Assessment of Neurobehavioral and Anti-Aging Effect of the Test Formulation in D-Galactose-Induced aging Dysfunction in Sprague Dawley Rats

The study aim was to evaluate the neurobehavioral effect of Consciousness Energy Healing Treated test formulation using behavioral test in male Sprague Dawley rats. The test formulation was divided into two parts, one was denoted as control, while the other was treated with Biofield Energy by Mr. Mahendra Kumar Trivedi (the Trivedi Effect -Consciousness Energy Healing) and defined as the Biofield Energy Treated sample. Besides, three group of animals were also received Biofield Energy Treatment by Mr. Trivedi. In Y-maze test, number of entries was significantly increased in the animals received Biofield Energy Treatment (-15 days) group (G6) by 176%, 47.53%, and 66.67% in the start arm, explored arm, and novel arm, respectively as compared to the untreated test formulation group (G4). Further, Biofield Treated animals

462 memory function like Alzheimer's disease [1]. Different behavioral test is used currently among them Y-maze, force swim, and Morris water maze tests. Y-maze experiment is used to find the capability of rodents in the recognition and memory ability and it provides a fruitful information that reflects the animal's ability to recognize and remember the new environment [2]. Stress is the causation of most prevalent psychiatric challenging disorders such as depression and anxiety [3]. The rodent forced swim test (FST) is mainly used as a preclinical model for depression [4,5]. There was a direct correlation between integration of aging and cognitive behaviour.
There is a numerous evidence suggesting that adults have more trouble learning new information, exhibit less efficient reasoning skills, are slower to respond on all types of cognitive tasks, and are more susceptible to disruption from interfering information than younger adults [6].
According to Hasher and Zacks [7], have reported that older adults are less effective at inhibiting irrelevant information than young adults. Based on the literature an importance of essential vitamins and minerals on stress-related disorders authors newly designed a proprietary test formulation consisting of zinc chloride, iron (II) sulfate, copper chloride, magnesium gluconate hydrate, cholecalciferol (vitamin D 3 ), vitamin B 6 (pyridoxine HCl), and vitamin B 12 (cyanocobalamin), which would be able to combat with stress-related disorders. Biofield Therapy has been established to be an alternative medicine approach effectively used on various properties of living organisms in a cost-effective manner [8]. Every living system bearing kind of unique quality vital force, an élan vital, which gives them special quality so called 'life' [9]. This ambiguous vital force defined as the 'Bioenergetics Field'. Many alternative therapies, medicines and practices such as Tai Chi, natural products, chiropractic/osteopathic manipulation, deep breathing, yoga, meditation, Qi Gong, special diets, massage, homeopathy, guided imagery, progressive relaxation, acupuncture, acupressure, relaxation techniques, hypnotherapy, movement therapy, healing touch, rolfing structural integration, pilates, mindfulness, traditional Chinese herbs and medicines, Ayurvedic medicine, naturopathy, aromatherapy, essential oils, cranial sacral, and Reiki therapy are extensively used as a complementary and alternative medicine (CAM) system. Although, a human has the ability to acquire the vital force energy from the universe and can transmit it into a useful way that is called Biofield Energy and the process is called as "Biofield Energy Treatment/Healing". Biofield (putative energy fields) or electromagnetic based energy therapies used to promote health and healing that had been exclusively reported by the National Institute of Health/National Center for Complementary and Alternative Medicine (NIH/NCCAM) [10]. The Trivedi Effect ® has been published in numerous peer-reviewed science journals with significant outcomes in many scientific fields such as cancer research [11,12], microbiology [13][14][15][16], biotechnology [17,18], pharmaceutical science [19][20][21][22], agricultural science [23][24][25][26], materials science [27][28][29][30], nutraceuticals [31,32]

Chemicals and reagents
Cholecalciferol (vitamin D 3 ), copper chloride, iron (II) sulfate, and sodium carboxymethyl cellulose (Na-CMC) were procured from Sigma-Aldrich, USA. Zinc chloride, vitamin B 6 (pyridoxine hydrochloride), cyanocobalamin (vitamin B 12 ), resveratrol, and magnesium (II) gluconate were purchased from TCI, Japan. D (+) Galactose obtained from Amresco, LLC. All the other chemicals used in this experiment were analytical grade procured from India.

Consciousness energy healing strategies
The test formulation was divided into two parts. One part of each ingredient was considered as control, where no Biofield Similarly, the control samples were subjected to "sham" healer under the same laboratory conditions for ~3 minutes. The "sham" healer did not have any knowledge about the Biofield Energy Treatment. After that, the Biofield Energy Treated samples were kept in the similar sealed condition and used as per the study plan.

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The Biofield Energy Treated animals were also be taken back to experimental room for further proceedings.

Experimental procedure
Five days after acclimatization, animals were randomized and grouped based on body weight. Dosing for group G7 and G8 was also initiated on day -15 till end of the experiment. However, G1 to G6 and G9 animals were dosed from day 1 till the end of experiment.
All the animals except G1 received D-Galactose, daily (500mg/ kg; i.p.) from day 1 to the end of the experiment. Body weight and clinical signs were taken daily throughout the experimental period.

Forced swimming test (FST):
The forced swim test was performed with slight modification as described [33,34]. In brief, all the animals except G1 were individually subjected to swim in a glass cylinder (40cm high, 18cm in diameter) filled with water (25 °C) up to height of 34cm for 10 minutes. In this cylinder, rats cannot touch the bottom or escape. For the first exposure, rats were placed in the water for 15 minutes of forced swimming (training session). Twenty-four hours later, rats were placed in the cylinder again for a 5-minute period (test session). In the test session rats were scored by an observer blind to the effect of test substance exposure of the animal for immobility time (floating with only small movements necessary to keep their head above water), swimming time (pedalling or making circular movements), and struggling time (climbing walls or diving attempts to escape). Water in the tank was changed after each session.

Morris water maze test:
The test was performed as per with slight modification [35]. Escape platform was sub-merged 1.0cm below the surface of the pool water, which was maintained at 23±2 °C, and mixed with milk powder to obscure the platform.
The location of the platform remained in the center of south west quadrant throughout the training period. Before the first session, each animal was put on the platform for 15 seconds, and then was given a 60 seconds free swim and then guided to the platform, where it remained for another 20 seconds. Latencies to escape from the water maze (finding the sub-merged escape platform), total distance travelled, and resting time of each animal were collected and the average value of each trial for a session was calculated.

Statistical analysis
The data were expressed as mean ± standard error of mean (SEM) and subjected to statistical analysis using Sigma Plot (Version 11.0) using one-way ANOVA. Student's t-test was performed for comparison of the individual treatment group with control. The p≤0.05 was considered as statistically significant (n=10).

Results and Discussion
Behavioral testing Y-maze test: The Y-maze test for cognitive function in laboratory animals is extensively used as a tool during the development of pharmaceutical formulations to the treatment of neurodegenerative diseases [36]. This behavioral test is used for the evaluation of cognitive functions like memory and learning ability of short-term work and is based on the natural instinct of rodents to explore unknown environments [37]. The effect of the test formulation on cognitive function using Y-Maze test in male Sprague Dawley rats is shown in Figure 1
Besides, in case of spontaneous alternation, where rats made alternations when they sequentially visited the three arms, without repeating any one arm [38]. The results indicated more alternation task because they spent more time to elucidate which was the last arm visited. Measurement of spontaneous alternation using a Y-maze is a test also utilized for the evaluation of habituation and spatial working memory [39]. Overall, Biofield Energy Treatment improved cognition function by significantly increasing number of entries, time spent and % alternation compared to the untreated test formulation group.

Forced swimming test (FST)
The results of FST after administration of the test formulation are shown in Figure 2. In this experiment, the number of climbing was significantly increased by 13.58%, 24.69%, 32.1%, 40.74%, and 3.70% in the G5, G6, G7, G8, and G9, respectively compared to the untreated test formulation control (G4) group. Additionally, the swimming time was significantly increased by 4.67%, 9.34%, and 10.31% in the G5, G6, and G7, respectively compared to the G4 group. Besides, the immobility time was significantly decreased by 27.91%, 55.81%, 61.63%, 5.81%, and 11.63% in the G5, G6, G7, G8, and G9, respectively compared to the G4 group ( Figure 2). The FST is another behavioral test battery normally used for rodent [40]. It is also called as Porsolt swim test developed for rats. It is used for the screening of antidepressant efficacy of new chemical entity and preventing depressive-like states [33,41]. It is also considered as an important tool in both academic research and drug discovery in industrial settings, where reliability and high throughput screening of novel compounds are essential [42]. Overall, the improvement of these parameters in the Biofield Energy Treated groups might be due to The Trivedi Effect ® -Consciousness Energy Healing Treatment.

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Morris water maze test: Many water mazes have been developed so far, but the 'the water maze' was developed by Richard Morris to assess spatial or place learning and defined as Morris water maze (MWM) [43]. For the assessment of spatial learning and memory Morris water maze test is widely used as a behavioral test [44,45]. The MWM is an important and dominant method has become more widely used than its predecessors such as radial-arm maze, passive avoidance, T-mazes and their variations, etc. [46]. The impact of the test formulation on activeness and spatial memory using Morris water maze test is shown in Figure 3. The escape latency was significantly reduced by 12.06% in session 2, in the G5 group compared to the G4 group. Moreover, escape latency was decrease by 6.33% and 13.04% in session 1 and 3, respectively in the G7 group compared to the G4 group. G8 group showed 14.88%

Volume 4 -Issue 4
Copyrights @ Mahendra showed the best results as a prophylactic/preventive treatment group compared to the other groups. Based on the overall data, it suggests that the Biofield Energy Healing Therapy was found to be most effective and benefited in order to prevent and protect from the occurrence of any type of diseases in rat model. It indicated that this therapy can act as a preventive maintenance therapy to prevent the occurrence of the disease, slow down the disease progression and disease related complications of the existing aliments that will ultimately improve the overall health and quality of life in human.

Conclusion
The result of Y-maze data suggested that number of entries was significantly elevated by 176% (in start arm), 47.53% (in explored arm), and 66.67% (novel arm) in the animals per se received Biofield Energy Treatment (-15 days) group (G6) compared to the untreated test formulation group (G4). The number of entries was also increased by 100% (in start arm) in the Biofield Treated animals per se + Biofield Treated test formulation (-15 days) group (G8) compared to the G4 group. Additionally, percent of alternation behaviour was increased by 7.26% in the G8 as compared to the G4 group. The results of force swim test showed that climbing frequency was increased by 24.69%, 32.1%, and 40.74% in the G6, G7, and G8, respectively compared to G4 group. Parameter like immobility time was significantly decreased by 27.91%, 55.81%, and 61.63% in the G5, G6, and G7, respectively compared to the G4 group. Besides, the results of the Morris water maze (MWM) data showed that escape latency was significantly decreased by 12.06% (session 2), 13.04% (session 3), 14.88% (session 3), and 17.52% (session 3) in the G5, G7, G8, G9 groups, respectively compared to the G4 group. Biofield Energy Healing Treatment (the Trivedi Effect ® ) per se showed best results with respect to different efficacy and biomarker parameters in the preventive maintenance group, G6 as compared to the other preventive maintenance groups (G7, G8, and G9) in rat model study. It also helped to slow down the disease progression and disease related complications of the overall animal's health. These data suggested that Biofield Energy Treatment per se and/or Biofield Energy Treated Test formulation in combination would be the best treatment strategies in order to prevent and protect from the occurrence of any type of diseases.
Therefore, the Biofield Energy Treatment might act as a preventive maintenance therapy in order to maintain good health, or full restoration of health or improve the overall health and quality of life in human. This therapy might also reduce the severity of any type of acute/chronic disease (auto-immune related and inflammatory disorders) progression rate and can be used in both before and after the manifestation of any disease symptoms in healthy, unhealthy, and ill peoples. Therefore, it is assumed that the Biofield Energy Treated test formulation could be more useful in various aging and neurobehavioral-related disorders such as stroke, occlusion, hemorrhagic, transient ischemic attack (TIA), arterialvenial malformation (AVM), phenylketonia (PKU), encephalitis,