Investigation of Impact of Biofield Energy Healing Treatment on Physicochemical, Thermal and Spectral Properties of Magnesium Gluconate

Chemicals and Reagents

Magnesium gluconate was procured from Tokyo Chemical Industry Co., Ltd., Japan. All other chemicals used in the experiment were of analytical grade available in India.

Consciousness Energy Healing Treatment Strategies

Magnesium gluconate i.e., the test compound was divided into two parts. Among both parts, one portion was denoted as control sample that did not receive the Biofield Energy Treatment. Besides, the other part of magnesium gluconate was considered as treated part that received the Energy of Consciousness Healing Treatment by the renowned Biofield Energy Healer, Mr. Mahendra Kumar Trivedi (USA), and named as the Biofield Energy Treated sample. In the process of Biofield Energy Treatment, the sample was kept under the standard laboratory conditions and the Biofield Energy Healer provided the Trivedi Effect® - Energy of Consciousness Healing Treatment to the sample, remotely, for 3 minutes through the Unique Energy Transmission process. On the other hand, the control magnesium gluconate was subjected to “sham” healer under the similar laboratory conditions, who did not have any knowledge about the Biofield Energy Healing Treatment. Consequently, the control as well as Biofield Energy Treated magnesium gluconate samples were kept in similar sealed conditions and further characterized by using PXRD, PSA, DSC, TGA/DTG, UV-Vis, and FT-IR techniques.

Characterization

Powder X-ray Diffraction (PXRD) Analysis: The PXRD analysis of the control and Biofield Energy Treated samples of magnesium gluconate was done with the help of PANalytical X'Pert3 powder X-ray diffractometer, UK. In this, the copper line was used as the radiation source for diffracting the analyte at 0.154 nm X-ray wavelengths, which is running at 40 mA current and 45 kV voltages. Also, the scanning rate for instrument was kept at 18.87°/second over a 20 range of 3-90° and the ratio of Kα-2 and Kα-1 was 0.5 (k, equipment constant). The data produced by the instrument was collected with the help of X'Pert data collector and X'Pert high score plus processing software. It provides the data in the form of a chart of the Bragg angle (20) vs. intensity (counts per second), and a table providing information regarding the peak intensity counts, d value (Å), full width half maximum (FWHM) (°20), relative intensity (%), and area (cts*°20). From this data, the crystallite size (G) was analyzed with the help of the Scherrer equation (1) [10, 11, 12] as follows:

$$G = k\lambda/(bCos\theta)$$ (1)

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

Later on, the percent change in crystallite size (G) of magnesium gluconate was calculated using following equation 2:

$$\% \text{change in crystallite size} = \frac{[G_{\text{Treated}} - G_{\text{control}}]}{G_{\text{control}}} \times 100$$ (2)

Where, $G_{\text{Control}}$ and $G_{\text{Treated}}$ are the crystallite size of the control and Biofield Energy Treated magnesium gluconate samples, respectively.

Particle Size Analysis (PSA): The particle size analysis was done using wet method, which involves the use of instrument, Malvern Mastersizer 3000, UK. The instrument has a detection range between 0.01 µm to 3000 µm [31], and the method involves the filling of sample unit (Hydro MV) with light liquid paraffin oil, which acts as dispersant medium. The refractive index values for dispersant medium and samples were 0.0 and 1.47, respectively. Later on, it was stirred at 2500 rpm, and the measurement was taken twice after reaching obscuration in between 10% and 20%, followed by averaging both measurements. The PS analysis provides data in the form of $d_{10}$ µm, $d_{50}$ µm, and $d_{90}$ µm, representing the particle diameter corresponding to 10% 50% and 90% of the cumulative distribution. D(4, 3) µm represents the average mass-volume diameter and SSA is the specific surface area (m²/Kg). The calculations were done by using software Mastersizer V3.50.

The percent change in particle size (d) for $d_{10}$, $d_{50}$, $d_{90}$ and D(4,3) was calculated using following equation 3:

$$\% \text{change in particle size} = \frac{[d_{\text{Treated}} - d_{\text{control}}]}{d_{\text{control}}} \times 100$$ (3)

Where, $d_{\text{control}}$ and $d_{\text{Treated}}$ are the particle size (µm) for at below 10% level ($d_{10}$) 50% level ($d_{50}$) and 90% level ($d_{90}$) of the control and Biofield Energy Treated samples, respectively.

Percent change in specific surface area (SSA) was calculated using following equation 4:

$$\% \text{change in specific surface area} = \frac{[d_{\text{Treated}} - d_{\text{control}}]}{d_{\text{control}}} \times 100$$ (4)

Where, $S_{\text{control}}$ and $S_{\text{Treated}}$ are the surface area of the control and Biofield Energy Treated magnesium gluconate, respectively.

Differential Scanning Calorimetry (Dsc): The DSC analysis of the samples was done under the dynamic nitrogen atmosphere with the help of DSC Q2000 differential scanning calorimeter, USA, at the flow rate of 50 mL/min. In this process, 2-4 mg sample was weighed and sealed in Aluminum pans. Later on, the sample was equilibrated at 30°C followed by heating up to 450°C at the rate of 10°C/min under nitrogen gas as purge atmosphere [32]. The thermogram reveals the value for onset, end set, peak temperature, peak height (mJ or mW), peak area, and change in heat (J/g) for each peak. Later on, the percent change in melting temperature (T) of the control and Biofield Energy Treated samples was calculated using following equation 5:

$$\% \text{change in melting temperature} = \frac{[T_{\text{Treated}} - T_{\text{control}}]}{T_{\text{control}}} \times 100$$ (5)

Where, $T_{\text{Tcontrol}}$ and $T_{\text{Treated}}$ are the melting temperature of the control and Biofield Energy Treated magnesium gluconate samples, respectively.

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

$$\% \text{change in latent heat of fusion} = \frac{[\Delta H_{\text{Treated}} - \Delta H_{\text{control}}]}{\Delta H_{\text{control}}} \times 100$$ (6)

Where, $\Delta H_{\text{control}}$ and $\Delta H_{\text{Treated}}$ are the latent heat of fusion of the control and Biofield Energy Treated magnesium gluconate, respectively.

Thermal Gravimetric Analysis (TGA) / Differential Thermogravimetric Analysis (DTG): TGA/DTG analysis was done using TGA Q500 themoanalyzer apparatus, USA under dynamic nitrogen atmosphere (50 mL/min). It involves heating the samples from 25°C to 800°C at the rate of 10ºC/min using platinum crucible [32]. In TGA analysis, the weight loss in gram as well as percent loss for each step was recorded from the thermogram with respect to the initial weight of the sample. Consequently, the DTG analysis revealed the thermogram from which, the onset, endset, peak temperature and integral area for each peak was recorded. The percent change in weight loss (W) was calculated using following equation 7:

[W - W ] %changein Weight loss = ×100 W

Treated control

(7) control Where, WControl and WTreated are the weight loss of the control and Biofield Energy Treated samples, respectively.

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

[M - M ] %changein T (M)= ×100 M

Treated control max control

(8) Where, MControl and MTreated are the Tmax values of the control and Biofield Energy Treated magnesium gluconate samples, respectively.

Ultraviolet-visible Spectroscopy (UV-Vis) Analysis: The UV-Vis spectral analysis of the control and Biofield Energy Treated magnesium gluconate samples was done with the help of 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 (λmax) were recorded.

Fourier Transform Infrared (FT-IR) Spectroscopy FT-IR spectroscopy of magnesium gluconate was done by using Spectrum ES Fourier transform infrared spectrometer (Perkin Elmer, USA) with the frequency array of 400-4000 cm-1. The process involves pressed KBr disk technique in which, ~2 mg of sample was taken along with about 300 mg of KBr as the diluent, followed by forming the pressed disk and running the sample in the spectrometer.

Powder X-ray Diffraction (PXRD) Analysis

The PXRD diffractograms of control and Biofield Energy Treated samples of magnesium gluconate are presented in Figure 1. The diffractograms showed sharp peaks that represents the crystalline nature of both samples. The PXRD data is presented in Table 1, which involves the details regarding Bragg angle (2θ), relative peak intensity (%), and crystallite size (G) for the control and Biofield Energy Treated magnesium gluconate samples. The crystallite size was calculated using Scherer equation [10, 11, 12].

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The analysis revealed that the crystallite sizes of the Biofield Energy Treated sample at 2θ equal to nearly 5.0°, 15.3°, 20.1°, and 22.4° (Table 2, entry 1, 6, 12, and 14) didn’t show any change as compared to the control sample. However, the crystallite sizes of the Biofield Energy Treated magnesium gluconate at 2θ equal to nearly 18.0°, 23.6°, 24.7°, 34.5°, and 37.6° (Table 2, entry 9, 15, 16, 20 and 22) were significantly decreased in the range from 7.16% to 25.01% with respect to the control sample. Consequently, the crystallite sizes at the other 2θ positions (Table 2, entry 2-5, 7, 8, 10, 11, 13, 17-19, 21, and 23-25) of the Biofield Energy Treated sample were significantly increased in the range from 7.14% to 185.79% as compared to the control sample. Also, the average crystallite size was significantly increased by 13.03% in the Biofield Energy Treated sample in comparison to the control sample. The possible reason behind this alteration may be that the Biofield Energy might induce the movement of crystallite boundaries that is responsible for the crystal growth and ultimately, the increased crystallite size [33]. Besides, the PXRD diffractogram of both samples showed highest peak intensity (100%) at Bragg’s angle (2θ) equal to 5.0° (Table 2, entry 1), but, the other peaks of Biofield Energy Treated sample’s diffractogram showed significant alteration in relative intensities as compared to control sample. The relative intensity of the peaks of Biofield Energy Treated sample was found to be increased in the range of 6.99% to 32.99% in comparison to the control sample, which indicated that the crystallinity of the Biofield Energy Treated sample was increased after treatment.

According to the literature, the alterations in the crystallite sizes and relative intensities indicated the changes in the morphology of the crystal and could be considered as the proof of polymorphic transition [34, 35, 36]. Since, the crystallite size of the Biofield Energy Treated sample was significantly altered as compared with the control sample; it may indicate the appearance of a new polymorphic form of magnesium gluconate after the Biofield Energy Treatment. The polymorphic form of a compound along with the crystal pattern, morphology and crystallite size plays the important role in its solubility, dissolution, and bioavailability [37]. Thus, the Biofield Energy Treatment could be considered as a very useful method for altering the crystal morphology of magnesium gluconate.

Particle Size Analysis (PSA)

The particle size and surface area of the control and Biofield Energy Treated magnesium gluconate was calculated and presented in Table 2. The particle size distribution of the control sample was observed at d10 (10.8 µm), d50 (58.5 µm), d90 (227 µm), and D(4,3) (91.7 µm). Consequently, the particle size distribution of the Biofield Energy Treated sample at d10, d50, d90, and D(4,3) was found as 10.4 µm, 45.7 µm, 177 µm, and 72.3 µm, respectively. The analysis revealed that the particle size values at d10, d50, d90, and D(4,3) in Biofield Energy Treated magnesium gluconate was significantly decreased by 3.70%, 21.88%, 22.03%, and 21.16%, respectively as compared with the control sample. However, the specific surface area (SSA) of Biofield Energy Treated magnesium gluconate (218.5m2/Kg) was significantly increased by 10.75% with respect to the control sample (197.3m2/Kg).

Table 2: Particle size distribution of the control and Biofield Energy Treated magnesium gluconate. d10, d50, and d90: particle diameter corresponding to 10%, 50%, and 90% of the cumulative distribution, D(4,3) : the average mass-volume diameter, SSA : the specific surface area; *denotes the percentage change in the particle size distribution of the Biofield Energy Treated sample with respect to the control sample.

The surface area plays an important role in the process of dissolution, as it influences the surface energy, which is the driving factor for dissolution efficiency. Moreover, it has been reported that decrease in particle size might increase the surface area [38], thus the Biofield Energy Treatment might be responsible for such alteration in the surface area that ultimately affects the dissolution profile of compound.

Differential Scanning Calorimetry (DSC) Analysis

The DSC thermograms of the control and Biofield Energy Treated magnesium gluconate exhibited two endothermic peaks (Figure 2). Since, the magnesium gluconate is generally present in the hydrate form, thus, the first peak (minor) of thermogram may be due to the evaporation of the water from the sample, whereas, the second sharp endothermic peak (major) was due to the melting point of the magnesium gluconate (Figure 2). The analysis revealed that there was a significant alteration in the vaporization temperature and latent heat of vaporization of the Biofield Energy Treated sample, as the values were decreased by 5.86% and 1.97%, respectively (Table 3), as compared to the control sample. Although, the melting point of the Biofield Energy Treated magnesium gluconate (169.80°C) was found almost similar as that of the control sample (169.97°C), but the latent heat of fusion (ΔHfusion) in the Biofield Energy Treated magnesium gluconate was slightly increased by 3.74% compared to the control sample (Table 3).

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The analysis indicated that the energy required by the Biofield Energy Treated magnesium gluconate in the form of ∆H differs from the control sample to undergo the process of melting. Hence, it is presumed that the Biofield Energy Treatment might induce some alterations in the intermolecular forces, which may be responsible for the change in ∆H. Moreover, it is also hypothesized that the Biofield Energy Treatment may cause emission of less energy in the process of phase transition from solid to liquid state, which may lead to increase in ∆H in the Biofield Energy Treated sample as compared to the control sample.

Thermal Gravimetric Analysis (TGA)/ Differential Thermogravimetric Analysis (DTG)

The TGA/DTG analysis was done for determining the thermal stability of the samples, with the help of thermograms of the control and Biofield Energy Treated magnesium gluconate (Figures 3 & 4).

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Apart from that, the TGA and DTG data for both, the control and Biofield Energy Treated samples are given in the Tables 4 & 5. The thermograms of both the samples showed three-step thermal degradation, in which, the weight losses of the Biofield Energy Treated sample at the 1st and 3rd steps were found to be increased by 10.14% and 4.86%, respectively, compared to the control sample. On the other hand, the Biofield Energy Treated magnesium gluconate showed decreased weight loss by 6.79% at the 2nd step of thermal degradation as compared to the control sample. The total weight loss of the Biofield Energy Treated sample showed a slight increase of 0.24% compared with the control sample (Table 4).

The DTG thermograms of the control and Biofield Energy Treated samples (Figure 4) exhibited four peaks. The analysis revealed that the maximum thermal degradation temperature (Tmax) of 1st and 2nd peaks of the Biofield Energy Treated sample were increased by 6.90% and 1.28%, as compared to the control sample (Table 5). Besides, the Tmax of 3rd and 4th peaks of the Biofield Energy Treated sample were slightly decreased by 0.57% and 0.24% compare to the control sample (Table 5). Overall, the TGA/DTG analysis revealed that the thermal stability of the Biofield Energy Treated magnesium gluconate was altered as compared to the control sample, which might be due to the alteration in particle size of the treated sample after the Biofield Energy Treatment.

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Ultraviolet-visible Spectroscopy (UV-Vis) Analysis

The UV-visible spectra of both, the control and Biofield Energy Treated magnesium gluconate samples are presented in Figure 5.

The spectra of both, the control and Biofield Energy Treated samples showed the maximum absorbance (λmax) at 202 nm, thus, no significant alteration was found in the absorbance maxima between the control and Biofield Energy Treated sample.

It reveals that there might not be any significant change in the electronic transitions between highest occupied molecular orbital and lowest unoccupied molecular orbital induced by the Biofield Energy Treatment.

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Fourier Transform Infrared (FT-IR) Spectroscopy

The FT-IR spectra of both the control and Biofield Energy Treated magnesium gluconate are shown in Figure 6 and it showed the clear stretching and bending peaks in both the functional group as well as the fingerprint region. The spectra of both the samples showed broad peaks in the functional group area near 3398 cm-1, which may be due to the O-H stretching present in the primary and secondary alcohol and two water molecules.

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The peak may arise due to the stretching vibrations of hydroxyl groups originating from the water present in magnesium gluconate in its hydrate form. Moreover, the functional region also involves the stretching vibrations due to the primary and secondary hydroxyl groups of the gluconate part, which may not be visible due to the intensive broad band of water [36]. Moreover, the peaks for the deformation vibration of the hydroxyl groups in the plane δ (OH) and out-of-plane γ (OH) regarding the primary and secondary hydroxyl groups were observed at 1435 cm-1 and 636 cm-1 and 577 cm-1, respectively in the spectrum of the control sample, whereas, at 1434 cm-1 and 636 cm-1 and 577 cm-1, respectively in the spectrum of Biofield Energy Treated magnesium gluconate hydrate. The spectra of both the samples showed C-H stretching at 2934 cm-1. Similarly, the intensive peaks at 1606 and 1380 cm-1 in the spectra of both the samples were may be due to the asymmetric and symmetric vibrations of C=O of a carbonyl group of carboxylate anion. Later on, the fingerprint region of the control sample and Biofield Energy Treated sample was observed to be same. The peak regarding C-O stretching vibrations of the primary alcohol group was perceived at 1056 cm-1 in the spectra of both the control and Biofield Energy Treated sample. Similarly, the absorption peaks due to the C-O stretching vibrations of the secondary alcohol groups were observed at 1229 cm-1 and 1142 cm-1 in the spectra of both the samples. The overall FT-IR analysis indicated that there was no significant alteration in the frequency of the characteristic peaks in the Biofield Energy Treated sample compared to the control samples. Hence, it can be concluded that the structure of the magnesium gluconate hydrate remained the same in both the Biofield Energy Treated and control samples.