Fig. 7 Difference spectrum of 200 μM ANA in 20 mM citrate buffer pH 3.5 with control added to the reference cuvette and Glycerol 50 M added to the sample cuvette. Spectra correspond to t = 0, t = 10minutes, t = 40 minutes, t = 100 minutes, t = 200 minutes and t = 4 days after mixing (see text for details). ANA, 6-amino-2-naphthoic acid.
Fig. 8 Difference spectrum of 70 μM DANDSA in 20 mM citrate buffer pH 4.0 with control added to the reference cuvette and Glycerol 50 M added to the sample cuvette. Spectra correspond to t = 0, t = 100 minutes, t = 220 minutes, t = 7 days and t = 18 days after mixing (see text for details). DANDSA, 5, 6-diamino-naphthalene-1,3-disulfonic acid.
DANDSA demonstrates the first clear evidence that potency is initially acting to change the pKa value of a molecular reporter, which is then followed by changes in aggregation levels, rather than by acting directly on aggregation levels. These results are discussed below in relation to a proposed common mechanism of action of potencies on all molecular reporters so far examined.
4-Aminobenzoic Acid (ABA) is the smallest and simplest molecule examined for the effects of potency. Surprisingly perhaps, despite its size, it also demonstrates changes in its UV spectrum in the presence of potency with a decrease in absorbance at c. 292 nm in 20 mM citrate buffer pH 3.5. This change constitutes c.1% of the total absorbance of ABA and is consistent with potency-induced protonation. It is significant that ANA, a molecule that differs from ABA only in the length of its aromatic bridge (), and consequently its dipole moment, should respond more strongly than ABA. The importance of this structural difference between ANA and ABA in relation to their ability to respond to potencies is discussed below.
4′-Amino-[1, 1′-Biphenyl]-4-Carboxylic Acid
4′-Amino-[1, 1′-Biphenyl]-4-Carboxylic Acid (ABPA) is a structural analogue of ANA in which the naphthalene ring is replaced by a biphenyl electron delocalised bridge. Difference spectra, ± potency in 20 mM citrate buffer pH 4.2 show a decrease at c.305 nm and an increase at c.270 nm, consistent with potency-induced protonation. Overall absorbance changes constitute c.2% of total absorbance. This lower number compared with that found for ANA may reflect the conformational mobility of ABPA compared with ANA, an issue already mentioned in relation to BM, and discussed in more detail below.
The above results from eight different molecular reporters demonstrate that potencies interact with a range of structural forms to produce significant spectral changes. Several compounds, and particularly DANDSA, have indicated that there are, however, at least two steps involved in the production of these spectral changes. The first involves potency-induced protonation. As solutions are buffered and it is known that ordinary pH indicators show no response to potencies, together with the slow appearance of spectral changes over hours, this suggests some kind of electron density shift occurs across the molecules resulting in altered pKa values.  This conclusion has already been deduced from results obtained with BDF in a previous study. The current study has provided further evidence that this indeed may be the case. If potencies are producing a pKa change in molecular detectors, then a preceding step involving some kind of electron density movement across the molecules may well be the primary form of the interaction between potencies and molecular detectors.
This possibility can be tested in the following way. If assays are performed at pH values well away from the pKa value of compounds, then protonation/deprotonation is not possible, and the putative step two is silenced. If molecular encapsulators such as β-CD or cucurbiturils are added to solutions to prevent any aggregation of compounds, then step three is also silenced. Any spectral changes in the presence of potencies are then likely to be attributable to an earlier, possibly primary, step.
The following results pertain to assays performed at pH values >> pKa values and in the presence of molecular encapsulators with dyes MV, BDF, BM, 4PP and ET33. It should be noted here that only solvatochromic dyes are capable of showing sufficient spectral changes due to spatial electron movement and so amino acids with an aromatic bridge cannot be tested for this step directly.
Assays at pH Values >> Dye pKas
Positively Solvatochromic Dyes MV and BDF shows difference spectra (right) obtained with 50 μM MV in 20 mM borate buffer pH 9.0/10 mM β-CD ± potency at t = 100 and 220 minutes. A peak at 629 nm is evident. The λmax of a control solution of MV in the same β-CD/borate buffer is at 617 nm (left) and this is attributable to monomer, with a shoulder at c.574 nm to dimer. The new peak at 629 nm in the presence of potency can, therefore, be confidently assigned to a form of monomer. Positively solvatochromic dyes display bathochromic shifts in their spectra with increasing stabilisation of the excited (more charged) state (). It seems reasonable to conclude, therefore, that potency is stabilising something similar to the excited state of MV in which the opposite ends of the molecule are becoming more formally charged.
Fig. 9 Difference spectra of MV in 20 mM borate buffer pH 9.0 containing 10 mM β-cyclodextrin with control added to the reference cuvette and Glycerol 50 M added to the sample cuvette. Spectra correspond to t = 100 minutes and t = 220 minutes after mixing. Maxima are at 629 nm (right-hand curves). Control solution of MV in the same β-cyclodextrin buffer (left-hand curve). Maximum is at 617 nm. See text for details. Spectra are not to scale. MV, methylene violet (Bernthsen). Fig. 10 Potencies are postulated to interact with and stabilise the ground (more polar) state of negatively solvatochromic dyes (left) and the excited (more polar) state of positively solvatochromic dyes (right).
A comparable result to that with MV is seen with BDF in 20mM borate buffer pH 9.0/10 mM β-CD ± potency. In this case, a new peak appears at 583 nm compared with the λmax of a control solution of BDF in the same buffer which is at 567 nm. Again, potency seems to be stabilising a more polar form of BDF. This can only occur if an electron density movement has occurred toward the carbonyl moiety of BDF, as previously suggested may be happening.
Negatively Solvatochromic Dyes BM, 4PP and ET33 shows a difference spectrum of 50 μM ET33 in 20 mM borate buffer pH 8.5/10 mM β-CD ± potency. While the differences are small, they nevertheless show a hypsochromic shift in the presence of potency with a decrease at 470 nm and an increase at 373 nm. The λmax of a control solution of ET33 in the same buffer is at 407 nm. In contrast to results seen with the positively solvatochromic dyes BDF and MV, potency is inducing a hypsochromic shift in the spectrum of ET33. Negatively solvatochromic dyes display hypsochromic shifts in their spectra with increasing stabilisation of their ground (more charged) state (). It seems, therefore, that in the presence of potency the ground state of ET33, which is already charged, is having its polarity increased even further.
Fig. 11 Difference spectrum of ET33 in 20 mM borate buffer pH 8.5 containing 10 mM β- cyclodextrin with control added to the reference cuvette and Glycerol 50 M added to the sample cuvette showing a decrease at 470 nm and an increase at 373 nm (bottom curve). Spectrum corresponds to t = 210 minutes after mixing (see text for details). Control spectrum of ET33 in the same buffer containing 10 mM β-cyclodextrin shows an absorbance maximum at c.407 nm (top curve). Spectra are not to scale. ET33, 2, 6-dichloro-4-(2, 4, 6-triphenyl-pyridinium-1-yl)-phenolate.
Similar results have been obtained with 4PP and BM.  shows a summary of the results obtained with all five dyes. It would appear from these results that potency is preferentially interacting with, and intensifying, the charged forms of both positively and negatively solvatochromic dyes.
The effect of potency on dye spectra in the presence of β-cyclodextrin and at pH values >> the pKa of dyes. Left-hand column gives dye maxima in control solutions of buffer/β-cyclodextrin; the right-hand column shows the effect of potency
|λmax 567 nm||New peak at c.583 nm|
|λmax 617 nm||New peak at c.629 nm|
|λmax 456.5 nm||Decrease at c.495 nm|
Increase at c.390 nm
|λmax 367 nm||Decrease at c.400 nm|
Increase at c. 330 nm
|λmax 406.5 nm||Decrease at c.470 nm|
Increase at c.373 nm
Abbreviations: β-CD, β-cyclodextrin; BDF, bis-dimethylaminofuchsone; BM, Brooker’s merocyanine; ET33, 2, 6-dichloro-4-(2, 4, 6-triphenyl-pyridinium-1-yl)-phenolate; MV, methylene violet (Bernthsen); 4PP, 4-pyridinium phenolate.
a Positively solvatochromic dyes.
b Negatively solvatochromic dyes.
c β-cyclodextrin concentration 10 mM/buffer concentration 20 mM (pH 8.5 and 9.0 borate; pH 11.0 CAPS).
The current study has considerably extended the range of compounds that respond to homeopathic potencies. Solvatochromic dyes now seem to be a sub-group of a larger class of compounds known as π-conjugated dipoles demonstrating interactions with serially diluted and succussed solutions. These include amino acids with an aromatic bridge (π-conjugated zwitterions). The presence of a large dipole moment, electron delocalisation, polarizability (the ability for electron density to shift across the molecule under an appropriate stimulus) and molecular rigidity seem to be general requirements in compounds for significant interactions with potencies to take place. Two particular compounds of this wider class which are readily available and provide significant spectroscopic responses to potencies are ANA and DANDSA, the latter demonstrating changes in its spectra of 8 to 10% over time. shows a plot of percentage change in dye spectra versus dye dipole moment. Some uncertainty exists over the ground or permanent dipole moment size for several compounds used in this study as their values are not available in the literature, but reasonable estimates can be made according to established principles. Despite these minor uncertainties in dipole moment size, the general trend is clear. The larger the size of the dipole moment of a compound, the larger the response to potencies appears to be. PB, examined previously, has the smallest ground dipole moment and produces the smallest response. Conversely MV, BM, and DANDSA have the largest dipole moments and produce the largest responses. In addition, comparing ABA with ANA, where the only difference is the distance between charged moieties, and hence dipole moment, a threefold increase in response is seen.
Fig. 12 Plot of percentage change in dye spectra versus dye ground or permanent dipole moment for solvatochromic compounds (upper plot) and non-solvatochromic compounds (lower plot) used in this study. 4ABA, 4-Aminobenzoic acid; ABPA, 4′-amino-[1,1′-biphenyl]-4-carboxylic acid; ANA, 6-amino-2-naphthoic acid; β-CD, β-cyclodextrin; BDF, bis-dimethylaminofuchsone; BM, Brooker’s merocyanine; C343, Coumarin 343; DANDSA, 5, 6-diamino-naphthalene-1,3-disulfonic acid; MV, methylene violet (Bernthsen); PB, phenol blue; 4PP, 4-pyridinium phenolate.
The slow appearance of spectra (     ) and the correlation between detector polarity and degree of response suggests some kind of synergistic process or resonant interaction taking place between detector and potency in which the polarity of both are gradually strengthened. The success and magnitude of such an interaction may well depend upon several factors (see below).
Several other insights emerge from . It is clear that in contrast to the ground or permanent dipole moment, the transition dipole moment (the difference between ground and excited states) is not an indicator of response to potencies and there does not appear to be any correlation. For instance, amino acids with an aromatic bridge such as ANA and DANDSA have negligible transition dipole moments, yet demonstrate responses. Pyridinium phenolates all have very large transition dipole moments of c. 20–22D  yet display modest spectral changes ().
Dipole moment size may not be the only determinant underlying response to potencies, however. MV is a conformationally rigid molecule compared with, for example, 4PP, which has a similar dipole moment, and yet there exists a significant difference in the magnitude of their responses to potency. Furthermore, BM is conformationally mobile and produces modest and very variable responses, and yet on encapsulation, which renders it more rigid, response increases considerably. These results may indicate that molecular rigidity is required for potencies to effectively engage with molecular detectors. Additional evidence for this proposition comes from results with C343 where response to potencies is greater than for 4PP and ET33 ().
Using a range of compounds, both solvatochromic and non-solvatochromic, has not only demonstrated that specific molecular features (large dipole moment, electron delocalisation, polarizability and molecular rigidity) appear to be important for interactions with potencies to take place, but it has also revealed that several steps are involved in the production of spectral changes indicative of these interactions.
Results pooled from all 10 compounds reported in this study indicate that there are three steps in the interaction between potencies and molecular detectors that produce the spectral changes seen. While not all steps are separately observable in all compounds so far tested, it seems likely that these steps are a common feature given the number of overlaps between results.  summarises results for all compounds investigated and reported herein.
Abbreviations: ABA, 4-aminobenzoic acid; ABPA, 4′-amino-[1,1′-biphenyl]-4-carboxylic acid; ANA, 6-amino-2-naphthoic acid; β -CD, β -cyclodextrin; BDF, bis-dimethylaminofuchsone; BM, Brooker’s merocyanine; ET33, 2, 6-dichloro-4-(2, 4, 6-triphenyl-pyridinium-1-yl)-phenolate; DANDSA, 5, 6-diamino-naphthalene-1,3-disulfonic acid; MV, methylene violet (Bernthsen); 4PP, 4-pyridinium phenolate.
a Not investigated.
b BDF pKa1 represents BDF + H+↔ BDF-H+ and pKa2 BDF + OH–↔ BDF-OH–.
c Relative aggregation levels observable by resonant light scattering (this study).
d Relative aggregation levels observable by fluorescence spectroscopy (this study).
e Relative aggregation levels observable by UV-vis and fluorescence spectroscopy (this study, see text for more details).
Step one appears to involve a primary interaction between potency and molecular detector, resulting in an electron density shift. This step is observable with positively and negatively solvatochromic dyes. Both are made more polar—the former having their excited (charged) state stabilised, and the latter having their ground (charged) state stabilised.
Step two follows on from step one and is a consequence of it. Any electron density shift in a delocalised system will cause a change in one or more of any ionisable groups attached to that system. For all compounds assayed, changes in pKa values are seen. For amino acids with an aromatic bridge, this is the first observable step as these compounds do not have the spectral characteristics of solvatochromic dyes and hence step one is silent. While not solvatochromic, aromatic bridged amino acids, which are zwitterionic, produce significant interactions with potencies. These results taken together suggest that potencies preferentially interact with polar species, rendering them more polar. Indeed, the more polar the detector, the more effect potency seems to have ().
Step three results from step two. Any change in pKa values and hence degree of protonation will affect aggregation levels, as the forces driving aggregation include ionic as well as hydrophobic interactions and hydrogen bonding. Step three is most clearly separately observable with DANDSA, but is also apparent with compounds such as BDF and MV.
A final observation may have some relevance to the discussion that follows. A previous study has found that sustained light exposure inhibits the BDF–potency interaction, and continuous irradiation results in no observable spectroscopic difference between dye–control and dye–potency solutions. This observation has now been extended to include 4PP, ET33, BM, MV and ANA. In all cases, no effect of potency is seen with any of the above compounds on continuous irradiation (usually at the absorbance maximum of the dye). Short exposure to light (i.e., during assay in the spectrophotometer) has little effect, so continuous exposure is necessary to fully inhibit the dye–potency interaction. In addition, any loss of spectroscopic differences from medium term light exposure to dye–potency solutions can be reversed by placing solutions in the dark.
Consequently, to avoid complications created by light exposure, and as described in the Materials and Methods, all dye–potency and dye–control incubations are performed in the dark and assays performed at intervals, with incubations being returned to black film canisters in between assays.
Curiously, however, irradiation of potency solutions themselves appears to have no deleterious effect, at least over the short term. In addition, all aromatic bridged amino acid–potency solutions are scanned down to at least 230 nm, and often 220 nm, when assayed. This is well into the UV region of the electromagnetic spectrum, and yet no obvious loss of potency strength has been observed. It may be that potencies are far more robust than previously thought and that any UV-induced inactivation is slow and requires multiple exposures.
Why potency solutions themselves appear to be immune to light exposure and yet dye–potency interactions are sensitive remains unclear at this stage. It is likely, however, to be saying something fundamental about the physico-chemical nature of potencies.
Returning to dipole moment size and degree of response in relation to the putative step one above, it would seem that the larger the dipole moment of molecular detectors, the larger the response to potencies. This implies that potencies require polarity to interact, and on interaction increase the polarity of detectors proportionately. This in turn suggests potencies themselves must have polarity to result in this kind of interaction, as has been put forward previously. Any proposal as to the possible physico-chemical nature of potencies must, therefore, include polarity in the proposal.
Several well-known facts about potencies are perhaps worth reconsidering at this point.