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Explaining Ultra-high Dilutions
Explaining Ultra-high Dilutions

In various publications, the terms 'highly diluted solutions' and 'ultra-low doses' describe solutions with an extremely low concentration of the active substance. In this article, we shall use the term ultra-high dilution (UHD) to describe such solutions. UHDs are obtained, for example, by the multiple sequential dilution of a solution of the initial substance. Since the initial substance cannot be detected in its UHD by modern methods of analysis, it is assumed that the properties of a UHD of any substance should not differ from the properties of the solvent. However, according to recent research, this is not the case.

Explaining Ultra-high Dilutions

One hypothesis about the possible mechanism of action of UHDs is the 'froth flotation hypothesis'. According to the hypothesis, the initial substance is retained in the solution due to the formation of nanoparticles. During succussion, the formation of air bubbles allows nanoparticles to 'float', thus a monolayer is formed on the surface of the highly diluted solution, and the nanoparticles are transferred to the next dilution. Source: (12).

Various studies cite experimental data on the effects of UHDs of various substances on biological systems (1, 2). In this regard, the question arises as to which mechanism could underlie this phenomenon. How do UHDs of substances differ from the solvent from a physicochemical point of view?

Theoretically, upon reaching a certain dilution level, the probability of detecting at least one molecule of the initial substance in the UHD becomes negligible. Let us go back to school chemistry lessons. One mole of a substance contains 6.02x1023 (or around 602 billion trillion) structural units (molecules, atoms, ions, etc.). This is Avogadro's number . Suppose we have 1 litre of a solution containing one mole of glucose (C6H12O6). If you dilute such a solution by 100 (take 10ml of the solution and add 990ml water), then the number of glucose molecules in it will also be 100 times less and will already be 6.02x1021. If you repeat this procedure 12 more times (the dilution becomes (102)12 = 1024), then only 0.6 molecules of glucose remain. Since there must be a whole number of molecules (this is called the principle of indivisibility), the probability of at least one glucose molecule being present in one litre of such a solution would be about 60% (that is, the probability that a solution with a dilution of 1024 times does not even contain one glucose molecule is 40%). Upon further dilution, the probability of finding a solute molecule in the resulting solution tends towards zero, making up less than 0.0001% when diluted to 1030 (or 1 to 100 for 15 times). Logically, a solution with a zero concentration of solute should be identical to the solvent.

Theoretically, if the dilution factor of the solution exceeds Avogadro's number (1024 or higher), the probability of detecting at least one molecule of the dissolved substance becomes less than one (almost surely), which indicates that the substance may not even be present in the solution. In practice, however, such solutions exhibit biological activity other than pure solvent. So how can UHDs affect biological systems?

Several hypotheses have been put forward which attempt to explain the nature of UHDs and their properties. Among them, one can highlight the water memory theory (3), the clathrate formation theory (4), the silicon hypothesis (5), as well as the hypothesis that UHDs carry specific signals or information that can act as a trigger to turn on or off certain genes in cells (6).

Scientists have demonstrated the difference between the physicochemical properties of UHDs and the properties of the solvent, explaining them by the presence of stable aqueous nanostructures (hundreds of nm in size) (7.8). To this end, the Italian scientist Vittorio Elia used both indirect methods (calorimetry, measuring thermal conductivity and pH) and direct methods (UV spectroscopy, fluorescence microscopy, Fourier-transform infrared spectroscopy and atomic force microscopy (7)). A group led by Alexander Konovalov investigated the specific electrical conductivity, surface tension and pH of UHDs, as well as their dielectric permittivity and optical activity. Dynamic light scattering was also used, making it possible to find small objects in solutions. Having studied UHDs of various compounds with concentrations from 10–2 to 10–20 M, the scientists concluded that under the influence of the dissolved substance and external electromagnetic fields, nanoassociates (nano-sized molecular ensembles) form in the UHDs. The specific structure of the soluble substance plays an important role in this process. In its absence, as in the absence of an external electromagnetic field, the formation of nanoassociates does not occur (8). Vittorio Elia and Alexander Konovalov believe that it is the formation of water nanoassociates that determines the aggregate properties of UHDs. However, their studies do not provide full answers to questions about the nature of nanoassociates, nor about what happens to them during the successive dilution of solutions.

In 2007, American scientists David Anick and John Ives proposed a 'silicon' theory of action for UHDs (5). According to this theory, during successive dilution of substances with succussion at each dilution, the solution interacts with the glass walls of the vessel. In this case, a small amount of ions and silicon dioxide (SiO2, of which glass is mainly composed) pass into the bulk of the solution, forming silicates, the structure of which is specific and depends on the structure of the solute. Silicates have polymerising properties, and their amount increases almost 100-fold with each dilution of the solution. What is more, the structure of previously formed silicates in the solution can change from dilution to dilution. Thus, depending on which initial substance and preparation procedure has been selected, a certain set or 'specific pattern' of silicon compounds is formed in the UHDs. The presence of such a pattern may be due to the specific properties of UHDs of various substances. At this stage, the work of Anick and Ives is a theory that requires experimental confirmation.

In 2010, a group of scientists from India conducted an extensive analysis of UHDs of various metals using modern methods: transmission electron microscopy, electron diffraction, and inductively coupled plasma atomic emission spectrometry (9). Researchers have shown that even with dilutions far exceeding Avogadro's number (1060 and higher), nanoparticles of the initial substance metals and their aggregates 5-10 nm in size are found in solutions. Furthermore, the concentration of these nanoparticles ceases to decrease with subsequent dilutions. The scientists conducted further studies using gold and zinc preparations. It transpired that during the production of UHDs, nanoclusters are formed from the metal particles and lactose, which is used as a carrier in 'dry' dilutions or 'triturations' in the early stages of preparing UHDs of metals. The concentration of such nanoclusters is greater on the surface of the solution than in its volume. The size of the resulting particles does not depend on the degree of dilution, and lactose contributes to the formation and stabilisation of the resulting nanoclusters. Based on these experimental data, the authors put forward the 'froth flotation' hypothesis (10). According to this hypothesis, air bubbles formed in the solution during succussion after each dilution contribute to the levitation of nanoparticles on the surface of the liquid, forming a monolayer, which is stored in successive dilutions. Thus, dilutions of the substance become illusory. Once the concentration of nanoparticles reaches a threshold of several ng/ml, further serial dilutions do not lead to a decrease in their number (10). Further developing their theory, the Indian team of scientists investigated drugs based on UHDs of inorganic salts (NaCl, KCl, CaSO4, Na2SO4) using high resolution transmission electron microscopy, scanning transmission electron microscopy and energy-dispersive X-ray analysis. The results of the study showed that in all the UHD samples, the initial elements (Na, K, Ca, S) are found along with Si and O. The source of silicon and oxygen is likely the glass walls of the tubes (11).

Considering the ‘silicon’ theory, the scientists believe that inorganic particles formed from the initial materials are coated with silicates during the preparation of UHDs. The authors refer to such silicon-compound membranes as 'micromesoporous', as they contain nanopores, which air bubbles penetrate when shaken. This, in turn, leads to the levitation of nanoparticles and the transfer of the active ingredient to the next dilution (11). Thus, UHDs of metals and inorganic salts retain the initial material in the form of nanoparticles encapsulated in a shell of silicon compounds, and the concentration of nanoparticles during subsequent dilutions is maintained due to the fact that such particles 'float' and are held on the surface of the solution. Stable gold and silver nanoparticles, which are also probably coated with silicon compounds, have been found (12, 13) in UHDs prepared in successive dilutions at 1012, 1060, and even 10400. In addition to metals, the presence of nanostructures in UHDs has also been shown for preparations containing substances of plant origin (14, 15). As for the size of the recorded nanoparticles, it varies in different studies from the size of quantum dots (no more than 15nm) (15-18) to hundreds of nanometres (13). In addition, the size of nanoparticles decreased during the preparation of dilutions in some studies (13, 14), while in others it was relatively constant (16-18).

It should be noted that there are a number of UHD studies whose data cast doubt on the 'foam flotation' hypothesis (15-18). In these studies, nanoparticles of the initial material (Fe, Au, NaCl) were found in high and ultrahigh dilutions (up to 10200000), however, it is mentioned that the solution was always taken from the centre of the tube for serial dilutions. Thus, the hypothesis of a 'monolayer' of nanoparticles on the surface of the solution is not adhered to. It is hypothesised that the concentration dependence on the dilution degree is not linear on a nano scale or during the preparation of the UHD. Succussion triggers chaotic processes, which cause the concentration of nanoparticles in the system to be maintained indefinitely (16). It follows from this that an approach involving a 'mathematical' assessment of the presence of the initial substance in the UHD is untenable.

Although there are still more questions than answers regarding the mechanisms of action for UHDs, it is possible to make a reasonable assumption based on the above experiments that ultrahigh dilutions of substances are complex systems with specific properties that are different from the properties of the solvent used to obtain such systems.


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