Immuno-modulatory Effects of a Herbo-mineral Ayurvedic Formulation in Experimental Models

Immuno-modulatory Effects of a Herbo-mineral Ayurvedic Formulation in Experimental Models | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Immuno-modulatory Effects of a Herbo-mineral Ayurvedic Formulation in Experimental Models Vaidya Prakash, Sneha Sati, Yashwant Rao, Shikha Prakash, Neha Negi This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3625156/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Ayurveda, an ancient Indian system of medicine, encapsulates comprehensive principles and formulations for disease prevention and treatment. A herbo-mineral Ayurvedic formulation, IMMBO, comprising Mandoor Bhasma and eighteen herbs has shown promising results in treating Allergic Rhinitis. The said efficacy was recently reinstated by a randomized controlled clinical trial conducted at a reputed hospital and IMMBO significantly reduced the IgE levels and Total nasal symptom scores in treated patients. This article discusses the findings of a series of experimental studies conducted to explore the immuno-modulatory potential of IMMBO. The studies reveal the immunomodulatory effects of IMMBO, evidenced by its effectiveness in reinstating neutrophil activation, stimulating cellular and humoral immunity, and counteracting immunosuppression at the molecular level. The modulation of key signalling molecules, including TNF-α, IFN-γ, IL-1β, ERK, PI3K, and NF-κB, showcases the formulation’s multifaceted impact. Additionally, its ability to block histamine release suggests potential in controlling allergic states, positioning it as a promising therapeutic candidate for immune-related disorders. However, the precise mode of action remains elusive, warranting further in-depth pharmacological studies. This research substantiates the ancient Ayurvedic wisdom using modern scientific parameters, endorsing IMMBO's potential as an immune-modulatory agent. Biological sciences/Biological techniques/Biological models/Immunological models Biological sciences/Immunology/Applied immunology/Primary immunodeficiency disorders Ayurveda Allergy Immuno-modulator Immunity IMMBO Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction: Ayurveda 1 , an ancient Indian system of medicine documented primarily in Sanskrit 2 , offers comprehensive definitions, principles, and procedures for disease prevention and treatment 1 . Its core components include dietary practices, lifestyle recommendations, and medicinal formulations (aahar, vihaar and aushadh) 3 . Rooted in centuries-old tradition, this knowledge has been nurtured in Gurukuls and Ashrams through the revered Guru-Shishya tradition 4 . Ayurvedic formulations, incorporating ingredients from plant, animal, and mineral origins, have gained global recognition for their healing potential 5 . Consequently, there has been a surge in efforts worldwide to subject Ayurvedic concepts, principles, and formulations to rigorous research methodologies. One specific Herbo-Mineral Ayurvedic Formulation (HMAF), a combination of eighteen herbs and Mandoor Bhasma, was initially pioneered by a registered Ayurvedic physician in North India, operating under the provisions of Section 33EEC of the Drug and Cosmetics Act of India 6 . HMAF has shown promising results in the treatment of patients suffering from Persistent and Intermittent forms of Allergic Rhinitis. Following successful outcomes, a manufacturing license was obtained from the State Drug Licensing Authority in India for its commercial production, and it was introduced into the market under the name IMMBO 7 . Simultaneously, IMMBO underwent a phase III prospective randomized controlled clinical trial to evaluate its efficacy in comparison to the standard treatment of FDC Levocetirizine 2.5 mg + Montelukast 4 mg in allergic rhinitis patients. The study yielded compelling evidence supporting IMMBO's effectiveness in treating allergic rhinitis 8 . Additionally, IMMBO underwent extensive in vivo and in vitro studies to explore its intriguing mechanism of action. Wistar albino rats were subjected to experiments involving Cyclophosphamide (CPH)-induced immune suppression 9 . These studies assessed IMMBO's impact on various parameters, including neutrophil adhesion, delayed-type hypersensitivity response, antibody levels, inflammatory and pro-inflammatory markers, and cell signaling pathways. This paper delves into the outcomes and interpretations derived from these studies. Materials & Methods Animals of the same sex, housed in groups of two to three per cage, were kept in a controlled environment within an air-conditioned room with a temperature maintained at 20 ± 3°C and a relative humidity ranging from 30–70%. The room had air changes occurring at a rate of 10–15 per hour, and a 12-hour light and 12-hour dark cycle was maintained. Temperature and humidity levels were continuously monitored and recorded once daily. The standard polycarbonate cages were equipped with stainless steel mesh top grills and provisions for pelleted food and water bottles. All the animals underwent a minimum five-day acclimatization period in a laboratory setting. Veterinary examinations were conducted on the day the animals were received and before the commencement of the treatment. Animals were fed with normal rodent feed and reverse osmosis water was always available to the animals during the experiments. Daily observations for any clinical signs were conducted. Study design The study was conducted in Southern India by an internationally accredited and DSIR, Government of India recognized research unit. Healthy Wistar albino rats (6–8 weeks old) were grouped based on body weight and randomly assigned to different treatment groups. In the main study, 48 rats were divided into seven experimental groups (G1 to G7) for immune modulator study. Additionally, a pilot set of rats (G7 to G10) was maintained separately to evaluate delayed-type hypersensitivity (DTH). Ethics This study was performed at a CPCSEA-approved and AAALAC accredited laboratory following all ethical practices as laid down in the guideline/s for animal care. The study was approved by the Institutional Animals Ethics Committee (IAEC) of the test facility. Materials Cyclophosphamide served as the negative control, functioning as an immunosuppressant, while Levamisole hydrochloride acted as the positive control, serving as an immunity stimulant 9 , 10 . Fresh sheep blood was collected and mixed with sterile Alsever's solution. The mixture was centrifuged at 3000 rpm for 5 minutes to obtain red blood cell sediment. The cell pellet was then resuspended in Phosphate Buffer Saline (PBS) with a pH of 7.2 and centrifuged at 1500 rpm. This washing process was repeated until a clear top layer was observed. The Sheep Red Blood Cells (SRBC) were adjusted to the desired levels based on haematocrit values and stored in the refrigerator for later use. RAW 264.7 macrophages and RBL-2H3 mast cells were obtained from ATCC, USA and maintained in DMEM with 5% FBS/1% penicillin-streptomycin at 37°C in a 5% CO2 environment. Both cell types were incubated in DMEM with 10% FBS, 2 mM glutamine, 100 U/mL penicillin, and 50 µg/mL streptomycin. Experimental protocol In set 1, rats in the normal control group (G1) received saline for two weeks. Experimental groups (G2 to G7) received cyclophosphamide (100 mg/kg) on Day 0, followed by sheep red blood cell (SRBC) challenge on Days 7 and 14. G4 received the immunostimulant levamisole (20 mpk). G5 to G7 received IMMBO at low (200 mpk), mid (400 mpk), and high (800 mpk) doses orally. In set 2, animals in G8 to G10 were subjected to delayed-type hypersensitivity (DTH) testing with SRBC on Days 7 and 14. G9 received levamisole (20 mpk) and G10 received high-dose IMMBO (800 mpk) from Day-1 to Day-14 as pre-treatment. Table 1 Work flow Test groups In-life experiments End point assessments Immune suppression SRBC (1–2%) Treatment Day 0 Day 7 & 14 Day 1 to 14 G1 Cyclophosphamide (100mpk) - Vehicle (0.9% saline) Body and lymphoid organs weight, Neutrophil adhesion, DTH, TNF- α , IFN-γ, IL-6, IL-1β, IL-10, IgG, IgA, IgM, MAP kinases (AKT, ERK, PI3K) G2 - - G3 SRBC (Control, IP) - G4 SRBC (1–2%, IP) Levamisole (20 mpk, po) G5 SRBC (1–2%, IP) IMMBO (200 mpk, po) G6 SRBC (1–2%, IP) IMMBO (400 mpk, po) G7 SRBC (1–2%, IP) IMMBO (800 mpk, po) G8 SRBC footpad control for DTH - DTH G9 SRBC footpad Levamisole (20 mpk, po) G10 SRBC footpad IMMBO (800 mpk, po) In vivo assessments Neutrophil adhesion : On day 14 after administering the test item, blood samples were taken from rats in Groups 5, 6, and 7. These samples were initially analyzed for total leukocyte counts (TLC) and differential leukocyte counts (DLC). Afterward, they were incubated with 80 mg/mL nylon fibers for 15 minutes at 37°C. The Neutrophil Index (NI) was calculated as the product of TLC and the percentage of neutrophils in the blood sample Neutrophil index (NI) = TLC x % neutrophil To determine the percentage of neutrophil adhesion, the following formula was used: Neutrophil Adhesion = [(NIu - NIt) / NIu] x 100, Where NI untreated (NIu) is the neutrophil index of the untreated blood sample; NI treated (NIt) is the neutrophil index of the treated blood sample. Determination of delayed-type hypersensitivity responses : On Day 7 of the study, animals in groups G8 to G10 received an injection of 0.1 mL containing 1x 10 8 SRBC into the right hind footpad, while the left footpad received 0.1% PBS as a control. Test group G9 received daily treatment with IMMBO at 800 mpk from Day 1 to 14, and reference group G10 received Levamisole at 20 mpk following the same schedule. On the 14th day, all animals were challenged with another subcutaneous injection of 0.1 mL containing 1 x 10 8 SRBCs into the left hind footpad. To assess delayed hypersensitivity (DTH), footpad thickness was measured using a Vernier caliper on both the right and left hind paws by using the formula: Left footpad challenged with antigen – Right foot pad control X 100 Determination of pro-inflammatory factors and total antibody levels : Major inflammatory cytokines namely NF-κB, TNF-α, IFN-γ, IL-6, IL-1β, IL-10, and iNOS (in spleen tissue) were assessed in specified matrices. The three major classes of immunoglobulins (IgG, IgA, and IgM) levels were assessed in serum. Western Blotting for Cell signaling markers / MAP kinases : Western blot experiments were conducted to assess MAP kinase expression levels, including AKT, ERK, and PI3K, using spleen tissue lysed with RIPA Buffer. The procedure involved: Tissue Collection: Spleen tissues were weighed, snap-frozen in liquid nitrogen, and homogenized in ice-cold RIPA Buffer containing protease and phosphatase inhibitors. Protein Extraction: Samples were centrifuged, and the supernatant was collected for analysis. Protein content was determined using the Bradford method with BSA as the standard. SDS-PAGE: Proteins were separated on 12.5% SDS-PAGE gels, with pre-stained markers included for reference. Membrane Transfer: Proteins were transferred to PVDF membranes using the wet blot method. Antibody Incubation: The membrane was blocked with 5% skimmed milk in TBST and then incubated with primary antibodies overnight at 4°C. After washing, secondary antibodies were applied for 1 hour at room temperature. Washing: Blots were washed three times with TBST. Detection: ALP-conjugated antibodies were used for color development, and blot images were captured. Band densities were quantified using Image J software. Normalization: Band densities were normalized to the GAPDH signal, serving as the loading control. In vitro assessments Stimulation of RAW cells with LPS and ELISA assay for cytokines : In this experiment, RAW 264.7 cells were seeded at 0.3 x 106 cells/mL in 6-well plates and treated with varying concentrations of IMMBO. The cells were also exposed to the inflammatory stimulant LPS at 1 µg/mL for 24 hours. After treatment, cell lysates were collected and analyzed to measure NF-κB and TLR-4 levels. NF-κB regulates immune and inflammatory genes, while TLR-4 detects LPS, a bacterial component triggering immune responses. This analysis assesses how IMMBO and LPS interact to affect NF-κB pathway activation and TLR-4 expression, providing insights into the potential anti-inflammatory or immunomodulatory effects of IMMBO in response to LPS stimulation. Assessment of IMMBO on RBL-2H3 cells degranulation : In this experiment, degranulation in RBL-2H3 cells was assessed by measuring β-hexosaminidase release. The process involved the following steps: Sensitization: RBL-2H3 cells were plated (1x10 5 cells/well in a 24-well plate) and sensitized with 0.2 µg/mL monoclonal anti-dinitrophenyl mouse Immunoglobulin-E (DNP-IgE) overnight at 37°C. Washing: The cells were washed with PIPES buffer to remove DNP-IgE. Test Item Treatment: The cells were incubated with different test item concentrations for 30 minutes at 37°C. Degranulation Induction: The cells were treated with 1 µg/mL human DNP-albumin and further incubated for 30 minutes at 37°C to induce degranulation. β-Hexosaminidase Release: After degranulation, 25 µL of cell supernatant was transferred to a 96-well plate containing 25 µL of 5 mM 4-nitrophenyl N-acetyl-β-D-glucosaminide in 0.1 M citrate buffer (pH 4.5) and incubated for 2 hours. Reaction Termination: The reaction was stopped by adding 200 µL of a stop buffer (0.05 M Na2CO3/0.05 M NaHCO3, pH 10). Measurement: The optical density was measured at 405 nm to assess degranulation. Statistical analysis The data was entered in the Microsoft Excel spreadsheet, where it was organized and then exported to the statistical software GraphPad Prism for further statistical analyses. It was observed that the data conformed to the assumptions of parametric data. One-way ANOVA was used to test the significance between means of respective control vs. treatment/exercise groups. The data was further subjected to Dunnett’s test for multiple comparisons. All results have been expressed as the Mean ± Standard Deviation (SD) and presented in tables and/or graphs. All differences were considered statistically significant if p ≤ 0.05. The GraphPad Prism software (Version 9.0, Sandiego, CA, USA) performed all statistical analyses. Results Determination of neutrophil adhesion CPH treatment completely suppressed neutrophil activation compared to untreated controls (G1 versus G2). However, treatment with the immune-stimulant reference compound Levamisole restored neutrophil activation (G1 versus G4). IMMBO was effective at mid- and high-dose levels in reinstating neutrophil activation to normal levels (G1, G6, and G7), but it had no significant effect at low doses (G5). IMMBO appears to be an effective method for activating the immune system in immune-suppressed animals. Table 2 Effect of IMMBO on neutrophil adhesion test in rats Groups Treatment % of Neutrophils adhered to nylon G1 Native control 6.06 G2 CPH alone -11.36 G4 CPH + SRBC + Levamisole 5.19 G5 CPH + SRBC + IMMBO 200mpk 1.95 G6 CPH + SRBC + IMMBO 400mpk 5.19 G7 CPH + SRBC + IMMBO 800mpk 6.06 Determination of delayed-type hypersensitivity responses The foot pad swelling assay in rats is used to assess DTH responses. In this model, rats' foot pads are sensitized with an antigen (SRBC), and after seven days a second dose of the antigen is given. The tissue responds in the form of swelling. Results show that CPH and IMMBO at 800mpk dose is effective in stimulating cellular immunity in an immune-suppressed condition (Fig. 1 ). Estimation of Antibody and Inflammatory Biomarkers in blood and serum The effect of the IMMBO on humoral immune response was assessed by measuring levels of IgG, IgM, and IgA antibodies in cyclophosphamide-induced immunosuppressed rats. The quantification of the antibodies was done by the ELISA kit method. Results are presented in Table 3 . Cyclophosphamide (CPH) treatment caused a rapid decline in the content of all three antibody classes, and treatment with Levamisole overcame this effect. Levamisole was found to increase circulating antibodies to significantly higher levels compared to untreated control animals. Treatment with IMMBO was found to be effective at all three dose levels, and for IgM and IgG, it compensated more than the control levels. The effect of IMMBO was similar to that achieved with Levamisole. This data demonstrates that IMMBO is a powerful stimulator of humoral response and has the potential for use in immune-suppressed states such as chemotherapy. Table 3 Effect of IMMBO on IgM, IgG, and IgA levels in sera in response to SRBC in Cyclophosphamide-induced Wister rat. Mean ± SD (n = 8). P < 0.0001, statistically significant as compared to a control group. P < 0.0001, statistically significant as compared to CPH alone Groups IgM (ng/mL) IgG (ng/mL) IgA (ng/mL) Control group 247 ± 12.3 320.74 ± 32.4 219.89 ± 33.2 CPHalone 172.22 ± 10.1 114.83 ± 21 168.46 ± 15.8 CPH + SRBC + Levamisole 455.29 ± 39.3 496.83 ± 38.3 659.87 ± 45.1 CPH + SRBC + Low dose IMMBO 437.81 ± 22.5 379.1 ± 56.3 215.7 ± 68.7 CPH + SRBC + Mid dose IMMBO 441.37 ± 32.1 314.53 ± 88 361.01 ± 47.2 CPH + SRBC + High dose IMMBO 460.26 ± 21.7 341.38 ± 91.8 502.82 ± 29.5 Groups IgM IgG IgA Mean SD P value Mean SD P value Mean SD P value Levamisole 455.29 39.3 496.83 38.3 659.87 45.1 Low dose IMMBO 437.81 22.5 0.2982 379.1 56.3 0.0003 215.7 68.7 < 0.0001 Mid dose IMMBO 441.37 32.1 0.4516 314.53 88.0 0.0003 361.01 47.2 < 0.0001 High dose IMMBO 460.26 21.7 0.7600 341.38 91.8 0.0016 502.82 29.5 0.000002 Level of significance is adjusted considering 4 groups. p value < = 0.05 considered statistically significant. Pro-inflammatory Cytokine levels after IMMBO treatment of immune-suppressed animals To evaluate the protective effect of IMMBO on immunosuppression in CPH-treated rats, IMMBO was orally administrated for 10 days at three different dose levels. The levels of cytokines and immunoglobulin in the serum and spleen were evaluated by ELISA. Immunomodulating cytokines namely NF-κB, TNF-α, IFN-γ, IL-6, IL-1β, IL-10, and iNOS in spleen tissue homogenate assessed as markers for effectiveness. Results are summarized in Fig. 2 . In comparison with the untreated control, the group treated with CPH alone showed a significant reduction in the expression of TNF-α, IFN-Ƴ, and IL-1β. (~ 2.6, 3.4, and 2.1 folds respectively compared to untreated animals). Levamisole corrected the expression levels to almost similar levels seen in untreated control animals. IMMBO treatment also rescued the cytokines expression levels, but a clear dose response was not seen. Incase of TNF-α and IFN-ϒ, the rescue was already high at a low dose of IMMBO (3.1-fold and 6.7-fold, respectively), and a further increase in dose level did not improve the rescue effect. Expression of iNOS (inducible nitric oxide synthase) was not affected by cyclophosphamide treatment (Panel D, Figure. 2). Levamisole or IMMBO treatment did not change the level of expression compared to untreated control animals. NFkB is a transcription factor, and controls expression of various genes coding both anti & pro-inflammatory cytokines. Treatment with cyclophosphamide caused a significant increase in expression of NF-kb and interestingly Levamisole treatment reduced the expression level to almost reaching that of control untreated naïve animals (Panel E, Fig. 2 ). Treatment with IMMBO also reduced its expression and a similar level of reduction was seen at the 3 dose levels used. Interestingly expression levels of IL-10, IL-6, and CRP were elevated in cyclophosphamide-treated animals, and their levels were suppressed to normal levels when Levamisole was used (Panels F, G, and H). Suppression of expression was also seen in IMMBO-treated animals, therefore demonstrating an effect similartothe reference drug Levamisole. These results are thought-provoking as immunosuppression generally down-regulates inflammatory cytokines and markers. The results obtained in this study are likely due to the combined use of SRBC antigen and cyclophosphamide in all test groups, which would have impacted immune system in an unknown manner. Cell signalling pathways in spleen tissue after IMMBO treatment of immune-suppressed animals The spleen is involved in a wide range of immunological functions and regulates both T and B cell responses to antigenic targets in the blood. Its role in haematopoiesis is well known. Components of MAPK and PI3K/AKT signalling pathways are well studied in spleen and immune regulation. Therefore, an attempt was made to study the effects of IMMBO treatment on the regulation of selected signal cascades in spleen tissue. The levels of MAPK (pERK1/2), PI3K, and AKT activation were determined using Western blot technique. At the end of the experimentation, animals were euthanized and spleen was collected. Tissue was homogenized using a lysis buffer containing protease and phosphatase inhibitors. After clarification by centrifugation, the supernatants were used for the western blot. Comparable protein amounts of homogenate were loaded in each gel. The results of this experiment are shown in Fig. 3 . The levels of ERK and PI3K were reduced significantly after treatment with cyclophosphamide (compare G1 and G2, Panel B). For ERK, the reduction was corrected to almost normal levels at the high dose of IMMBO (compare G1 and G7, Panel B). For PI3K, the correction to normal levels was seen at the lowest dose of IMMBO tested and did not improve any more at the next dose levels. We were unable to see clear changes in expression patterns for other targets such as Akt1, pAkt1, and pERK1, probably due to the lack of sensitivity of primary antibody preparations used in the study. However, the positive effect of IMMBO on key signalling molecules (ERK and PI3K) is an interesting observation. Both these proteins are known to be involved in immune modulation and the western blot data corroborates earlier data presented in this report on immune modulation with IMMBO. Regulation of NF-κB expression by IMMBO in RAW264.7 cells Stimulation with E. coli LPS triggers the expression of pro-inflammatory cytokines and biomarkers in cells. These can be measured using ELISA assays. To study the effect of IMMBO, RAW cells were seeded in 6 well plates (0.3 x 106 cells/mL). The cells were pre-treated with IMMBO at different concentrations, with or without LPS (1 µg/mL) for 24 h. Cell supernatant was collected by centrifugation and analyzed for the expression of NF-κB (Fig. 4 ). The result showed that LPS significantly increased the NF-κB level (~ 10 folds compared to untreated control cells). IMMBO treatment resulted in a concentration-dependent inhibition of NFkb expression, and at the highest dose of IMMBO, NFkb levels were found similar to untreated cells. Effect of IMMBO treatment on degranulation of RBL-2H3 cells IgE-activated cells release histamine, measured with ELISA assays. These cells were used to test IMMBO's ability to block histamine release in allergies. Degranulation was detected by measuring β-hexosaminidase release in the medium. Results are shown in Fig. 5 . IgE-DNP HSA stimulated cells showed a ~ 6-fold increase in β hexosaminidase release compared to unstimulated control cells. Control compound Dexamethasone (100µM) significantly reduced the histamine level. IMMBO treatment was effective albeit at low levels, and the effect was clearly seen at higher concentrations (25ug/mL and above). Higher concentrations of IMMBO could not be tested due to challenges of media dilution etc. that impacted assay performance. Overall, results indicate that IMMBO at higher concentrations can block histamine release and thus has the potential to be used in controlling allergic states. Discussion The Ayurvedic pharmacopeia is enriched with an extensive array of classical formulations, incorporating elements from plant, animal, and mineral sources, either individually or in combination 11 . These formulations, crafted over time by revered Rishis and Munis of ancient India, were rooted in visionary and intuitive wisdom, complemented by keen observations. The sages documented the formulas along with brief descriptions of preparation methodologies and recommended usage with doses. However, applied aspects of Ayurvedic therapy were more hands-on back then, guided by a Guru and the knowledge was transmitted through generations, adhering to the Guru Shishya Tradition of the era. Present-day Ayurveda primarily relies on these ancient descriptions, lacking modern pharmacological explanations. IMMBO is a modified and standardized form of a classical Ayurvedic formulation called Punarnavadi Mandoor 12 . IMMBO was first found effective in treating patients suffering from intermittent and persistent Allergic Rhinitis in a North India-based Ayurvedic clinical practice. IMMBO, which is prepared using eighteen herbs and Mandoor Bhasma , could easily be explained for its therapeutics following Ayurvedic concepts of Vata, Pitta, Kapha , and other essential parameters derived from Ayurvedic principles of diagnosis and treatment. However, it is difficult to explain it in today's scientific terminology. Hence, proof of efficacy study was carried out on IMMBO as a Phase III Randomised Controlled Comparative Clinical under subject experts of modern medicines. IMMBO depicted promising clinical efficacy in reducing IgE levels and total nasal symptom scores in the study 8 . IMMBO is a complex compound containing herbs and minerals. None of the ingredients possesses properties to treat Allergic Rhinitis when used alone, but act differently when used in a compound form, indicating the synergistic effect of herbs and minerals. In the above background, exploratory experimental studies were carried out on IMMBO to understand the science behind the seen effect in alleviating the symptoms of Allergic Rhinitis. The findings of the experimental studies shed light on the immuno-modulatory potential of IMMBO. The observed effectiveness of IMMBO in reinstating neutrophil activation under immune-suppressed conditions is a key indicator of its immunostimulant properties. The foot pad swelling assay reveals the ability of IMMBO to stimulate cellular immunity in immune-suppressed conditions induced by cyclophosphamide. The ability of IMMBO to compensate for the decline in antibody levels induced by cyclophosphamide, akin to the reference drug Levamisole, positions it as a potent stimulator of humoral response. The modulation of TNF-α, IFN-γ, IL-1β, and other cytokines demonstrates IMMBO's potential in counteracting immunosuppression at the molecular level. The observed reduction in ERK and PI3K levels after cyclophosphamide treatment and the subsequent correction by IMMBO suggests that IMMBO may exert its effects through the regulation of key signalling molecules involved in immune modulation. The study on NF-κB expression in RAW264.7 cells adds another layer of evidence to IMMBO's anti-inflammatory potential. The ability of IMMBO to block histamine release in IgE-activated cells suggests its potential in controlling allergic states. The results of the present study are very encouraging and advocate Ayurvedic knowledge by using modern scientific parameters and technology. The comprehensive exploration of various immunological parameters provides valuable insights into its potential applications. However, the exact mode of action of IMMBO is unclear. It will be worthwhile to carry forward in-depth pharmacological studies to develop intriguing science behind the immune-enhancing characteristics of IMMBO starting its administration into the human body and its pathways from buccal mucosa to cellular metabolism. Conclusion In conclusion, the results presented in this study collectively underscore the immunomodulatory potential of IMMBO, a Herbo-Mineral Ayurvedic Formulation. The observed effectiveness of IMMBO in reinstating immune responses under various experimental conditions positions it as a promising candidate for further research and potential therapeutic applications in immune-related disorders. 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Le lévamisole, stimulant du système immunitaire chez l'animal et chez l'homme [Levamisole, stimulant of the immune system in animal and man (author's transl)]. Pathol Biol (Paris). 1977;25(1):57–66. Kumar S, Dobos GJ, Rampp T. The Significance of Ayurvedic Medicinal Plants. J Evid Based Complementary Altern Med. 2017; 22(3):494–501. doi: 10.1177/2156587216671392 . Samal J and Dehury RK. A Review of Literature on Punarnavadi Mandura: An Ayurvedic Herbo-Mineral Preparation. Pharmacognosy Journal. 2016;8(3):180–184. doi: 10.5530/pj.2016.3.2 Additional Declarations There is NO Competing Interest. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3625156","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":250409736,"identity":"40d2d930-7280-4982-8daf-f83a96b76382","order_by":0,"name":"Vaidya Prakash","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/klEQVRIiWNgGAWjYBACAwbmBgYGNhATSDA22AAZjI0H8GthRNGSBtLSQJKWw2BRvFrM2Q82Pi4oO5xn3n4s8ePPHeft1rYfBtpSYxONS4tlT2Kz8Yxzh4tlzqQdluY9czt525lEoJZjabkNuBx2ILFNmrftcOIMhvQGaca228lmB4BagC7EreX8w/bfYC38z5t//mw7l2x2/iEBLTcS25jBWiTSjknwth2wM7tBwBbLGQ+bpXnOpRdLSDxLs+ZtS04wuwG0JQGPX8z5kw9+5imzzpPgTzO++bPNzt7sfPrDBx9qbHBqgYEEGCOxAYVLjBZ7IhSPglEwCkbBCAMAP11olAOMcLIAAAAASUVORK5CYII=","orcid":"","institution":"VCPC Research Foundation","correspondingAuthor":true,"prefix":"","firstName":"Vaidya","middleName":"","lastName":"Prakash","suffix":""},{"id":250409737,"identity":"00c4faa2-fac0-49ff-86fe-bf74e2a2597f","order_by":1,"name":"Sneha Sati","email":"","orcid":"","institution":"VCPC Research Foundation","correspondingAuthor":false,"prefix":"","firstName":"Sneha","middleName":"","lastName":"Sati","suffix":""},{"id":250409738,"identity":"8b204ec5-db19-45eb-ac97-ce6f7fb9aa69","order_by":2,"name":"Yashwant Rao","email":"","orcid":"","institution":"GSVM Medical College","correspondingAuthor":false,"prefix":"","firstName":"Yashwant","middleName":"","lastName":"Rao","suffix":""},{"id":250409739,"identity":"d403c2d8-b15c-4e61-99eb-8469c4e648c0","order_by":3,"name":"Shikha Prakash","email":"","orcid":"","institution":"Padaav - A Specialty Ayurvedic Treatment Centre","correspondingAuthor":false,"prefix":"","firstName":"Shikha","middleName":"","lastName":"Prakash","suffix":""},{"id":250409740,"identity":"1ff09821-4e3b-47a0-b0b6-55b9e23c0292","order_by":4,"name":"Neha Negi","email":"","orcid":"","institution":"Padaav - A Specialty Ayurvedic Treatment Centre","correspondingAuthor":false,"prefix":"","firstName":"Neha","middleName":"","lastName":"Negi","suffix":""}],"badges":[],"createdAt":"2023-11-17 10:40:54","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3625156/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3625156/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":50225813,"identity":"054188cc-2a94-43b0-b398-2f854d5d10f7","added_by":"auto","created_at":"2024-01-26 17:55:15","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":39357,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOutcome of the delayed type hypersensitivity study; Figure shows increased paw thickness in the animals; Graph denotes mean paw thickness (in mm) in the three study groups\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"f1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3625156/v1/79a0288f31db5d193a0c3163.jpg"},{"id":50225817,"identity":"23d1e085-cd83-49c1-bbae-88d73ae784bc","added_by":"auto","created_at":"2024-01-26 17:55:15","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":81928,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eModulation of cytokine expression in spleen of immune-suppressed rat after treatment with IMMBO. Rats (n=8/group) were rendered immune-suppressed by treatment with Cyclophosphamide and stimulated with SRBC as an external antigen. Immune modulatory effect of IMMBO was tested at 3 different dose levels after repeated oral dosing for 10 days. Spleen was collected from the animals, and the homogenates were assayed for expression of various biomarkers by specific ELISA kits. Statistical analysis was performed using GraphPad prism. Statistical differences between groups were evaluated using a one-way analysis of variance followed by Dunnett's test or t-test\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"f2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3625156/v1/685988c8638b96400a7addb5.jpg"},{"id":50225814,"identity":"0d734074-7455-4ee6-b611-28ff7b750ff2","added_by":"auto","created_at":"2024-01-26 17:55:15","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":48353,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(A) Western blot analysis of spleen tissue homogenates for expression of selected signalling proteins. Proteins were separated on a 12.5% SDS-PAGE and after transfer to a nitrocellulose membrane, the blots were developed using specific primary and secondary antibodies; (B) The density of bands was measured using Image J software\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"f3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3625156/v1/50119ce101072e7d75dc7afd.jpg"},{"id":50225815,"identity":"7d8f38d0-7c4a-4065-9472-bc4d169f616e","added_by":"auto","created_at":"2024-01-26 17:55:15","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":16356,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect if IMMBO on NF-kb in LPS-treated RAW 264.7 cells\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"f4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3625156/v1/a0ac61ba6bb9c9b39c8cbd29.jpg"},{"id":50226310,"identity":"f68d8daa-453b-4e3c-ac60-39fb8b388623","added_by":"auto","created_at":"2024-01-26 18:03:15","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":18718,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of IMMBO on degranulation in stimulated RBL 2H3 cell line\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"f5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3625156/v1/4df9b8803f90300f5278bd77.jpg"},{"id":50226549,"identity":"63893879-28af-4470-8e7b-02b283dfeb6c","added_by":"auto","created_at":"2024-01-26 18:11:15","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":832271,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3625156/v1/ad2afc0b-d6d1-48e5-b07b-b59ec4774094.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Immuno-modulatory Effects of a Herbo-mineral Ayurvedic Formulation in Experimental Models","fulltext":[{"header":"Introduction:","content":"\u003cp\u003eAyurveda \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e, an ancient Indian system of medicine documented primarily in Sanskrit \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, offers comprehensive definitions, principles, and procedures for disease prevention and treatment \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Its core components include dietary practices, lifestyle recommendations, and medicinal formulations \u003cem\u003e(aahar, vihaar\u003c/em\u003e and \u003cem\u003eaushadh)\u003c/em\u003e \u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e. Rooted in centuries-old tradition, this knowledge has been nurtured in Gurukuls and Ashrams through the revered Guru-Shishya tradition \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Ayurvedic formulations, incorporating ingredients from plant, animal, and mineral origins, have gained global recognition for their healing potential \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Consequently, there has been a surge in efforts worldwide to subject Ayurvedic concepts, principles, and formulations to rigorous research methodologies.\u003c/p\u003e \u003cp\u003eOne specific Herbo-Mineral Ayurvedic Formulation (HMAF), a combination of eighteen herbs and Mandoor Bhasma, was initially pioneered by a registered Ayurvedic physician in North India, operating under the provisions of Section 33EEC of the Drug and Cosmetics Act of India \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. HMAF has shown promising results in the treatment of patients suffering from Persistent and Intermittent forms of Allergic Rhinitis. Following successful outcomes, a manufacturing license was obtained from the State Drug Licensing Authority in India for its commercial production, and it was introduced into the market under the name IMMBO \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eSimultaneously, IMMBO underwent a phase III prospective randomized controlled clinical trial to evaluate its efficacy in comparison to the standard treatment of FDC Levocetirizine 2.5 mg\u0026thinsp;+\u0026thinsp;Montelukast 4 mg in allergic rhinitis patients. The study yielded compelling evidence supporting IMMBO's effectiveness in treating allergic rhinitis \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAdditionally, IMMBO underwent extensive in vivo and in vitro studies to explore its intriguing mechanism of action. Wistar albino rats were subjected to experiments involving Cyclophosphamide (CPH)-induced immune suppression \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. These studies assessed IMMBO's impact on various parameters, including neutrophil adhesion, delayed-type hypersensitivity response, antibody levels, inflammatory and pro-inflammatory markers, and cell signaling pathways. This paper delves into the outcomes and interpretations derived from these studies.\u003c/p\u003e"},{"header":"Materials \u0026 Methods","content":"\u003cp\u003eAnimals of the same sex, housed in groups of two to three per cage, were kept in a controlled environment within an air-conditioned room with a temperature maintained at 20\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u0026deg;C and a relative humidity ranging from 30\u0026ndash;70%. The room had air changes occurring at a rate of 10\u0026ndash;15 per hour, and a 12-hour light and 12-hour dark cycle was maintained. Temperature and humidity levels were continuously monitored and recorded once daily.\u003c/p\u003e \u003cp\u003eThe standard polycarbonate cages were equipped with stainless steel mesh top grills and provisions for pelleted food and water bottles. All the animals underwent a minimum five-day acclimatization period in a laboratory setting. Veterinary examinations were conducted on the day the animals were received and before the commencement of the treatment.\u003c/p\u003e \u003cp\u003eAnimals were fed with normal rodent feed and reverse osmosis water was always available to the animals during the experiments. Daily observations for any clinical signs were conducted.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy design\u003c/h2\u003e \u003cp\u003eThe study was conducted in Southern India by an internationally accredited and DSIR, Government of India recognized research unit. Healthy Wistar albino rats (6\u0026ndash;8 weeks old) were grouped based on body weight and randomly assigned to different treatment groups. In the main study, 48 rats were divided into seven experimental groups (G1 to G7) for immune modulator study. Additionally, a pilot set of rats (G7 to G10) was maintained separately to evaluate delayed-type hypersensitivity (DTH).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eEthics\u003c/h2\u003e \u003cp\u003e This study was performed at a CPCSEA-approved and AAALAC accredited laboratory following all ethical practices as laid down in the guideline/s for animal care. The study was approved by the Institutional Animals Ethics Committee (IAEC) of the test facility.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eMaterials\u003c/h2\u003e \u003cp\u003eCyclophosphamide served as the negative control, functioning as an immunosuppressant, while Levamisole hydrochloride acted as the positive control, serving as an immunity stimulant \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFresh sheep blood was collected and mixed with sterile Alsever's solution. The mixture was centrifuged at 3000 rpm for 5 minutes to obtain red blood cell sediment. The cell pellet was then resuspended in Phosphate Buffer Saline (PBS) with a pH of 7.2 and centrifuged at 1500 rpm. This washing process was repeated until a clear top layer was observed. The Sheep Red Blood Cells (SRBC) were adjusted to the desired levels based on haematocrit values and stored in the refrigerator for later use.\u003c/p\u003e \u003cp\u003eRAW 264.7 macrophages and RBL-2H3 mast cells were obtained from ATCC, USA and maintained in DMEM with 5% FBS/1% penicillin-streptomycin at 37\u0026deg;C in a 5% CO2 environment. Both cell types were incubated in DMEM with 10% FBS, 2 mM glutamine, 100 U/mL penicillin, and 50 \u0026micro;g/mL streptomycin.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eExperimental protocol\u003c/h2\u003e \u003cp\u003eIn set 1, rats in the normal control group (G1) received saline for two weeks. Experimental groups (G2 to G7) received cyclophosphamide (100 mg/kg) on Day 0, followed by sheep red blood cell (SRBC) challenge on Days 7 and 14. G4 received the immunostimulant levamisole (20 mpk). G5 to G7 received IMMBO at low (200 mpk), mid (400 mpk), and high (800 mpk) doses orally.\u003c/p\u003e \u003cp\u003eIn set 2, animals in G8 to G10 were subjected to delayed-type hypersensitivity (DTH) testing with SRBC on Days 7 and 14. G9 received levamisole (20 mpk) and G10 received high-dose IMMBO (800 mpk) from Day-1 to Day-14 as pre-treatment.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eWork flow\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eTest groups\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eIn-life experiments\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eEnd point assessments\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eImmune suppression\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSRBC (1\u0026ndash;2%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTreatment\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDay 0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDay 7 \u0026amp; 14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDay 1 to 14\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eG1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"9\" rowspan=\"10\"\u003e \u003cp\u003eCyclophosphamide (100mpk)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eVehicle (0.9% saline)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"6\" rowspan=\"7\"\u003e \u003cp\u003eBody and lymphoid organs weight, Neutrophil adhesion, DTH, TNF-\u003cb\u003eα\u003c/b\u003e, IFN-γ, IL-6, IL-1β, IL-10, IgG, IgA, IgM, MAP kinases (AKT, ERK, PI3K)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eG2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eG3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSRBC (Control, IP)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eG4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSRBC (1\u0026ndash;2%, IP)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLevamisole (20 mpk, po)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eG5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSRBC (1\u0026ndash;2%, IP)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIMMBO (200 mpk, po)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eG6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSRBC (1\u0026ndash;2%, IP)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIMMBO (400 mpk, po)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eG7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSRBC (1\u0026ndash;2%, IP)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIMMBO (800 mpk, po)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eG8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSRBC footpad control for DTH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eDTH\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eG9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSRBC footpad\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLevamisole (20 mpk, po)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eG10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSRBC footpad\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIMMBO (800 mpk, po)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"In vivo assessments","content":"\u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003e \u003cem\u003eNeutrophil adhesion\u003c/em\u003e: On day 14 after administering the test item, blood samples were taken from rats in Groups 5, 6, and 7. These samples were initially analyzed for total leukocyte counts (TLC) and differential leukocyte counts (DLC). Afterward, they were incubated with 80 mg/mL nylon fibers for 15 minutes at 37\u0026deg;C.\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003eThe Neutrophil Index (NI) was calculated as the product of TLC and the percentage of neutrophils in the blood sample\u003c/p\u003e \u003cp\u003eNeutrophil index (NI)\u0026thinsp;=\u0026thinsp;TLC x % neutrophil\u003c/p\u003e \u003cp\u003eTo determine the percentage of neutrophil adhesion, the following formula was used:\u003c/p\u003e \u003cp\u003eNeutrophil Adhesion = [(NIu - NIt) / NIu] x 100,\u003c/p\u003e \u003cp\u003eWhere NI untreated (NIu) is the neutrophil index of the untreated blood sample; NI treated (NIt) is the neutrophil index of the treated blood sample.\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003e \u003cem\u003eDetermination of delayed-type hypersensitivity responses\u003c/em\u003e: On Day 7 of the study, animals in groups G8 to G10 received an injection of 0.1 mL containing 1x 10\u003csup\u003e8\u003c/sup\u003e SRBC into the right hind footpad, while the left footpad received 0.1% PBS as a control. Test group G9 received daily treatment with IMMBO at 800 mpk from Day 1 to 14, and reference group G10 received Levamisole at 20 mpk following the same schedule. On the 14th day, all animals were challenged with another subcutaneous injection of 0.1 mL containing 1 x 10\u003csup\u003e8\u003c/sup\u003e SRBCs into the left hind footpad. To assess delayed hypersensitivity (DTH), footpad thickness was measured using a Vernier caliper on both the right and left hind paws by using the formula:\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003eLeft footpad challenged with antigen \u0026ndash; Right foot pad control X 100\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003e \u003cem\u003eDetermination of pro-inflammatory factors and total antibody levels\u003c/em\u003e: Major inflammatory cytokines namely NF-κB, TNF-α, IFN-γ, IL-6, IL-1β, IL-10, and iNOS (in spleen tissue) were assessed in specified matrices. The three major classes of immunoglobulins (IgG, IgA, and IgM) levels were assessed in serum.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003e \u003cem\u003eWestern Blotting for Cell signaling markers / MAP kinases\u003c/em\u003e: Western blot experiments were conducted to assess MAP kinase expression levels, including AKT, ERK, and PI3K, using spleen tissue lysed with RIPA Buffer. The procedure involved:\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003e \u003col\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eTissue Collection: Spleen tissues were weighed, snap-frozen in liquid nitrogen, and homogenized in ice-cold RIPA Buffer containing protease and phosphatase inhibitors.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eProtein Extraction: Samples were centrifuged, and the supernatant was collected for analysis. Protein content was determined using the Bradford method with BSA as the standard.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eSDS-PAGE: Proteins were separated on 12.5% SDS-PAGE gels, with pre-stained markers included for reference.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eMembrane Transfer: Proteins were transferred to PVDF membranes using the wet blot method.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eAntibody Incubation: The membrane was blocked with 5% skimmed milk in TBST and then incubated with primary antibodies overnight at 4\u0026deg;C. After washing, secondary antibodies were applied for 1 hour at room temperature.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eWashing: Blots were washed three times with TBST.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eDetection: ALP-conjugated antibodies were used for color development, and blot images were captured. Band densities were quantified using Image J software.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eNormalization: Band densities were normalized to the GAPDH signal, serving as the loading control.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eIn vitro assessments\u003c/h2\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003e \u003cem\u003eStimulation of RAW cells with LPS and ELISA assay for cytokines\u003c/em\u003e: In this experiment, RAW 264.7 cells were seeded at 0.3 x 106 cells/mL in 6-well plates and treated with varying concentrations of IMMBO. The cells were also exposed to the inflammatory stimulant LPS at 1 \u0026micro;g/mL for 24 hours. After treatment, cell lysates were collected and analyzed to measure NF-κB and TLR-4 levels. NF-κB regulates immune and inflammatory genes, while TLR-4 detects LPS, a bacterial component triggering immune responses. This analysis assesses how IMMBO and LPS interact to affect NF-κB pathway activation and TLR-4 expression, providing insights into the potential anti-inflammatory or immunomodulatory effects of IMMBO in response to LPS stimulation.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003e \u003cem\u003eAssessment of IMMBO on RBL-2H3 cells degranulation\u003c/em\u003e: In this experiment, degranulation in RBL-2H3 cells was assessed by measuring β-hexosaminidase release. The process involved the following steps:\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eSensitization: RBL-2H3 cells were plated (1x10\u003csup\u003e5\u003c/sup\u003e cells/well in a 24-well plate) and sensitized with 0.2 \u0026micro;g/mL monoclonal anti-dinitrophenyl mouse Immunoglobulin-E (DNP-IgE) overnight at 37\u0026deg;C.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eWashing: The cells were washed with PIPES buffer to remove DNP-IgE.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eTest Item Treatment: The cells were incubated with different test item concentrations for 30 minutes at 37\u0026deg;C.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eDegranulation Induction: The cells were treated with 1 \u0026micro;g/mL human DNP-albumin and further incubated for 30 minutes at 37\u0026deg;C to induce degranulation.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eβ-Hexosaminidase Release: After degranulation, 25 \u0026micro;L of cell supernatant was transferred to a 96-well plate containing 25 \u0026micro;L of 5 mM 4-nitrophenyl N-acetyl-β-D-glucosaminide in 0.1 M citrate buffer (pH 4.5) and incubated for 2 hours.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eReaction Termination: The reaction was stopped by adding 200 \u0026micro;L of a stop buffer (0.05 M Na2CO3/0.05 M NaHCO3, pH 10).\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eMeasurement: The optical density was measured at 405 nm to assess degranulation.\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe data was entered in the Microsoft Excel spreadsheet, where it was organized and then exported to the statistical software GraphPad Prism for further statistical analyses. It was observed that the data conformed to the assumptions of parametric data. One-way ANOVA was used to test the significance between means of respective control vs. treatment/exercise groups. The data was further subjected to Dunnett\u0026rsquo;s test for multiple comparisons. All results have been expressed as the Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;Standard Deviation (SD) and presented in tables and/or graphs. All differences were considered statistically significant if p\u0026thinsp;\u0026le;\u0026thinsp;0.05. The GraphPad Prism software (Version 9.0, Sandiego, CA, USA) performed all statistical analyses.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of neutrophil adhesion\u003c/h2\u003e \u003cp\u003eCPH treatment completely suppressed neutrophil activation compared to untreated controls (G1 versus G2). However, treatment with the immune-stimulant reference compound Levamisole restored neutrophil activation (G1 versus G4). IMMBO was effective at mid- and high-dose levels in reinstating neutrophil activation to normal levels (G1, G6, and G7), but it had no significant effect at low doses (G5). IMMBO appears to be an effective method for activating the immune system in immune-suppressed animals.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffect of IMMBO on neutrophil adhesion test in rats\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroups\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTreatment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e% of Neutrophils adhered to nylon\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eG1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNative control\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6.06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eG2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCPH alone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e-11.36\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eG4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCPH\u0026thinsp;+\u0026thinsp;SRBC\u0026thinsp;+\u0026thinsp;Levamisole\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5.19\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eG5\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCPH\u0026thinsp;+\u0026thinsp;SRBC\u0026thinsp;+\u0026thinsp;IMMBO 200mpk\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.95\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eG6\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCPH\u0026thinsp;+\u0026thinsp;SRBC\u0026thinsp;+\u0026thinsp;IMMBO 400mpk\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5.19\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eG7\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCPH\u0026thinsp;+\u0026thinsp;SRBC\u0026thinsp;+\u0026thinsp;IMMBO 800mpk\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6.06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of delayed-type hypersensitivity responses\u003c/h2\u003e \u003cp\u003eThe foot pad swelling assay in rats is used to assess DTH responses. In this model, rats' foot pads are sensitized with an antigen (SRBC), and after seven days a second dose of the antigen is given. The tissue responds in the form of swelling. Results show that CPH and IMMBO at 800mpk dose is effective in stimulating cellular immunity in an immune-suppressed condition (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eEstimation of Antibody and Inflammatory Biomarkers in blood and serum\u003c/h2\u003e \u003cp\u003eThe effect of the IMMBO on humoral immune response was assessed by measuring levels of IgG, IgM, and IgA antibodies in cyclophosphamide-induced immunosuppressed rats. The quantification of the antibodies was done by the ELISA kit method. Results are presented in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Cyclophosphamide (CPH) treatment caused a rapid decline in the content of all three antibody classes, and treatment with Levamisole overcame this effect. Levamisole was found to increase circulating antibodies to significantly higher levels compared to untreated control animals. Treatment with IMMBO was found to be effective at all three dose levels, and for IgM and IgG, it compensated more than the control levels. The effect of IMMBO was similar to that achieved with Levamisole. This data demonstrates that IMMBO is a powerful stimulator of humoral response and has the potential for use in immune-suppressed states such as chemotherapy.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffect of IMMBO on IgM, IgG, and IgA levels in sera in response to SRBC in Cyclophosphamide-induced Wister rat. Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD (n\u0026thinsp;=\u0026thinsp;8). P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, statistically significant as compared to a control group. P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, statistically significant as compared to CPH alone\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroups\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIgM (ng/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIgG (ng/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIgA (ng/mL)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl group\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e247\u0026thinsp;\u0026plusmn;\u0026thinsp;12.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e320.74\u0026thinsp;\u0026plusmn;\u0026thinsp;32.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e219.89\u0026thinsp;\u0026plusmn;\u0026thinsp;33.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCPHalone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e172.22\u0026thinsp;\u0026plusmn;\u0026thinsp;10.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e114.83\u0026thinsp;\u0026plusmn;\u0026thinsp;21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e168.46\u0026thinsp;\u0026plusmn;\u0026thinsp;15.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCPH\u0026thinsp;+\u0026thinsp;SRBC\u0026thinsp;+\u0026thinsp;Levamisole\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e455.29\u0026thinsp;\u0026plusmn;\u0026thinsp;39.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e496.83\u0026thinsp;\u0026plusmn;\u0026thinsp;38.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e659.87\u0026thinsp;\u0026plusmn;\u0026thinsp;45.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCPH\u0026thinsp;+\u0026thinsp;SRBC\u0026thinsp;+\u0026thinsp;Low dose IMMBO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e437.81\u0026thinsp;\u0026plusmn;\u0026thinsp;22.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e379.1\u0026thinsp;\u0026plusmn;\u0026thinsp;56.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e215.7\u0026thinsp;\u0026plusmn;\u0026thinsp;68.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCPH\u0026thinsp;+\u0026thinsp;SRBC\u0026thinsp;+\u0026thinsp;Mid dose IMMBO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e441.37\u0026thinsp;\u0026plusmn;\u0026thinsp;32.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e314.53\u0026thinsp;\u0026plusmn;\u0026thinsp;88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e361.01\u0026thinsp;\u0026plusmn;\u0026thinsp;47.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCPH\u0026thinsp;+\u0026thinsp;SRBC\u0026thinsp;+\u0026thinsp;High dose IMMBO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e460.26\u0026thinsp;\u0026plusmn;\u0026thinsp;21.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e341.38\u0026thinsp;\u0026plusmn;\u0026thinsp;91.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e502.82\u0026thinsp;\u0026plusmn;\u0026thinsp;29.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e \u003ccolgroup cols=\"10\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eGroups\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eIgM\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e \u003cp\u003eIgG\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c10\" namest=\"c8\"\u003e \u003cp\u003eIgA\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMean\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eP value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMean\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eP value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eMean\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eSD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eP value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLevamisole\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e455.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e39.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e496.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e38.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e659.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e45.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLow dose IMMBO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e437.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e22.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.2982\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e379.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e56.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.0003\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e215.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e68.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMid dose IMMBO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e441.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e32.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.4516\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e314.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e88.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.0003\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e361.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e47.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHigh dose IMMBO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e460.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e21.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.7600\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e341.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e91.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.0016\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e502.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e29.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.000002\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eLevel of significance is adjusted considering 4 groups. p value\u0026thinsp;\u0026lt;\u0026thinsp;=\u0026thinsp;0.05 considered statistically significant.\u003c/b\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003ePro-inflammatory Cytokine levels after IMMBO treatment of immune-suppressed animals\u003c/h2\u003e \u003cp\u003eTo evaluate the protective effect of IMMBO on immunosuppression in CPH-treated rats, IMMBO was orally administrated for 10 days at three different dose levels. The levels of cytokines and immunoglobulin in the serum and spleen were evaluated by ELISA. Immunomodulating cytokines namely NF-κB, TNF-α, IFN-γ, IL-6, IL-1β, IL-10, and iNOS in spleen tissue homogenate assessed as markers for effectiveness. Results are summarized in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. In comparison with the untreated control, the group treated with CPH alone showed a significant reduction in the expression of TNF-α, IFN-Ƴ, and IL-1β. (~\u0026thinsp;2.6, 3.4, and 2.1 folds respectively compared to untreated animals). Levamisole corrected the expression levels to almost similar levels seen in untreated control animals. IMMBO treatment also rescued the cytokines expression levels, but a clear dose response was not seen. Incase of TNF-α and IFN-ϒ, the rescue was already high at a low dose of IMMBO (3.1-fold and 6.7-fold, respectively), and a further increase in dose level did not improve the rescue effect. Expression of iNOS (inducible nitric oxide synthase) was not affected by cyclophosphamide treatment (Panel D, Figure. 2). Levamisole or IMMBO treatment did not change the level of expression compared to untreated control animals. NFkB is a transcription factor, and controls expression of various genes coding both anti \u0026amp; pro-inflammatory cytokines. Treatment with cyclophosphamide caused a significant increase in expression of NF-kb and interestingly Levamisole treatment reduced the expression level to almost reaching that of control untreated na\u0026iuml;ve animals (Panel E, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Treatment with IMMBO also reduced its expression and a similar level of reduction was seen at the 3 dose levels used. Interestingly expression levels of IL-10, IL-6, and CRP were elevated in cyclophosphamide-treated animals, and their levels were suppressed to normal levels when Levamisole was used (Panels F, G, and H). Suppression of expression was also seen in IMMBO-treated animals, therefore demonstrating an effect similartothe reference drug Levamisole. These results are thought-provoking as immunosuppression generally down-regulates inflammatory cytokines and markers. The results obtained in this study are likely due to the combined use of SRBC antigen and cyclophosphamide in all test groups, which would have impacted immune system in an unknown manner.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eCell signalling pathways in spleen tissue after IMMBO treatment of immune-suppressed animals\u003c/h2\u003e \u003cp\u003eThe spleen is involved in a wide range of immunological functions and regulates both T and B cell responses to antigenic targets in the blood. Its role in haematopoiesis is well known. Components of MAPK and PI3K/AKT signalling pathways are well studied in spleen and immune regulation. Therefore, an attempt was made to study the effects of IMMBO treatment on the regulation of selected signal cascades in spleen tissue. The levels of MAPK (pERK1/2), PI3K, and AKT activation were determined using Western blot technique. At the end of the experimentation, animals were euthanized and spleen was collected. Tissue was homogenized using a lysis buffer containing protease and phosphatase inhibitors. After clarification by centrifugation, the supernatants were used for the western blot. Comparable protein amounts of homogenate were loaded in each gel. The results of this experiment are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. The levels of ERK and PI3K were reduced significantly after treatment with cyclophosphamide (compare G1 and G2, Panel B). For ERK, the reduction was corrected to almost normal levels at the high dose of IMMBO (compare G1 and G7, Panel B). For PI3K, the correction to normal levels was seen at the lowest dose of IMMBO tested and did not improve any more at the next dose levels. We were unable to see clear changes in expression patterns for other targets such as Akt1, pAkt1, and pERK1, probably due to the lack of sensitivity of primary antibody preparations used in the study. However, the positive effect of IMMBO on key signalling molecules (ERK and PI3K) is an interesting observation. Both these proteins are known to be involved in immune modulation and the western blot data corroborates earlier data presented in this report on immune modulation with IMMBO.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eRegulation of NF-κB expression by IMMBO in RAW264.7 cells\u003c/h2\u003e \u003cp\u003eStimulation with E. coli LPS triggers the expression of pro-inflammatory cytokines and biomarkers in cells. These can be measured using ELISA assays. To study the effect of IMMBO, RAW cells were seeded in 6 well plates (0.3 x 106 cells/mL). The cells were pre-treated with IMMBO at different concentrations, with or without LPS (1 \u0026micro;g/mL) for 24 h. Cell supernatant was collected by centrifugation and analyzed for the expression of NF-κB (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The result showed that LPS significantly increased the NF-κB level (~\u0026thinsp;10 folds compared to untreated control cells). IMMBO treatment resulted in a concentration-dependent inhibition of NFkb expression, and at the highest dose of IMMBO, NFkb levels were found similar to untreated cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eEffect of IMMBO treatment on degranulation of RBL-2H3 cells\u003c/h2\u003e \u003cp\u003eIgE-activated cells release histamine, measured with ELISA assays. These cells were used to test IMMBO's ability to block histamine release in allergies. Degranulation was detected by measuring β-hexosaminidase release in the medium. Results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. IgE-DNP HSA stimulated cells showed a\u0026thinsp;~\u0026thinsp;6-fold increase in β hexosaminidase release compared to unstimulated control cells. Control compound Dexamethasone (100\u0026micro;M) significantly reduced the histamine level. IMMBO treatment was effective albeit at low levels, and the effect was clearly seen at higher concentrations (25ug/mL and above). Higher concentrations of IMMBO could not be tested due to challenges of media dilution etc. that impacted assay performance. Overall, results indicate that IMMBO at higher concentrations can block histamine release and thus has the potential to be used in controlling allergic states.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe Ayurvedic pharmacopeia is enriched with an extensive array of classical formulations, incorporating elements from plant, animal, and mineral sources, either individually or in combination \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. These formulations, crafted over time by revered Rishis and Munis of ancient India, were rooted in visionary and intuitive wisdom, complemented by keen observations. The sages documented the formulas along with brief descriptions of preparation methodologies and recommended usage with doses. However, applied aspects of Ayurvedic therapy were more hands-on back then, guided by a Guru and the knowledge was transmitted through generations, adhering to the Guru Shishya Tradition of the era. Present-day Ayurveda primarily relies on these ancient descriptions, lacking modern pharmacological explanations.\u003c/p\u003e \u003cp\u003eIMMBO is a modified and standardized form of a classical Ayurvedic formulation called \u003cem\u003ePunarnavadi Mandoor\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. IMMBO was first found effective in treating patients suffering from intermittent and persistent Allergic Rhinitis in a North India-based Ayurvedic clinical practice. IMMBO, which is prepared using eighteen herbs and \u003cem\u003eMandoor Bhasma\u003c/em\u003e, could easily be explained for its therapeutics following Ayurvedic concepts of \u003cem\u003eVata, Pitta, Kapha\u003c/em\u003e, and other essential parameters derived from Ayurvedic principles of diagnosis and treatment. However, it is difficult to explain it in today's scientific terminology. Hence, proof of efficacy study was carried out on IMMBO as a Phase III Randomised Controlled Comparative Clinical under subject experts of modern medicines. IMMBO depicted promising clinical efficacy in reducing IgE levels and total nasal symptom scores in the study \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIMMBO is a complex compound containing herbs and minerals. None of the ingredients possesses properties to treat Allergic Rhinitis when used alone, but act differently when used in a compound form, indicating the synergistic effect of herbs and minerals. In the above background, exploratory experimental studies were carried out on IMMBO to understand the science behind the seen effect in alleviating the symptoms of Allergic Rhinitis.\u003c/p\u003e \u003cp\u003eThe findings of the experimental studies shed light on the immuno-modulatory potential of IMMBO. The observed effectiveness of IMMBO in reinstating neutrophil activation under immune-suppressed conditions is a key indicator of its immunostimulant properties. The foot pad swelling assay reveals the ability of IMMBO to stimulate cellular immunity in immune-suppressed conditions induced by cyclophosphamide. The ability of IMMBO to compensate for the decline in antibody levels induced by cyclophosphamide, akin to the reference drug Levamisole, positions it as a potent stimulator of humoral response. The modulation of TNF-α, IFN-γ, IL-1β, and other cytokines demonstrates IMMBO's potential in counteracting immunosuppression at the molecular level. The observed reduction in ERK and PI3K levels after cyclophosphamide treatment and the subsequent correction by IMMBO suggests that IMMBO may exert its effects through the regulation of key signalling molecules involved in immune modulation. The study on NF-κB expression in RAW264.7 cells adds another layer of evidence to IMMBO's anti-inflammatory potential. The ability of IMMBO to block histamine release in IgE-activated cells suggests its potential in controlling allergic states. The results of the present study are very encouraging and advocate Ayurvedic knowledge by using modern scientific parameters and technology.\u003c/p\u003e \u003cp\u003eThe comprehensive exploration of various immunological parameters provides valuable insights into its potential applications. However, the exact mode of action of IMMBO is unclear. It will be worthwhile to carry forward in-depth pharmacological studies to develop intriguing science behind the immune-enhancing characteristics of IMMBO starting its administration into the human body and its pathways from buccal mucosa to cellular metabolism.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, the results presented in this study collectively underscore the immunomodulatory potential of IMMBO, a Herbo-Mineral Ayurvedic Formulation. The observed effectiveness of IMMBO in reinstating immune responses under various experimental conditions positions it as a promising candidate for further research and potential therapeutic applications in immune-related disorders.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eJaiswal YS, Williams LL. A glimpse of Ayurveda - The forgotten history and principles of Indian traditional medicine. J Tradit Complement Med. 2016;7(1):50\u0026ndash;53. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.jtcme.2016.02.002\u003c/span\u003e\u003cspan address=\"10.1016/j.jtcme.2016.02.002\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ehttps://en.wikipedia.org/wiki/Sanskrit#:~:text=Sanskrit%20belongs%20to%20the%20Indo,.%20750%E2%80%93400%20BCE. Last Accessed on 4-11-2023\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMishra D and Sinha M. Ayurvedic Management Of Polycystic Ovarian Syndrome(Infertility Queen). J Res Educ Indian Med. 2008; 14(1): 33\u0026ndash;40.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDr. Chaithanya, Dr. Venandana, \u0026amp; Dr. Chetan M. (2019). Guru and Shishya: A unique coordinate. Journal of Ayurveda and Integrated Medical Sciences, 4(05), 337\u0026ndash;343. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.21760/jaims.v4i05.745\u003c/span\u003e\u003cspan address=\"10.21760/jaims.v4i05.745\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChauhan A, Semwal DK, Mishra SP, Semwal RB. Ayurvedic research and methodology: Present status and future strategies. Ayu. 2015 Oct-Dec;36(4):364\u0026ndash;369. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.4103/0974-8520.190699\u003c/span\u003e\u003cspan address=\"10.4103/0974-8520.190699\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eProvisions relating to Ayurvedic Siddha and Unani drugs. In The Drugs and Cosmetics Act, of 1940. 2003. Government of India Ministry of health and family welfare. Available at: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cdsco.gov.in/opencms/export/sites/CDSCO_WEB/Pdf-documents/acts_rules/2016DrugsandCosmeticsAct1940Rules1945.pdf\u003c/span\u003e\u003cspan address=\"https://cdsco.gov.in/opencms/export/sites/CDSCO_WEB/Pdf-documents/acts_rules/2016DrugsandCosmeticsAct1940Rules1945.pdf\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. Last accessed on: 4-11-2023.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eManufacturing License No. A-3077/2000 for Bharat Bhaishajya Shala Pvt Ltd, Uttarakhand\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePrakash V B, Rao Y K, Prakash S, et al. (October 08, 2023) Proof of Efficacy Study to Evaluate an Ayurvedic Formulation in the Treatment of Allergic Rhinitis: An Open Label Randomized Controlled Clinical Trial. \u003cem\u003eCureus\u003c/em\u003e 15(10): e46663. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.7759/cureus.46663\u003c/span\u003e\u003cspan address=\"10.7759/cureus.46663\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKolathingal-Thodika N, Usha PTA, Sujarani S, Suresh NN, Priya PM, Naseef PP, Kuruniyan MS, Ollakkode S, Elayadeth-Meethal M. A cyclophosphamide-induced immunosuppression Swiss Albino mouse model unveils a potential role for cow urine distillate as a feed additive. J Ayurveda Integr Med. 2023;14(5):100784. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.jaim.2023.100784\u003c/span\u003e\u003cspan address=\"10.1016/j.jaim.2023.100784\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAndrieu JM. Le l\u0026eacute;vamisole, stimulant du syst\u0026egrave;me immunitaire chez l'animal et chez l'homme [Levamisole, stimulant of the immune system in animal and man (author's transl)]. Pathol Biol (Paris). 1977;25(1):57\u0026ndash;66.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKumar S, Dobos GJ, Rampp T. The Significance of Ayurvedic Medicinal Plants. J Evid Based Complementary Altern Med. 2017; 22(3):494\u0026ndash;501. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1177/2156587216671392\u003c/span\u003e\u003cspan address=\"10.1177/2156587216671392\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSamal J and Dehury RK. A Review of Literature on Punarnavadi Mandura: An Ayurvedic Herbo-Mineral Preparation. Pharmacognosy Journal. 2016;8(3):180\u0026ndash;184. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.5530/pj.2016.3.2\u003c/span\u003e\u003cspan address=\"10.5530/pj.2016.3.2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Ayurveda, Allergy, Immuno-modulator, Immunity, IMMBO","lastPublishedDoi":"10.21203/rs.3.rs-3625156/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3625156/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAyurveda, an ancient Indian system of medicine, encapsulates comprehensive principles and formulations for disease prevention and treatment. A herbo-mineral Ayurvedic formulation, IMMBO, comprising \u003cem\u003eMandoor Bhasma\u003c/em\u003e and eighteen herbs has shown promising results in treating Allergic Rhinitis. The said efficacy was recently reinstated by a randomized controlled clinical trial conducted at a reputed hospital and IMMBO significantly reduced the IgE levels and Total nasal symptom scores in treated patients. This article discusses the findings of a series of experimental studies conducted to explore the immuno-modulatory potential of IMMBO. The studies reveal the immunomodulatory effects of IMMBO, evidenced by its effectiveness in reinstating neutrophil activation, stimulating cellular and humoral immunity, and counteracting immunosuppression at the molecular level. The modulation of key signalling molecules, including TNF-α, IFN-γ, IL-1β, ERK, PI3K, and NF-κB, showcases the formulation\u0026rsquo;s multifaceted impact. Additionally, its ability to block histamine release suggests potential in controlling allergic states, positioning it as a promising therapeutic candidate for immune-related disorders. However, the precise mode of action remains elusive, warranting further in-depth pharmacological studies. This research substantiates the ancient Ayurvedic wisdom using modern scientific parameters, endorsing IMMBO's potential as an immune-modulatory agent.\u003c/p\u003e","manuscriptTitle":"Immuno-modulatory Effects of a Herbo-mineral Ayurvedic Formulation in Experimental Models","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-26 17:55:10","doi":"10.21203/rs.3.rs-3625156/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9041475b-de0a-4f24-9bbc-6e93d1233b14","owner":[],"postedDate":"January 26th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":26447470,"name":"Biological sciences/Biological techniques/Biological models/Immunological models"},{"id":26447471,"name":"Biological sciences/Immunology/Applied immunology/Primary immunodeficiency disorders"}],"tags":[],"updatedAt":"2024-01-26T17:55:10+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-26 17:55:10","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3625156","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3625156","identity":"rs-3625156","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}