Authors: Sukul , Nirmal C. Since the subject of high dilution effects is still a subject for debate, this volume provides evidence in support of effects from control clinical studies, clinical records from veteran physicians, controlled experiments on animals and plants, and in vitro tests without any organisms Chapter II. An overview of the methods for preparing drugs at ultra high dilution is also provided as well as the basic principles of homeopathy, which has been alleviating human suffering through the use of these drugs for several hundred years Chapter I.
Since water is used as the diluents media, the structure and dynamics of water polymers in relation to high dilution are discussed in order to facilitate easy comprehension of this physical aspect, the basic principles of spectroscopy are also described. Chapter IV focuses on the mechanism of action of potentized drugs in the living system, discussing the structure of the cell, the plasma membrane, the integral proteins on the membrane, the interaction between these proteins and high dilutions and the manifestations of the therapeutic effects of high dilutions.
Some aspects, peculiar to homeopathy, such as the chief miasm psora, and the literalities and time modalities of symptoms and drug action are interpreted from a scientific perspective. Chapter IV ends with a brief discussion on water structures and the origin of life to show the natural evolution of high dilution effects. The book not only helps in understanding the physical basis of high dilutions and their mechanism of action in organisms but provides many new avenues of investigation into this interdisciplinary field of science.
High dilutions of endogenous substances such as thymulin, a hormone from thymus, and bursin from the bursa of Fabricius of chickens, produced immunomodulation in mice and bursectomised chickens, respectively Bastide and Boudard, High dilutions of Silicea accelerated healing of perforations of mouse ear Oberbaum et al. High dilutions of caffeine and adenine prevented teratogenic effect induced by those drugs on mice Taddei-Ferretti and Cotugno, Burlakova observed high dilution effects of several biologically active substances at organismic, cellular and macromolecular level of both plants and animals.
She concluded that the effect could not be linked to any definite substance structure or biological organization level. These drugs enhanced restraint-induced catalepsy in rats. Catalepsy is a transitory state of immobility in which the animals fail to correct the imposed posture. It can be induced by restraint on movement or by drugs. It is a central nervous system phenomenon involving serveral neurotransmitter systems Sanberg, , Hartgraves and Kelly, ; Klemm , ; Sukul et al. We improved the technique and observed that potentized Agaricus could significantly influence catalepsy in albino mice induced by neurotransmitter drugs.
While apomorphine does not produce catalepsy by itself, bromocriptine produces a very mild catalepsy Sukul and Klemm, This is a good model, but care should be taken that animals are not stressed before starting an experiment. The results show that homeopathic potencies can interact with neurotransmitter drugs. Oral route effective We further observed that Agaricus 30 could reverse haloperidol catalepsy only when it was administered through the oral route Sukul, Here also Agaricus was effective only through the oral route and not through the intraperitoneal route Ghosh et al.
The results suggest that receptors in oral mucosa play a role in mediating the effect of homeopathic potencies. Higher dilutions like and did not produce any anticataleptic effect. However, the dilution could be made effective if it was subjected to agitations, in this case, by sonication. The anticataleptic effect was observed when Agaricus was administered before and immediately after haloperidol Sukul et al.
This shows that there is a limit to dilution of a homeopathic potency and that mechanical agitation plays a role in maintaining the efficacy of a diluted drug. In a recent study we have observed that mere successive dilution without mechanical agitation could produce an effective homeopathic potency.
So, higher dilution requires mechanical agitation. We know that drunk persons move to and fro due to loss of righting reflex. Righting reflexes maintain the normal erect posture of an animal through a series of responses which are integrated mostly in the mid brain Ganong, Ethanol can induce loss of righting reflex in mice which can be measured in terms of sleep time Draski et al.
Because of the loss of righting reflex due to alcohol the animals cannot move but lie motionless as if they are sleeping. Nux vomica is known to be effective against the effects of alcoholism Kent, ; Boericke, We Sukul et al. This simple model can be easily tested in any laboratory.
Aqueous ethanol, the effective medium We prepared Nux vom 30 in our laboratory from the ethanolic extract of the ground seeds of Strychnos nuxvomica L. But here instead of succussion by hand we sonicated each dilution at 20 kHz for 30 sec. We observed that neither pure ethanol nor pure water made an effective potency of Nux showing antihypnotic effect in albino mice Sukul et al.
This experiment shows that aqueous ethanol is the suitable medium for the preparation of an effective homeopathic potency. The potentized medicines were stored in the laboratory for about 6 months before testing on animals. Pure water preparations might have lost their efficacy during storage. This experiment needs to be repeated with other models. The results are important and linked to the physical basis of a potency.
Biological effects on amphibians The common laboratory animal toad has long been used for biological and pharmacological experiments. We conducted experiments on toads to develop suitable animal models for demonstrating the biological effects of homeopathic potencies. Controls for the liquid potencies were prepared in the same way in 30 successive dilution and sonication without using the mother tincture of Nux at the initial level.
Control for the solid potency was prepared by trituration of lactose without Nux T at any stage. The drug or control was diluted with distilled water and the toadlets were exposed to the mixture by immersion for 5 min. The treatment was repeated twice everyday for 3 consecutive days. On the 4th day they were exposed to mM ethanol solution by partial immersion. Every 10 min the toadlets were transiently removed from the ethanol solution and placed in a supine position on a dry flat surface.
Failure to right within 60 sec was considered loss of righting reflex. Nux 30 prepared with other media showed no difference from the control with respect to the loss of righting reflex in toadlets. This shows once again that aqueous ethanol is the suitable medium for the preparation of a homeopathic potency Sukul et al. The potentized medicines were stored for some months before use.
Here Nux and were administered orally on freshly collected adult toads 30 in each group. Six hours later they were injected i.
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The duration of sleep time was measured as in the case of mice. Toads treated with either of the two potencies of Nux regained righting reflex more quickly than those of the control group. This shows that adult toads can serve as good animal models for demonstrating the biological effect of a homeopathic potency Sukul et al. Thus we see that potentized Nux vomica produces antialcoholic effect both in amphibians as well as in mammals. Effects on chronic alcoholism Chronic alcoholism is a major health problem all over the world and Homeopathy has some effective remedies to tackle the problem.
We tested one of these remedies, Nux vomica on rats for demonstrating its antialcoholic effects. They swam actively, paused for sometime and continued swimming. The period of immobilization with the head seldom protruded over the surface of water was recorded for each rat. The duration of immobilization is directly proportional to the depressive like state. The highly active rats are characterized by a period shorter than sec and the lowly active ones by the period longer than sec. It is the lowly active rats that are potential alcoholics Paul et al. These rats were selected for tests involving ethanol consumption.
So the potential alcoholics among the rat population would be suitable for experiments on alcohol consumption. The rats were then treated orally with Nux vom 30 and Nux vom While Nux vom was prepared by usual succussion, Nux vom 30 was prepared by sonication. One batch was given Nux vom 30 through the intraperitoneal route. One batch served as the untreated control. While Nux was given one dose everyday, Nux 30 was given one dose every 15 days. Treatment and daily assessment of ethanol and water consumption were continued for one month.
Both Nux 30 and Nux reduced ethanol consumption in rats significantly. However, Nux 30 given through the intraperitoneal route did not show any difference in ethanol consumption from the control. Nux 30 oral was more efefctive than Nux Figure 2. This shows that sonication makes a more powerful potency than manual succussion. This experiment once again shows that oral route is the effective route for a homeopathic potency. It is thought that oral receptors mediate the action of a homeopathic potency Sukul et al.
In another experiment we observed that both Nux vom 30 and Nux vom mother tincture reduced alcohol consumption in rats significantly Sukul et al. Degenerative changes in the vagus and sympathetic nerves were reported in patients with alcoholic neuropathy.
We have made an experiment to see whether Nux vom 30, prepared by sonication, could reverse the peripheral neuropathy in alcoholic rats. Then they were treated with Nux 30, one dose every 15th day for 4 months. The daily consumption of ethanol and water was recorded for each rat for the entire period of 6 months. Besides the treated and untreated batches of alcoholic rats, there was one batch without ethanol. All the rats were sacrificed after 6 months and their atrioventricular valves were examined for any degenerative changes in the adrenergic nerve plexuses.
The latter showed significant degeneration in alcoholic untreated rats.
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Treatment with Nux 30 not only reduced ethanol consumption in rats but also brought about considerable regeneration of most of the nerve terminals. This experiment shows that a homoepathic potency can produce visible anatomical changes in treated animals Sukul et al. Effects on parasitic diseases An early report shows that cholera epidemics in Europe were effectively tackled by homeopathic medicines Leary, The work refers to the epidemic in The results may not be reliable because modern diagnostic techniques were not available at that time and adequate placebo controls were not kept.
Abrotanum D1 was tested on 27 dogs and 26 cats infected with intestinal nematodes in a Veterinary clinic of Germany and the results were positive Krause, Besides diseases due to metabolic and immunological diorders or injuries, and toxic substances, diseases caused by parasites or pathogens are also effectively treated by homeopathic medicines. Some homeopathic drugs showed antiviral effect against chicken embryo virus Singh and Gupta, Trichinosis or trichinellosis is a disease caused by the nematode parasite, Trichinella spiralis in man.
Adult worms live in intestinal mucosa, but the juveniles migrate to muscles and get encysted there. The worm also infects rats, mice, bears, pigs etc. Human infection results mostly from consumption of poorly cooked pork containing encysted juveniles. There is no effective remedy for the elimination of T. We conducted an experiment on albino mice inoculated with T. Hydrocortisone was given to obtain a more or less uniform infection in mice. Thirty days after inoculation, mice were treated first with Cina 1M for 3 days followed by Calcarea fluor CM in one batch.
There was one more batch in which the second medicine was Thuja CM. Since trichinosis involves cysts in muscles Calcarea fluor and Thuja were used. Cina was used initially because the exciting cause for the cyst was a round worm. Larval densities in the muscle were determined on days 10, 17 and 24 following the last date of treatment after sacrificing 6 mice from each group. The untreated control showed Treatments significantly reduced juvenile densities Nux vom oral 10 Nux vom 30s oral 1 3 5 7 9 11 13 15 17 19 21 23 25 27 30 Days of observation Figure 2.
Effect of potentized Nux Vomica on alcohol intake in rats. Nux vom 30 prepared by sonication s and given by oral route once every 15 days reduced alcohol consumption relative to water in albino rats as compared to the control. Nux vom prepared by succussion and given one dose daily also reduced alcohol consumption in rats as compared to the control. Reproduced, with permission, from Sukul et al. The action of the drugs is thought to be mediated directly through the nervous system which in turn triggers the necessary inflammatory response to the parasitic invasion.
Since the drugs were administered through the oral route, they might have acted through oral afferents and the impulses reached the cortex through nucleus tractus solitarius and thalamus. There in the cortex the message was processed and the autonomic outflow might have influenced the immune system in such a way that the juveniles of T. There is evidence that neural mechanisms influence immune responses and inflammation Barnes, , Roitt et al. Release of histamine from mast cells is brought about by neurotransmitters.
Uninfected mice have mast cells and basophils which bear receptor-bound IgE. The binding of specific antigen to the receptor-bound IgE activates the cell. Antigen-IgE stimulation of mast cells is considered to play a role in the elimination of helminth parasites Foreman, This may be the possible way by which potentized drugs reduce T.
The work has been reproduced recently with similar results. Dirofilaria immitis is a nematode parasite living in the aorta of dogs. Infected dogs carry microfilaria in their blood. Since Cina is used for worm infection we selected this remedy for treatment of canine dirofilariasis. An examination of blood of an infected dog shows large number of microfilaria. Cina is prepared from the extract of the flowering top of the plant Artemisia nilagirica. We used the mother tincture of Cina and its two potencies like Cina and Cina Treatment of dogs 4 in each batch continued for 30 days.
The daily dose for each potency was 0. Cina T, Cina and Cina reduced microfilarial density in treated dogs by Filarial worms are known to cause immunosuppression Ottesen It is possible that potentized Cina might have removed immunosuppression resulting in vigorous responsiveness of the host to parasite antigens thereby clearing microfilaria from the blood Sukul et al. In another experiment on microfilaraemic dogs we used Cina 30 prepared by succesive dilution and sonication at 20 kHz for 30 sec in each step. Treatment continued for 30 days. Electropysiological Studies In the foregoing paragraphs we have seen that biological effects of homeopathic drugs manifest themselves after a time interval which may vary from a few hours to several days.
The information of drug molecules is conveyed to the brain by the oral afferents. Since autonomic functions are centrally controlled in the hypothalamus we can expect a change in the electrical activity of hypothalamic neurons immediately after a homeopathic drug is put on oral mucosa. An experiment was designed just to verify this idea. Dogs 4 naturally infected with Dirofilaria immitis were treated with Cina 30, one dose every day for 30 days.
Four dogs served as controls. Mf density on day 0 served as standard. The horizontal bar at the base indicates the period of treatment. Blood was sampled from dogs every 15 days. Single unit discharges were recorded from 9 cats of either sex with a tungsten microelectrode tip 1. After the application of a drug into the mouth, the activity was recorded for min.
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The drug was repeated once and the recording was made for the same period. We applied three drugs, namely Arnica montana 30, Hypericum and Arsenicum album Arnica is normally used for an injury, Hypercium for a punctured wound affecting nerves and Arsenic for some toxic effects of drugs. The test animals suffered injuries and punctured wounds when their skull was exposed and electrodes driven into their brain. They received anaesthesia which had some toxicity. The drug produced a significant reduction in firing rate in case of 5 neurons.
The effect lasted from 1. In one neuron the firing rate increased within 30 sec and the effect lasted for 3 min. The 3 neurons of the left side and one from the right did not show any change in the firing rate after Arnica Hypericum produced a decline in firing rate and the effect lasted from 3 to 14 min with a latency period varying from 10 sec to 6 min. In one neuron a transient increase in firing rate preceded the decline. One neuron did not show any response to Hypericum Arsenic 30 produced an instantaneous latency 1 sec to 1 min increase in firing rate in case of 5 neurons, but the effect was transitory 50 sec to 1 min Figure 4.
In one neuron the drug produced decline in firing rate after the second application and the effect lasted for 30 sec with a latency of 1 min. One neuron did not respond to Arsenic 30 Sukul et al.
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A glass-coated silver microelectrode tip 3. Recording was started 4 days after operation when the rat behaved normally, and continued for several days. After the last session the rat brain was examined histologically for the recording site. The effect of anaesthesia could be avoided in the study on an awake rat.
The rat readily took the control solution or the drug. Here Arnica 30 produced a decrease in firing rate in neurons and the effect lasted for more than 20 min with a latency period of 1. In one neuron the response was discernible only after the second application of the drug. Arnica 30 produced an increase in firing rate in one neuron within 1. Hypericum produced a decrease in firing rate in case of 5 neurons and the effect lasted from 1.
In two neurons there was a transient potentiation followed by inhibition. In one neuron there was an increase in firing rate within 2 min and the effect lasted for 11 min. Arsenic 30 produced a marked increase in firing rate within 1 sec and the effect lasted for 20 sec. In all the cases the control did not show any marked change in the firing rate Sukul et al.
Inhibitory effect of Arnica 30 and Hypericum , and excitatory effect of Arsenic 30 on the lateral hypothalmic area LHA neurons of cats. Arrows indicate the time of application of drugs on the tongue of the test animal. Indian Biologist ; 23 : 19 and excitatory effect on different neurons. Some neurons may even be totally nonresponsive to a drug. It is evident that drugs are effective through oral receptors.
go site The final therapeutic efefct of a drug depends on the interaction of different neurons in different areas of the brain. Rats used in the electrophysiological tests all belong to Charles Foster CF strain. They were given food and water ad lib. Rats were anaesthetized with peraldehyde 1. A hole was trefined on skull in the LHA according to specific stereotaxic coordinates Konig and Klippel, A glass-coated silver electrode tip 0. In case of gross activity a spike of fixed amplitude was selected and its frequency per 20 sec was counted.
In case of unit activity, impulses per 20 sec were counted. Drugs or control solutions were put on the tongue of the rats and the effect was recorded. Single unit discharges in the LHA were recorded by means of glass-coated silver microelectrode tip 3. Nux vom and Nux vom increased the neuronal activity in the LHA of the alcoholic rats. This increase in neuronal activity was more pronounced with Nux vom than with Nux vom Application of Nux vom T mother tincture also reduced the activity. The effect of the drugs was instantaneous occurring in a few seconds.
The effect lasted from a few secs to a few min Paul et al. In this experiment the rats were conditioned in the sense that they were continuously given ethanol. This may be the reason that the neuronal responses to drugs were more or less uniform. It might be that neurons of alcoholic rats have assumed a specific state so that they can respond to drugs uniformly. We can assume that neurons in a diseased state undergo specific changes so that they become responsive to an appropriate remedy in a non-random way.
The changes are manifest in the symptoms of the disease. Here enhanced susceptibility has been produced in rats. Electrophysiological responses appear to constitute holistic processes. A stimulus applied by homeopathic drugs elicited a magnified response in the form of electrocardiogram ECG and electroencephalogram EEG from rats and man Torres, We also conducted another electrophysiological experiment where rats were conditioned in a different way and appropriate remedies were applied.
They were anaesthetized and fixed on a stereotaxic table. A glass coated silver microelectrode tip 3. Natrum mur 30 produced a reduction in the frequency of spikes - If the electrical activity of hypothalamic neurons undergoes a change due to the application of a homeopathic potency, it is quite probable that synaptic transmission in the LHA would also undergo a change.
The latter change would be reflected at the level of neurotransmitters and their metabolites in the hypothalamus. In fact, these changes were detected in another experiment. We have already noted that potentized Agaricus muscarius influences catalepsy in mice. We, therefore, wanted to study whether a potency of Agaricus muscarius alters the level of dopamine and serotonin and their metabolites in the mice.
Agaricus 12 was administered orally on 6 mice at the rate of 3. All the mice 30 - 35 g were sacrificed by decapitation 6 hr after the administration of the drug or the control. The brain was quickly removed, kept over ice and the hypothalamus scooped out by means of a sharp needle, placed in a microcentrifuge tube, weighed, and then frozen at C. Hypothalamic mass ranged from 7 to 12 mg for the controls and 8. A, B gross and C unit activity Reproduced, with permission, from Sukul et al : High dilution effects of Strychnos nuxvomica L on hypothalamic neurons and adrenergic nerve endings of alcoholic rats.
In : High dilution effects on cells and integrated systems. C Taddei Ferretti, P Marotta [editors]. The sample was centrifuged 2 min at xg and the amine determinations were accomplished by injecting the supernatant of the centrifuged brain homogenate on to a reversed phase C column. After elution of the amines with mobile phase 0. The column was heated to C; flow rate of the mobile phase was 0. The internal standard was used to accommodate for changes in extraction efficiency and detector sensitivity.
This was done by comparing the peak height of the known amount of epinine added to a tissue sample with that of its standard. Unextracted standards were also used to rectify any differential loss in amines during extraction. Sample values were calculated as amine content per gm of wet weight of brain. Experimental evidences from plants Kolisko cited by Righetti, observed enhanced growth of germinated seeds with homeopathic dilutions of silver nitrate Argentum nitricum.
Pongratz and Endler and Pongratz et al. The effect of homeopathic drugs on plants was reported by other workers Boiron and Zervouacki, ; Boiron and Marin, ; Auquiere et al. Effect on parasites and pathogens of plants As in the case of man and animals homeopathic drugs can also ameliorate diseases of plants caused by parasites and pathogens. Arsenicum album c and Kali iodide c reduced fruit rot of guava and mango Khanna and Chandra, , This work, however, did not have controls.
Cina, Santonin and Filix at 1 ppm concentration partially inhibited hatching of eggs of root-knot nematodes Meloidogyne incognita of vegetable crops Sen and Dasgupta, The mother tinctures of the drugs were used in this experiment. Meloidogyne incognita is a nematode parasite of vegetables occurring all over the world. It also attacks cereals, pulses, tea, coffee, fruit trees etc. Since Cina is effective against nematode parasites of man and animals, this drug has been selected for the treatment of plant diseases caused by nematode parasites.
Cina is produced from the ethanol extract of the flowering tops of Artemisia sp. Cina c was purchased from C. Ringer, Calcutta. The drugs were produced according to the homeopathic pharmacopoeia of India Sucrose globules, soaked with the liquid drug, were mixed with sterile distilled water in the proportion of 7.
Cowpea plants Vigna unguiculata, grown in pots, were inoculcated with the 2nd-stage larvae of M. Four days after inoculation, one of the inoculated groups was treated by foliar spray, once daily for 10 days, with Cina c solution. Treatment was given in such a way that all the leaves of the plant were completely drenched in the solution. The other inoculated group was similarly treated with the control solution. All the plants were uprooted 30 days after inoculation. Biomass of the test plants, root-gall numbers, root-nodule numbers, nematode population in roots and rhizospheric soil and root-protein content were estimated.
Treatment with Cina c increased the plant growth and reduced nematode infection significantly Table 2. This experiment was repeated twice Sukul and Sukul, In another experiment Cina c and Cina c were applied by foliar spray on tomato plants inoculated with root-knot nematodes. Here also both the potencies reduced nematode infection and improved plant growth significantly Sukul, Values are means with S.
Different letters a, b, c against the values in a column indicate significant difference P Shoot Shoot length wt cm g Uninoculated r9a r 7a untreated Inoculated r10a r8a untreated Inoculated, r6b r6b treated with Cina These potencies are thought to have stimulated natural defense response in plants which in turn reduced nematode infection.
Plants have no specialized immunocompetent cells. All the cells in a plant have the ability to recognise the self from the non-self. The results of these molecular recognition events in response to a local stimulus are also transmitted to distant regions of the plant Bowles, Pathogenesis-related proteins PR proteins are known to accumulate in the leaves of potato plants following root invasion by cyst nematodes Hammond-kosack et al. Prior inoculation of tomato and pyrethrum plants with M. Salicylic acid increases throughout the plant after only one part of the plant is infected by a pathogen.
Following this increase in salicylic acid PR proteins are expressed in the plants Malamy et al. Application of salicylic acid or its analogue aspirin acetyl salicylic acid induces rapid expression of PR genes Ward et al. It is quite probable that potentized Cina induced systemic resistance in plants thereby reducing infection of root-knot nematodes.
Betti et al. Control of plant diseases by homeopathic potencies would not only eliminate pesticidal residues in treated plants but also do away with environmental pollution in the agroecosystem. For this, new materia medica in relation to plants should be developed by drug proving on plants and by merging plant pathology with toxicology. Evidences from in vitro tests In vitro tests were conducted on isolated organs, cell cultures or macromolecules such as proteins. High dilutions of anti-IgE antibodies promoted human basophil degranulation in vitro Davenas et al.
The work, published in Nature, generated a lot of controversy among scientists. Belon et al. High dilutions of drugs showed positive effects on rat liver microsomes Kretschmer, Van Wijk and Wiegant observed that rat hepatoma cells, pretreated with heat shock, sodium arsenite or cadmium chloride, developed a charcteristic set of heat shock proteins hsps. When the sensitized cell populations were exposed to very low doses of each of the three types of stressors, a set of hsps characteristic of the stressor developed, thereby providing experimental evidences for the specificity of the similia principle.
Cambar et al. Cristea employed a classical pharmacological technique to assess the efficacy of potentized Belladonna. The principal alkaloid in the Belladonna mother tincture is atropine which serves as an antagonist against the neurotransmitter acetylcholine Ach. High dilutions of Belladonna upto the th potency was applied on the isolated rat duodenum mounted in an isolated organ bath. The effect of Belladonna potencies on the rat duodenum was bidirectional and multiphasic. Bastide and Boudard reported immunomodulating effects of high dilutions of such endogenous substances as thymulin, bursin and cytokinins on mice and birds.
The experiments conducted were in vitro, in vivo and ex vivo. Here a potentized homeopathic drug was considered as a distinct chemical differing in chemical reaction from its vehicle such as water or aqueous ethanol. Boyd , , , first conducted this type of in vitro tests using Mercuric chloride He prepared Merc cor 30 in double-distilled water and tested it on the activity of disastase which is a mixture of starch-digesting enzymes. He observed that Merc cor 30 accelerated the rate of hydrolysis of starch by disastase.
More than comparisons between the tests and the control were carried out and the difference was statistically significant at 0. The experiments were conducted with utmost care. The purpose was to see whether the metal ion or the halide ion could play any individual role in altering the enzyme activity. The efficacy of the two media, water and aqueous ethanol, was also tested by these experiments. It is the experience of homeopathic physicians and pharmacists that potentized homeopathic drugs prepared in aqueous ethanol keep their activity for a pretty long time.
For this, we tested Merc cor 30 Mercurius corrosives 30 in aqueous preparation of 3 different ages, one 4-day old, the 2nd one-month old and the third twelve-month old. The hydrolysis of soluble starch by D-amylase was measured by the standard biochemical procedure Bernfield, Pancreatic Damylase broke down starch yielding mainly maltose, a disaccharide of D- glucose Nelson and Cox, The breakdown product maltose was quantified from a standard curve prepared with the help of a Jasco spectrophotometer model There were 10 replicates for each drug and its corresponding control.
In vitro tests with enzymes show that potentized drugs can act on chemicals without any mediation of living organisms. Here we see that both water and aqueous ethanol can serve as good media for the preparation of potentized drugs. Boyd also observed that the efficacy of Merc cor 30 in water deteriorated with the passage of time. Let us examine whether the metal ion in a salt or the halide ion plays any individual role in altering the enzyme activity.
The hydration stabilizes the ions Watanabe and Iwata, Mercury ions usually inhibit the enzyme activity by binding to cysteine, histidine and threonine residues Muller and Saenger, Two residues known to be essential in substrate binding in D-amylase are His and His Qian et al. In the potentized form, as in our tests with Merc cor 30 and Merc iod 30, mercury served as a promoter of the activity of D-amylase.
Chloride ions are known to activate D-amylase, but the activity is not dependent on this ion Kabuto et al. Three residues involved in chloride binding are Arg, Arg and Asn Qian et al. In our experiment both Mercuric chloride 30 and Mercuric iodide 30 enhanced the enzyme activity. If idodide had played an independant role Merc iod 30 would have retarded the D-amylase activity.
But this did not happen. It appears that the compound as a whole, rather than its individual ions, induces specific activity in the diluent medium during dynamization and the medium itself plays a role in acquiring and retaining the activity. The solution structure of the diluent medium appears to have altered the structure of the enzyme thereby changing its activity. The functionality of biological molecules is strongly influenced by their three-dimensional structures, which are primarily determined through non-covalent interactions with metal ions, hydrogen-bonding interactions and solvation Rodgers and Armentrout, In this case H-bonding interaction and solvation by structured water of Merc cor 30 or Merc iod 30 altered the activity of D-amylase Sukul et al.
The results further show that water molecules carry the specific information of drug molecules during the process of dynamization. Ethanol molecules having a large non-polar part stabilize the specific water structure acquired during the process of successive dilution and mechanical agitation. In vitro experiment on red blood cells Based on previous works on Homeopathy we have hypothesized that the primary target of a homeopathic potency in an organism is the water-channel protein or aquaporin Sukul and Sukul, Aquaporins occur in all life forms and facilitate permeation of water across biological membranes.
We have discussed in details about the structure and function of aquaporins and their relation to health and disease in chapter IV. If the primary target of a homeopathic potency is aquaporin, application of a homeopathic potency on cell membranes would affect water flow into the cells. In order to test this hypothesis we treated red blood cells of a fresh water fish Clarius batrachus with Mercuric chloride 30 Merc cor 30 and Nux vomica 30 Nux vom 30 separately in a hypotonic medium.
In the control red cells were treated with Ethanol The tests were performed on blood cells collected from two groups of fishes, one given i. After treatment blood samples were centrifuged, supernatant fluid part removed and erythrocyte pellets dried in a BOD incubator at C for 12 hrs. The difference between dry weight and wet weight of erythrocyte pelletes gave the weight of intracellullar water in red cells. Erythrocytes from ethanol-injected fish permeated more water than those from normal fish.
Water permeation was significantly enhanced with Merc cor 30 and Nux vom 30 as compared to the control. RBCs from fish pretreated with Nux vom 30 inbibed more water in in vitro treatments than those from fish pre-treated with Ethanol Since aquaporins are mainly responsible for water transport through the plasma membrane of red blood cells it is thought that potentized drugs such as Merc cor 30 and Nux vom 30 acted upon these proteins and facilitated water influx into the cells Sukul et al. Summary Evidences in support of high dilution effects come from clinical records by practising physicians and scientists, and results of experiments conducted in laboratories.
Unlike crude drugs, the therapeutic effect of a high dilution depends not on the common symptoms of a particular disease but on the totality of the symptoms of an individual patient. Thus clinical trials would yield positive results if the objective is to see whether diseases, whatever their name is, are cured by high dilutions. Case records of homeopathic physicians show that diseases such as uterine tumours, leishmaniasis, anal fistula, bronchial asthma, viral encephalitis, hepatitis, cerebral haemorrhage, heart block etc.
Occasional failures in clinical trials result from specific inherent condition of patients called miasms. High dilutions of drugs have been used on human patients for a couple of centuries, and animal experimentation has been done only to confirm their therapeutic effects and study their mode of action. High dilutions have been found to produce effects on such animals as rats, mice, birds, toads and fishes.
The basic principle is to create a disease in the animals and test appropriate remedies on them. Some models like catalepsy and righting reflex ones are non-sacrifice animal models which can be easily used to test the biological effects of potentized drugs. Potentized Cina, Calcarea fluor and Thuja reduced infection of Trichinella spiralis in albino mice. Potentized Cina reduced Dirofilaria immitis infection in dogs. High dilutions did not kill the nematode parasites directly but worked through the immune system of the host animals.
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In electrophysiological experiments potentized Arnica, Hypericum and Arsenicum album, given on the tongue, altered the rate of discharge of the lateral hypothalamic neurons in anaesthetized cats. Arnica 30, Hypercium and Arsenic 30, administered orally, altered the firing rate of the medial frontal cortex neurons of awake rats. Distilled water showed an excitatory effect on those neurons. Natrum mur 30 and Phosphorus produced inhibitory effect on hypothalamic neurons of rats given Naturam mur 30, one dose daily for 18 days.
Here also distilled water produced the excitatory effect. Agaricus muscarius 12, administered orally on mice, increased the level of dihydroxyphenyl acetic acid dopamine metabolite and 5-hydroxyindol acetic acid serotonin metabolite in the mouse hypothalamus indicating increased transmission in the dopaminergic and serotonergic neurons there. These results indicate that hypothalamic and cortical neurons play an important role in mediating the therapeutic action of potentized drugs. Arsenicum album c and Kali iodide c reduced fruit rot of guava and mango. Cina c, applied by foliar spray, reduced root-knot disease, caused by nematode parasites, of cowpea plants.
The drugs augmented the natural defense response in plants and thus reduced the parasite infection. High dilution effects were observed in in vitro tests involving isolated organs, cell cultures or macromolecules such as proteins. Mercuric chloride 30c and Mercuric iodide 30c enhanced an enzyme activity in vitro. Unlike high dilutions in aqueous ethanol, efficacy of high dilutions in water deteriorates over time.
Mere cor 30 and Nux vom 30 altered water permeation in fish erythrocytes in vitro thereby indicating the influence of high dilution on water channel proteins or aquaporins on plasma membranes. In practice, homeopathic physicians usually prescribe 30th potency of a drug or its higher potencies for an ailment. We have also noted that these potencies work on patients. There are also evidences from experiments on animals and plants that potentized drugs are effective. Drugs in high potencies also differ from each other with respect to their efficacy on organisms. Let us examine what physical entities are present in potentized drugs.
Are the entities molecules of the concerned drug or something else? It is obvious that when the solution or suspension of a drug in a solvent is diluted with the solvent, the dilution would contain fewer molecules of the concerned drug. Avogadro put forward his hypothesis in In a classical experiment Jean Baptiste Perrin determined the Avogadro number which was confirmed later by other physicists Alberty and Silbey, ; Vemulapalli, Hahnemann could not have known that his potencies actually crossed the Avogadro number.
If the dilution is prepared, as usual, from an amount of substance less than one mole, the number of dilutions necessary to obtain the absence of drug particles is still less than In compliance with the Avogadro number, all higher potencies of a drug contain only the molecules of the diluent medium, i. We have noted in the foregoing chapter that potencies, prepared with pure water, are also effective although the efficacy of the aqueous preparation declines with the passage of time.
There are several hypotheses that have attempted to unravel the mystery of the physical basis of potentized drugs. All these hypotheses are based on the molecules of the diluent medium. These works have been reviewed earlier Rubik, ; Jacobs and Moskowitz, ; Sukul, According to Barnard , polymers of water appear during the process of succussion. These polymers are thought to assume specific configuration in a potency depending on the chemical nature of the concerned drug. Callinan proposed that vibrational energy of water molecules is raised much above the ground state by succussion.
According to Sharma , , resonant promotion of the lone pair of electrons of the -OH groups of diluent molecules contributes to the physical basis of a potentized drug. Different diluent media water, alcohol, lactose , used in potentizing drugs, have in common the -OH groups in their molecules. An oxygen atom has four equivalent valency orbitals, two of which are occupied by bond pair and the rest by a lone pair of electrons. During succussion or trituration the outermost electron shell of the drug molecule comes close to the outermost electron shells of the diluent molecules repeatedly.
This induces resonant promotion of the lone pair of electrons of the -OH groups of the diluent molecules to energy levels equal to those of chemically active electrons in the drug molecules. The process imprints chemical specificity of drug molecules on the molecules of the diluent medium or vehicle.
According to Dutta , , light isotopes of drug molecules are formed during the process of dynamization of a drug. These isotopes have chemical properties similar to those of the drug but negligible mass.
These isotopes continue to exist in the medium when the original drug molecules disappear during the process of successive dilution. As for example, positronium, which is made up of an electron, the elementary negative charge, and a positron the elementary positive charge, is the light isotope of hydrogen. Light isotopes of helium and lithium have been reported. These isotopes are thought to carry the message of original drug molecules and produce the necessary biological effects.
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- High Dilution Effects: Physical and Biochemical Basis?
Light isotopes increase in number proportionately with the incerasing degree of dynamization due to autocatalytic effect. The higher is a potency, the more numerous are the light isotopes in the medium. This makes the higher potencies of a drug produce stronger and more prolonged effect on a patient. Water molecules may form clusters or clathrates around drug molecules or particles Wei et al. Anagnostatos et al. The vacant clathrates remain and are immediately surrounded by water molecules.
The dislodged drug molecules are also surrounded by water molecules. All these clathrates are thought to carry the message of drug molecules when the latter disappear in course of successive dilution and succussion. Experiments have been conducted on the diluent media to find out the physical basis of potentized drugs. The data on the spectroscopic analyses of homeopathic potencies are presented below.
NMR spectroscopy Before describing the application of Nuclear magnetic resonance NMR spectroscopy to potentized homeopathic drugs we would first discuss the basic principles of NMR spectroscopy. This spectroscopy is a powerful tool providing structural information about molecules. Like UV-visible and infra red spectrometry, NMR spectrometry is also a form of absorption spectrometry. Nuclei of some isotopes possess a mechanical spin and the total angular momentum depends on the nuclear spin, or spin number 1.
Obviously, the medium is made up of molecules of hydrogen proton , oxygen and carbon. Each nucleus in which the value of I is greater than 0 has a characteristic magnetic moment. Since an electric charge is associated with an atomic nucleus, the spinning nucleus produces a magnetic field with its axis in line with the axis of the spin. The magnetic moments of nuclei are oriented in random directions in a sample of H2O. If the sample is placed in a uniform magnetic field Ho, the nuclei will tend to align themselves along the field. But because the nucleus is spinning, its axis would not be parallel or antiparallel to the applied magnetic field.
There would be a finite angle between the spin axis and Ho with the result of a circular motion of spin axis around Ho. This motion of the nucleus is called precession Figure 6. The frequency of precession is most important in NMR. The low energy orientation corresponds to that state in which the nuclear magnetic moment is aligned parallel to the external magnetic field, and the high energy orientation corresponds to that state in which the nuclear magnetic moment is antiparallel opposed to the external magnetic field Ho.
There is, however, a slight excess population of nuclei in the lower energy state lower spin state. It is possible to induce transitions between these two orientations of the nuclei. The precessional frequency of a spinning nucleus is exactly equal to the frequency of electromagnetic radiation necessary to induce a transition from one nuclear spin state to another.
Electromagnetic radiation is an oscillating electric field E in space which is propagated with the velocity of light. It has an electric vector E, directed along the displacement direction of the wave. The associated magnetic field vector H lies perpendicular to the electric vector and perpendicular to the direction of propagation.
The radio frequency electromagnetic energy is applied in such a way that its magnetic component H1 is at right angles to the axis of the main external magnetic field Ho and is rotating with the precessing proton. When the frequency of the rotating magnetic component is equal to that of the precessing nucleus they are said to be in resonance, and the absorption or emission of energy by the nucleus can occur.
It is the excess of muclei in the lower energy state that gives rise to net absorption of energy in the radio-frequency region. At the point of resonance the absorbed energy is at a maximum, and the precessing nuclei are tilted away from alignment with Ho toward the horizontal plane Figure 7. The magnetic component thus generated in that plane can be detected. The resonance can be produced by keeping Ho constant and changing the frequency of the rotating coil with its axis at right angles to the main magnetic field Ho.
Or, the oscillator frequency is kept constant and Ho is varied over a narrow range. As the excess population of nuclei in the lower energy are raised to the high energy state by absorption of energy, the intensity of absorption signal diminishes and finally disappears. The phenomenon is known as saturation in which the populations of muclei in the two spin states become equal.
However, there exists a mechanism by which the nucleus in the higher energy state can lose energy to its environment and thus returns to its lower energy state. The mechanism is called a spin-lattice or longitudinal relaxation T1. It involves transfer of energy from the nucleus from its high-energy state to the lattice.
The term lattice refers to the framework of molecules say drug and solvent or diluent containing the precessing nuclei. All these molecules have transational, rotational and vibrational motions and have magnetic properties. Thus, a variety of small magnetic fields are present in the molecular lattice.
A particular small magnetic field, properly oriented in the lattice, can induce a transition in a particular precessing nucleus from a higher to a lower energy state. The spin-lattice relaxation, therefore, maintains an excess of nuclei in the lower energy state, a condition necessary for the observation of NMR phenomenon.
Another type of relaxation process known as spin-spin or transverse relaxation T2 involves transfer of energy from one high energy nucleus to another. This relaxation does not contribute to the maintenance of the excess population of nuclei in a lower energy state. Magnetic component H of the applied radiofrequency electromagnetic energy rotating with the precessing proton 1 The natural width of a spectral line is inversely proportional to the lifetime of the excited state.
Thus, sharp resonance lines are observed for states of prolonged excitation, and broad lines are observed for short-lived excited states. Both spin-spin and spin-lattice relaxations contribute to the width of a spectral line. Most solids and viscous liquids show very long spin-lattice relaxation and very short spin-spin relaxation.
In case of nonviscous organic liquids and solids in solution, resonance lines are broadened and, therefore, relaxation times are short. Two other factors influence the width of a spectral line. The presence of paramagnetic molecules, such as dissolved oxygen, results in reduction of T1 due to the large magnetic fields associated with the paramagnetic lattice components. Because the electron magnetic moment is about times greater than nuclear magnetic moment, nuclear relaxation will be rapid, being dominated by the unpaired electron of oxygen.
It is not known whether degassing results in any alteration of the efficacy of a homeopathic potency in water or aqueous ethanol, the usual diluent medium of a homeopathic drug. However, for purposes of comparison between a homeopahtic potency and its diluent medium no degassing is necessary.
This is because of the fact that both the medium and the potentized drug are likely to contain an almost equal amount of the dissolved oxygen. These nuclei have short spin-lattice relaxation times. In the NMR experiment, as stated earlier, an excess spin population is moved from one energy level to another by electromagnetic radiation of the appropriate frequency.
This is excitation. The radiationless return to equilibrium is called spinlatice relaxation because the excess energy passes from the spins to the lattice as heat. The relaxation requires magnetic fields which are fluctuating at the appropriate frequency. The dominant fields arise from the magnetic moments of protons in the same molecules as they tumble in solution. This is the dipole-dipole interaction. The rate of dipole-dipole relaxation depends on the strength and frequency of the fluctuating magnetic fields. These in turn depend on three factors.
These factors are : i the distance between the nuclei involved, ii the effective correlation time, Wc, of the vector that joins the nuclei, and iii the nature of the nuclei themselves. Wc is the average time taken to rotate through one radian or roughly the reciprocal of the rate of tumbling in solution of the relevant piece of the molecule Sanders and Hunter, Only a single peak should appear from the interaction of radio-frequency energy and strong magnetic field on a proton.
The peak area, as measured by an integrator, is proportional to the number of protons it represents. However, the nucleus is shielded to some extent by its electron cloud whose density varies with the environment. Protons in different environments are shielded by circulations of surrounding electrons to different extents. The result is usually expressed as a shifting of the resonance frequency of the proton by the electron cloud, and the position of the signal of the particular proton as the chemical shift of the proton. The chemical shifts are measured by using a proton in a suitable compound as a reference.
The most commonly used compound for this purpose is tetramethylsilane TMS. It is chemically inert, magnetically isotropic, volatile b. It absorbs at a higher field than almost all organic protons. Homeopathic potencies are prepared in distilled water or aqueous ethanol. When water or deuterium oxide is the solvent, TMS can be used as an external reference. Here TMS is kept in a sealed capillary immersed in the sample. DSS, acetonitrile and dioxane are used as references in aqueous solution Silverstein et al. The position of the NMR signal is recorded in Hertz Hz as a difference between the positions of the observed and reference signals.
The range is normally Hz, which is called the sweep width. When the chemical shifts are given in Hz designated X , the applied frequency must be specified. This can be done by dividing X by the applied frequency and multiplying by Since the resultant term is too small, it is expressed as parts per million ppm. The delta G system assigns 0 to the reference signal and expresses the positions of other signals in ppm.
Structure of tetramethylsilane a lower resultant magnetic field around the proton. An alternative unit tau W assigns a value of 10 to the reference signal TMS. For shifts at a higher field than TMS G 0. In homeopathic potencies the molecular formula of the diluent medium, water or ethanol-water, is known. Thus height ratios of peaks can be converted into the number of protons giving each peak. The factors that influence the width are: i instrumental factors, ii factors relating to the environment in which the molecules exist and iii factors which depend on the molecules themselves Dani, The group ii factors are important in Homeopathy because other factors i and iii would remain unaltered when a potentized drug, say Nux vom 30, is compared with its diluent medium aqueous ethanol or water using the same instrument under the same experimental conditions.
The environmental factors like collision between molecules, exchange reactions and hydrogen bonding introduce slight variations in the transition energies which result in broadening of the signal. The principle states that there is a minimum uncertainty involved in the estimation of any two simultaneous dependent variables. The two variables here are 'E energy and 't life time of excited state. The lifetimes vary but a common value in NMR is s. The shorter is the life time the broader is the signal. Broadening due to this effect is insignificant in UV and IR spectra where higher frequencies - are employed.
The life time is related to the rate of relaxation. Factors that influence chemical shifts are hydrogen bonding, dipole moment and bulk magnetic susceptibility of the solvent used.