Pyridoxine hydrochloride (vitamin B₆), zinc chloride, magnesium (II) gluconate, and β-carotene (retinol, provit A) were purchased from TCI, Japan. Cyanocobalamin (vitamin B₁₂), calcium chloride, vitamin E (Alpha-Tocopherol), cholecalciferol (vitamin D₃), iron (II) sulfate, and Carboxymethyl Cellulose Sodium were procured from Sigma-Aldrich, USA. Ascorbic acid (vitamin C) and sodium selenate were obtained from Alfa Aesar, India. Panax ginseng extract and Cannabidiol Isolate were obtained from Panacea Phytoextracts, India and Standard Hemp Company, USA, respectively. Dexamethasone was obtained from Clear synth, India. For the estimation of serum inflammatory biomarker panel, cytokines estimation, specific ELISA kits were used such as for detection of TNF-α, IL-1β, IL-6, IL-10, IL-12, IL-17, and IFN-γ were procured from CUSABIO, USA.

All the animals were handled humanely with due regard for their welfare. The animal care were comply with the regulations of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Environment and Forest, Govt. of India. The test facility is registered (registration no. 64/PO/RcBi/S/99/CPCSEA) for experiments on animals with the CPCSEA. The animals were procured using the protocol approved (IAEC/42/533) by the Institutional Animal Ethics Committee (IAEC) and the husbandry conditions were maintained as per the recommendations of the CPCSEA.

Randomly breed maleSprague Dawley (SD) rats with body weight ranges from 200 to 300 gm were used in this study. The animals were purchased from M/s. Vivo Bio Tech, Hyderabad, India. Animals were randomly divided into nine groups based on their body weights consist of 10-12 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.

Each ingredient of the novel test formulation was divided into two parts. One part of the test compound did not receive any sort of treatment and were defined as the untreated or control sample. The second part of the test formulation was treated with the Trivedi Effect^® - Energy of Consciousness Healing Treatment (Biofield Energy Treatment) by a renowned Biofield Energy Healer, Mr. Mahendra Kumar Trivedi under laboratory conditions for ~3 minutes. Besides, three group of animals also received Biofield Energy Healing Treatment (known as the Trivedi Effect^®) by Mr. Mahendra Kumar Trivedi under similar laboratory conditions for ~3 minutes. The Biofield Energy Healer was located in the USA, however the test formulation were located in the research laboratory of Dabur Research Foundation, New Delhi, India. The energy transmission/Blessing (prayer) was given remotely to the samples or animals for about 3 minutes via online web-conferencing platform. After that, the Biofield Energy Treated samples was kept in the similar sealed condition and used as per the study plan. In the same manner, the control test formulation group was subjected to “sham” healer for ~3 minutes treatment, under the same laboratory conditions. The “sham” healer did not has any knowledge about the Biofield Energy Treatment. The Biofield Energy Treated animals were also taken back to experimental room for further proceedings.

Seven days after acclimatization, animals were randomized and grouped based on the body weight. The test formulation was prepared freshly prior to dosing and administered to the animals using an oral intubation needle attached to an appropriately graduated disposable syringe. The dose volume was 10 mL/kg in morning and evening based on body weight. The experimental groups were divided as G1 as normal control (vehicle, 0.5% w/v CMC-Na); G2 as disease control (Cecal Slurry, LPS and E. coli + 0.5% CMC-Na); G3 as reference item (Cecal Slurry, LPS and E. coli + Dexamethasone); G4 includes Cecal Slurry, LPS and E. coli along with untreated test formulation; G5 as Cecal Slurry, LPS and E. coli along with the Biofield Energy Treated test formulation; G6 group includes Cecal Slurry, LPS and E. coli along with Biofield Energy Treatment per se to animals from day -15; G7 as Cecal Slurry, LPS and E. coli along with the Biofield Energy Treated test formulation from day -15; G8 group includes Cecal Slurry, LPS and E. coli along with Biofield Energy Treatment per se plus the Biofield Energy Treated test formulation from day -15, and G9 group denoted Cecal Slurry, LPS and E. coli along with Biofield Energy Treatment per se animals plus the untreated test formulation. Dosing for groups G7 and G8 were started on Day -15 and continued till end of the experiment. However, Group G1 to G5 and G9 animals were dosed with respective formulations from Day 1 and continued till the end of the experiment. Group G6 animals received Biofield Energy Treatment on Day-15 and were not dosed throughout the experimental period. At the end of the experimental period (8 weeks treatment), the animals were sacrifice and blood was collected and separate serum subjected for cytokines (TNF-α, IL-1β, IL-6, IL-10, IL-12, IL-17, and IFN-γ) estimation.

A combination model of sepsis was developed in SD rats by administering Cecal slurry (from donor animals, intraperitoneally, at the dose of 400 mg/kg) in combination with LPS (at the dose of 100 µg/animal) and E. coli [Escherichia coli; 0.2 mL (2M CFU)/animal]). The animals were monitored for various parameters for up to 56 days after disease (SIRS) induction. Ten Donor (~20 weeks old) rats were anesthetized. A midline laparotomy was performed on them and the cecum was extruded. A 0.5 cm incision was made on the anti-mesenteric surface of the cecum, and the cecum was squeezed to expel the feces. The feces from different donor animals was collected and weighed. Immediately after collection, the feces were pooled, diluted 1:3 with 5% dextrose solution and filtered to get a homogeneous suspension. Bacterial viability in the cecal slurry was analyzed. Cecal slurry prepared from donor rats was injected intraperitoneally into experimental rats (G2 to G9) at the dose of 400 mg/kg within 2 hours of preparation. After 3 hours, lipopolysaccharide (LPS) at the dose of 100 µg/animal, and gram-negative viable bacteria such as E. coli (0.2 mL (2M CFU)/animal) were injected, intraperitoneally (G2 to G9).

Animals were observed daily to check for SIRS related mortality. Mortality along with survival proportion across time was presented in Kaplan-Meier Graph. Percentage mortality, percentage survival and the increase in life span was calculated 29.

With the continued treatment to the respective groups of 8^th week of the experimental period, all the animals were individually subjected for blood collection using retro-orbital route and the blood was collected in the plain vial, which was used for the separation of serum in all the animals of different experimental groups. The serum from all the groups was stored at -20°C for further estimation. Alternatively, aliquot all the samples and store samples at -20°C or -80°C. Avoid repeated freeze-thaw cycles, which may alter the level of cytokines during final calculations.

The serum from all the groups was subjected for the estimation of level of cytokines such as TNF-α (CSB-E11987r), IL-1β (CSB-E08055r), IL-6 (CSB-E04640r), IL-10 (CSB-E04595r), IL-12 (CSB-E07364r), IL-17 (CSB-E07451r), and IFN-γ (CSB-E04579r). All the serum biomarker panel was estimation using ELISA method as per manufacturer’s recommended standard procedure. This was a quantitative method and the principle was based on the binding of antigen and antibody in sandwich manner assay.

The data were represented as mean (n=6 to 9) ± standard error of mean (SEM) and subjected to statistical analysis using Sigma-Plot statistical software (Version 11.0). For multiple comparison One-way analysis of variance (ANOVA) followed by post-hoc analysis by Dunnett’s test and for between two groups comparison Student’s t-test was performed. The p≤0.05 was considered as statistically significant.

Mortality, particularly within a few hours/days of SIRS induction, was observed in all the disease induced groups, indicating the onset and severity of SIRS induction. The survival rates varied according to the treatment. A % increase in the proportion of surviving animals was observed in the groups G6, G7, and G8 when compared to disease control (G2) group, indicating an increased survival rate (Figure 1 and Table 1).

Serum cytokine, TNF-α was estimated in the presence of the effect of the test formulation, which was measured in all the experimental groups and was graphically presented in the Figure 2. The data suggested that the disease control (Cecal Slurry, LPS and E. coli + 0.5% CMC-Na) + 0.5% CMC) group (G2) showed value of tumor necrosis factor-alpha (TNF-α) as 32.68 ± 5.71 pg/mL, which was increased by 831.27% as compared with the normal control (G1, 3.51 ± 0.33 pg/mL). However, positive control (Dexamethasone) treatment (G3) showed the level of serum TNF-αi.e., 12 ± 2.4 pg/mL, which was significantly (p≤0.01) decreased by 63.27% as compared to the G2 group. The level of serum proinflammatory cytokine (TNF-α) was significantly decreased by 37.06%, 40.50%, 85.36% (p≤0.01), 50.66% (p≤0.01), 87.38% (p≤0.01), and 58.63% (p≤0.01) in the G4 (Cecal Slurry, LPS and E. coli along with untreated test formulation); G5 (Cecal Slurry, LPS and E. coli along with the Biofield Energy Treated test formulation); G6 (Cecal Slurry, LPS and E. coli along with Biofield Energy Treatment per se to animals from day -15); G7 (Cecal Slurry, LPS and E. coli along with the Biofield Energy Treated test formulation from day -15); G8 (Cecal Slurry, LPS and E. coli along with Biofield Energy Treatment per se plus the Biofield Energy Treated test formulation from day -15), and G9 (Cecal Slurry, LPS and E. coli along with Biofield Energy Treatment per se animals plus the untreated test formulation) groups, respectively as compared to the disease control (G2) group. On the other hand, the expression of TNF-α was reduced by 5.47%, 76.74%, 21.61%, 79.95%, and 34.27% in the G5, G6, G7, G8, and G9 groups, respectively as compared to the untreated test formulation (G4) group (Figure 2). TNF-α, a pro-inflammatory cytokines play a wide role in the human body. These cell signaling protein (cytokine) significantly involved in systemic inflammation along with acute phase reaction. It mediates and regulates immune responses and inflammation. It also produce widespread deleterious effects when expressed in large amounts. It is produced in the heart by both cardiac myocytes and resident macrophages under conditions of cardiac stress, and responsible for various cardiac disease, elevated in congestive heart failure and potentiates heart failure 30. TNF-alpha may also triggering and perpetuation of atherosclerosis. Treatment with biologic agents that inhibits TNF-alpha expression has various clinical benefits in inflammatory diseases such as rheumatoid arthritis (RA) and may be able to reduce cardiovascular risk 31. Overall, in this experiment the Biofield Energy Treated test formulation and Biofield Energy Treatment per se reduced the level of TNF-alpha which might be helpful for the management of various inflammatory disorders.

The effect of the test formulation and Biofield Energy Treatment per se was estimated using the level of serum IL-1β, and the results were graphically presented in the Figure 3. The disease control (Cecal Slurry, LPS and E. coli + 0.5% CMC-Na) + 0.5% CMC) group (G2) showed value of IL-1β as 90 ± 7.2 pg/mL, which was increased by 63.64% as compared with the normal control (G1, 55 ± 3.66 pg/mL). Further, the positive control (Dexamethasone) treatment (G3) showed decreased serum IL-1β level by 15.87% i.e., 75.71 ± 6.31 pg/mL as compared to the G2 group. The level of IL-1β was decreased by 3.47%, 11.11%, 9.52%, and 6.35% in the G5 (Cecal Slurry, LPS and E. coli along with the Biofield Energy Treated test formulation); G6 (Cecal Slurry, LPS and E. coli along with Biofield Energy Treatment per se to animals from day -15); G8 (Cecal Slurry, LPS and E. coli along with Biofield Energy Treatment per se plus the Biofield Energy Treated test formulation from day -15), and G9 (Cecal Slurry, LPS and E. coli along with Biofield Energy Treatment per se animals plus the untreated test formulation) groups, respectively, as compared to the disease control group (G2). Similarly, IL-1β level was decreased by 9.91%, 17.04%, 5.52%, 15.56%, and 12.59% in the G5, G6, G7, G8, and G9 groups, respectively as compared to the untreated test formulation (G4) group (Figure 3). The experimental and clinical evidence reported that interleukin-1 beta (IL-1β) plays both vascular and systemic contributor against conventional risk factors to atherosclerosis 32. Another studies evidence in humans, suggests that IL-1β plays a role in insulin resistance, both T2DM and pre-diabetic states 33. Therefore, in this experiment the Biofield Energy Treated test formulation and Biofield Energy Treatment per se reduced the level of IL-1β, which could be beneficial in the inflammatory disease conditions.

The level of serum IL-6 was detected in all the experimental groups and was presented in Figure 4. The data suggested that disease control (Cecal Slurry, LPS and E. coli + 0.5% CMC-Na) group (G2) showed value of IL-6 as 22.44 ± 0.21 pg/mL, which was increased by 94.36% as compared with the normal control (G1, 11.54 ± 0.64 pg/mL) group. While, the positive control (Dexamethasone) treatment (G3) significantly (p≤0.001) decreased the level of IL-6 by 43.02% i.e. 12.79 ± 0.36 pg/mL as compared to the G2 group. The level of interleukin-6 (IL-6) was significantly (p≤0.001) decreased by 34.50%, 36.18%, 50.24%, 43.25%, 52.69%, and 38.23% in the G4 (Cecal Slurry, LPS and E. coli along with untreated test formulation); G5 (Cecal Slurry, LPS and E. coli along with the Biofield Energy Treated test formulation); G6 (Cecal Slurry, LPS and E. coli along with Biofield Energy Treatment per se to animals from day -15); G7 (Cecal Slurry, LPS and E. coli along with the Biofield Energy Treated test formulation from day -15); G8 (Cecal Slurry, LPS and E. coli along with Biofield Energy Treatment per se plus the Biofield Energy Treated test formulation from day -15), and G9 (Cecal Slurry, LPS and E. coli along with Biofield Energy Treatment per se animals plus the untreated test formulation) groups, respectively as compared to the disease control (G2) group. Moreover, the level of IL-6 was reduced by 2.58%, 24.04%, 13.36%, 27.78%, and 5.71% in the G5, G6, G7, G8, and G9 groups, respectively as compared to the untreated test formulation (G4) group (Figure 4). There is increasing evidence that the metabolic syndrome is associated with a proinflammatory state. IL-6 has a dual effect; at some levels it acts as a defence mechanism but in chronic inflammation it is rather proinflammatory. Literature stated that IL-6 can be utilised as a treatment approach effectively for rheumatoid arthritis and other chronic inflammatory diseases 34. Overall, here the Biofield Energy Treated test formulation and Biofield Energy Treatment per se reduced the level of IL-6, which could be beneficial in the inflammatory symptoms.

The effect of the test formulation and Biofield Energy Treatment per se was estimated using the level of serum IL-10, and the results were graphically presented in the Figure 5. The disease control (Cecal Slurry, LPS and E. coli + 0.5% CMC-Na) group (G2) showed value of IL-10 as 8.71 ± 1.86 pg/mL, which was increased by 199.04% as compared with the normal control (G1, 2.91 ± 0.04 pg/mL). Further, the positive control (Dexamethasone) treatment (G3) showed decreased serum IL-1β level by 86.54% i.e., 1.17 ± 0.57 pg/mL as compared to the G2 group. The level of IL-10 was decreased by 57.61%, 70.53%, 49.25%, 60.18%, 41.54%, and 58.89% in the G4 (Cecal Slurry, LPS and E. coli along with untreated test formulation); G5 (Cecal Slurry, LPS and E. coli along with the Biofield Energy Treated test formulation); G6 (Cecal Slurry, LPS and E. coli along with Biofield Energy Treatment per se to animals from day -15); G7 (Cecal Slurry, LPS and E. coli along with the Biofield Energy Treated test formulation from day -15); G8 (Cecal Slurry, LPS and E. coli along with Biofield Energy Treatment per se plus the Biofield Energy Treated test formulation from day -15), and G9 (Cecal Slurry, LPS and E. coli along with Biofield Energy Treatment per se animals plus the untreated test formulation) groups, respectively, as compared to the disease control group (G2). Similarly, IL-10 level was decreased by 30.43%, 19.80%, 6.01%, 38%, and 2.96% in the G5, G6, G7, G8, and G9 groups, respectively as compared to the untreated test formulation (G4) group (Figure 5). The experimental and clinical evidence reported that interleukin-10 (IL-10) plays an important role in the control of inflammation 35. Another study suggests that IL-10 can regulates the transcriptional modification in inflammation and autoimmune disease 36. Therefore, in this experiment the Biofield Energy Treated test formulation and Biofield Energy Treatment per se reduced the level of IL-10, which could be beneficial in the inflammatory disease conditions.

The effect of the test formulation and Biofield Energy Treatment per se on the level of serum interleukin-12, and the results were graphically presented in the Figure 6. The disease control (Cecal Slurry, LPS and E. coli + 0.5% CMC-Na) group (G2) showed value of IL-12 as 9.51 ± 0.80 pg/mL, which was increased by 141.71% as compared with the normal control (G1, 3.93 ± 0.23 pg/mL). Further, the positive control (Dexamethasone) treatment (G3) showed decreased serum IL-12 level by 62.37% i.e., 3.58 ± 0.41 pg/mL as compared to the G2 group. The level of IL-12 was decreased by 13.4%, 30.24%, 31.67%, 29.82%, 45.77%, and 50.54% in the G4 (Cecal Slurry, LPS and E. coli along with untreated test formulation); G5 (Cecal Slurry, LPS and E. coli along with the Biofield Energy Treated test formulation); G6 (Cecal Slurry, LPS and E. coli along with Biofield Energy Treatment per se to animals from day -15); G7 (Cecal Slurry, LPS and E. coli along with the Biofield Energy Treated test formulation from day -15); G8 (Cecal Slurry, LPS and E. coli along with Biofield Energy Treatment per se plus the Biofield Energy Treated test formulation from day -15), and G9 (Cecal Slurry, LPS and E. coli along with Biofield Energy Treatment per se animals plus the untreated test formulation) groups, respectively, as compared to the disease control group (G2). Similarly, IL-12 level was decreased by 19.49%, 21.13%, 19%, 37.42%, and 42.91% in the G5, G6, G7, G8, and G9 groups, respectively as compared to the untreated test formulation (G4) group (Figure 6). Overall, in this experiment the Biofield Energy Treated test formulation and Biofield Energy Treatment per se significantly reduced the level of IL-12, which could be suppressed inflammatory conditions and simultaneously reduce the risks of inflammatory diseases.

The level of serum IL-17 was detected in all the experimental groups and the data were presented in Figure 7. The disease control (Cecal Slurry, LPS and E. coli + 0.5% CMC-Na) group (G2) showed value of IL-17 as 1546.74 ± 249.27 pg/mL, which was increased by 155.57% as compared with the normal control (G1, 605.21 ± 56.18 pg/mL). Further, the positive control (Dexamethasone) treatment (G3) showed decreased serum IL-17 level by 60.37% i.e., 612.94 ± 36.14 pg/mL as compared to the G2 group. The level of IL-17 was decreased by 17.87%, 48.75%, 59.61%, 59.28%, 62.49%, and 58.65% in the G4 (Cecal Slurry, LPS and E. coli along with untreated test formulation); G5 (Cecal Slurry, LPS and E. coli along with the Biofield Energy Treated test formulation); G6 (Cecal Slurry, LPS and E. coli along with Biofield Energy Treatment per se to animals from day -15); G7 (Cecal Slurry, LPS and E. coli along with the Biofield Energy Treated test formulation from day -15); G8 (Cecal Slurry, LPS and E. coli along with Biofield Energy Treatment per se plus the Biofield Energy Treated test formulation from day -15), and G9 (Cecal Slurry, LPS and E. coli along with Biofield Energy Treatment per se animals plus the untreated test formulation) groups, respectively, as compared to the disease control group (G2). Similarly, IL-17 level was decreased by 37.59%, 50.83%, 50.42%, 54.33%, and 49.66% in the G5, G6, G7, G8, and G9 groups, respectively as compared to the untreated test formulation (G4) group (Figure 7). Interleukin-17 (IL-17) is a cytokine which elicits protection against extracellular bacterial and fungal infections and which plays important roles in inflammation 37. Overall, here the Biofield Energy Treated test formulation and Biofield Energy Treatment per se reduced the level of IL-17, which could be beneficial in the inflammatory symptoms.

The level of serum interferon-γ (IFN-γ) was detected in all the experimental groups and the data were presented in Figure 8. The disease control (Cecal Slurry, LPS and E. coli + 0.5% CMC-Na) group (G2) showed value of IFN-γ as 2.03 ± 0.13 pg/mL, which was increased by 317.88% as compared with the normal control (G1, 0.49 ± 0.08 pg/mL). Further, the positive control (Dexamethasone) treatment (G3) showed decreased serum IFN-γ level by 67.91% i.e., 0.65 ± 0.18 pg/mL as compared to the G2 group. The level of IFN-γ was decreased by 2.05%, 49.56%, 24.09%, 23.7%, 56.98%, and 44.94% in the G4 (Cecal Slurry, LPS and E. coli along with untreated test formulation); G5 (Cecal Slurry, LPS and E. coli along with the Biofield Energy Treated test formulation); G6 (Cecal Slurry, LPS and E. coli along with Biofield Energy Treatment per se to animals from day -15); G7 (Cecal Slurry, LPS and E. coli along with the Biofield Energy Treated test formulation from day -15); G8 (Cecal Slurry, LPS and E. coli along with Biofield Energy Treatment per se plus the Biofield Energy Treated test formulation from day -15), and G9 (Cecal Slurry, LPS and E. coli along with Biofield Energy Treatment per se animals plus the untreated test formulation) groups, respectively, as compared to the disease control group (G2). Similarly, IFN-γ level was decreased by 48.54%, 22.56%, 22.17%, 56.11%, and 43.84% in the G5, G6, G7, G8, and G9 groups, respectively as compared to the untreated test formulation (G4) group (Figure 8). Interferon-gamma (IFN-γ) is a hallmark of innate and adaptive immunity. It also plays a vital role in host defense, its excessive release has been associated with the pathogenesis of chronic inflammatory and autoimmune diseases 38. Overall, here the Biofield Energy Treated test formulation and Biofield Energy Treatment per se reduced the level of IFN-γ, which could be beneficial for the treatment of various inflammatory disorders.

Experiment includes four preventive maintenance groups (G6, G7, G8 and G9). The findings showed the significant slowdown of inflammation-related symptoms and also reduced the chances of disease susceptibility. All-inclusive, it indicate that the Trivedi Effect^® was found to be most effective and benefited to protect different kinds of diseases and also improve the overall health and quality of life.