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

1. Santu Kumar Saha (Cytogenetics and Molecular Biology Laboratory, Department of Zoology, University of Kalyani, Kalyani 741235, India ) 2. Sreemanti Das (Cytogenetics and Molecular Biology Laboratory, Department of Zoology, University of Kalyani, Kalyani 741235, India ) 3. Anisur Rahman Khuda-Bukhsh (Cytogenetics and Molecular Biology Laboratory, Department of Zoology, University of Kalyani, Kalyani 741235, India E-mail: E-mail: [email protected])

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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
1. Santu Kumar Saha (Cytogenetics and Molecular Biology Laboratory, Department of Zoology, University of Kalyani, Kalyani 741235, India )
2. Sreemanti Das (Cytogenetics and Molecular Biology Laboratory, Department of Zoology, University of Kalyani, Kalyani 741235, India )
3. Anisur Rahman Khuda-Bukhsh (Cytogenetics and Molecular Biology Laboratory, Department of Zoology, University of Kalyani, Kalyani 741235, India E-mail: E-mail: [email protected])

OBJECTIVE: To explore if some ultra-highly diluted homeopathic remedies claimed to have antiviral effects can demonstrate any discernible action in the bacteria Escherichia 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.
Received November 11, 2011; accepted December 21, 2011; published online April 15, 2012.
Full-text LinkOut at PubMed. Journal title in PubMed: Zhong Xi Yi Jie He Xue Bao.

Correspondence: Anisur Rahman Khuda-Bukhsh, PhD, Professor; Tel: +91-33-25828750-315; E-mail: [email protected], [email protected]

The phages (or bacteriophages) are group of viruses that grow in bacterial cells. ФX174 (ФX) is the prototypical minute icosahedral bacteriophage that was first characterized in the Sinsheimer laboratory beginning in the 1950s[1]. The ФX virion contains a circular single-stranded DNA that replicates through a double-stranded replicative form of DNA during intracellular development[2]. ФX DNA has been used in many landmark experiments. For the first time it was demonstrated that ФX DNA could be synthesized with DNA polymerase, using the intact genome as a template[3]. This feat was hailed as “life in the test tube”[4]. The ФX174 genome was also the first DNA that could be completely sequenced[5]. The sequence described a remarkably compact gene organization with several cases of genes expressed by translating overlapping regions of the DNA in two reading frames. The first synthetic genome by whole genome assembly from synthetic oligonuleotides was also prepared for ФX174[6].
Further, ФX174 accomplishes host lysis (plaque formation) through activation of a single gene E and its product does not have any other enzymatic activity[7], contrary to what happens in some other phages like lambda (λ) and T4, which accomplish their infectivity by using more than one gene-regulatory circuit/system (both lytic and lysogenic cycles)[8,9]. Therefore, ФX174 phage infectivity in Escherichia coli can be utilized as a good experimental model to study the expression of gene E. Any phenotypic change involving plaque number in the bacterial colony due to ФX174 infection should logically be attributed to the altered expression of gene E inside the bacteria, either as a result of repressed activity of the gene E or to breaking down of the virus DNA, thereby inactivating proper functioning of viral genome, specifically inactivating the expression potential of the gene E.
Switching on of lysogenic or lytic state is tightly dependent on nutrients, chemical compounds and also environmental stimuli such as ultraviolet radiation and X-ray [10]. Though ФX174 does not carry any lysogenic gene, it has only lytic gene of action, the lytic profile of ФX is also known to be greatly altered by external factors[11-13].
ФX infection is usually not known to interrupt protein, DNA, or RNA synthesis by the host cell[14]; it is also not known to prevent induction of an inducible enzyme[15] in the host. ФX mRNA made in the infected cell has a base sequence identical to the single stranded DNA contained in the virus[15].
The use of crude ethanolic extracts derived from various parts of plants such as root, shoot, and leaves is common in complementary and alternative medicine practice including homeopathy. In homeopathy, two forms of drug are used, one is the mother tincture, mostly being crude extract of medicinal plants, minerals or of animal origin, and the other is a much diluted “potentized” form, diluted in serial steps with a fixed number of jerks or succussions[16]. When the homeopathic dilution crosses 12C potency, it crosses Avogadro’s limit, thereby making the existence of the original drug substance highly improbable in the remedies above 12C. This is one of the areas of concern for the rationalists and scientists who question the efficacy of such ultra-highly diluted remedies. While clinical effects of some homeopathic drugs could be convincingly shown, one of the greatest objections to this science lays in its inability to explain the mechanism of action of the ultra-highly diluted remedies based on proper scientific experimentations and proofs[16].
The above challenging phenomenon led us to design an experiment in such a way to be able to address several questions: if ultra-highly diluted homeopathic remedies could elicit protective responses in the lower animals like bacteria (prokaryote); if certain homeopathic drugs claimed to have antiviral effects and clinically used effectively against viral diseases could really elicit antiviral responses in E. coli; if it could, whether it was possible to elucidate the plausible mechanism at the molecular level; and if the ultra-highly diluted homeopathic remedies could actually show discernible changes depicting their action on the expression of certain relevant genes of the bacteria or bacteriophage.

1 Materials and methods
1.1 Bacteria and bacteriophage The bacteriophage ФX174 wild type and its host bacterial strain E. coli C/1 (a spontaneous phage T1 resistant mutant) used in this study were obtained from Dr. Tarakdas Basu, Department of Biochemistry and Biophysics, University of Kalyani, Kalyani, India, where these were maintained.
1.2 Media and reagents Luria-Bertani (LB) broth was obtained from Himedia, Mumbai, India. Starvation buffer (SB) contained 5.0 g potassium chloride (KCl), 1.0 g sodium chloride (NaCl), 1.2 g hydroxymethyl aminomethane (Tris), 0.1 g magnesium sulphate (MgSO4) and 1.0 mL of 1.0 mol calcium chloride (CaCl2) in 1 L distilled water; pH was adjusted to 8.1. Borate buffer contained 0.05 mol Tris sodium borohydrate. For phage viability assay, “bottom layer agar” consisted of 10.0 g agar (Himedia, Mumbai), 10.0 g bactotryptone (Himedia, Mumbai), 11.25 g glucose, and 2.5 mL of 1.0 mol CaCl2 to make 1 L with pH adjusted to 7.5 and “top layer agar” 0.8 g agar, 1.0 g bactotryptone, 0.5 g NaCl and 0.25 mL of 1.0 mol CaCl2 to make 100 mL with pH adjusted to 7.5. All chemicals were of analytical grade and were procured from Merck (Germany) except for the ones mentioned above from other sources.
1.3 Phage stock preparation Stock of phage was prepared[17] with slight modification: bacteria (E. coli C/1) were grown in LB with aeration and the culture was infected with phage when it was in the logarithmic phase of growth (about 108 cells/mL), then 25 mmol/L CaCl2 was added to the media for better adsorption before inoculum phage was added. Infection was allowed for 2 h in a shaker incubator at 37 ℃ for progeny phage generation in a sufficient amount. The infected cell lysates obtained were subjected to two successive centrifugations (12 000×g for 20 min at 4 ℃). The phage particle was resuspended in 0.05 mol borate buffer.
1.4 Cell synchronization Since these experiments entail an analysis of the behaviour of phage-bacteria complexes when subjected to a particular perturbation at defined stages in their development, it is necessary to ensure that all of the cells in the population are at a similar stage of development. Hence all the experiments were done with starved synchronized cells, made in the following way[18] with little modification: (1) overnight cells of bacterium E. coli C/1 were first grown at 37 ℃ to the logarithmic phase (about 108 cells/mL corresponding to the bacterial optical density (OD) at 600 nm which equals to 0.2 determined in Shimadzu Spectrophotometer UV 1700 model) in LB medium; (2) the grown cells were washed with and suspended in SB; and (3) the cells were allowed to starve with shaking at 37 ℃ for 1 h for synchronization.
1.5 Plaque assay The numbers of infective centers, namely, the plaque-forming units (PFU) of the bacteriophage were measured by double-layer agar plating method[19]. To study the phage infectivity, starved synchronized cells were infected with the phage at a multiplicity of infection (MOI) value of 1.0 (which can be roughly estimated as the ratio of equal number of phage and the bacteria) and was allowed to adsorb to the cell surface at room temperature for 10 min. The adsorbed complex was then serially diluted in SB. Then 0.1 mL of each of the properly diluted samples was mixed with 0.2 mL of an overnight culture of E. coli C/1 (as seed bacteria) and 2.7 mL of molten “top layer agar”. The homogeneous mixture was then spread over the “bottom layer agar” plates and the plates were then kept upside down in an incubator at 37 ℃ for 2 to 3 h to obtain the PFU.
1.6 The homeopathic drugs and their source Rhus toxicodendron (Rhus Tox 30C; batch No: 7800) and Atropa belladonna (Belladonna 30C; batch No: 7800), are claimed to have antiviral effects[20-22] in the sense that they are used in viral illnesses and thought to stimulate host reaction; Arnica montana (Arnica 30C; batch No: 7800) is claimed to have mainly effects on shock, trauma and injury. The homeopathic remedies, prepared as per standard Indian Homeopathic Pharmacopaea, were procured from Hahnemann Publishing Company, BB Ganguly Street, Kolkata, India. The drugs prepared in the ethanolic media (and the placebo or positive control prepared from the same stock as the drug and succussed appropriately to make it succussed alcohol 30C) were suitably diluted to 1% ethanolic strength to minimize alcoholic effect on the microbes.
1.7 Replication Different parameters of study mentioned below have been conducted in two subsets of experiments each and for each subset of experiments three replicates were studied. For normalization of data against the standard growth of bacteria and phage, mean values were presented.
1.8 Homeopathic remedies on bacteria Experiment was done according to the following steps: (1) the starved synchronized cells were allowed to grow in fresh LB in presence of 1% (volume ratio) of different homeopathic remedies; (2) the cells were further shifted to SB by centrifugation; (3) normal phage was added to such cells at multiplicity of infection ratio of 1.0; (4) the phage-adsorbed cells were then serially diluted in SB; (5) subsequent steps to obtain PFU were done as described above.
1.9 Homeopathic remedies on phage Experiment was done according to the following steps: (1) phage was allowed to incubate in presence of 1% (volume ratio) of different homeopathic remedies; (2) the starved synchronized cells were mixed with drug-treated phage at MOI of 1.0; (3) the phage-adsorbed cells were then serially diluted in SB; (4) subsequent steps to obtain PFU were done as described above.
1.10 Blinding The vials containing the drugs and the placebo were coded and not known to the observers as to which contained the drug and which contained the placebo. The codes were later deciphered after the observation and counting of plaques.
1.11 Statistical analysis All data were expressed as mean±standard error of mean and based on means of five sets of experiments, taking three replicates for each. For statistical analysis, the analysis of variance followed by post hoc analysis was adopted using SPSS software. P0111santu%e5%9b%be1

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.

0111santu%e5%9b%be3
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; *P0111santu%e5%9b%be2
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.
0111santu%e5%9b%be4
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.

3 Discussion
The dilution of the potentized remedies used in the present study was 30C, which means that it was diluted 1060 times, well above the Avogadro’s limit (6.023×1023) (dilutions of homeopathic drugs at 12C and above exceed/cross this limit[16]). The results of the present study indicated that all of these remedies had abilities at a varying degree to render protection to the bacteria against the infection of the phage. Interestingly, except for Arnica Montana 30C used mainly against shock and injury and which showed lesser protective effect, the three other remedies are claimed in the homeopathic literature to have antiviral potentials[20], and are generally used, depending on the totality of symptoms, against some common viral diseases like influenza and rhinitis, with great benefits[20-22]; all of these three rendered considerable additional ability to the bacteria to thwart the phage attack, as revealed from their respective plaque numbers. On the other hand, the placebo-treated bacteria failed to show any such protective effect against the viral infection, confirming that it was not just the alcohol effect. However, when the phage was pre-treated with these remedies individually, the virus hardly showed any modulation in their ability to attack the bacteria and formed nearly similar number of plaques by their lytic effect comparable to when they were drug-untreated. Thus, the homeopathic drugs which showed their antiviral effects when treated to the bacteria did not show the direct antiviral effect on the phage by way of reducing their ability to infect the bacteria. So the homeopathic remedy by itself did not apparently show its ability to directly repress or inactivate the gene E of the phage responsible for the synthesis of product that helps them in the bacterial lysis. But when the bacteria were exposed to the drug, the plaque numbers were clearly reduced; this would reveal strong circumstantial evidence that some genes of the bacteria, responsible for production of the repressor proteins might have been activated and additional amount of the “repressor” molecules were produced to block the gene E expression of the phage. Since the phage DNA only entered into the bacterial cell leaving aside the protein coat at the bacterial outer membrane, it was only possible for the bacteria to stop the phage attack by blocking the expression of the lytic gene E expression in the phage by some way after it had entered the bacterial cell. Alternatively, the phage DNA could have been totally annihilated as and when they entered, by some specific gene products (like restriction endonucleases) of the bacteria. Many microbes rely on diverse defence mechanisms that allow them to withstand viral predation and exposure to invading nucleic acid. In many bacteria, clustered regularly interspaced short palindromic repeats (CRISPR) form peculiar genetic loci, which provide acquired immunity against viruses and plasmids by targeting nucleic acid in a sequence-specific manner[23,24]. These hypervariable loci take up genetic material from invasive elements and build up inheritable DNA-encoded immunity over time. The homeopathic remedies apparently could have enhanced the activity of these genetic clustered elements, so that the bacteria could alternatively annihilate the phage as they entered.
The significance of choosing bacteriophage ФX174, from a pool of other bacteriophages is that the other phages like lambda phage, T4, T2, etc. have more than one gene to control the lytic cycle; they also have a lysogenic cycle controlled by some specific genes. This makes ФX174 rather unique in the evolutionary sense, having only an exclusive gene E, the product of which is solely responsible for successfully infecting the bacterial genome and causing bacterial lysis. The phage is able to produce the phenotype of the plaque only when the lytic gene product is allowed to be synthesized. Therefore, it is implied that when the plaque numbers were reduced, less number of phages had their lytic gene E operative inside the bacterial cell, and that could only happen when the protective genes of the bacteria were alerted and triggered for the synthesis of the gene products to inactivate the viral genome inside the bacteria. Since this entire process of phage infection (and lysis) is solely controlled by the activity of the single gene E, the introduction of any compound/drug must have to target to inactivate that single gene E that could stop it from synthesizing the lytic element to bring about a concomitant alteration/change in their phenotypic expression (that should obviously be reflected in the plaque number). Several reports from our earlier studies[25-27] have already established the fact through reverse-transcriptase polymerase chain reaction studies that introduction of homeopathic drugs can bring about changes in the gene expression of the bacterial system, both at the mRNA and DNA levels. The results of the present study also give us a scope to use the prokaryotic organism as an experimental model, because at the one hand, the phage is evolutionarily equipped to attack the bacteria with the help of expression of a single gene E, and at the other, the bacteria, in order to save them from the phage attack, also have been trying to develop an effective genetic machinery to inactivate the phage, by activating certain genes they acquired by inserting part of the viral genome in course of time[23,24].
Further, the lytic gene E in bacteriophage is the only gene that is well identified and characterized for its active infection to accomplish. The insertion of gene E into bacterial host genome produced products that readily killed all but a few bacteria. The surviving bacteria genome was found to have modified (mutated) gene E that conferred the resistance to the bacteria. Cloning studies revealed the mutated gene in the bacteria to be “sly D” gene (sensitivity to lysis gene)[28]. It has already been mentioned that ФX174 infection is usually not known to interrupt protein, DNA, or RNA synthesis by the host cell under normal circumstances, but result of our present study would reveal that the host DNA under the influence of the homeopathic drugs could trigger the synthesis of a “product”, presently of unknown nature, that inactivated the lytic gene E expression in the phage.
Since these experiments entail an analysis of the behaviour of phage-bacteria complexes when subjected to a particular perturbation at defined stages in their development, it was necessary to ensure that all of the cells in the population were at a similar stage of development. Hence all the experiments were done with starved synchronized cells, which were necessary and important to yield dependable and repeatable results[29].
Khuda-Bukhsh and his group have long been advocating[16,30-34] that the potentized homeopathic drugs could possibly act through regulation of gene expression, with exhaustive experimental evidences from both the higher organisms like mammals[35] and lower organisms like yeast and bacteria[26,36]. The results of the present study provide yet another strong evidence of the capability of the potentized homeopathic drugs to trigger specific gene activity in the bacteria to render protective effects against phage attack. Since there is hardly any antiviral drug without toxicity in the orthodox regimen, the results of the study assume special significance. The present findings would give support for the use of these potentized drugs more assuredly against influenza and other viral diseases. Further studies are needed to pinpoint the epigenetic changes, if any, in the bacterial genome, after the drug-pretreated bacteria are subjected to phage attack.

4 Acknowledgements
The authors express their sincere thanks to Dr. Philippe Belon, Ex-Director, and Dr. N. Boujedaini, Research Manager of Boiron Laboratories, France, for their encouragements and to Dr. Tarakdas Basu of the Department of Biochemistry and Biophysics, University of Kalyani, India for providing the stock of the bacteriophage and bacteria for our experimental use. The authors are also thankful to Dr. P.K. Das, former Director, Central Vector Control Research Centre, Puducherry, India, for going through the manuscript critically.

5 Competing interests
The authors declare that they have no competing interests.

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  • DrPramod Kumar

    Homoeo dilution has great power .can do miracle.