Evaluation of Structural Properties and Isotopic Abundance Ratio of Biofield Energy Treated (The Trivedi Effect®) Magnesium Gluconate Using LC-MS and NMR

The current research work was designed to explore the impact of The Trivedi Effect Energy of Consciousness Healing Treatment (Biofield Energy Healing Treatment) on magnesium gluconate for the change in the structural properties and isotopic abundance ratio (PM+1/PM and PM+2/PM) by using LC-MS and NMR spectroscopy. Magnesium gluconate was divided into two parts – one part was control, and another part was treated with The Trivedi Effect ® Biofield Energy Healing Treatment remotely by seven renowned Biofield Energy Healers and defined as The Trivedi Effect ® Treated sample. The LCMS analysis of the both control and treated samples revealed the presence of the mass of the protonated magnesium gluconate at m/z 415 at the retention time of 1.53 min with similar fragmentation pattern. The relative peak intensities of the fragment ions of the treated sample were significantly changed compared with the control sample. The proton and carbon signals for CH, CH2 and CO groups in the proton and carbon NMR spectra were found almost similar for the control and the treated samples. The isotopic abundance ratio analysis revealed that the isotopic abundance ratio of PM+1/PM ( 2 H/ 1 H or 13 C/ 12 C or 17 O/ 16 O or 25 Mg/ 24 Mg) in the treated sample was significantly increased by 34.33% compared with the control sample. Subsequently, the percentage change of the isotopic abundance ratio of PM+2/PM ( 18 O/ 16 O or 26 Mg/ 24 Mg) was significantly decreased in the treated sample by 64.08% as compared to the control sample. Briefly, 13 C, 2 H, 17 O, and 25 Mg contributions from (C12H23MgO14) + to m/z 416; 18 O and 26 Mg contributions from (C12H23MgO14) + to m/z 417 in the treated sample were significantly changed compared with the control sample. Thus, the treated magnesium gluconate could be valuable for designing better pharmaceutical and/or nutraceutical formulations through its changed physicochemical and thermal properties, which might be providing better therapeutic response against various diseases such as diabetes mellitus, allergy, aging, inflammatory diseases, immunological disorders, and other chronic infections. The Biofield Energy Treated magnesium gluconate might be supportive to design the novel potent enzyme inhibitors by using its kinetic isotope effects.


Introduction
Magnesium ion (Mg 2+ ) is a major intracellular divalent cation. It is an essential mineral for several enzymes, DNA and RNA synthesis, reproduction and protein synthesis as well as a vital coherent controller of glycolysis and the Krebs cycle [1,2]. Magnesium gluconate (C 12 H 22 MgO 14 ) is the organometallic salt of magnesium with gluconic acid produced from glucose catalyzed by glucose oxidase [3]. Magnesium gluconate is found to be the most powerful antioxidant than other magnesium salts and it is useful for the prevention and treatment of many diseases such as cardiovascular diseases, diabetes mellitus, allergy, inflammatory diseases, immunological disorders, Alzheimer's disease, asthma, pre-eclampsia and eclampsia, cancer, etc. [4][5][6][7][8]. It can be used as neuroprotective [9], for the treatment of oxidative stress induced ischemia/reperfusion injury [10] and also labor in women arrested initially with intravenous therapy as an oral tocolytic agent [11]. This organometallic salt can be used in a skintightening cosmetic composition [12]. Magnesium gluconate showed the highest bioavailability and most physiologically acceptable salt among other magnesium salts like chloride, sulfate, carbonate, acetate, citrate, lactate, aspartate, etc. [8,13]. Therefore, magnesium gluconate was considered as one of the components in a novel proprietary herbomineral formulation for the source of magnesium ion. This herbomineral formulation which is designed as nutraceutical supplement can be used for the prevention and treatment of various human diseases.
Since ancient times, many different cultures, religions and systems of belief have recognized a living force that preserves and inhabits every living organism. This force is the source of 'life' and has been called various names, such as prana by the Hindus, qi or chi by the Chinese, and ki by the Japanese. This is believed to co-relate with the soul, spirit and mind. This hypothetical vital force has been scientifically evaluated and is now considered the Bioenergetics Field. The Biofield Energy is a dynamic electromagnetic field surrounding the human body, resulting from the continuous emission of low-level light, heat, and acoustical energy from the body. Biofield Energy is infinite, paradimensional and can freely flow between the human and environment [14,15]. So, a human has the ability to harness energy from the ionosphere of the earth, the "universal energy field", and transmit it to any living organism(s) or nonliving object(s) around the globe. The object or recipient always receives the energy and responds in a useful way. This process is known as The Trivedi Effect ® -Biofield Energy Healing Treatment [16,17]. Biofield (Putative Energy Field) based Energy Therapies are used worldwide to promote health and healing. The National Center of Complementary and Integrative Health (NCCIH) has recognized and accepted Biofield Energy Healing as a Complementary and Alternative Medicine (CAM) health care approach in addition to other therapies, medicines and practices such as natural products, deep breathing, yoga, Tai  Chi,  Qi  Gong,  chiropractic/osteopathic manipulation, meditation, massage,  special diets, homeopathy, progressive relaxation, guided  imagery, acupressure, acupuncture, relaxation techniques,  hypnotherapy, healing touch, movement therapy, pilates, rolfing structural integration, mindfulness, Ayurvedic medicine, traditional Chinese herbs and medicines, naturopathy, essential oils, aromatherapy, Reiki, cranial sacral therapy and applied prayer (as is common in all religions, like Christianity, Hinduism, Buddhism and Judaism) [18]. Biofield Energy Treatment (The Trivedi Effect ® ) has been extensively studied with significant outcomes in many scientific fields such as cancer research [19], altered antimicrobial sensitivity of pathogenic microbes in microbiology [20,21], genetics [22,23], biotechnology [24,25], altered physical and chemical properties of pharmaceuticals [26,27], nutraceuticals [28,29], organic compounds [30,31], improved overall growth and yield of plants in agricultural science [32,33], and changing the structure of the atom in relation to various metals, ceramics, polymers and chemicals in materials science [34,35]. The scientific study indicated that Biofield Energy Healing Treatment (The Trivedi Effect ® ) might be an alternate method for increasing or decreasing the natural isotopic abundance ratio of the substances [36][37][38]. The stable isotope ratio analysis has the wide applications in several scientific fields for understanding the isotope effects resulting from the variation of the isotopic composition of the molecule [39,40]. Conventional mass spectrometry (MS) techniques such as liquid chromatography -mass spectrometry (LC-MS), gas chromatography -mass spectrometry (GC-MS) are widely used for isotope ratio analysis with sufficient precision [41]. Hence, LC-MS and NMR (Nuclear Magnetic Resonance) were used in this study to characterize the structural properties of the Biofield Energy Treated and untreated magnesium gluconate qualitatively for the purpose of the pharmaceutical and nutraceutical industrial applications. Consequently, LC-MS based isotopic abundance ratio (P M+1 /P M and P M+2 /P M ) analysis in both the Biofield Energy Treated and untreated samples was designed to investigate the influence of The Trivedi Effect ® -Energy of Consciousness Healing Treatment on the isotopic abundance ratio in magnesium gluconate.

Chemicals and Reagents
Magnesium gluconate hydrate was purchased from Tokyo Chemical Industry Co., Ltd. (TCI), Japan. All other chemicals used in the experiment were of analytical grade available in India.

Energy of Consciousness Healing Treatment Strategies
Magnesium gluconate hydrate was one of the components of the new proprietary herbomineral formulation, which was developed by our research team and was used per se as the test compound for the current study. The test compound was divided into two parts, one part of the test compound did not receive any sort of treatment and was defined as the untreated or control magnesium gluconate sample. The second part of the test compound, which was denoted as the Biofield Energy Treated sample, was treated with the Biofield Energy by the group of seven renowned Biofield Energy Healers (The Trivedi Effect ® ) remotely. Six Biofield Energy Healers were remotely located in the U.S.A. and one of which was remotely located in Canada, while the test compound was located in the research laboratory of GVK Biosciences Pvt. Ltd., Hyderabad, India. This Biofield Treatment was provided for 5 minutes through the Healer's Unique Energy Transmission process remotely to the test compound, which was kept under laboratory conditions. None of the Biofield Energy Healers in this study visited the laboratory in person, nor had any contact with the compounds. Similarly, the control compound was subjected to "sham" healer for 5 minutes, under the same laboratory conditions. The sham healer did not have any knowledge about the Biofield Energy Treatment. After that, the Biofield Energy Treated and untreated samples were kept in similar sealed conditions and characterized thoroughly by LC-MS and NMR spectroscopy.

Liquid Chromatography Mass Spectrometry (LC-MS) Analysis
Liquid chromatography was performed using The Waters ® ACQUITY UPLC, Milford, MA, USA equipped with a binary pump (The Waters ® BSM HPLC pump), autosampler, column heater and a photo-diode array (PDA) detector. The column used for the study was a reversed phase Acquity BEH shield RP C18 (150 X 3.0 mm, 2.5 µm). The column temperature was kept constant at 40˚C. The mobile phase was 2mM ammonium acetate in water as mobile phase A and acetonitrile as mobile phase B. Chromatographic separation was achieved with following gradient program: 0 min -5%B; 1 min -5%B; 15 min -97%B; 20 min -97%B; 21 min -5%B; 25 min -5%B. The flow rate was at a constant flow rate of 0.4 mL/min. The control and Biofield Energy Treated samples were dissolved in a mixture of water and methanol (60:40 v/v) to prepare a 1 mg/mL stock solution. An aliquot of 2 µL of the stock solution was used for analysis by LC-ESI-MS and the total run time was 25 min.
Mass spectrometric analysis was accompanied on a Triple Quad (Waters Quattro Premier XE, USA) mass spectrometer equipped with an electrospray ionization (ESI) source with the following parameters: electrospray capillary voltage 3.5 kV; source temperature 100°C; desolvation temperature 350°C; cone voltage 30 V; desolvation gas flow 1000 L/h and cone gas flow 60 L/h. Nitrogen was used in the electrospray ionization source. The multiplier voltage was set at 650 V. LC-MS was taken in positive ionization mode and with the full scan (m/z 50-1400). The total ion chromatogram, % peak area and mass spectrum of the individual peak (appeared in LC) were recorded.

Isotopic Abundance Ratio Analysis
The relative intensity of the peak in the mass spectra is directly proportional to the relative isotopic abundance of the molecule and the isotopic abundance ratio analysis was followed the scientific literature reported [36][37][38]42] method described as below: P M stands for the relative peak intensity of the parent molecular ion [M + ] expressed in percentage. In other way, it indicates the probability to A elements having only one natural isotope in appreciable abundance (for e.g. 12 C, 1 H, 16 O, 24  i.e. the probability to A + 1 elements having an isotope that has one mass unit heavier than the most abundant isotope (for e.g. 13  i.e. the probability to have A + 2 elements having an isotope that has two mass unit heavier than the most abundant isotope (for e.g. 18 O, 26 Mg, etc.) contributions to the mass of isotopic molecular ion [(M+2) + ].

Nuclear Magnetic Resonance (NMR) Analysis
1 H NMR spectra were recorded in a 400 MHZ VARIAN FT-NMR spectrometer at room temperature. Data refer to solutions in D 2 O with the residual solvent protons as internal references. 1 H NMR multiplicities were designated as singlet (s), doublet (d), triplet (t), multiplet (m), and broad (br). 13 C NMR spectra were measured at 100 MHz on a VARIAN FT-NMR spectrometer at room temperature. Chemical shifts (δ) were in parts per million (ppm) relative to the solvent's residual proton chemical shift (D 2 O, δ = 4.65 ppm) and solvent's residual carbon chemical shift (D 2 O, δ = 0 ppm).

Liquid Chromatography-Mass Spectrometry (LC-MS) Analysis
The liquid chromatograms of both the control and Biofield Energy Treated magnesium gluconate ( Figure 1) exhibited a sharp peak at the retention time (R t ) of 1.53 min. This result clearly indicated that the polarity/affinity of the Biofield Energy Treated sample remained unchanged compared with the control sample. The ESI-MS spectra of both the control and Biofield Energy Treated magnesium gluconate at R t of 1.53 min as shown in the Figure 2     , 342) as shown in the Figure 3. By this way removing water along with other groups like alkyl, magnesium gluconate produces different fragmentation ion peaks at m/z 320, 307, 284, 279, 271, 254, 225, 206, 183, 179, 165, 142, 135, 123, 114, and 100 which were observed in the ESI-MS spectra of both the control and Biofield Energy Treated samples ( Figure 2). These ions correspond to the following proposed molecular formula C 10  , respectively as shown in Figure 3. The ESI-MS spectra of both the control and Biofield Energy Treated samples (Figure 2) exhibited that the fragmentation pattern of both the control and Biofield Energy Treated samples were the same. The fragmented ion at m/z 165 corresponding to C 5 H 9 O 6 + exhibited 100% relative peak intensity in the control sample, while fragment ion at m/z 135 corresponding to C 5 H 11 O 4 + showed 100% relative peak intensity in the Biofield Energy Treated sample (Figure 2). The relative peak intensities of the Biofield Energy Treated sample were significantly altered compared with the control sample.

Isotopic Abundance Ratio Analysis
The molecular formula of magnesium gluconate is C 12 Table 2.  The isotopic abundance ratio of P M+1 /P M in the Biofield Energy Treated sample was significantly increased by 34.33% with respect to the control sample (Table 2). Consequently, the percentage change of the isotopic abundance ratio of P M+2 /P M was remarkably decreased by 64.08% in the Biofield Energy Treated sample compared with the control sample (Table 2). So, 13   Scientific literature [40][41][42]45] reported that the vibrational energy is closely related with the reduced mass (µ) of the compound and the alteration of the vibrational energy can affect the several properties like physicochemical, thermal properties of the molecule. The relation between the vibrational energy and the reduced mass (µ) for a diatomic molecule is expressed as below (Equation 1) [40,45] Where E 0 = the vibrational energy of a harmonic oscillator at absolute zero or zero point energy f = force constant μ = reduced mass = + Where m a and m b are the masses of the constituent atoms. The alteration in the isotopic abundance ratios of 13  Mass spectroscopic analysis of the several organic compounds revealed that the isotopic abundance of [M+1] + and [M+2] + ions were increased or decreased, thereby suggesting the change in number of neutrons in the molecule. It was then postulated to the alterations in atomic mass and atomic charge through possible mediation of neutrino oscillation [46,47]. It is then assumed that The Trivedi Effect ® -Energy of Consciousness Healing Treatment might provide the required energy for the neutrino oscillations. The changes of neutrinos inside the molecule in turn modified the particle size, chemical reactivity, density, thermal behavior, selectivity, binding energy etc. [46]. Kinetic isotope effect that is resultant from the variation in the isotopic abundance ratio of one of the atoms in the reactants in a chemical reaction is very useful to study the reaction mechanism as well as for understanding the enzymatic transition state and all aspects of enzyme mechanism that is supportive for designing enormously effective and specific inhibitors [40,45,48]. As magnesium is an essential cofactor for various enzymatic reactions, Biofield Energy Treated magnesium gluconate that had altered isotopic abundance ratio might be advantageous for the study of enzyme mechanism as well as support in the designing of novel potent enzyme inhibitors.

Nuclear Magnetic Resonance (NMR) Analysis
The 1 H and 13 C NMR spectra of the control and Biofield Energy Treated magnesium gluconate are presented in the Figures 4 and 5, respectively. NMR assignments of the control and Biofield Energy Treated magnesium gluconate are presented in the Table 4.   br-broad, s-singlet, and m-multiplet, * These assignments can be switched.
Although magnesium gluconate contains a large number of hydroxyl (OH) groups, the proton spectra of both the control and Biofield Energy Treated samples did not show any signal for the hydroxyl protons. The scientific literature mentioned that when deuterated water was used as solvent for spectra recording, the hydroxyl protons were replaced by deuterium from deuterated water. The signals for the protons coupling of CH 2 group and adjacent CH protons (2)(3)(4)(5) in the gluconic acid portion were observed in the range of δ 3.60-4.20 ppm in the proton spectrum of sodium gluconate [49]. The proton signals for CH 2 and CH groups for the control sample were observed in the range of δ 3.47-4.015 ppm (Table 4), whereas these signals were noticed in the Biofield Energy Treated in the range of δ 3.43-3.995 ppm. Similarly, the carbon signals for CO group, CH 2 and CH groups in the 13 C NMR spectrum of the Biofield Energy Treated sample were slightly decreased compared with the control sample (Table 4). These results indicated that the structure of the magnesium gluconate remained unchanged due to the Biofield Energy Healing Treatment.

Conclusions
The current study successfully demonstrated the structural characterization of magnesium gluconate by using LC-MS and NMR techniques and a significantly impact of The Trivedi Effect ® -Energy of Consciousness Healing Treatment (Biofield Energy Healing Treatment) on the isotopic abundance ratios of P M+1 /P M and P M+2 /P M . The LC-MS analysis of the both control and Biofield Energy Treated samples revealed the presence of the mass of the protonated magnesium gluconate at m/z 415 at the retention time of 1.53 min with similar fragmentation pattern. The relative peak intensities of the fragment ions of the treated sample were significantly changed compared with the control sample. The isotopic abundance ratio of P M+1 /P M ( 2 H/ 1 H or 13 26 Mg contributions from (C 12 H 23 MgO 14 ) + to m/z 417 in the treated sample were significantly altered with respect to the control sample. The treated sample might exhibit isotope effects such as altered physicochemical and thermal properties, rate of the reaction, selectivity and binding energy due to its changed isotopic abundance ratios of P M+1 /P M and P M+2 /P M as compared to the control sample. The treated magnesium gluconate might be helpful to understand the enzymatic reactions as well as design the novel potent enzyme inhibitors by using its kinetic isotope effects. Besides, The Trivedi Effect ® -Energy of Consciousness Healing Treatment, could be a useful approach in the design of better nutraceutical and/or pharmaceutical formulations