A Comprehensive Physical , Spectroscopic , and Thermal Characterization of Withania somnifera ( Ashwagandha ) Root Extract Treated with the Energy of Consciousness ( The Trivedi Effect ® )

Withania somnifera (Ashwagandha) root extract possesses a broad range of pharmacological activities. The aim of current study was to explore the impact of The Trivedi Effect Energy of Consciousness Healing Treatment (Biofield Energy Healing Treatment) on the physical, spectroscopic, and thermal properties of ashwagandha root extract using PXRD, PSD, FTIR, UV-vis spectroscopy, TGA, and DSC analysis. Ashwagandha root extract was divided into two parts. One part was denoted as the control, while the other part was defined as The Trivedi Effect Biofield Energy Treated sample, which received The Trivedi Effect Treatment remotely from eighteen renowned Biofield Energy Healers. The PXRD analysis exhibited that both the control and The Trivedi Effect Biofield Energy Treated samples were amorphous in nature. The particle size values at d10 and d50 of the treated sample were decreased by 4.66% and 6.02% and at d90 slightly increased by 0.99% compared with the control sample. The average surface area of the treated (0.130 m/g) sample was increased by 4.84% compared with the control (0.124 m/g) sample. The FT-IR results suggested that the force constant of O-H (stretching), and C-O (stretching) functional groups of the treated sample were reduced compared with the control sample. The UV-vis analysis revealed that the wavelength for the maximum absorbance of the control and treated samples were at 205.4 nm and 205.6 nm, respectively. The TGA thermograms exhibited three steps of the degradation process, and the total weight loss of the treated (86.45%) sample was increased by 1.44% compared with the control (85.22%) sample. The DSC analysis revealed that melting point of the treated (201.67°C) sample was reduced by 0.87% compared with the control (203.43°C) sample. The latent heat of fusion of the treated (6.77 J/g) sample was significantly decreased by 21.82% compared to the control (8.67 J/g) sample. Hence, the treated sample would be more advantageous for the better therapeutic responses compared to the control sample. These findings suggest that The Trivedi Effect Energy of Consciousness Healing Treatment might have the astounding capacity to 6 Mahendra Kumar Trivedi et al.: A Comprehensive Physical, Spectroscopic, and Thermal Characterization of Withania somnifera (Ashwagandha) Root Extract Treated with the Energy of Consciousness (The Trivedi Effect) enhance the solubility, dissolution, absorption, and bioavailability of ashwagandha root extract in various forms of pharmaceutical/nutraceutical formulations by modifying its particle size and surface area. Thus, The Trivedi Effect Energy of Consciousness Healing Treated ashwagandha root extract might provide better therapeutic response against inflammatory diseases, immunological disorders, stress, arthritis, cancer, diabetes, sexual disorders, aging and other chronic infections.


Introduction
The importance of the herbal medicines in the prevention and treatment of the various diseases has been increased dayby-day throughout the world due to its impressive therapeutic effects and fewer side effects compared to the modern medicines [1]. The roots of Withania somnifera (W. somnifera) (L.) Dunal (Family-Solanaceae) -a vital Rasayana herb is traditionally known as 'Ashwagandha' or winter cherry. In Ayurveda, it is called as 'Indian ginseng'. Ashwagandha is widely used in the most of the Indian herbal drugs and nutraceuticals for the treatment of various diseases include nervous and sexual disorders, infectious diseases, diabetes, cancer, ulcer, immunological disorders, stress, arthritis, etc. From the ancient time, it is used as a tonic to arrest the ageing process, rejuvenate the body and boost the defence against infectious disorders and also promote the longevity [2][3][4][5][6][7][8][9][10][11]. Ashwagandha root extract contains a wide array of nutrients and phytochemicals. Hence, it is used as a dietary supplement for the health restoration [3]. The major active phytoconstituents of ashwagandha root extract are highly oxygenated withanolides. Withanolides have C28 steroidal nucleus with C9 side chain, having a six membered lactone ring (Figure 1). The oxidation at various sites of the skeleton is responsible for the structural deviations in different classes of withanolides [11][12][13][14]. Literatures reported that withanolides such as withaferin A, withanolide D, withanolide E, etc. possesses various pharmacological activities include antioxidant, anticancer, immunomodulating, neuroprotective, antiepileptic, antibacterial, adaptogenic, spermatogenic, antidepressant, antianxiety, hepatoprotective, anti-inflammatory, antiarthritic, antimicrobial, hypoglycemic, hypolipidemic, aphrodisiac, antiulcer, radiosensitizing, etc. [11,[14][15][16][17][18][19]. Therefore, a new proprietary herbomineral formulation was formulated that consisted of the herbal product like ashwagandha root extract along with minerals such as zinc, magnesium, and selenium. This herbomineral formulation is designed as nutraceutical supplement and can be used for the prevention and treatment of various human disorders.
Every living organism preserves some kind of unique quality, an élan vital or vital force, which contributes the 'life'. From the ancient-time, this living force is known as Prana by the Hindus, qi or chi by the Chinese, and ki by the Japanese and is usually believed to create the source of life that is related with soul, spirit, and mind. Now-a-days, this hypothetical vital force is considered as the Bioenergetics Field. This energy field is a dynamic electromagnetic field existing surround of the human body. The Biofield Energy is infinite and paradimensional. It can freely flow between the human and the environment that leads to the continuous movement or matter of energy [20,21]. Thus, the human can harness energy from the earth, the "universal energy field" and transmit it to any living or nonliving object (s) around the globe. The objects always receive the energy and respond in a useful way. This process is known as Biofield Energy Healing Treatment [22,23]. Biofield (Putative Energy Fields) based Energy Therapies are used worldwide to promote health and healing [24]. The National Center of Complementary and Integrative Health (NCCIH) has been 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) [25]. The Biofield Energy Treatment (The Trivedi Effect ® ) has been extensively studied with significant outcomes in many scientific fields such as cancer research [26], altered antimicrobial sensitivity of pathogenic microbes in microbiology [27][28][29], biotechnology [30,31], genetics [32,33], changing the structure of the atom in relation to the various metals, ceramics, polymers and chemicals materials science [34][35][36], altered physical and chemical properties of pharmaceuticals [37,38], nutraceuticals [39,40], organic compounds [41][42][43], and improved overall growth and yield of plants in agricultural science [44,45]. Herbal extracts and herbal drugs despite of their outstanding in vitro results exhibited poor or negligible in vivo activity, because of their reduced lipid solubility or improper molecular size, causing deprived absorption and thus poor bioavailability [1]. According to the recent study on the bioavailability of major withanolides of W. somnifera, Devkar et al. demonstrated that the nonpolar and low molecular weight withanolides were highly permeable, whereas the high glycosylated and polar withanolides displayed low permeability in their in vitro absorption model system [46]. As ashwagandha root extract has the outstanding nutritional and medicinal values, researchers are still working on to find out an optimal dosage range for reproducing the desired effects in human as well as to determine the safe, non-toxic, and effective dosage form [7]. The physicochemical properties such as particle size, crystalline structure, crystallite size, surface area, etc. and thermal properties of a drug have a vital role in bioavailability as well as the stability of the drug during processing, formulation, storage, and packaging [47,48]. Biofield Energy Treatment (The Trivedi Effect ® ) has been reported to change the particle size, specific surface area, crystalline, chemical and thermal behaviour of an atom/ion through the possible mediation of neutrinos [49]. By considering all these aspects, the objective of the current study was to examine whether the Biofield Energy Treatment can change the physical, structural, and thermal properties in such a way that might be assisting in the improvement of the solubility, absorption and also help in designing of any suitable pharmaceutical formulation. The physical, spectroscopic, and thermal properties of both the control and Biofield Energy Treated ashwagandha root extracts were evaluated using various analytical techniques include Fourier transform infrared (FT-IR) spectrometry, ultraviolet-visible (UV-vis) spectroscopy, powder X-ray diffraction (PXRD), particle size distribution analysis (PSD), thermogravimetric analysis (TGA), and differential scanning calorimetry (DSC).

Chemicals and Reagents
Withania somnifera (ashwagandha) hydroalcoholic root extract was purchased from Sanat Product Ltd, India. All the other chemicals used in this experiment were analytical grade procured from the local vendors.

Energy of Consciousness Healing Treatment Strategy
Ashwagandha root extract powder was one of the components of the new proprietary herbomineral formulation, developed by our research team, and it 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 was treated with The Trivedi Effect ® -Energy of Consciousness Healing Treatment by renowned Biofield Energy Healers and defined as The Trivedi Effect ® sample, while the second part of the test compound did not receive any sort of treatment and defined as untreated or control ashwagandha root extract sample. The group of eighteen Biofield Energy Healers who participated in this study performed The Trivedi Effect ® -Energy of Consciousness Healing Treatment remotely to the test compound. Eleven of the Biofield Energy Healers were located in the U. S. A., four in Canada, one in Ireland, one in the United Kingdom, and one in Russia performed The Trivedi Effect ® on the test compound that was located in the research laboratory of GVK Biosciences Pvt. Ltd., Hyderabad, India. This Biofield Energy Treatment was provided for 5 minutes through Healer's Unique Energy Transmission process remotely to the test compound under the 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" healers for 5 minutes, under the same laboratory conditions. The sham healer did not have any knowledge about The Trivedi Effect ® -Biofield Energy Treatment. After that, the treated and untreated samples were kept in similar sealed conditions and were characterized thoroughly by PXRD, PSD, FT-IR, UV-visible spectroscopy, TGA, and DSC analysis.

Powder X-Ray Diffraction (PXRD) Analysis
The PXRD analysis was accomplished on PANalytical X'pert Pro powder X-ray diffractometer system. The X-ray of wavelength 1.54056 Å was used. The data was collected in the form of a chart of the Bragg angle (2θ) vs. intensity, and a detailed table containing information on peak intensity counts, d value (Å), relative intensity (%), full width half maximum (FWHM) (θ°). From the XRD results, the crystallite size (G) was calculated using X'pert data collector and X'pert high score plus processing software. A total of 500.19 mg of control and 500.23 mg of the treated samples individually used for the analysis and prepared by back loading technique using the sample preparation kit. The sample was spread on the holder ring in sufficient quantity to fill the ring cavity. It was then pressed down using powder press block and scrap the powder that was in surplus using a glass slide to get densely packed specimen. Consequently, the bottom plate was placed onto the holder ring and clamp in position. The sample holder was then removed from the sample preparation table by turning it upside down. A smooth surface of the sample was obtained to ensure the optimum results.

Particle Size Distribution (PSD) Analysis
The average particle size and particle size distribution were analyzed using Malvern Mastersizer 2000, UK with a detection range between 0.01 µm to 3000 µm. The sample unit was filled with the dispersant medium and operated the stirrer at 2500 rpm. Alignment of the optics was done and the background measurement was taken. After the background measurement, the sample was added in to the sample unit with constant monitoring the obscuration and stopped the addition of sample when the obscuration reached in between 15% to 20%. When the obscuration was stable, the measurement was taken twice, and the average was taken of two measurements. The average histogram of the two measurements was recorded. Along with histogram, the data was presented in a table format which includes particle size (µm). Also, the values at below 10% level (d 10 ), 50% level (d 50 ), and 90% level (d 90 ) were calculated from the histogram, and the calculations such as surface area (m 2 /g) were done by using software Mastersizer 2000. Percent change in particle size (d) for at below 10% level (d 10 ), 50% level (d 50 ), and 90% level (d 90 ) was calculated using following equation 1: % change in particle size 100 (1) Where, d Control and d 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 treated samples, respectively.
Percent change in surface area (S) was calculated using following equation 2: Where, S Control and S Treated are the surface area of the control and treated samples, respectively.

Fourier Transform Infrared (FT-IR) Spectroscopy
FT-IR spectroscopy of ashwagandha root extract was performed on Spectrum two (Perkin Elmer, USA) Fourier transform infrared spectrometer with the frequency array of 400-4000 cm -1 by using pressed KBr disk technique.

Ultra Violet-Visible Spectroscopy (UV-Vis) Analysis
The UV-Vis spectral analysis was carried out using Shimadzu UV-2450 with UV Probe, Japan. The spectrum was recorded using 1. cm quartz cell that has a slit width of 1.0 nm. The wavelength ranges chosen for recording the spectra was 190-800 nm. The absorbance spectra (in the range of 0.2 to 0.9) and the wavelength of maximum absorbance (λ max ) were recorded.

Thermal Gravimetric Analysis (TGA)
The TGA analysis was performed using Instrument TGA Q50 (TA Instruments, USA) at a heating rate of 10°C/min from room temperature i.e. 30°C to 900°C under nitrogen atmosphere. A total of 11.86 mg of sample was used for the analysis and was taken on the platinum pan. In TGA, the weight loss for each step was recorded in grams as well as in percent loss with respect to the initial weight. Also, the onset, endset, and peak temperature for each step were recorded in TGA. Percent change in weight loss (W) was calculated using following equation 3: % change in weight loss ' ' ' 100 (3) Where, W Control and W Treated are the weight loss of the control and Biofield Energy Treated samples, respectively.

Differential Scanning Calorimetry (DSC)
Analysis was performed using the DSC Q20 (TA Instruments, USA) differential scanning calorimeter. A total of 8.10 mg sample was weighed and sealed in aluminium pan and equilibrated at 25°C and heated up to 450°C at the heating rate of 10°C/min under nitrogen gas as purge atmosphere with the flow rate of 50 mL/min. The value for onset, endset, peak temperature, peak height (mJ or mW), peak area, and change in heat (J/g) for each peak were recorded. Percent change in melting point (T) was calculated using following equation 4: Where, ∆H Control and ∆H Treated are the latent heat of fusion of the control and treated samples, respectively.

Powder X-ray Diffraction (PXRD) Analysis
The powder X-ray diffraction study was conducted to explore the crystalline pattern of the control and treated samples of ashwagandha root extract ( Figure 2). The PXRD diffractograms of the control and treated samples of ashwagandha did not contribute any diffraction peaks, indicated that both the samples were amorphous in nature. The X-rays of the amorphous compound scatter in many directions leading to a large bump distribute in a wide range (2θ) instead of high concentration narrower peaks [50,51].

Particle Size Distribution (PSD) Analysis
The particle size (d 10 , d 50 and d 90 ) of the control and treated samples were investigated, and the results are shown in Table  1. The particle size of the treated sample showed at d 10 and d 50 was decreased by 4.66% and 6.02%, respectively compared with the control sample. The particle size of the treated sample at d 90 was increased slightly by 0.99% compared with the control sample. The surface area of the treated sample (0.130 m 2 /g) was increased by 4.84% compared with the control sample (0.124 m 2 /g) ( Table 1). The reduction in particle size leads to the increase in surface area in the treated sample. The results revealed that there might be an effect of the high energy milling induced by The Trivedi Effect ® treatment to ashwagandha root extract. The decrease in the particle size and increase in the surface area has the significant influence on the increase in drug release, dissolution, absorption, drug action and dose uniformity of any substance in various drug delivery system [52].

Fourier Transform Infrared (FT-IR) Spectroscopy
The FT-IR spectra displayed the prominent peaks correspond to the different functional groups of phytoconstituents present in both the control and treated ashwagandha root extract (Figure 3). The wavenumber of the absorbance (ν) of a diatomic can be calculated from the Hooke's law (Eq.6). The following equation gives the frequency of radiation (IR) that a molecule absorb and gives the frequency of vibration of the normal mode being excited by that radiation [53]. Where, v = vibrational frequency (cm -1 ), c = the velocity of light (cm/s), m 1 and m 2 = the mass of atoms 1 and 2, respectively in g, f = the force constant of the bond (dyne/cm).
From the above equation 6, it has been shown that if other factors remain constant, the vibrational frequency (wavenumber) is directly proportional to the force constant.
The FT-IR spectra ( Figure 3) indicated the O-H stretching of the treated sample (3367 cm -1 ) was shifted downward compared with the control sample (3392 cm -1 ) ( Table 2, entry 1). The broad peak at 3600-3200 cm -1 may be due to the O-H functional group of the phytoconstituents present in the ashwagandha root extract [10,53,54]. Similarly, the C-O stretching of the treated sample (1268 cm -1 ) was shifted downward compared with the control sample (1291 cm -1 ). The results indicated that the force constant of the O-H functional group of the treated sample was reduced compared with the control sample. The FT-IR exhibited similar aliphatic C-H stretching at 2927-2856 cm -1 , and C=O stretching at 1684 cm -1 for aldehyde/ketonic functional group in both the control and treated samples ( Table 2,   Altogether, FT-IR results suggested that the force constant of O-H (stretching), and C-O (stretching) functional groups of the phytoconstituents present in the treated ashwagandha root extract were reduced compared with the control sample. Hence, it is assumed that The Trivedi Effect ® treatment might be responsible for changing the structural features of the phytoconstituents present in the ashwagandha root powder extract.

Ultra Violet-Visible Spectroscopy (UV-Vis) Analysis
The UV-visible spectra of the control and The Trivedi Effect ® treated ashwagandha root extract are shown in Figure  4. The wavelength for the maximum absorbance (λ max ) of the both the control and treated ashwagandha root extract were at 205.4 and 205.6 nm, respectively. There was a minor shift of (Ashwagandha) Root Extract Treated with the Energy of Consciousness (The Trivedi Effect ® ) absorbance maxima from the control sample (1.8559) to the treated sample (1.8361). It has been reported that the wavelength for the maximum absorbance for the ashwagandha root extract was at 208.50 nm [55]. However, no significant change in the λ max of the treated sample was observed compared with the control sample. It is reported that the UV absorbance occurred due to excitation of electrons from highest energy occupied molecular orbital (HOMO) to lowest energy unoccupied molecular orbital (LUMO) [56]. It indicated that energy gap between the HOMO and LUMO in the treated sample was similar compared with the control sample.

Thermogravimetric Analysis (TGA)
The TGA thermograms of both the control and treated samples showed the three steps of thermal degradation ( Figure 5) and the results are mentioned in Table 3. The control sample lost weight about 10.51%, 57.69%, and 17.02% in the 1 st , 2 nd , and 3 rd steps of degradation, respectively from its original weight. Similarly, the treated sample lost weight about 10.49%, 58.74%, and 17.22% in the three steps of degradation from its original total weight. The total mass loss in the control and treated sample was 85.22%, and 86.45%, respectively. The total weight loss was increased by 1.44% in the case of the treated sample compared with the control sample. The current study showed there was a decrease in the thermal stability of the treated ashwagandha compared with the control sample.

Differential Scanning Calorimetry (DSC) Analysis
Measurement of the melting point and latent heat of fusion was performed using DSC analysis ( Figure 6. and Table 4). The DSC thermograms of the control and treated samples revealed the sharp endothermic inflection at 96.71°C and 91.08°C respectively, which was due to the evaporation of the bounded water molecule present in the sample. However, the control and treated ashwagandha indicated the endothermic peak at 203.43°C and 201.67°C, respectively. Several small endothermic peaks also observed in the thermograms of both in the control and treated samples, which may be due to the multiple phytoconstituents present in the ashwagandha root extract in a very small concentration [57,58]. The analysis revealed that there was a lowering of the melting point by 0.87% of the treated sample than the control sample.   The latent heat of fusion (∆H) of the treated (6.77 J/g) sample was significantly decreased by 21.82% compared with the control (8.67 J/g) sample. The results indicated that the treated ashwagandha needed the less latent heat of fusion compared with the control sample for the process of melting.
It is anticipated from the thermal analysis that The Trivedi Effect ® -Energy of Consciousness Healing Treatment might alter the stored potential energy of the ashwagandha root extract which may lead to the reduction in the melting point and latent heat of fusion (∆H), which may influence the volatility of the ashwagandha root extract. Since the sample has to be in the vapour phase to be transported, Gas Chromatography is advantageous to thermally volatile compounds [59,60]. Hence, the treated sample would be more advantageous in the chromatography point of view for the better metabolite profiling of ashwagandha root extract by the Gas Chromatography-Mass spectroscopy (GC-MS).

Conclusions
The current research work revealed that The Trivedi Effect ® -Energy of Consciousness Healing Treatment (Biofield Energy Healing) Treatment has the outstanding capability for the reduction of the particle size, along with enhanced surface area and altered the thermal properties of W. somnifera (ashwagandha) root extract. The PXRD analysis exhibited that both the control and treated samples were amorphous in nature. The particle size at d 10 and d 50 values of the treated sample were decreased by 4.66% and 6.02% and at d 90 slightly increased by 0.99% compared with the control sample The average surface area of the treated (0.130 m 2 /g) sample was increased by 4.84% compared with the control (0.124 m 2 /g) sample. The FT-IR results suggested that the force constant of O-H (stretching), and C-O (stretching) functional groups of the phytoconstituents present in the treated ashwagandha root extract were reduced compared with the control sample. The UV-vis analysis revealed that the wavelength for the maximum absorbance of the control and treated samples were at 205.4 and 205.6 nm, respectively. The TGA thermograms exhibited three steps of the degradation process, and the total weight loss of the treated (86.45%) sample was increased by 1.44% compared with the control (85.22%) sample. The DSC analysis revealed that the melting point of the treated (201.67) sample was reduced by 0.87% compared with the control (203.43°C) sample. The latent heat of fusion of the treated (6.77 J/g) sample was significantly decreased by 21.82% compared with the control (8.67 J/g) sample. Hence, the treated ashwagandha root extract could be more soluble, absorbable from the gut and more bioavailable compared with the untreated compound and be more suitable for any type of oral pharmaceutical and nutraceutical formulation which might be providing better therapeutic response against various diseases such as diabetes mellitus, allergies and septic shock; stress-related disorders like sleep disorder, insomnia, anxiety, depression, Attention Deficit Disorder (ADD), Attention Deficit Hyperactive Disorder (ADHD), mental restlessness (mind chattering), brain frog, low libido, impotency, lack of motivation, mood swings, fear of the future, confusion, migraines, headaches, forgetfulness, overwhelm, loneliness, worthlessness, indecisiveness, frustration, irritability, chronic fatigue, obsessive/compulsive behavior and panic attacks; inflammatory diseases and immunological disorders like