Copper chloride, cholecalciferol (vitamin D₃), sodium carboxymethyl cellulose (Na-CMC), sulphasalazine, and iron (II) sulfate were procured from Sigma-Aldrich, USA. Pyridoxine hydrochloride (vitamin B₆), zinc chloride, cyanocobalamin (vitamin B₁₂), and magnesium (II) gluconate were purchased from TCI, Japan. TNBS (Trinitrobenzenesulphonic acid) was purchased from HiMedia, India. All the other chemicals used in this experiment were analytical grade procured from India. 

Randomly breed male Sprague Dawley (SD) rats with body weight around 220 to 350 gm were used in this study. The animals were purchased from M/s. National Institute of Biologicals, India. Animals were randomly divided into nine groups based on their body weights consist of eight animals of each group. They were kept individually in sterilized polypropylene cages with stainless steel top grill having provision for holding pellet feed and drinking water bottle fitted with stainless steel sipper tube. The animals were maintained as per standard protocol throughout the experiment.

The each ingredient of the novel test formulation was divided into two parts. One part of each ingredient was considered as control, where no Biofield Energy Treatment was provided. Another part of each ingredient received Biofield Energy Treatment by Mr. Mahendra Kumar Trivedi (known as the Trivedi Effect^®) under laboratory conditions for ~3 minutes. In addition, three different test group as per experimental protocol, the animals were also received Biofield Energy Treatment under laboratory conditions for ~3 minutes. The blessing/treatment was given to the remotely without touching in the laboratory of Dabur Research Foundation, near New Delhi, India. 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. The Biofield Energy Treated animals were also taken back to the animal experimental room for further proceedings.

The gut health oxidative stress biomarkers experiment was divided into 9 animals per groups from G1 to G9. G1 denoted as normal control with vehicle (0.5% CMC), G2 group defined as colitis control, with 50% TNBS in ethanol using intra colonic route, G3 group include reference compound i.e. sulfasalazine (250 mg/kg), G4 group included administration of untreated test formulation, G5 include Biofield Energy Treated test formulation, G6 group denoted as Biofield Energy Treatment per se to the animals (Day -15) along with vehicle (0.5% CMC), G7 group defined as Biofield Energy Treated test formulation from day -15, G8 group included Biofield Energy Treatment per se to the animals along with Biofield Energy Treated test formulation from day -15, and G9 group animals were administered with the Biofield Energy Treatment per se (day -15) to the animals along with the untreated test formulation. 50% TNBS in ethanol was given to the entire test group except G1.

Five days after acclimatization, animals were randomized and grouped based on their body weight. Groups G1, G2 and G6 were treated with 0.5% w/v CMC-Na in distilled water for 8 weeks (Day 1 to 56). Group 3 was treated orally with sulphasalazine (reference item) at a dose of 250 mg/kg body weight for 8 weeks. The freshly prepared suspensions of untreated and Biofield Energy Treated Test formulation were administered orally to the G4 and G5 groups at a dose of 130.525 mg/kg for 8 weeks. Similarly, group G7 and G8 group were treated with the test formulation at a dose of 130.525 mg/kg from the day of Biofield Energy Treatment (day -15 to 56), while in group G9, Biofield Energy Treated animals were treated with untreated test formulation for 8 weeks. Before the induction of experimental colitis, a short fasting preceded. The duration ranged from 12 to 18 hours, while the chronic colitis was induced by intra colonic administration of TNBS-50% ethanol in a total volume of 400 μL, at a dose of 10 mg/rat. TNBS was instilled by a suitable medical-grade polyurethane catheter for enteral feeding approximately 8 cm proximal to the anal verge. Accordingly, TNBS injection was given on day 1, 8, 15, 21, 27, 34, 40, 48, and 54. After the end of the experiment, the colon was subsequently divided longitudinally into different pieces, homogenized and stored in -80°C for estimation of oxidative stress biomarker such as LPO, anti-oxidant enzymes such as MPO, SOD, Catalase, GSH, and GPx.

All the animals in various groups after the treatment with the untreated and Biofield Energy Treated test formulation till 8 weeks were examined for antioxidant assay. In order to test the major oxidative stress biomarkers, colon of all the animals was subsequently collected in each animal of various groups. The colon was divided longitudinally into different pieces, homogenized, and stored in -80°C for further testing of oxidative biomarkers using standards kits. The colon homogenate (100 mg tissue/mL) was subjected to the analyses of biomarkers testing. The detailed test procedure of manufactured instructions were used for the identification of oxidative stress biomarkers such as LPO along with anti-oxidant enzymes viz. MPO, SOD, Catalase, GSH, and GPx. Following kits such as rat myeloperoxidase (MPO) ELISA, LPO TBARS (TCA Method) Assay Kit, glutathione assay kit, glutathione peroxidase assay kit, superoxide dismutase assay kit, and catalase assay kit as per manufacturer instructions.

The data were expressed as mean ± standard error of mean (SEM) and subjected to statistical analysis using SigmaPlot (Version 11.0). For between two groups comparison Student’s t-test was performed, while multiple groups analysis one-way ANOVA was performed followed by post hoc analysis by Dunnett’s test. The p≤0.05 was considered as statistically significant (n=8).

The results of CAT activity after treatment using novel test formulation are presented in Figure 1. The catalase activity in the colon of rats treated with TNBS (G2) was 111.45 ± 16.07 nM/min/mL, which was significantly (p<0.05) lower than that of the control (G1) group 162.90 ± 13.59 nM/min/mL. However, the animals treated with the Biofield Energy Treatment per se, different combinations of Biofield Energy Treated and untreated test formulation to the Biofield Energy Treated and untreated TNBS induced animals including reference compound (sulphasalazine, G3) significantly altered the CAT activity. The reference compound, sulphasalazine that was treated with animals in G3 group treatment significantly decreased the catalase activity (68.78 ± 7.20 nM/min/mL). In addition, the untreated test formulation to the untreated animals in G4 group showed decreased CAT activity (58.94 ± 11.77 nM/min/mL). The results showed that the level of catalase was reduced by 29.3%, 37.7%, 73.9%, 46.6%, and 58.8% in G5, G6, G7, G8, and G9 groups, respectively as compared with the diseases control group (G2). However, the level of CAT was significantly increased by 95.4% (p≤0.001), 72.3%, 47.6%, and 13.9% in the G5, G6, G8, and G9 groups, respectively as compared to the untreated test formulation group (G4). CAT is the powerful antioxidant enzyme, also known as the longevity enzyme, which protect from free radicals in the body. CAT also play a significant role in ulcerative colitis, gut health, along with various powerful action as anti-aging and anti-degenerative effects, fat reduction, improve quality of life, and also helps in prevention of DNA damage 33. Therefore, the Trivedi Effect^®-Biofield Energy Treated novel test formulation has significant powerful antioxidant action, which leads to improve gut health and its related inflammatory disorders.

The data suggested that GSH level was depleted after intestinal injury in rat’s induced by TNBS. The level of GSH in the animal colon treated with the TNBS (G2) was 89.87 ± 6.11 µM, which was similar than that of the control (G1) group 90.03 ± 4.09 µM (Figure 2). Slightly increased in GSH level was observed in G3, G4, G5, G6 and G9 groups except group G7 and G8, which showed decreased of GSH level.In our study, we found significant changes in the activity of GSH in the colonic tissue of the TNBS-induced colitis group. GSH activity was found to be significantly increased by 23.2% (p≤0.05) 15.4%, and 15.5%, in groups G5, G6, and G9 groups, respectively as compared with the G2 group. However, GSH activity was found to be increased by 16%, 8.7%, and 8.8% in groups G5, G6, and G9 groups, respectively with respect to untreated test formulation, G4.

GSH regulates the antioxidant mechanism of the body and helps to develop gut immunity, which would improve the pathogenic condition of ulcerative colitis 34. Thus, Biofield Energy Treated test formulation would be a promising candidate for ulcerative colitis and improve the antioxidant activity.

The elevated LPO levels in the colon has been found to be related with triggering the cascade of reactions, which initiate more and more free radical generation that wear out the antioxidant defense and results in the development of ulceration and inflammation. The experimental data suggested that malondialdehyde (MDA) level was significantly higher in the colitis control group (G2) suggesting an oxidative stress in colon. The level of LPO in the colon of rats was found to be significantly decreased in all the treatment groups, indicating less free radical generation. The LPO level in the colon of rats treated with TNBS (G2) was 3.94 ± 0.19 µM, which was significantly higher (p<0.001) than that of the control (G1) group 2.74 ± 0.13 µM (Figure 3). In addition, data suggested significant decreased (p<0.05, 3.01 ± 0.32 µM) in the level of LPO in sulphasalazine treated group (G3) as compared to the G2 group. However, the animals showed decreased LPO by 12%, 17%, 18%, 19.1%, and 3.2% in G5, G6, G7, G8, and G9 groups, respectively as compared with the G2 group.

LPO affects the cellular membranes, lipoproteins, and other related molecules containing the lipids in accordance with the oxidative stress. A cellular membrane lipid represents most often substrates of oxidative attack and is regarded as one the vital step in the pathogenesis of various disease states. Thus, Biofield Energy Treated test formulation would improve the antioxidant activity by reducing the LPO level in colon.

Neutrophil infiltration is one of the most remarkable histological findings in the inflamed colonic tissue of IBD. The MPO is an indicator for leukocyte infiltration, which is commonly found in inflamed tissue such as chronic processes like neurodegenerative diseases and atherosclerosis 35. MPO activity was significantly induced by TNBS administration, while the MPO level in the rat colon was found to be significantly decreased in all the treatment groups and well corroborated by the significant decrease in inflammatory cell infiltration in the G4, G7, G8 and G9 groups (Figure 4), which indicated that Biofield Energy Treated and the novel test formulation ameliorates the colon inflammation. The experimental results showed that MPO level in the colon of rats treated with TNBS (G2) was 45.97 ± 1.19 ng/mL, which was significantly higher than that of the control (G1) group 24.45 ± 0.66 ng/mL (p<0.001). Sulphasalazine treatment (G3) significantly reduced (p<0.001) MPO level (37.80 ± 0.91 ng/mL) by 17.8% as compared by G2. However, G4 showed significantly decreased the MPO level (33.00 ± 0.95 ng/mL) by 28.2% as compared with G2. G5 group showed a significantly decreased (p<0.001) MPO level (33.13 ± 1.02 ng/mL), while G6 group also revealed significant decreased (p<0.001) MPO level (35.84 ± 1.33 ng/mL) by 27.9% and 22% respectively, as compared with G2. Further, 15 days pre-treatment of the Biofield Energy Treated test formulation (G7) significantly decreased MPO level (39.31 ± 2.30 ng/mL) by 14.5% as compared with G2, while G8 and G9 group showed a significant decreased level of MPO by 16.6% and 25.3% respectively, as compared with the G2.

The experimental data suggested significant increased levels of SOD in colitis control rats (G2) validating the results of oxidative stress in the colon (Figure 5). The SOD activity in the colon rats treated with TNBS (G2) was 0.587 ± 0.021 U/mL, which was significantly higher than that of the control (G1) group 0.358 ± 0.037 U/mL (p<0.001). Sulphasalazine treatment (G3) decreased SOD level (0.525 ± 0.021 U/mL), G4 group showed decreased SOD level (0.483 ± 0.049 U/mL), G5 also showed decreased SOD level as compare to the colitis control (0.564 ± 0.033 U/mL), G6 group showed a decreased the SOD level (0.415 ± 0.040 U/mL) as compared with G2. G7 group showed a decreased the SOD level (0.365 ± 0.072 U/mL), G8 also revealed a decreased SOD level (0.397 ± 0.058 U/mL) and G9 also showed a decreased SOD level (0.552 ± 0.023 U/mL) as compared with the G2 group. The level of SOD in the colon of rats was found to be decreased by 10.6%, 17.6%, 3.8%, 29.3%, 37.8%, 32.2%, and 5.9% in the G3, G4, G5, G6, G7, G8, and G9 groups respectively, as compared with the G2 group. In addition, SOD level was increased by 16.7% and 14.2% in the G5 and G9 groups, respectively as compared with the untreated test formulation, G4 group.

SOD is the antioxidant enzyme, which constitute the first line of defense against deleterious effects of oxy-radicals in all the living cells. SOD breaks down the most dangerous free radical superoxide anion to molecular oxygen and hydrogen peroxide and prevents subsequent formation of hydroxyl radicals and plays an important role in the cellular antioxidant mechanism. It also prevents the oxidative hazards and prevents the generation of hydroxyl radical and protects cellular constituents from oxidative damage. It possesses a powerful anti-inflammatory activity against chronic inflammation. The scientific data suggested that SOD enzyme supplement treatment reduced the ROS generation, oxidative stress and also inhibits the endothelial activation 36. Thus, Biofield Energy Treatment would be the best alternative treatment approach to treat ulcerative colitis using improved anti-oxidation action.

The GPx activity in the colon of rats treated with TNBS (G2) was 222.86 ± 101.39 nM/min/mL, which was lower than that of the control (G1) group 477.55 ± 131.27 nM/min/mL (Figure 6). Sulphasalazine (G3) group showed an increased GPx activity (382.04 ± 117.90 nM/min/mL). G4 group showed an increased GPx activity (445.71 ± 93.21 nM/min/mL), G5 group showed an increased GPx activity (618.54 ± 155.79 nM/min/mL), G6 group showed an increased GPx activity (382.04 ± 83.37 nM/min/mL) as compared with G2. G7 group showed an increased GPx activity (382.04 ± 83.37 nM/min/mL), G8 group showed an increased GPx activity (582.15 ± 173.22 nM/min/mL), and G9 group data showed an increased GPx activity (477.55 ± 101.39 nM/min/mL) as compared with the G2 group. Overall, the data suggested that GPx level was increased by 177.6%, 71.4%, 71.4%, 161.2%, and 114.3% in G5, G6, G7, G8, and G9 groups, respectively as compared with the G2 group. In addition, GPx level was significantly increased by 38.8%, 30.6%, and 7.1% in G5, G8, and G9 groups, respectively as compared with the untreated test formulation, G4 group.

The scientific data suggested that an improved GPx level reduced the free radicals, ROS generation, oxidative stress, which results in inhibition of inflammatory ulcerative colitis condition 36. Thus, Biofield Energy Treatment would be the best alternative treatment approach to treat ulcerative colitis using improved anti-oxidation action. This would lead to improve various diseases conditions such as atherosclerosis, inflammatory bowel disease, retinopathy of prematurity (ROP), borderline personality disorder (BPD), asthma, Parkinson's disease, kidney damage, preeclampsia and many more 37383940. Thus, it can be suggested that the Biofield Energy Treated novel test formulation showed a significant improved antioxidant profile, which suggested the use of test formulation against various autoimmune disorders, anti-inflammatory diseases, anti-aging, and many more.