Differences in Median Ultraviolet Light Transmissions of Serial Homeopathic Dilutions of Copper Sulfate, Hypericum perforatum, and Sulfur

Homeopathic remedies are produced by potentising, that is, the serial logarithmic dilution and succussion of a mother tincture. Techniques like ultraviolet spectroscopy, nuclear magnetic resonance, calorimetry, or thermoluminescence have been used to investigate their physical properties. In this study, homeopathic centesimal (c) potencies (6c to 30c) of copper sulfate, Hypericum perforatum, and sulfur as well as succussed water controls were prepared. Samples of these preparations were exposed to external physical factors like heat, pressure, ultraviolet radiation, or electromagnetic fields to mimic possible everyday storage conditions. The median transmissions from 190 nm to 340 nm and 220 nm to 340 nm were determined by ultraviolet light spectroscopy on five measurement days distributed over several months. Transmissions of controls and potencies of sulfur differed significantly on two of five measurement days and after exposure to physical factors. Transmissions of potencies exposed to ultraviolet light and unexposed potencies of copper sulfate and Hypericum perforatum differed significantly. Potency levels 6c to 30c were also compared, and wavelike patterns of higher and lower transmissions were found. The Kruskal-Wallis test yielded significant differences for the potency levels of all three substances. Aiming at understanding the physical properties of homeopathic preparations, this study confirmed and expanded the findings of previous studies.


Introduction
Homeopathic preparations (hp) are used in complementary medicine worldwide, but homeopathy has been and is still vigorously debated [1][2][3][4], and these debates are based on prior believes [5]. While there seems to be good preclinical and clinical evidence for speci�c e�ects of hp [6][7][8][9][10][11][12][13][14][15], the underlying mode of action is yet unclear. Our aim is to determine potential physical properties of hp, which eventually may allow a scienti�c understanding of hp.
In preceding studies, we observed signi�cant di�erences in the transmission of UV light between hp and controls [23,24], and between hp exposed to physical factors and unexposed hp [23]. e aim of the present study was (i) to target reproducing our results and (ii) to expand the nature of starting materials and external physical factors. We included a dilution of Hypericum perforatum, a plant oen used in homeopathy, anthroposophically extended medicine, and phytotherapy. Since many questions about the stability of hp remain unsettled, for example, regarding storage conditions, sterilisation procedures, or exposure to radiation from mobile phones and scanners, we exposed hp to elevated temperature, pressure in an autoclave, UV light, and non-ionising radiation.
Cleaning of the vessels for potentisation, autoclavation and rinsing the tubes and cuvettes during the measurements was performed by using 18 MΩ distilled sterile water (puri�ed water by Arium 61316 reverse Osmosis System, Satorius Stedim AG, Aubagne, France). For the preparation of controls and hp, 18 MΩ autoclaved distilled deionised water (Hiscia Institute, Arlesheim, Switzerland) was used, delivered in 10 L Schott Duran bottles (VWR International Dietikon, Switzerland).
All of the hp and controls were stored and potentised in 500 mL narrow necked bottles with standard ground joint and a conical shoulder, made from borosilicate glass with hydrolytic class 1, that is, highly resistant against corrosion in neutral, basic, and acid environments (Schott Duran, VWR International Dietikon, Switzerland), closed with standard ground Duran �at-head stoppers.
Potentisation vessels and stoppers as well as UV measurement test tubes were reused from former experiments [23,24]. Preceding potentization all vessels were cleaned by rinsing three times with 18 MΩ water in order to decrease potential ion leaching from the vessel wall. e same procedure was applied to the test tubes used in the autosampler of the UV-spectrometer (see below). e process of cleaning, drying, and �lling all vessels and test tubes was performed in a laboratory under laminar �ow (Prettl GmbH, Pfullingen, Germany). e test tubes for UV measurements of the samples were 18 mL tubes, made from hydrolytic glass (Schott Fiolax, Mitterteich, Germany) and were �lled with the homeopathic samples by one-way 20 mL, sterile, polystyrene pipettes (Pipetboy acu, Integra Bioscience AG, Zizers, Switzerland).

Sample
Preparation. e hp were prepared according to the legal regulation for homeopathic remedies [35] by using the multiple glass method. Potentisation was performed by hand through horizontally shaking the vessel at a rate of about 2.7 Hz for 4 min for CuSO 4 and S 8 , and for 2.5 min for hypericum prior to each dilution step.
All hp were made as c preparations (i.e., centesimal potency means 100-fold dilution with each step) up to 30c. CuSO 4 and hypericum had each 10 independent succussed water controls, while S 8 had 12 controls, prepared with each 5 (6) vessels before and 5 (6) aer the potentisation process to examine possible cross contamination. Controls were produced by shaking the potentisation medium (water) at the same duration as the hp, but controls were not diluted.
All steps of preparation and handling with open vessels were performed under a laminar �ow box wearing sterile examination gloves and a lab coat to prevent unwanted contamination of the samples. Vessels were shielded with aluminium foil and stored closed in boxes at stable temperature and humidity.
Computer-generated random codes were used for randomisation. Blinding of the vessels was performed by an unbiased person, lists of the allocation of contents to the vessels were kept closed until the end of data attainment and data reduction.

Exposure to External Physical Factors.
Samples of hp and controls were exposed to one of the following external physical factors: (1) incubation (Incubator, Sauter, Switzerland) at 37 ∘ C for 24 hours, (2) UV light at 252 nm of a sterilisation lamp for 12 hours (CAMAG Reprostar, Switzerland), (3) heat under pressure by autoclave (Fedegari Autoclavi, Vitaris AG, Baar, Switzerland) at a temperature of 90 ∘ C for 20 minutes, �lled into autoclavable Duran vessels (Schott Fiolax), or (4) an electromagnetic �eld of a mobile phone (Philips, Savvy Dual Band) at 900 MHz with an output of 2 W for 120 minutes while the test tubes with samples were placed on a turning plate under the laminar �ow.

UV Spectroscopy.
Data were acquired by a Shimadzu UV PC 1650 spectrometer (Kyoto, Japan) with a wavelength range from 190 to 1100 nm, equipped with an auto sampler CETAC ASX-260 (Omaha, USA), and a sipper.
Comprehensive preparatory measurements were achieved in previous investigations to identify the impact of instrumental parameters on reproducibility such as wavelength of lamp change from visible (VIS) to UV lamp and scan speed, instrumental dri, warm-up time, number of repetitions, sip and purge time. Prior to the measurements, a baseline calibration was completed with the cuvette �lled with 18 MΩ water. Light transmission was measured from 190 to 1100 nm. Each measurement was repeated four times with the �rst run including �ve samples of 18 MΩ water as a run-in before the actual samples. e unit of equipment was engaged 10 hours prior to actual measurements to achieve an efficient warm-up and to decrease the instrumental dri. Room temperature and humidity were kept constant. Heated samples were allowed to regain room temperature before the measurements. Figure 1 shows the timeline of preparation of the samples and measurements.

Data Analysis.
To compare measurements performed on different days, the common daily variations of a UV spectrophotometer that occur due to a new calibration on each measurement day had to be corrected for. us, transmissions of the samples (controls or hp) were divided by transmissions of the pooled controls for each day and wavelength (nm).
Median transmission values were calculated for the ranges of 190 nm-340 nm and 220 nm-340 nm as in [23].
Since not all data were normally distributed, non-parametric tests (Mann-Whitney-U, Kruskal-Wallis and Jonckheere-Terpstra) were used to compare controls and hp on the same measurement day or hp on different measurement days. Effect sizes ( ) were calculated and results were reported according to [36].
In order to compare the present study to the previous ones performed by our group [23,24], differences of means (transmission of controls − transmission of hp) in % and 95% con�dence intervals were calculated. All measurements of CuSO 4 and of S 8 were �nally combined with the number of measurement days as weight.
SPSS Statistics 17.0 and 20.0 (IBM, Armonk, USA) was used for statistical analyses.

Controls Prepared before and aer the hp.
When controls 1 (prepared before the series of potencies) and controls 2 (prepared aer the series of potencies) were compared by Mann-Whitney-U test, only in measurement 1 (of 5) of the CuSO 4 measurements a statistically signi�cant difference was found, but in none of the measurements of hypericum or S 8 . erefore, it was concluded that the order of preparation did not have an effect on the transmissions measured, and, consequently, controls 1 and 2 for each substance were combined for further calculations.

Differences between Controls and hp.
Controls and hp of each measurement day were compared separately. In unexposed samples, controls and hp differed signi�cantly for S 8 on measurement days 2 and 3 ( Table 1). While controls of CuSO 4 and hypericum tended to have higher transmissions than hp, controls of S 8 had lower transmissions than hp.
Controls and hp of samples exposed to physical factors showed signi�cant differences in transmission for CuSO 4 aer incubation, for hypericum aer exposure to UV, and for S 8 aer all of the 4 factors (Table 1). Both ranges of transmission (190 nm-340 nm and 220 nm-340 nm) yielded similar results with respect to signi�cant differences between groups.

�n�uence of ��ein�.
To investigate the possible in�uence of ageing on hp, measurements 1 to 5 of hp (without controls) were compared using a Kruskal-Wallis test. Signi�cant differences between the 5 measurement days were found for S 8 (190 nm-340 nm: P = 0.002; 220 nm-340 nm: P = 0.004). Jonckheere's test revealed no signi�cant trend in the data. For CuSO 4 and hypericum, no differences between the measurement days were found.

Effect of Exposure to External Physical Factors.
It was investigated whether exposure of the hp to external physical factors had an effect on transmission compared to nonexposed hp. For that purpose, non-exposed hp of measurements 2 and 3 were combined and compared to exposed hp ( Table 2). Signi�cant differences were found for CuSO 4 aer incubation and UV as well as for hypericum aer UV, where transmissions of hp aer exposure to these physical factors were reduced compared to transmissions of non-exposed hp. �o signi�cant changes have been observed for S 8 .

Differences between Potency Levels.
Potency levels 6c to 30c of non-exposed hp were also compared among one another.   Mann-Whitney-test, mean normalised transmission with standard deviation (SD) is shown, statistically signi�cant results ( ) are displayed in bold, = effect size. b Measurements 2 and 3 were combined, since these two measurements were closest in time to the measurements of the exposed samples. c for CuSO 4 and for hypericum and S 8 (unexposed), for CuSO 4 and for hypericum and S 8 (exposed).
When every single potency level was compared to the respective controls by Mann-Whitney-U test, only 2 potency levels of the S series (16c, 29c) showed a signi�cant difference to the controls aer Bonferroni correction for multiple testing (Figure 2(c)). Table 3 compares previous works of others and our group that investigated hp with UV, visible and/or near infrared light spectroscopy. In Figure  3, all results from our previous [23,24] and present study are combined.

Development of Light Spectroscopy
Studies. �e �rst studies that investigated hp with light spectroscopy compared whole spectra of hp and controls in mixtures of ethanol and water (Table 3). Zacharias [22] observed differences between hp prepared in pharmacies and under rigorous conditions of cleanness and concluded that changes in the spectra were caused by the introduction of contaminants during preparation. Rao et al. [19] found that the UV spectrum of succussed solvent (ethanol) differed from that of unsuccussed solvent. Korenbaum et al. [17] (comparing homeopathic nosodes and placebos) applied statistical tests in their comparisons and registered distinct wavelengths with signi�cant differences between nosodes and placebos. Works from our group [23,24] introduced series of hp, from 10c to 30c, and compared hp to succussed controls or different potencies of the same original substance. We used water as the solvent and no longer visually compared whole spectra, but applied statistical tests. Absorbance 600-800 nm centering of spectra, comparison of electronic-homeopathic copies (EHC) to every of the 3 placebo groups, registration of all wavelengths between 700-800 nm with signi�cant differences, Mann-Whitney-test e spectra of each placebo group did not essentially differ from those of the other placebo groups.
e spectrum of EHC manus differed signi�cantly from all three placebo groups. e spectra of EHCs DNA-tox and toxic metal differed signi�cantly from two placebo groups. e spectra of EHCs bacteria and vanilmandelic acid differed signi�cantly from only one of the placebo groups. e spectra of EHCs fungus and virus did not differ from any of the placebo groups. Rao

Reproducibility of Our Experiments.
Difficulties in reproducing experimental results are sometimes used as arguments against speci�c actions of hp. In the present study, we investigated UV transmissions of hp for the third time. In the �rst study, hp of CuSO 4 were found to have signi�cantly lower UV transmissions than controls [24]. In the second study, slightly aged but not fresh hp of CuSO 4 were found to have signi�cantly lower UV transmissions than controls [23]. No differences were found between decimal serial dilutions of S 8 and controls in either of these two studies.
For the present (third) study, modi�cations in the experimental setup and data analysis were made: the samples were not measured immediately aer production (because in earlier studies, no signi�cant differences could be observed at that time point), centesimal instead of decimal dilutions of S 8 were prepared (to enable a comparison between different substances but of the same dilution category), and non-parametric statistical tests were used. Now signi�cant differences between hp of S 8 (but not of CuSO 4 ) and controls were found. In both the second and third study, incubation to 37 ∘ C for 24 h led to differences between hp of CuSO 4 and controls, and exposure of hp to 37 ∘ C or UV radiation led to reduced transmissions compared to non-exposed hp.
Hp of S 8 had higher transmissions than controls, unlike hp of CuSO 4 and hypericum. In the present study, we investigated centesimal potencies of S 8 (S 8 c) in contrast to decimal potencies in earlier studies in order to be able to compare centesimal potencies of different starting substances. erefore, it may well be to that we obtained different results because S 8 c may exhibit different features than S 8 x. In fact, in clinical use for some substances, such as sulfur and phosphor reciprocal effects depending on the potency level are known. If lower transmission was an indicator of a less structured state, higher transmission could be an indicator of a more structured state of the S 8 hp. Additionally, in one of our previous studies [23], S 8 also showed a different behaviour than CuSO 4 : when exposed to external factors, the variance of CuSO 4 hp was increased, whereas the opposite was the case for S 8 .
Overall, hp and controls showed comparable differences in these three studies, indicating speci�c characteristics of hp. When these studies are combined, hp of CuSO 4 have signi�cantly lower transmissions than controls. Heat and ageing seem not only to change the physical properties of hp, but also their efficacy, as observed in a wheat germination model [37].

Possibility of Contaminations in hp.
Earlier publications by other groups suggested contaminations to occur during the potentisation process [21,22,38]. In one of our previous studies, however, we showed that hp can be prepared with a minimum of inorganic contaminants, and differences in transmission of hp and controls are not due to contaminants [24]. According to the conclusions of a previous study [30], importance was attached in the experiments presented in this article to the cleaning of the bottles, the preparation of hp and controls (handling under a laminar �ow, potentisation with water only, controls were succussed but not potentised) as well as the storage conditions (equal for hp and controls).

Models Assume Changes in Water
Structure. So far, several models have been proposed to explain the different properties of hp and controls, including supramolecular states of dissolved gases and hydrogen-bonded supramolecular water structures [31] or dynamisation [30]. Most models assume the absence of traces of the starting material and focus on water structure, although it was reported that nanoparticles of metal starting materials may be found in high potencies [39]. Important questions remain how various starting materials can give rise to distinguishable physicochemical properties of the hp, for example the response to external physical factors that differed between CuSO 4 , hypericum and S 8 in our experiments. It is a common criticism about homeopathic remedies, that if water had a memory of the original substances it came in contact with, it would be full of memories and would exert unpredictable effects [1,4]. However, it has been shown that exposure of hp to external physical factors may reverse the properties of hp towards the properties of the solvents [31].

Limitations of is Work.
Since the UV spectrophotometer was calibrated before each measurement series, there were small differences in the level of the absolute transmission values (in the order of <1%). ese daily differences affected the controls and hp in the same way and are therefore not the reason for differences between controls and hp. As can be seen in Figure 2(a), not only the potency levels of CuSO 4 showed variations in transmission, but also the controls deviated from each other. is may be the reason why the differences shown in previous studies between hp and controls of CuSO 4 [23,24] were not found in this study. Further differences were the person producing and measuring the hp and controls, as well as the location of the production and measurement. Exposures to external physical factors were done only once per factor (autoclave, EMF, incubation at 37 ∘ C, UV light) and per starting material (CuSO 4 , hypericum, S 8 ) due to the limited total amount of our hp samples. In future studies repeating of exposure should be considered to obtain more indicative results. Additionally, it would be worthwhile investigating in future studies how repetitive exposure to physical factors would affect the results.
Trivial artefacts such as a cause for the differences between homeopathic preparations and controls can be ruled out due to the rigorous study design including randomisation and blinding of the samples.

Conclusions
is study con�rmed and expanded some of our previous �ndings. By demonstrating differences in UV transmission between hp and controls, the study contributes to the understanding of physical properties of hp. It also shows that hp are not inert to for example heat and UV light and that their properties may change, which might be relevant for production, storage, and handling of hp.

Con�ict of �nterests
e authors declare that they have no con�ict of interests.