Effect of the Energy of Consciousness (The Trivedi Effect®) on the Structural Properties and Isotopic Abundance Ratio of Magnesium Gluconate Using LC-MS and NMR Spectroscopy

Magnesium gluconate has the wide application for the prevention and treatment of hypomagnesemia. The objective of the current study was to investigate the effect 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) 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 LC-MS analysis of both the 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 minutes with almost similar fragmentation pattern. The relative peak intensities of the fragment ions of the treated sample were significantly altered compared with the control sample. The proton and carbon signals for CH, CH2 and CO groups in the proton and carbon NMR spectra were observed almost similar for the control and the treated samples. The isotopic abundance ratio analysis revealed that the percentage of 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) was significantly increased in treated sample by 80.38%, compared with 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 increased 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 Trivedi Effect ® treated magnesium gluconate might be supportive to design the novel potent enzyme inhibitors using its kinetic isotope effects.


Introduction
Magnesium is a second most abundant mineral in the human body. It is an essential mineral for more than 300 enzymes, DNA and RNA synthesis, reproduction and protein synthesis as well as a vital coherent controller of glycolysis and the Krebs cycle [1][2][3]. Low level of magnesium i. e. hypomagnesemia may cause several diseases and disorders such as diabetes mellitus, allergies, septic shock, inflammatory diseases, immunological disorders, asthma, arrhythmias, acute myocardial infarction, gestational hypertension, preeclampsia, eclampsia, Alzheimer's disease, cancer, etc. [4][5][6]. Magnesium gluconate (C 12 H 22 MgO 14 ) is the organometallic salt of magnesium with gluconic acid produced from glucose catalyzed by glucose oxidase [7]. Magnesium gluconate is found to be the most powerful antioxidant than other magnesium salts [8]. Magnesium gluconate exhibits the highest level of bioavailability of magnesium and is more physiologically acceptable salt among the commercially available magnesium salts such as chloride, sulfate, citrate, lactate, aspartate, etc. [9,10]. Thus, magnesium gluconate has the important application for the prevention and treatment of hypomagnesemia and also oxidative stress induced ischemia/reperfusion injury [8,9,11]. It can be used as neuroprotective [12], an oral tocolytic agent [13], and also in a skin-tightening cosmetic composition [14]. Subsequently, magnesium gluconate was considered as one of the components in a novel proprietary herbomineral formulation, which is designed as nutraceutical for the source of magnesium ion.
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, para-dimensional and can freely flow between the human and environment [15]. F. Sances et al. reported that Biofield Energy can be transmitted into any living organism(s) or nonliving object(s) around the globe with scientifically measurable effect through the intentional mental energies by specific energy healers [16]. The object or recipient always receives the energy from the ionosphere of the earth, the "universal energy field" and responds in a useful way. This process is known as The Trivedi Effect ® -Biofield Energy Healing Treatment [17,18]. 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) [19]. Biofield Energy Treatment (The Trivedi Effect ® ) has been drawn attention more in the recent times for its scientifically measurable capability to transform the characteristic properties of a wide varieties living and non-living substances such as animals [20], cancer cells [21], microbes [22,23], plants [24,25], medium [26,27], materials [28,29], pharmaceuticals [30,31], nutraceuticals [32,33], organic compounds [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][39]. 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 [40,41]. 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 [42]. Hence, LC-MS and NMR (Nuclear Magnetic Resonance) methods were applied 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 ) analysis in both the Biofield Energy Treated and untreated samples was designed to explore the effect 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 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 Advances in Biochemistry 2017; 5(1): 7-15 9 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 was denoted as The Trivedi Effect ® -Energy of Consciousness Healing Treatment (Biofield Energy Healing Treatment) treated sample. The treated magnesium gluconate was subjected to The Trivedi Effect ® by the group of seven renowned Biofield Energy Healers remotely. All seven Biofield Energy Healers were remotely located in the U. S. A., while the test compound was located in the research laboratory of GVK Biosciences Pvt. Ltd., Hyderabad, India. The Trivedi Effect ® -Energy of Consciousness Healing Treatment (Biofield Energy Healing 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 Trivedi Effect ® -Energy of Consciousness Healing Treatment (Biofield Energy Healing Treatment). After that, 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 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 minutes. 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][39] 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  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 Where, IAR Treated = isotopic abundance ratio in the treated sample and IAR Control = isotopic abundance ratio in the control sample.

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 Isotopic Abundance Ratio of Magnesium Gluconate Using LC-MS and NMR Spectroscopy 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 The Trivedi Effect ® Treated (Biofield Energy Treated) magnesium gluconate as shown in Figure 1 showed a sharp and narrow peak at the retention time (R t ) of 1.53 minutes.

Isotopic Abundance Ratio Analysis
The molecular formula of magnesium gluconate is The calculated isotopic abundance of P M+1 value was near to the observed value (Table 2). Thus, the probability of A + 1 elements having an isotope having one mass unit heavier than the most abundant isotope (e. g. 13 C, 2 H, 17 O, and 25 Mg) contributions to the mass of the isotopic molecular ion [M+1] + . Deuterium did not contribute much any isotopic m/z ratios because of less natural abundance relative to the natural abundances of carbon, oxygen and magnesium isotopes [43,44]. The calculation indicated that 13 C and 25 Mg have the major contributions from magnesium gluconate to the isotopic peak at m/z 416.
LC-MS spectra of the control and treated samples indicated the presence of the mass for the protonated magnesium gluconate (m/z 415). Hence, P M and P M+1 for magnesium gluconate at m/z 415 and 416 of the control and treated samples were obtained from the observed relative peak intensities of [M + ] and [(M+1) + ] peaks, respectively in the respective ESI-MS spectra are presented in Table 2. Isotopic Abundance Ratio of Magnesium Gluconate Using LC-MS and NMR Spectroscopy  The isotopic abundance ratio of P M+1 /P M in the treated sample was significantly increased by 80.38% compared with the control sample (Table 2). So, 13 C, 2 H, 17 O, and 25 Mg contributions from (C 12 H 23 MgO 14 ) + to m/z 416 in the treated sample was significantly increased compared with the control sample.
Scientific literature [37][38][39]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 [42,45]: 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.  Table 3 that leads to the isotope effects of the molecule.
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 [37][38][39]46]. Thus, it is assumed that The Trivedi Effect ® -Energy of Consciousness Healing Treatment might offer 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 [42,45,47]. As magnesium is an essential cofactor for various enzymatic reactions, The Trivedi Effect ® 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 treated magnesium gluconate are presented in the Figures 4 and 5, respectively. NMR assignments of the control and treated magnesium gluconate are presented in the Table 4. Although magnesium gluconate contains a large number of hydroxyl (OH) groups, the proton spectra of both the control and treated samples did not show any signal for the hydroxyl protons due to the replacement of the hydroxyl protons by deuterium from deuterated water, which was used as solvent for spectra recording.
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 control and treated samples in the range of δ 3.48 to 4.02 ppm (Table 4), which was almost in accordance with the proton spectrum of sodium gluconate [48].
br-broad, s-singlet, d-doublet, and dd-doublet of doublet, * These assignments can be switched.
Similarly, the carbon signals for CO group, CH 2 and CH groups in the 13 C NMR spectrum of the treated sample were almost similar compared with the control sample (Table 4).
So, the structure of the magnesium gluconate in the treated samples remained same with the control sample.

Conclusions
The current research work envisaged the structural characterization of magnesium gluconate using LC-MS and NMR techniques and a significant influence of The Trivedi Effect ® -Energy of Consciousness Healing Treatment (Biofield Energy Healing Treatment) on the isotopic abundance ratio of P M+1 /P M . The LC-MS 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 minutes with the nearly same type of fragmentation. The relative peak intensities of the fragment ions of The Trivedi Effect ® 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 C/ 12 C or 17 O/ 16 O or 25 Mg/ 24 Mg) was significantly increased in the treated sample by 80.38% compared with the control sample. Briefly, 13 C, 2 H, 17 O, and 25 Mg contributions from (C 12 H 23 MgO 14 ) + to m/z 416 in the treated sample was significantly increased compared to the control sample. The Trivedi Effect ® treated sample might exhibit isotope effects such as altered physicochemical and thermal properties, rate of the reaction, selectivity and binding energy due to its reduced isotopic abundance ratios of P M+1 /P M and P M+2 /P M compared with the control sample. The Trivedi Effect ® treated magnesium gluconate might be helpful to understand the enzymatic reactions as well as design the novel potent enzyme