Evaluation of the Physico-chemical, Thermal and Behavioral Properties of Ashwagandha Root Extract: Effects of Consciousness Energy Healing Treatment

Withania somnifera (Ashwagandha) root extract has a broad range of pharmacological activities due to the presence of many biologically active metabolites. The current study was designed to evaluate the influence of The Trivedi Effect Consciousness Energy Healing Treatment (Biofield Energy Healing) on the physical, spectroscopic, thermal and behavioral properties of ashwagandha root extract using PXRD, PSD, FT-IR, UV-vis spectroscopy, TGA, and DSC analysis. Ashwagandha root extract was divided into two parts – one part was control without any Biofield Energy Treatment and another part was treated with the Consciousness Energy Healing Treatment remotely by twenty renowned Biofield Energy Healers and defined as Biofield Energy Treated sample. The PXRD analysis exhibited that both the control and treated samples were amorphous in nature. The particle size values at d10, d50, and d90 of the treated sample were increased significantly by 8.69%, 7.32%, and 6.50%, respectively compared with the control sample. Similarly, the surface area of the treated sample was significantly decreased by 12.69% compared to the control sample. The FT-IR analysis indicated no significant alteration of the force constant for the functional groups in the treated sample compared to the control sample. The UV-vis analysis revealed that the wavelength for the maximum absorbance of the control and treated samples were at 207.5 and 205.2 nm, respectively. The TGA revealed the two major thermal degradation steps and the total weight loss was decreased by 0.86% in the treated sample compared with the control sample. Consequently, the maximum thermal degradation temperature at 273.53°C and 389.39°C for two broad peaks in treated sample was increased by 0.78% and 0.31%, respectively compared to the control the sample (271.40°C and 388.48°C). The DSC analysis indicated that the evaporation temperature was significantly increased by 10.95% in the Biofield Energy Treated sample with a 5.34% reduced the latent heat of vaporization compared with the control sample. The overall thermal analysis indicated that the treated sample was thermally more stable as compared to the control sample. The Trivedi Effect Energy of Consciousness Healing Treatment might lead to have better powder flowability and long-term storage stability compared with the control sample. Thus, the treated ashwagandha root extract might provide better therapeutic response against inflammatory diseases, immunological disorders, stress, arthritis, 42 Mahendra Kumar Trivedi et al.: Evaluation of the Physico-chemical, Thermal and Behavioral Properties of Ashwagandha Root Extract: Effects of Consciousness Energy Healing Treatment cancer, diabetes, sexual disorders, ageing, and other chronic infections.


Introduction
The use of the herbal medicines are increasing day-by-day throughout the world due to its impressive therapeutic effects and fewer side effects as compared to the allopathic medicines [1]. Withania somnifera is a vital herb, traditionally known as 'Ashwagandha' or winter cherry. In Ayurveda, it is defined as 'Indian ginseng'. Ashwagandha is widely used in the most of the Indian herbal medicine and nutraceuticals for the treatment of various diseases including nervous and sexual disorders, infectious diseases, diabetes, cancer, ulcer, stress, immunological disorders, arthritis, etc. From the ancient time, it is used as a tonic to arrest the ageing process, rejuvenate the body and boost the defense against infectious disorders and also promote the longevity [2][3][4][5][6][7][8][9][10][11]. As ashwagandha root extract contains a wide array of nutrients and phytochemicals, it is used as a dietary supplement for the better quality of life [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. possess various pharmacological activities including antioxidant, anticancer, immunomodulating, neuroprotective, antiepileptic, antibacterial, adaptogenic, spermatogenic, antianxiety, antidepression, hepatoprotective, anti-inflammatory, antiarthritic, antimicrobial, hypoglycaemic, hypolipidemic, aphrodisiac, antiulcer, radiosensitizing, etc. [11,[14][15][16][17][18][19].
Therefore, a new proprietary herbomineral formulation was formulated that consisted of 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.
From the ancient-time, the living force preserved by every living organisms that contributes the 'life' is defined 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 is considered now as the Bioenergetics Field. The Biofield 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 environment that leads to the continuous movement or matter of energy [20,21]. Thus, the human has the ability to 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]; altered 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 it's formulations despite of their outstanding in vitro results exhibited poor or low in vivo activity, because of their low lipid solubility or improper molecular size, causing in deprived absorption and thus poor bioavailability [1]. According to the recent study on the bioavailability of major withanolides of Withania somnifera, Devkar et al. demonstrated that the nonpolar and low molecular weight withanolides are highly permeable, whereas the high glycosylated and polar withanolides displayed low permeability in their in vitro absorption model system [46]. For instance, ashwagandha root extract has the outstanding nutrition 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 [47]. The physicochemical properties such as particle size, crystalline structure, crystallite size, surface area, etc. and thermal properties of a drug play a vital role in bioavailability as well as stability of the drug during processing, formulation, storage, and packaging [48,49]. Biofield Energy Treatment (The Trivedi Effect ® ) has been reported to change the particle size, specific surface area, crystalline, chemical and thermal behavior of an atom/ion through possible mediation of the neutrinos [50]. By considering all these aspect, the objective of the current study was to examine whether the Biofield Energy Treatment can change the physical, structural, and thermal properties of ashwagandha root extract in such a way that might be assist in the improvement of the solubility and absorption of ashwagandha root extract and also help in designing of any suitable pharmaceutical formulation. The physical, spectroscopic, and thermal properties of both control and the 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) root hydroalcoholic extract was purchased from Sanat Product Ltd., India. All other chemicals used in the experiment were of analytical grade available in India.

Consciousness Energy Healing Treatment Strategies
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 ® -Consciousness Energy Healing Treatment (Biofield Energy Healing) by renowned Biofield Energy Healers and defined as Biofield Energy Treated sample, while the second part of the test compound did not receive any sort of such treatment and defined as untreated or control ashwagandha root extract sample. The Trivedi Effect ® treatment was provided by the group of twenty renowned Biofield Energy Healers who participated in this study and performed the Biofield Energy Treatment (The Trivedi Effect ® ) remotely. Eighteen Biofield Energy Healers were remotely located in the U.S.A. and two of which were remotely located in Canada, while the test compound was located in the research laboratory of GVK Biosciences Pvt. Ltd., Hyderabad, India. The Trivedi Effect ® -Consciousness Energy Healing 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 sample was subjected to "sham" healers for 5 minutes, under the same laboratory conditions. The sham healer did not have any knowledge about Biofield Energy Treatment. After that, the treated and untreated samples were kept in similar sealed conditions and 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 mg of the control and treated samples individually were 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 sample was obtained to ensure 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 table format which include 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 range chosen for recording the spectra was 190-800 nm. The absorbance spectra (in the range of 0.2 to 0.9) and wavelength of maximum absorbance (λ max ) were recorded.

Thermal Gravimetric Analysis (TGA)
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 ~14 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: Where, W Control and W Treated are the weight loss of the control and The Trivedi Effect ® treated samples, respectively.

Differential Scanning Calorimetry (DSC)
Analysis was performed using the DSC Q20 (TA Instruments, USA) differential scanning calorimeter. A total of ~4 mg sample was weighed and sealed in aluminum 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 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
Powder X-ray diffraction study was conducted to examine the crystalline pattern of the control and treated ashwagandha root extract. Figures 2 showed the PXRD diffractograms of both the control and Biofield Energy Treated did not contribute any diffraction peaks. It was then concluded that both samples are amorphous in nature and the Biofield Energy Treatment has no effect compared to the control sample.

Particle Size Distribution (PSD) Analysis
Particle size data of both the control and Biofield Energy Treated ashwagandha root extract was investigated and the results are presented in Table 1. The particle size values of the Biofield Energy Treated ashwagandha root extract at d 10 , d 50 , and d 90 were significantly increased by 8.69%, 7.32%, and 6.50%, respectively compared with the control sample (Table 1). Similarly, the surface area of the Biofield Energy Treated ashwagandha root extract (0.075 m 2 /g) was significantly decreased by 12.69% compared with the control sample (0.086 m 2 /g). The results revealed that there might be an effect of Biofield Energy Treatment on the intrinsic properties of ashwagandha root extract. Numerous literatures mentioned that particle size, shape and surface area of the pharmaceuticals/nutraceuticals have an important impact on solubility, dissolution rate, in vivo bioavailability, dose uniformity and therapeutic efficacy as well as assist in the design of the new drug delivery systems [48,51]. Introduction of the external force leads to the transform the small particles into larger particles. The various reasons to increase the particle size are enhanced flowability, improved product shape and appearance [52]. It is assumed that Biofield Energy Healing Treatment (i.e. consider as external force) might improve the flowability, shape and appearance of ashwagandha root extract.

Fourier Transform Infrared (FT-IR) Spectroscopy
The FT-IR spectra of both the control and Consciousness Energy Healing Treated ashwagandha root extract are presented in Figure 3. The FT-IR spectral data are presented in Table 2. The wavenumber of the absorbance (ν) of a diatomic can be calculated from the following equation  It has been observed that the vibrational frequencies for the O-H, C-H, C-C, C-O, and C=O groups of the Biofield Energy Treated ashwagandha were almost similar to the control sample ( Table 2, entry 1-7). It indicated that the force constant of these groups was remained unchanged. The presence of epoxide, unsaturated lactone, 1-keto-2-ene functions play a vital role to elicit the pharmacological activities of withanolides [14,[55][56][57]. In summary, the structural properties of the phytoconstituents present in the ashwagandha root powder extract remained unaltered due to the Biofield Energy Healing Treatment.  The UV-visible spectra of both the control and Biofield Energy Treated ashwagandha root extracts are shown in the Figure 4. The wavelength for the maximum absorbance (λ max ) of both the control and Biofield Energy Treated ashwagandha root extracts were at 207.50 and 207.20 nm, respectively and there was a minor shift of absorbance maxima from 1.5548 in the control sample to 1.4372 in the Biofield Energy Treated sample. The UV absorbance occurs due to the different type of energy transitions from the singlet to the singlet excited state such as σ → σ * , n → π * , and π → π * . These type of electronic transitions are happened when the difference in energy between the lowest unoccupied molecular orbital (LUMO) and the highest occupied molecular orbital (HOMO) is significantly higher than the activation energy of the compound [59]. It has been reported that the wavelength for the maximum absorbance for the ashwagandha root extract was at 208.50 nm. Thus, no significant change in the λ max of the Biofield Energy Treated sample was observed as compared to the control sample. Hence, it is anticipated that the structure of the phytoconstituents in the treated sample was remained unaffected as compared to the control sample.

Thermal Gravimetric Analysis (TGA)
TGA was applied to investigate the thermal stability of the control and Biofield Energy Treated ashwagandha root extracts. The TGA analysis exhibited three steps of thermal degradation as mentioned in Table 3 and Figure 5. In the 1 st , 2 nd , and 3 rd steps of thermal degradation, the weight loss of the Biofield Energy Treated ashwagandha root extract was decreased by 2.54%, 0.49% and 1.63%, respectively compared with the control sample as shown in the Table 3. However, the total weight loss of the Biofield Energy Treated sample (79.77%) was reduced by 0.86% compared with the control sample (79.08%). It is then anticipated that Biofield Energy Healing Treatment might enhance the thermal stability of ashwagandha root extract. The DTG thermograms of the control and Biofield Energy Treated samples ( Figure 6) displayed two broad peaks. The maximum thermal degradation temperature at 273.53°C and 389.39°C for two broad peaks in the Biofield Energy Treated ashwagandha root extract was increased by 0.78% and 0.31%, respectively compared to the control the sample (271.40°C and 388.48°C). Overall, an improvement in the thermal stability of the Biofield Energy Treated ashwagandha was increased compared to the control sample.

Differential Scanning Calorimetry (DSC) Analysis
The DSC thermograms of both the control and Biofield Energy Treated ashwagandha root extract are presented in the Figure 7 and Table 4. The DSC thermograms of the control and Biofield Energy Treated samples indicated the presence of a broad endothermic inflection at 80.83°C and 89.68°C, respectively which is not the true melting point of the Biofield Energy Treated ashwagandha root extract powder. It may be the evaporation of the bounded water present in the sample. This evaporation temperature was significantly increased by 10.95% in the Biofield Energy Treated sample with a 5.34% reduced latent heat of vaporization compared with the control sample (Table 4).  It is assumed that the Biofield Energy Healing Treatment might altered the intermolecular force in the Biofield Energy Treated sample, which probably increase the heat change. These results suggest that the temperature for water removal of the Biofield Energy Treated sample was significantly increased as compared to the control sample. However, the Biofield Energy Treated, and control W. somnifera indicated several endothermic peak around 200°C (Figure 7). Several small endothermic peaks were also observed in the thermogram of both in control and treated samples, which may be due to the multiple phytoconstituents present in the root extract in a very small concentration [60,61]. In the Biofield Energy Treated sample the endothermic peak was sharp and significantly prominent as compared to the control sample.

Conclusions
The present research work revealed that The Trivedi Effect ® -Consciousness Energy Healing Treatment (Biofield Energy Healing Treatment) has the outstanding capability for increase the thermal stability and particle size of the ashwagandha root extract along with decreased surface area. The PXRD analysis exhibited that both the control and treated samples were amorphous in nature. The particle size values of the Biofield Energy Treated sample at d 10 , d 50 , and d 90 were significantly increased by 8.69%, 7.32%, and 6.50%, respectively compared with the control sample. Similarly, the surface area of the treated sample was significantly decreased by 12.69% compared to the control sample. The TGA revealed the two major thermal degradation steps and the total weight loss was decreased by 0.86% in the treated sample compared with the control sample. Consequently, the maximum thermal degradation temperature at 273.53°C and 389.39°C for two broad peaks in treated sample was increased by 0.78% and 0.31%, respectively compared to the control the sample (271.40°C and 388.48°C). The DSC analysis indicated that the evaporation temperature was significantly increased by 10.95% in the Biofield Energy Treated sample with a 5.34% reduced latent heat of vaporization compared with the control sample. The overall thermal analysis indicated that the treated sample was thermally more stable as compared to the control sample. In summary, The Trivedi Effect ® -Energy of Consciousness Healing Treated ashwagandha root extract could have better powder flowability and long-term storage stability compared with the control sample and could be suitable for any 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 occupied molecular orbital, LUMO: Lowest energy unoccupied molecular orbital, TGA: Thermal gravimetric analysis, T peak = Peak vaporization temperature, ∆H: Latent heat of vaporization, UV-vis: Ultraviolet-visible spectroscopy, PSD: Particle size distribution; PXRD: Powder X-ray diffraction.