Cholecalciferol was purchased from Sigma-Aldrich, India. All other chemicals used during the experiments were of analytical grade available in India.

The vitamin D₃ sample was divided into two parts. One part of the vitamin D₃ was considered as a control/untreated sample, which was not received the Biofield Energy Treatment. The second part of the vitamin D₃ sample was received the Trivedi Effect^®-Consciousness Energy Treatment remotely under standard laboratory conditions for ~3 minutes by Mr. Mahendra Kumar Trivedi and termed as the Biofield Energy Treated vitamin D₃. Mr. Trivedi was located in the USA, while the test samples were located in the research laboratory in India. This Biofield Energy Treatment was provided through the Mahendra Trivedi’s unique energy transmission process to the test item. Consequently, the Control sample was subjected to a “sham” healer (who did not have any knowledge about the Biofield Energy Treatment) under similar laboratory conditions. Later on, the control and the Biofield Energy Treated samples were kept in similar sealed conditions and characterized with the help of PSA, PXRD, DSC, TGA/DTG, FTIR, and UV-Vis techniques.

The PXRD analysis of samples of cholecalciferol was done using PANalytical X’Pert3 powder X-ray diffractometer, UK. The copper line was used as the source of radiation for diffraction of the analyte at 0.154 nm X-ray wavelength that is running at 40 mA current and 45 kV voltage. The instrument uses a scanning rate of 18.87°/second over a 2q range of 3-90° and the ratio of Kα-2 and Kα-1 was 0.5 (k, equipment constant). The data was collected using X’Pert data collector and X’Pert high score plus processing software in the form of a chart of the Bragg angle (2θ) vs. intensity (counts per second), and a detailed table containing information on peak intensity counts, d value (Å), full width half maximum (FWHM) (°2θ), relative intensity (%), and area (cts*°2θ). The crystallite size (G) was calculated by using the Scherrer equation (3) as follows:

G = kλ/(bCosθ) ...(3)

Where, k is the equipment constant (0.5), λ is the X-ray wavelength (0.154 nm); b in radians is the full-width at half of the peaks, and θ is the corresponding Bragg angle.

Percent change in crystallite size (G) of cholecalciferol was calculated using the following equation 4:

...(4)

Where, G_Control and G_Treated are the crystallite size of the control and the Biofield Energy Treated cholecalciferol samples, respectively.

The DSC analysis of the samples was performed using DSC Q2000 differential scanning calorimeter, USA, under the dynamic nitrogen atmosphere with a flow rate of 50 mL/min. For analysis, 2-4 mg sample was weighed and sealed in Aluminum pans. Further, it was equilibrated at 30°C and heated up to 450ºC at the heating rate of 10º C/min under Nitrogen gas as a purge atmosphere 33. The value for onset, end set, peak temperature, peak height (mJ or mW), peak area, and change in heat (J/g) for each peak was recorded. Later on, the percent change in melting temperature (T) of the control and the Biofield Energy Treated samples were calculated using the following equation 5:

...(5)

Where, T_Control and T_Treated are the melting temperatures of the control and the Biofield Energy Treated cholecalciferol samples, respectively.

Also, the percent change in the latent heat of fusion (ΔH) was calculated using the following equation 6:

...(6)

Where, ΔH_Controland ΔH_Treatedare the latent heat of fusion of the control and treated cholecalciferol, respectively.

TGA/DTG thermograms of control and the Biofield Energy Treated cholecalciferol samples were obtained using TGA Q500 themoanalyzer apparatus, USA under dynamic nitrogen atmosphere (50 mL/min). It involves the heating rate of 10 ºC/min from 25 °C to 800 °C and uses platinum crucible 33. In TGA analysis, the weight loss in gram as well as percent loss for each step was recorded with respect to the initial weight of the sample. Later on, in DTG analysis, the onset, endset, peak temperature, and integral area for each peak was recorded. The percent change in weight loss (W) was calculated using the following equation 7:

...(7)

Where, W_Control and W_Treated are the weight loss of the control and the Biofield Energy Treated samples, respectively.

Also, the percent change in maximum thermal degradation temperature (T_max) (M) was calculated using the following equation 8:

...(8)

Where, M_Control and M_Treated are the T_max values of the control and the Biofield Energy Treated samples, respectively.

FT-IR spectroscopy of cholecalciferol was performed on Spectrum ES Fourier transform infrared spectrometer (Perkin Elmer, USA) by using pressed KBr disk technique with the frequency array of 400-4000 cm^-1. The technique uses ~2 mg of the control sample and about 300 mg of KBr as the diluent to form the pressed disk followed by running the sample in the spectrometer. The same procedure was used for the Biofield Energy Treated sample 3435.

The UV-Vis spectral analysis of the control and the Biofield Energy Treated cholecalciferol samples was carried out using Shimadzu UV-2400PC SERIES with UV Probe (Shimadzu, JAPAN). The spectrum was recorded in the wavelength range of 190-800 nm using 1 cm quartz cell having a slit width of 0.5 nm. The absorbance spectra (in the range of 0.2 to 0.9) and wavelength of maximum absorbance (λ_max) were recorded 3637.

PSA analysis was used to determine the particle sizes {d₁₀, d₅₀, d₉₀,and D(4, 3)} and the surface area of the control and the Biofield Energy Treated cholecalciferol samples, and the results are mentioned in Table 1. The particle size distribution of the control sample was observed at d₁₀ = 103.37µm, d₅₀ = 328.16µm, d₉₀ = 685.18µm, and D(4, 3) = 365.12µm. On the other hand, the particle size distribution of the Biofield Energy Treated sample was observed at d₁₀ = 97.37µm, d₅₀ = 274.06µm, d₉₀ = 565.12µm, and D(4, 3) = 305.85µm. The analysis (Table 1) showed that the particle size values at d₁₀, d₅₀, and d₉₀, and D(4, 3) in the Biofield Energy Treated sample were significantly decreased by 5.80%, 16.49%, 17.52%, and 16.23%, respectively compared to the control sample. Besides, the specific surface area (SSA) of the Biofield Energy Treated sample (35.18 m2/Kg) was found to be significantly increased by 7.26% as compared to the control sample (32.80 m2/Kg).

The possible reason behind such alterations in the particle sizes might be that the Trivedi Effect^® might act as an external force that helps in reducing the particle size of vitamin D₃ as done in case of treatment under ball mill. Moreover, according to scientific literature, the reduced particle size and thereby, increased the surface area of any pharmaceutical solid compound further helps in improving the dissolution rate, absorption, and bioavailability profile of drugs 3839. Thus, it is presumed that the Trivedi Effect^® - Consciousness Energy Treated cholecalciferol might produce better bioavailability as compared to the untreated sample.

The XRD diffractograms of both the samples, i.e., control and the Biofield Energy Treated samples showed sharp and intense peaks. Since there were no broadening of peaks observed in diffractograms, thus it suggested the crystalline nature of both the samples. Also, the Bragg angle and other PXRD data from the diffractogram of the control sample were observed to be similar to the reported literature 40. The PXRD data that was collected from the diffractograms, such as Bragg angle (2θ) and relative peak intensity (%) were analyzed for the determination of crystallite size (G) of both the samples. Moreover, the Scherer equation 40 was used for calculating the crystallite sizes of both the samples.

The PXRD diffractogram of the control sample showed highest peak intensity (100%) at Bragg’s angle (2θ) equal to 5.0° (Table 2, entry 1); whereas, it was observed at 18.1° (Table 2, entry 11) in the diffractogram of the Biofield Energy Treated sample. Apart from that, the relative intensities of the PXRD peaks at 2θ equal to 13.7°, 18.1° and 27.0° (Table 2, entry 7, 11 and 14) in the Biofield Energy Treated sample were significantly increased by 7.38, 20.19, and 22.42%, respectively compared to the control sample. However, the relative intensities of the other PXRD peaks (Table 2, entry 1-6, 8-10, 12, and 13) in the Biofield Energy Treated sample were significantly decreased in that range from 3.16% to 42.56%, compared to the control sample.

Besides, the crystallite sizes of the Biofield Energy Treated sample at 2θ equal to 8.7°, 13.0°, 16.7°, 21.8°, and 23.7° (Table 2, entry 4, 6, 10, 12, and 13) were observed to be significantly increased in the range from 16.68% to 72.71%, with respect to the control sample. Although, the crystallite sizes of the control and the Biofield Energy Treated samples at 2θ equal to 13.7° and 14.9° (Table 2, entry 7 and 8) remained unaltered; however, the crystallite sizes of the Biofield Energy Treated sample at position 2θ equal to 5.0°, 5.1°, 6.7°, 9.0°, 15.5°, 18.1°, and 27.0° (Table 2, entry 1-3, 5, 7, 9, 11, and 14) were observed to be significantly decreased in the range of 11.12% to 41.69%, compared with the control sample. Also, the average crystallite size of the Biofield Energy Treated sample was observed to be 34.72 nm, in comparison to the control sample (35.70 nm), thus showed 2.80% reduction in the crystallite size. Such changes in the relative intensities, as well as the crystallite size, indicated some alterations in the crystal morphology of the Biofield Energy Treated cholecalciferol, compared to the control sample. It could be presumed that the Trivedi Effect^® - Consciousness Energy Treatment might introduce a novel polymorphic form of the cholecalciferol by using the process of energy transfer. Also, the compound’s polymorph may affect the physicochemical and thermodynamic properties and thereby the drug performance, such as bioavailability and therapeutic efficacy 4142. Thus, the Biofield Energy Treatment might be considered as an approach for introducing the new polymorph of cholecalciferol that may help in improving its performance. Figure 1.

The DSC analysis was used for the control and the Biofield Energy Treated samples to determine their melting and decomposition pattern. The data and further analysis from the DSC thermograms (Figure 2) of control and the Biofield Energy Treated samples are presented in Table 3. The scientific literature reported that the cholecalciferol exhibited sharp endothermic peak near 86.0°C as a result of the melting; whereas, the exothermic peak near 220°C might appear due to the decomposition of cholecalciferol 43. The DSC thermograms of both, the control and the Biofield Energy Treated cholecalciferol samples (Figure 2) showed two endothermic and one exothermic peak. It was observed from the thermograms that the Biofield Energy Treated sample showed a slight increase in the melting point (86.06°C) by 0.24% as compared to the control sample (85.85°C). However, the latent heat of fusion (ΔH) of the Biofield Energy Treated sample was observed to be significantly increased by 3.68% compared with the control sample. Hence, it might be assumed that the Biofield Energy Treated sample needs more energy to undergo the melting process. Later on, the decomposition temperature (2^nd peak) in the thermogram of the Biofield Energy Treated cholecalciferol was found to be reduced by 0.29%, but the enthalpy of decomposition showed 5.79% increase as compared with the control sample.

Moreover, the 2^nd broad endothermic peak (3^rd peak), which was observed at 308.34°C in the control sample and 309.0°C in the Biofield Energy Treated sample, might appear as a result of the slow degradation of non-volatile intermediates developing in the process of thermal reaction. The temperature and ΔH corresponding to this peak of the Biofield Energy Treated sample was found to be increased by 0.21% and 3.62%, respectively (Table 3) compared to the control sample. The overall results suggested that the thermodynamic stability of the Biofield Energy Treated cholecalciferol was significantly increased as compared to the untreated sample.

The TGA/DTG analysis is used here to analyze the thermal stability of the control and the Biofield Energy Treated samples with the help of their thermograms (Figure 3 and Figure 4). Also, the TGA and DTG data and their further analysis regarding the control and the Biofield Energy Treated samples are presented in Table 4. The scientific literature reported that the TGA thermogram of cholecalciferol showed a significant weight loss at 128°C that may occur due to the boiling and possible splattering of the sample 43. In this study, the TGA thermograms of the control and the Biofield Energy Treated samples showed three steps of thermal degradation (Figure 3). The analysis showed that the percentage weight loss in the Biofield Energy Treated cholecalciferol sample wassignificantly decreased by 18.58% and 89.81% in the 1^st and 3^rd step of degradation, respectively; however, there was a slight increase in weight loss in the 2^nd step by 0.57%, compared with the control sample (Table 4).

Besides, the DTG thermograms of the control and the Biofield Energy Treated samples (Figure 4) showed a single peak. The control sample showed maximum thermal degradation temperature (T_max) as 299.06°C, while the Biofield Energy Treated sample showed more stability, and T_max was observed at 304.53°C. Thus, the analysis reported that the T_max of the Biofield Energy Treated cholecalciferol was significantly increased by 1.83% as compared to the control sample. Overall, the TGA/DTG analysis represented that the thermal stability of the Biofield Energy Treated cholecalciferol was significantly increased as compared to the control sample.

The FT-IR spectra of both the control as well as the Biofield Energy Treated cholecalciferol samples are presented in Figure 5. The FT-IR spectra of both the samples showed a clear stretching and bending peak in the functional group as well as the fingerprint region.

The spectra of both the samples showed broad peaks near 3305 cm^-1 in the functional group area and were assigned to O-H stretching. Furthermore, the aromatic C-H stretching was observed at 3080 cm^-1 in case of both, the control and the Biofield Energy Treated samples. The peaks due to aliphatic C-H stretching were observed at 2935 cm^-1 and 2869 cm^-1 in the spectra of both, the control and the Biofield Energy Treated sample. Besides, there was the presence of variable peak at 1647cm^-1 in the spectra of control and the Biofield Energy Treated cholecalciferol that was due to C=C stretching. Moreover, the spectra of control sample showed aromatic C=C stretching frequency at 1440 and 1460 cm^-1, which was observed to be similar in the Biofield Energy Treated sample (1439 and 1460 cm^-1). The FTIR techniques can be used to monitor the stability, durability and to understand the chemical and surface chemistry in various types of membrane toward their performance. FTIR analysis is crucial to support the justification of the changes in their properties and performance in various applications 343544. The overall FT-IR analysis showed that the fingerprint region of the spectra of control and the Biofield Energy Treated sample was similar and no alteration in the vibrational frequencies was observed among both the spectra. Thus, it is presumed that the structural properties of the Biofield Energy Treated sample remained the same as that of the control sample.

The UV-visible spectra of both the control and the Biofield Energy Treated cholecalciferol samples are presented in Figure 6. The UV spectrum of both the samples i.e., the control and the Biofield Energy Treated cholecalciferol sample showed the maximum absorbance at 213 nm (λ_max) and 264 nm (λ_max). Thus, it is assumed that the electronic transitions between the highest occupied molecular orbital and lowest unoccupied molecular orbital of the Biofield Energy Treated sample were the same as that of the untreated cholecalciferol sample. UV–visible spectrophotometry is an important for the detection of various naturally occurring groups of substances like flavonoids, phenolic acids, anthraquinones, and coumarins due to their sharp characteristic UV spectra 4546.