外线照射后的大肠杆菌DNA损伤.中西医结合学报. 2012; 10(3):337-346.
[28] Maratea D, Young K, Young R . Deletion and fusion analysis of the phage <pX174 lysis gene E. Gene. 1985; 40(1) : 39-46.
doi: 10.1016/0378-1119(85)90022-8
[29] Kawakami AP, Osugui L, Cesar AT, Priven SW, de Carvalho VM, Bonamin LV . In vitro growth of uropathogenic Escherichia coli isolated from a snow leopard treated with homeopathic and isopathic remedies: a pilot study. Int J High Dilution Res. 2009; 8(27) : 41-44.
[30] Khuda-Bukhsh AR . Potentized homeopathic drugs act through regulation of gene-expression: a hypothesis to explain their mechanism and pathways of action in vivo. Complement Ther Med. 1997; 5(1) : 43-46.
doi: 10.1016/S0965-2299(97)80090-8
[31] Khuda-Bukhsh AR . Laboratory research in homeopathy: pro. Integr Cancer Ther. 2006 ; 5(4) : 320-332.
doi: 10.1177/1534735406294794
[32] Khuda-Bukhsh AR . Mice as a model for homeopathy research. Homeopathy. 2009; 98(4):267-279.
doi: 10.1016/j.homp.2009.09.007
[33] Khuda-Bukhsh AR, Pathak S . Homeopathic drug discovery: theory update and methodological aspect. Expert Opin Drug Discov. 2008; 3(8) : 979-990.
doi: 10.1517/17460441.3.8.979
[34] Mallick P, Mallick JC, Guha B, Khuda-Bukhsh AR . Ameliorating effect of microdoses of a potentized homeopathic drug, Arsenicum Album, on arsenic-induced toxicity in mice. BMC Complement Altern Med. 2003; 3:7.
doi: 10.1186/1472-6882-3-7
[35] Khuda-Bukhsh AR, Bhattacharyya SS, Paul S, Dutta S, Boujedaini N, Belon P . Modulation of signal proteins: a plausible mechanism to explain how a potentized drug Secale Cor 30C diluted beyond Avogadro’s limit combats skin papilloma in mice. Evid Based Complement Alternat Med. 2011: 286320.
[36] 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. English with abstract in Chinese.
Das D, De A, Dutta S, Biswas R, Boujedaini N, Khuda- BukhshAR.顺势疗法药物白砷剂对暴露于砷的酵母菌的蛋白标志物及基因表达的影响.中西医结合学报. 2011; 9(7) : 752-760.
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