Understanding of potentization process of homoeopathic medicine of Aurum Metallicum and its characterisation

Synthesis of sample

Gold (Au) of 99.9% purity was used to prepare Aurum met. The sample was prepared in glass and plastic vials following the standard protocol of Homoeopathic Pharmacopoeia, India.^[15,16] The sample and procedure were performed in the HAPCO Pharmaceutical laboratory. The detailed processing step is described in the supplementary file. A schematic representation of the process steps is shown in Figure 1. Aurum met and its potencies (up to 200C in glass and plastic vials) are prepared and standardised in GMP certified company (HAPCO, Kolkata). Every measurement was performed thrice and the average result was used.

Close

Figure 1:

Schematic diagram of the trituration process of homoeopathic medicine, Au: Aurum Metallicum.

Export to PPT

Characterizations

The structural analysis of the powder sample (3X and 6X, X = powder sample potency) was performed using a PANalytical X’Pert diffractometer X-ray diffraction (XRD) with Cu-Kα incident radiation (λ = 0.15406 nm) in the scan range of 10°– 90° with a scan rate of 1°/min. Ultraviolet-visible (UV-Vis) absorption spectra (Analytical Instrument) were recorded with a double-beam spectrophotometer. Raman spectroscopy measurements were performed using the WITec UHTS Raman Spectrometer (WITec, UHTS 300 VIS, Germany) at an excitation wavelength of 532 nm. Transmission electron microscopy (JEOL JEM 2200FS) was carried out to know the particle size in the diluted sample (24C and 200C, C = Liquid sample potency). Zeta potential and particle size were measured using the Zeta and particle size analyser, respectively (Model No: Horiba Scientific SZ-100).

XRD pattern of triturated powder samples (3X and 6X)

To characterize (the presence of phases, size, and strain) the Au and sugar of milk (lactose) mixture during mechanical grinding, XRD analysis was performed. Figure 2a shows the XRD pattern of the powder sample with different concentrations of Au and lactose. The diffraction peaks confirm the presence of Au NPs with lactose peaks. The peak at 38° corresponds to the (111) plane of Au NPs and matches with the standard JCPDS card.^[18] Along with the Au NPs peak, we also observed other peaks corresponding to lactose. Furthermore, no other impurity peaks were observed. Figure 2b shows the XRD peak of only (111) Au NPs for two different triturations (3X and 6X). For higher trituration (i.e., 6X), we observe a higher angle shift with a line broadening than 3X trituration. The shift of the XRD pattern reveals the presence of strain (~4%) in the materials. The origin of the strain is due to the mechanical grinding during trituration. We used the Debye Scherrer equation for calculating the crystalline size of samples.^[19] The 3X and 6X triturated sample shows a crystalline size of ~2.6 and 2.4 nm, respectively. The reduction in crystalline size is due to the continuous mechanical grinding of metallic Au in the presence of covalently bonded lactose crystals. The mechanical grinding results in lattice strain, and after critical stress, the Au particles fracture and form nano-sized crystalline materials. It is important to note that, as per previous observations,^[6,20] NPs are found in higher dilution medicine samples, but the origin of NPs formation has not been reported so far. Based on the current observation, we can see that the nanocrystalline Au NPs are formed during the trituration process itself. This highlights a unique observation that the initial preparation (starting material) for the high homoeopathic dilution is different from the other conventional medicinal preparation, which was never noticed so far, leading to erroneous conclusions.

Close

Figure 2:

(a) XRD pattern of power sample with two different grindings. (b) XRD peak of gold nanoparticles, Red colour: Indicates Aurum Metallicum 6X potency, Black colour: Indicates Aurum Metallicum 3X potency, XRD: X-Ray diffraction.

Export to PPT

UV-Vis absorption spectra of liquid colloidal suspension samples (all diluted samples: 6C–200C)

The UV-Vis absorption spectroscopy measurement is an essential tool that can provide helpful information about materials in the homoeopathic potencies (different dilutions). Figure 3a shows a schematic diagram of a borosilicate glass bottle containing homoeopathic potency. The absorption spectra of the solution have been taken from two different portions of the bottle (upper and lower part), as shown in Figure 3a. Figure 3b and c shows the UV-Vis absorption spectra of the top and bottom part solution with different potencies. The presence of three absorption bands is observed for all samples. To know the exact positions of the bands, we have plotted the second derivative of a spectrum as a function of wavelength [Figure 3d]. The occurrence of the absorption band at ~557 nm and ~721 nm is due to the presence of an anisotropic structure of different-sized Au NPs with localised surface plasmon resonance (LSPR).^[3,21] In addition, it shows the shape and absorption intensity of the spectra change with potencies. A broad absorption band has been observed at higher potency (i.e., concentration-10⁶ –10¹⁷ times dilution), which gradually became narrow/sharp after 24C. Similar variation has been observed for both the upper and lower part of the potencies.

Close

Figure 3:

(a) Schematic representation of a sample bottle with liquid dispersion. Ultraviolet-visible absorption spectra of the sample with different dilutions. Samples are taken from the bottom (b) and bottom and top (c) parts of the borosilicate glass bottle. (d) Second-order derivative of the absorption spectra as a function of wavelength. Indicates the presence of localised surface plasmon resonance and different oscillation modes of Au nanoparticles. (e) Baseline corrected fitted absorption spectra with three Gaussian peaks of the 200C sample. (f) Variation of full-width half maximum as a function of dilution at two different portions of the bottle. (g) Variation of the relative intensity of absorbance of gold nanoparticles.

Export to PPT

The obtained absorption spectra have been fitted by the Gaussian multiple peak fitting method to know the presence of NPs in the potencies [Figure S1]. A representative Gaussian fitted plot of 6C potency is shown in Figure 3e, and other diluted samples are shown in supporting figures. From the fitting, we have observed that three major peaks are present. The first broad peak, at ~300–450 nm, is associated with the presence of silicates ([SiO₄]^4-) with the hydrocarbon chain. Previously, it was reported that the origin of silicates is the chemical reaction between ethanol solution and sodium silicate, which discharge from the borosilicate glass bottle.^[7] We also measured the UV-Vis absorption spectra of the samples kept in plastic bottles to validate the results. The absorption peak is absent in samples processed in plastic bottles; the details are described in the supporting information [Figure S2]. The other two peaks originate due to the LSPR of Au NPs. The narrowing of the absorption peaks in the absorption spectra [Figure 3a and b] of the samples indicated the relatively narrower size distribution of the Au NPs. To know the exact origin of the narrowing of the Au absorption edge, we have plotted the full-width half maximum (FWHM) as a function of potencies, as shown in Figure 3f. The FWHM decreases with increased potencies, which means the deviation in Au NP size distribution decreases with higher potency. The distribution of particle size is different for the top and bottom parts of the container.

Moreover, the Au NPs size variation is different for the top and bottom parts of the potency, as shown in Figure 3f. The absorption peak of Au NPs becomes more prominent in the higher potency samples, as shown in Figure 3g. Hence, we can conclude that the ratio of Au NP to lactose increases as the potency increases. The three critical observations during these current characterizations are as follows:

1. The distribution of particles is not uniform across the triturations (top and bottom parts are different)

2. The distribution of the particles changes as a function of potency

3. The ratio of Au/lactose increases due to homoeopathic potentization.

The succussion process was studied in detail using optical measurements. The UV-Vis absorption spectra have been taken in different succussion for different potencies. A representative spectrum of 200C potency after different succussions is shown in Figure 4a; other spectra are shown in supplementary file [Figure S3]. Figure 4a(I) shows an enlarged view of the first absorption band (~210–260 nm), denoted as I. The peak intensity increases with the increase of succussion. This peak is due to the presence of hydrocarbon sources in the solution.^[22,23] [Figure 4a(II)] presents the second absorption band, as denoted II, and this band also shows similar nature as the succussion number increases; peak intensity increases. This peak originated from the aromatic π-system and hydrocarbon chain with silicate.^[23] During the succussion process, the lactose layers disintegrate and lead to an increase in hydrocarbon-silicate groups. In Figure 4a(III), we present the absorption spectra of Au NPs as a function of succussion. The intensity of Au NPs peak increases as a function of succussion. The layers of lactose dissolve in the solvent (water/alcohol mixture) during the process, leading to exposure of the NP surface. Based on the absorption spectra, the absorption intensity was calculated for different potencies (6C, 50C, and 200C) as a function of succussion, as shown in Figure 4b-d. It shows that the absorption intensity (defined as P1 and P2) increases with succussion. The trend remains similar for different potencies (6C, 50C, and 200C). Therefore, in subsequent succussion, the fraction of active NPs (surface of NPs without the lactose matrix) increases, stimulating the response of the medicines in human health.

Close

Figure 4:

(a) Ultraviolet-visible absorption spectra of the sample (200C) after and before succussion. I, II and II show the enlarged portion of the marked area. I, II and III denotes three different region in the absorption spectra. Region I denotes the first absorption band and the peak intensity increases with succussion. Region II denotes the second absorption band originated from hydrocarbon-silicate groups. Region III denotes absorption band of Au NPs and the peak intensity varies with succussion. The variation of absorption intensity of Peak I (P1) and Peak II (P2) for (b) 6C, (c) 50C and (d) 200C samples as a function of succussion.

Export to PPT

Particle size and zeta potential analysis

Quantitative measurement of NPs present in the different potencies is performed using dynamic light scattering (DLS), as shown in Figure 5a-d. As described earlier, we took the solution in two parts of the borosilicate glass bottle (top and bottom) to measure particle size distribution. Most of the particles are in the size of ~200–600 nm (upper portion of the glass bottle) [Figure 5a]. However, primarily large particles (~6 µm) are found [Figure 5b]. The particle size distribution decreases (size range of particles present) as the potency increases. It is important to note that at lower potency, the size distribution of particles in the solution is non-uniform (top part). However, after a particular potency, it shows a more uniform size of NPs. Therefore, potentization results in solutions with smaller particles, which improves the effectiveness of the medicine on human health. In addition, the size distribution for the lower part sample is similar for all potencies. Hence, we take the solution from the top part during the potentization process, that is, smaller particles. We have also measured the particle size in different succession, as shown in Figure 5c and d (upper and lower parts of the borosilicate bottle).

Close

Figure 5:

The variation of particle size in two different positions of the sample container (a) Top and (b) Bottom, respectively. Variation of particle size after different succussions in blank solution in (c) Top and (d) Bottom portion. (e) Zeta potential of all the diluted samples. (f) Variation of zeta potential with succussions.

Export to PPT

Interestingly, we have seen that in the upper part of the bottle, the average size of the particle decreases with an increase in the succussion number. Hence, more active NPs are exposed to the surface. However, in the lower part, the size of the particle increases with succussion during potentization. This result indicates increased air-bubble formation and a mixture of NPs with bubbles. Zeta potential is also an essential parameter for knowing the stability of colloidal systems. It also helps to quantify the surface charge of the particle. The zeta potential of all potency has been measured using the Zeta analyser measurement system, as shown in Figure 5e and f. The plot of zeta potential as a function of potency shows a change in the value from negative to positive, which means the nature of the solutions is unstable. Such experimental values suggest the un-stability of the solution, that is, the activity of the charged particles changes during succussion and potentization, which is consistent with our optical measurements.

TEM and Raman measurement of low and high diluted sample

The above discussions are, further, supported using the TEM study, as shown in Figure 6. Here, two different potencies (low and high) are used to understand the formation of thin layer and chemical composition of the particles. Figure 6a-c shows the TEM and high-resolution TEM (HRTEM) images of a low-potency sample with different sizes. Figure 6b shows that the Au NPs are distributed on the silicate chain with the hydrocarbon layer. The silicate chains are assembled from the discharge of silicate from the borosilicate bottle. With gradual dilution, the lactose disintegrates and forms a hydrocarbon layer. The HRTEM image of Au NPs, shown in Figure 6c, confirms the crystalline nature of the NPs. However, well-separated monodispersed Au NPs are observed in higher potency, as shown in Figure 6d-g. Figure 6e and f shows a single particle of Au coated with a thin silicate chain and hydrocarbon layer. HRTEM image shows [Figure 6e] that the surface of Au NPs is affected by electron beam irradiation. These results confirm that the surface of Au NPs is more reactive at a higher potency. In Figure 6g, we present the reactive edge of the Au NPs. We have also schematically presented the TEM observation in Figure 6h and i. It is observed that a layer of hydrocarbon and silicate is formed at low potency, and the Au NPs are embedded/encapsulated on that. However, the layer may disintegrate after trituration and form a thin coating on every NP. In addition, at very high potency, the surface of the particles is more active, and this active surface can react with humans. We have also taken the Raman spectra of different samples to confirm our assumption, and obtained results are well-matched with the TEM results. As shown in Figure 6j, the Raman vibrations are well-matched with previously reported data.^[24] It is found that several Raman peaks have appeared related to sucrose, lactose and glucose, which strongly supports the TEM results. In addition, it was found that the intensity of the Raman peaks varied with dilution. Previously, it was reported that the Raman intensity strongly depends on the sample concentration when other excitation source parameters are constant.^[25] Therefore, this clearly indicates effective silicate coating with a hydrocarbon layer on Au NPs. The spectra show that lactose/fructose/glucose molecules firmly stabilize the Au NPs by layer over the NPs.

Close

Figure 6:

(a) Transmission electron microscopy (TEM) image of a low potency sample. Red Square indicates presence of medicinal Au NPs in low potency. (b) High-resolution TEM (HRTEM) image of the sample. (c) HRTEM image of gold nanoparticles (Au NPs). (d) TEM image of high potency sample. (e) Single Au NPs with a thin layer at high potency. Red Square indicates presence of thin layer on the surface of Au NPs in high potency. (f) The enlarged portion of Figure e. Red Square indicates enlarged version of 6e and presence of reactive surface edges in high potency. (g) Reactive surface edge of Au nanoparticle. Schematic representation of (h) low potency and (i) high potency samples. (j) Raman spectra of both samples.

Export to PPT

We would like to propose a numerical definition of the sample in a liquid medium so that our findings can be compared to the experimental data that are currently accessible. Our initial step was to study the interaction dynamics between Au NPs and the amorphous carbon (a-C) layer. We were able to analyse the dynamics of the interaction between the two by distributing Au NPs close to the a-C layer. Figure 7 shows the computational calculation of the interaction. Here, we observe that a-C was seen to interact with the Au NPs surface and causes a-C bond length increases with mean, µ = 0.033 Å and standard deviation, standard deviation (SD) = 0.05 Å [Figure 7]. To deepen our understanding of the interaction mechanism, we also calculate the mean interaction energy using the equation given below,

Moreover, we observed E_interaction = −25.901385 kcal/mol, where a negative value means a-C was binding to the surface of Au NPs cluster; E_aC-Au = 116,041.185 kcal/mol, E_Au = 6,316.252 kcal/mol, E_aC = 109,750.834 kcal/mol. This suggests that within 0.1 ns, the addition of a-C interacts fast with the Au NPs surface, but this does not cause any breaking of the Au atoms bond and a-C bond. Further, we have studied the dynamics of the interaction of a-C and Au NPs with ethanol. Similar to the previous configuration, Au NPs and ethanol are dispersed close to the a-C. Further, we calculated using the interaction energy Equ-2 and found ~E_interaction = 403.83 kcal/mol, where a positive value signifies that a-C in ethanol was not binding to the surface of Au NPs. In addition, we calculate the other interaction energy, E_{aC-Au-ethanol} = 116,064.761 kcal/mol, E_ethanol = 27 kcal/mol, E_Au-np = 109,248.187 kcal/mol, E_aC = 6,385.741 kcal/mol

Figure 7 shows that the mean a-C bond length is μ =0.019, and the SD is ~0.04 due to the interaction between Au NPs. However, we observe a shift between two configurations, Δ _mean = 0.014 Å and Δ_SD = 0.01 Å, and this indicates the effect of a-C in the presence of ethanol.

Close

Figure 7:

(a) Amorphous C- Gold nanoparticles (Au NPs) Configuration (Left-Bottom) Amorphous C - Au NPs - Ethanol Configuration; Carbon is represented in the grey colour sphere, Gold in yellow, Hydrogen in White, Oxygen in Red; (b) Change in Bond length of Carbon distribution with interaction with Au nanocluster and with ethanol and Au NPs.

Export to PPT

Tyndall effect of all diluted samples

Digital photographs of the sample (before and after succussion) are fully transparent at different potencies (6C, 50C and 200C). However, they show light scattering properties under laser beam excitation due to the Tyndall effect [Figure 8a-d]. The Tyndall effect confirms the presence of particles in the solution. For further analysis of the presence of air-bubble-particle, line scans were performed using ImageJ software, as shown in Figure 8b-d. Some spikes are noted, which are due to the presence of particles. The inset shows the digital photographs of the samples (before and after succussion) under Laser excitation. Interestingly, after succussion, drastic change is noted. The appearance of the new spikes indicates the formation of air-bubble-particle mixtures. The spike changes’ size and shape are associated with forming different size floated air-bubble-particle mixtures in the solution.^[26,27]

Close

Figure 8:

(a) The schematic shows the Tyndall effect in a low-potency sample. Digital photographs with line scan profiles of the samples before and after succussion (b) 6C, (c) 50C and (d) 200C.

Export to PPT