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Back to Journal »Journal of Experimental Pharmacology» Volume 13

Divya-Herbal-Peya soup coordinates the inflammatory response in a zebrafish model induced by lipopolysaccharide

Authors: Balkrishna A, Lochab S, Joshi M, Srivastava J, Varshney A 

Published on December 2, 2021, Volume 2021: 13 pages, 937-955 pages

DOI https://doi.org/10.2147/JEP.S328864

Single anonymous peer review

Editor who approved for publication: Professor Paola Rogliani

Acharya Balkrishna,1–3 Savita Lochab,4 Monali Joshi,5 Jyotish Srivastava,5 Anurag Varshney1,2,6 1 Patanjali Institute of Drug Discovery and Development, Haridwar, 249405, Uttarakhand, India; 2 Department of Joint and Applied Sciences, Patanjali University, Yog Peeth, Patanjali, Haridwar, 249405, Uttarakhand, India; 3 Patanjali Yog Peeth (UK) Trust, Glasgow, G41 1AU, UK; 4 Department of Biology, Medicine Department of Discovery and Development, Patanjali Institute, Haridwar, 249405, Uttarakhand, India; 5 Department of Chemistry, Department of Drug Discovery and Development, Patanjali Institute, Haridwar, 249405, Uttarakhand, India Germany; 6 Special Center for Systems Medicine, Jawaharlal Nehru University, New Delhi, India Corresponding author: Anurag Varshney Department of Drug Discovery and Development, Patanjali Institute, NH-58, Haridwar, 249405, Uttarakhand, India Email [Email protection] Background: Divya-Herbal- Peya (DHP) is a botanical decoction that contains 14 herbs in precise quantities; it is usually prescribed by Ayurvedic practitioners to reduce stress and minimize repeated infections Annoying symptoms. Our research aims to provide experimental verification for the immunomodulatory properties of DHP. Method: Physical and chemical analysis of DHP to assess the presence of secondary metabolites. Then use HPTLC, UHPLC and GC-MS techniques to identify and quantify phytochemicals. In order to solve the scientific principles behind DHP, lipopolysaccharide (LPS) is injected intraperitoneally into adult zebrafish to produce an inflammatory response. After LPS induction, the relative swimming speed and turning rate of the experimental zebrafish were evaluated to determine the abnormality of the movement behavior. The pathophysiological effects are determined by gill cover frequency, behavioral fever, and caudal fin injury. LPS-mediated inflammation was measured by RT-PCR to study the expression of pro-inflammatory cytokines, TNFα, IL-6 and IL-1β in animal serum. Results: Our research carried out phytochemical characterization and determination of glycyrrhizic acid, rosmarinic acid, gingerol, cinnamic acid, protocatechuic acid, gallic acid, ellagic acid, piperine and cinnamaldehyde in DHP decoction Its existence. LPS induces abnormal movement patterns, behavioral fever, and caudal fin damage in zebrafish. It was also determined that the gene expression levels of pro-inflammatory cytokines, TNFα, IL-6 and IL-1β increased significantly. However, these movement deviations and behavioral fevers were pre-administered with DHP or dexamethasone (DEX) in a dose-dependent manner in the zebrafish group, which can be ignored. In the group pretreated with DHP and DEX, changes in the rate of operculum, caudal fin damage and increased transcription levels of pro-inflammatory genes after LPS induction were avoided. Conclusion: DHP preventively prevents LPS-induced abnormal zebrafish behavior and inflammation-related pathophysiology. The immunomodulatory properties of DHP may not have therapeutic intervention, but it does endow nutraceuticals with health benefits against mild infections. Keywords: immune regulation, anti-inflammatory, zebrafish, plant metabolites, Divya-Herbal-Peya, herbal soup

Ayurveda is a traditional form of medicine that has been used in India for centuries and has made a significant contribution to the health maintenance system. It encourages the use of specific herbs, whether raw or in decoction form, to prevent repeated infections. 1,2 However, scientific evidence on its safety, effectiveness, and mechanism of action is limited, which has formed a mystery in the mainstream healthcare system. Similarly, Divya-Herbal-Peya (hereinafter referred to as DHP) is an example of a herbal decoction that contains a variety of herbs in precise quantities. It is prescribed by Ayurveda to reduce stress and minimize irritating symptoms such as fever, cold and cough. Considering the benefits of DHP, it is necessary to analyze the scientific principles behind this evidence-based herbal beverage, albeit through experiments. We aim to verify the nutritional properties of DHP to prevent infection and reduce related symptoms.

The ingredients of DHP follow the concept of "comprehensive herbal medicine" and have additional therapeutic effects compared to single herbs. 3 It contains herbs with anti-microbial and anti-inflammatory properties. These herbs are rich in natural polyphenols, flavonoids, saponins, tannins and essential oils with therapeutic potential. Thorough component analysis prompted us to determine possible immunomodulatory properties that enable it to reduce symptoms associated with mild infection, cough or fever. In order to verify our hypothesis, we first used high-performance thin liquid chromatography (HPTLC), ultra-high performance liquid chromatography (UHPLC) and gas chromatography-mass spectrometry (GC-MS) to chemically characterize DHP, and determined the plant metabolites Exist, namely, glycyrrhizic acid, rosmarinic acid, gingerol, cinnamic acid, protocatechuic acid, gallic acid, ellagic acid, piperine and cinnamaldehyde. The identified plant metabolites have anti-inflammatory and anti-microbial properties.

Our research further explored the preventive role of DHP in the process of fighting inflammation. We developed an inflammation model system by inducing zebrafish (Danio rerio) with lipopolysaccharide (LPS). Zebrafish are consistent with mammals in terms of physiological signaling pathways and functions. 4 Zebrafish is an established model of human immunology related to the pathogenesis of bacterial and viral infections. 5 Previously, lipopolysaccharide (LPS)-induced adult zebrafish has been shown to be an in vivo inflammation model for pharmacological research. 6,7

Therefore, we evaluated the immunomodulatory effect of DHP on the LPS-mediated inflammatory response in zebrafish. LPS induces behavioral diseases and abnormal movement patterns in zebrafish. However, zebrafish that were given DHP in advance maintained their normal behavior and movement patterns. We show that DHP significantly normalizes the abnormal movement patterns caused by LPS in the zebrafish model. DHP also modulated the exacerbated opercular rate and behavioral fever in the zebrafish model stimulated by LPS. Phenotypic screening showed that DHP offset the damage of the zebrafish caudal fin caused by LPS. In addition, we evaluated the gene expression levels of inflammatory markers (ie CRP, TNFα, IL-6 and IL-1β) in zebrafish attacked by LPS to resolve the anti-inflammatory activity of DHP. In conclusion, our research provides experimental evidence that DHP is a plant-derived immunomodulatory beverage.

We purchased DHP from the manufacturer of Divya Pharmacy in Haridwar, India (batch number B-DHP109, which will expire in May 2022). Lipopolysaccharides from E. coli O111:B4 (L2630) and dexamethasone (PHR1526) were purchased from Sigma-Aldrich, Bangalore. Standards for HPTLC, UHPLC and GC-MS were purchased from Natural Remedies Private Ltd. in Bangalore; Sigma Aldridge, Bangalore; Cayman Chemical Industry, USA; and SISCO Research Laboratory (SRL) in India. HPLC grade chemicals are mainly purchased from Merck and Sigma Aldrich.

DHP can be purchased as a dry powder packaged in filter paper bags similar to tea bags. Each bag contains 2 grams of DHP, which simplifies the preparation of decoctions in a convenient and labor-saving way. These porous bags help to properly extract the plant metabolites of DHP in hot water. The decoction rate of a 5.5 g DHP sample was evaluated by boiling in water for 120 minutes. The extract was then vacuum filtered and dried under reduced pressure. Weigh the obtained residue and calculate the percentage of water decoction yield as follows:

Decoction yield = (residue weight obtained / sample weight) × 100.

The purity of DHP is determined by calculating total ash and acid-insoluble ash. The dried DHP is incinerated in a crucible at 600°C for 3-4 hours to obtain carbon-free ash, and weighed to calculate the total ash content, using the starting amount as a reference percentage. The total ash was then heated in 25 mL of 5N HCl for 10 minutes and filtered. The filtrate was washed several times with water and then returned to the muffle furnace in the crucible. The obtained residue was cooled in a desiccator for 30 minutes and weighed to determine the insoluble ash value.

The Folin Ciocalteu method was used to determine the total phenol content in DHP. 8,9 Using the calibration curve generated by gallic acid as a standard, calculate the total phenol content in gallic acid equivalent (GAE) per mg of DHP dry weight. 10 Use the aluminum chloride extraction method described previously to quantify the flavonoids. A calibration curve of quercetin was generated to calculate the total flavonoid content in quercetin equivalent (QE) per milligram of DHP dry weight. Saponin was estimated by adding vanillin-ethanol (2 mL, 8% w/v) and 75% sulfuric acid to the water extract of DHP. The mixture was then incubated at 60°C for 10 minutes, and then the absorbance at 544 nm was recorded. The content of saponins is determined according to the absorbance standard curve generated for dioscin. For tannin quantification, the aqueous extract of DHP was titrated with 0.1N potassium permanganate solution. Procyanidins were estimated by adding 4% valine-methanol and hydrochloric acid to the water extract of DHP. The mixture was then incubated at room temperature for 15 minutes, and then the absorbance was recorded at 500 nm. The proanthocyanidin content was evaluated according to the catechin standard. 11

The HPTLC system (Camargue, Switzerland) is equipped with an automatic TLC sampler (ATS4) and scanner 4 to identify phytochemicals in DHP. Use the integrated software Win-CATS for analysis. Dissolve 350 mg of DHP in 6 mL of water: methanol (9:1), then sonicate and centrifuge to obtain a clear solution. The separation of the solution was carried out on pre-coated silica gel 60F254 plates. The plant components in the DHP sample are identified and quantified by equating the Rf value of the observed band with the standard Rf value. The mobile phase-A (toluene: ethyl acetate: formic acid (9:10:1 v/v/v)) is used to separate rosmarinic acid (RA), piperine (PE) and cinnamic acid (CA) while flowing Phase-B (ethyl acetate: formic acid: acetic acid: water (10:1:1:1:2.3 v/v/v)) is used to detect glycyrrhizin (GA). The TLC plate was air-dried and scanned at 254 nm to show bands, and scanned at 280 nm to generate 3-D overlay chromatograms. The standards used in HPTLC are the same as those introduced in UHPLC.

Analyze the presence of characteristic plant metabolites in DHP by UHPLC (Shimadzu Prominence XR, Japan) equipped with a quaternary pump (Nexera XR LC-20AD XR), which includes a degassing device (DGU-20A 5R) and a DAD detector (SPD-M20 A) and autosampler (Nexera XR SIL-20 AC XR).

0.5 g DHP was diluted with 10 mL methanol:water (10:90) and sonicated for 30 minutes. The sample is then centrifuged at high speed and filtered with a 0.45 µ nylon filter before being injected into the HPLC unit. Standard products include gallic acid (potency-97.30%, Sigma Aldrich), protocatechuic acid (potency-99.50%, natural medicine), glycyrrhizic acid (potency-93.0%, natural medicine), ellagic acid (potency-99.60%, Sigma Aldrich), Rosmarinic Acid (Efficacy-98.0%, Sigma Aldrich), Cinnamic Acid (Efficacy-99.70%, SRL), 6-Gingerol (Efficacy-98.90%, Cayman Chemicals) and Piperine (Efficacy-97.0%) , Sigma Aldrich). Separately inject 10 µL of DHP sample and standard into a Shodex C18-4E (5 µm, 4.6 × 250 mm) chromatographic column to properly separate the components through binary gradient elution. Solvent A (0.1% orthophosphoric acid in water, adjusted to pH 2.5 with diethylamine) and solvent B (acetonitrile). The gradient is achieved by mixing solvent B and A in a specific ratio for 30-40 minutes, 25-40% 40-50 minutes, 40-70% 50-60 minutes, 70-90% 60-65 minutes, 90- The flow rate of all samples for 95% 65-60 minutes and 95% 66-70 minutes was 1.0 mL/min, maintained at 35 °C in the column. The peak in the chromatogram is recorded at 250 nm. The plant components in DHP are determined according to the standard retention time.

To identify the plant components in DHP, the same hexane extract was prepared by incubating a 106 mg DHP sample in 5.0 mL of hexane, then centrifuging at high speed and filtering through a 0.22 µ nylon filter. The cinnamaldehyde stock solution (potency-99.5%, Sigma Aldrich) is prepared by dissolving 32.3 mg in 25 mL methanol, and then further diluting it 10 times in methanol. The analysis was performed on a 7000D GC/MS triple quadrupole and a 7890B GC system (Agilent, USA), equipped with Mass Hunter software. The HP-INNOWAX capillary column (30 mx 0.25 mm, 0.25 µm) is used to separate DHP samples and standards. The flow rate of the carrier gas (helium) is maintained at 1 mL/min. The injection volume of the DHP sample is 1 μL, the temperature of the split injector is adjusted at 250 °C, and the split ratio is 20:1. Set the column temperature to 80 °C (hold for 2 minutes), then increase the temperature to 160 °C at a rate of 10 °C/min (hold for 3 minutes), and then increase the temperature to 230 °C at a rate of 2 °C/min (hold for 4 minute). The GC-MS ion source temperature and ionization potential were set to 230 °C and 70 eV, respectively. The identified compounds were taken from the NIST14.L library.

The quantification of cinnamaldehyde is carried out using the response factor of the reference standard, while the quantification of other molecules takes into account the area percentage obtained in the chromatogram, uses the content of cinnamaldehyde as the internal standard, and applies the molecular weight correction factor of a single molecule.

All wild-type AB strains of zebrafish regardless of sex, weight (0.5 g) and age (1.2-1.5 years) come from Pentagrit's internal breeding facilities. We randomly assigned 24 adult zebrafish to each group. Zebrafish kept at 27±1°C for 14 hours: 10 hours (light: dark) cycle. Maintain water quality and housing units in accordance with standard agreements. The zebrafish were fed commercial feed (TetraBit, Spectrum Brands Pet LLC, Blacksburg, VA, USA) at 5 mg per gram of body weight per day. Euthanize the fish by the rapid cooling method discussed earlier. 12

According to the relative weight and surface area of ​​the zebrafish, the DHP dose of the zebrafish model was optimized to make it 1000 times smaller than the human equivalent dose. Therefore, for zebrafish, the human equivalent dose of DHP (2 g/day; 26.6 mg/kg) translates to 28 μg/kg. Similarly, the human prescribed dose of dexamethasone (DEX) is 6 mg/day (100 μg/kg), compared to 0.08 μg/kg for zebrafish. 13 After acclimating the zebrafish for 7 days under standard laboratory conditions, they were divided into six groups (group I-VI) with 24 fish each. On day 0, groups I and II were fed normal fish feed, while group III was fed a feed containing DEX at a dose of 0.08 µg/kg. Groups IV, V, and VI were fed fish feed injected with DHP at doses of 6 µg/kg (0.2x), 28 µg/kg (1x), and 142 µg/kg (5x). For 14 days, the fish were fed their respective feeds every 24 hours (Table 1, Figure 1). Table 1 Zebrafish experimental group settings and their respective dosing schedules Figure 1 Schematic diagram of the experimental design. The zebrafish were divided into six groups (group I-VI) with 24 fish in each group, and were acclimatized under standard laboratory conditions for 7 days. From day 0 to day 13, feed the I-VI group as shown. DEX and DHP are incorporated into commercial feeds in specified doses. On day 13, the zebrafish were anesthetized with 10 μg LPS or 1% saline. After 24 hours, after injection and returning to their respective recovery tanks, the zebrafish were analyzed for indicated clinical endpoints. Abbreviations: DEX, dexamethasone; DHP, Divya-Herbal-Peya.

Table 1 Zebrafish experimental group settings and their respective dosing regimens

Figure 1 Schematic diagram of experimental design. The zebrafish were divided into six groups (group I-VI) with 24 fish in each group, and were acclimatized under standard laboratory conditions for 7 days. From day 0 to day 13, feed the I-VI group as shown. DEX and DHP are incorporated into commercial feeds at specified doses. On day 13, the zebrafish were anesthetized with 10 μg LPS or 1% saline. After 24 hours, after injection and returning to their respective recovery tanks, the zebrafish were analyzed for indicated clinical endpoints.

Abbreviations: DEX, dexamethasone; DHP, Divya-Herbal-Peya.

On the 13th day, all groups except group I (normal control group) were induced with LPS. For this purpose, zebrafish are individually anesthetized using progressive cold water treatment. Initially, the fish was placed in water at 17°C until the movement of the gill cover was drastically reduced. The zebrafish were then transferred to water at 12°C until a noticeable loss of response to caudal fin contact was noticed; soon after, the zebrafish were placed in the injection stage for LPS injection. LPS is prepared in 1% saline and the final concentration is 3.33 µg/µL. A Hamilton syringe was used to inject 3 µL of the LPS solution between the lateral line and the anal orifice of all group II-VI zebrafish. Similarly, in the normal control zebrafish, group I was injected with 3 µL of 1% saline. Immediately after the injection, the zebrafish were transferred back to the recovery tank containing 28°C water. 24 hours after LPS induction or saline injection, various behavioral and pathological parameters of zebrafish were analyzed.

Control and treated zebrafish were placed in adjacent experimental tanks and allowed to acclimatize for 3 minutes before assessing motor activity. At the same time, experiments were carried out to eliminate circadian rhythm deviations between subjects. Calculate the swimming speed (mm/sec) of individual zebrafish by recording the end point of the displacement within a specific time (second). Thereafter, the average swimming speed of the entire group is calculated. Calculate the number of turns per minute by counting the number of times the zebrafish changes its swimming direction continuously in 3 minutes. Similarly, once the temperament movement of the zebrafish ceases, the movement of the operculum is continuously recorded for 3 minutes. After all individual zebrafish have adapted to the environment, the data is analyzed from the 3-minute video recorded in the DSLR camera. A method based on Image J software is used to evaluate sports activity. 14

Determine the behavioral fever of zebrafish as described earlier. 15 In short, an experimental glass jar with interconnected chambers is used to study the behavioral heat of zebrafish. These perforated, interconnected chambers are maintained at 23°C, 29°C and 37°C with the help of adjacent storage tanks, which are adjusted at 18°C ​​(low) and 40° by continuous heating or cooling C (high). Each zebrafish from each group was introduced individually into the interconnected chambers, allowed to acclimate for 3 minutes, and then the time they spent at each temperature for 3 minutes was continuously recorded. Figure 2 Composition and physicochemical analysis revealed DHP as a rich source of secondary metabolites. (A) Heat map visualization of the percentage contribution of each plant family in DHP. The legend to the right represents the color intensity and the corresponding percentage (%) contribution. (B) The pie chart depicts the percentage (%) of the contribution of plant parts used to formulate DHP. (C) The bar graph shows the content (% w/w) of secondary metabolites identified in DHP. Abbreviations: DHP, Divya-Herbal-Peya.

Figure 2 Composition and physical and chemical analysis show that DHP is a rich source of secondary metabolites. (A) Heat map visualization of the percentage contribution of each plant family in DHP. The legend to the right represents the color intensity and the corresponding percentage (%) contribution. (B) The pie chart depicts the percentage (%) of the contribution of plant parts used to formulate DHP. (C) The bar graph shows the content (% w/w) of secondary metabolites identified in DHP.

At the end of the experiment, the zebrafish's caudal fin damage was carefully monitored. Before observation under a microscope, the caudal fin was taken out and fixed in PBS. The anatomical image of the caudal fin was taken using a stereo microscope with 10x magnification and a 14MP Labomed camera.

Total RNA was extracted from fresh fin tissue of zebrafish. Use the Primer-BLAST tool to design forward and reverse primers for the target gene sequence. The 16 primers used in this study were synthesized from Sigma Aldrich and are briefly introduced in Table 2. Use RNAqueous®-Micro Kit (Thermo Fisher Scientific, Massachusetts, USA) and use the Transcriptor First Strand cDNA Synthesis Kit (Roche) for reverse transcription in accordance with the manufacturer's prescribed guidelines and protocols. Perform RT-PCR to study the relative quantification of TNFα, IL-6, and IL-1β mRNA expression levels, and standardize against GAPDH. Table 2 RT-PCR primer sequence

Table 2 RT-PCR primer sequence

GraphPad Prism 7.0 software and MS office Excel 2010 are used to perform statistical calculations. The data set of each group is expressed as the mean ± standard error of the mean (SEM). If p<0.05 (*p<0.05, **p<0.005, ***p<0.0005), the p-value of the data set is considered significant, if p>0.05, it is considered insignificant (ns) . To determine the p-value, Dunnett's multiple comparison test was used to analyze the mean by one-way or two-way analysis of variance (ANOVA).

All experimental procedures and protocols for zebrafish (Danio rerio) were approved by the Institutional Animal Ethics Committee (IAEC Approval Number-226/Go072020/IAEC), and animal experiments were carried out in accordance with the guidelines of the Control and Supervision Committee (CPCSEA), India Government; and in line with ICH uniform principles of animal husbandry and handling.

We traversed the plant families and their respective parts used to formulate DHP. 44% of the herbs in DHP belong to the family Zingiberaceae and Leguminosae, while 33% belong to the family Lauraceae, Gramineae and Labiatae (Figure 2A, Table 3). In addition, a specific part of the plant was selected to prepare the DHP meal (Figure 2B, Table 3) to produce a water decoction percentage of 38.18 w/w with a pH of 5.08. We determined a total ash content of 12.59% w/w and an acid-insoluble ash content of 0.04% w/w, indicating that the inorganic and silicate impurities in DHP are negligible. 17 In addition, we conducted various calorimetric tests to evaluate the presence of plant metabolites in DHP. Quantitative analysis based on the respective standards revealed a large amount of polyphenols (10.71% w/w), proanthocyanidins (2.76% w/w), flavonoids (1.23% w/w), saponins (8.13% w/w) and monopolyphenols (8.13% w/w). Ning (8.66% w/w) (Figure 2C). We also assessed that DHP samples were not contaminated by harmful pathogens, heavy metals, or aflatoxins (Table S1). In summary, we believe that DHP is a scientific combination of herbs with a rich source of metabolites. Table 3 The weight percentage of herbal ingredients used to formulate DHP. Figure 3 The identification and quantification of phytochemicals in DHP based on HPTLC, UHPLC and GC-MS. (A) (Left) The digital fingerprint of the TLC plate captured at 254 nm shows the bands corresponding to GA and glycyrrhizin in the DHP sample. (Right) The digital fingerprint of the TLC plate captured at 254 nm shows that the bands identified in the DHP sample correspond to RA, CA, and PE, where RA is rosmarinic acid, CA is cinnamic acid, and PE is piperine. (B) A graph showing the content (in μg/mg) of designated plant metabolites identified by HPTLC. (C) The 3-D superimposed density chart recorded at 280 nm shows the peaks of RA, CA and PE in the DHP sample, reference standard. (D) The 3-D superimposed density plot monitored at 280 nm shows the peak of GA, which is the glycyrrhizin in the DHP sample and standard. (E) The chromatogram generated at 250 nm shows the superposition of the DHP sample (pink) and the standard (blue). The number in the chromatogram indicates the number of peaks determined by the retention time of the reference standard. Peak numbers Figures 5 and 7 have been enlarged to provide a clear illustration of overlapping peaks in DHP samples and standards. (F) The bar graph shows the quantification (in μg/mg) of phytochemicals identified by UHPLC. (G) The GC-MS chromatogram shows the superposition of the DHP sample (green) and the cinnamaldehyde standard (blue). Table 5 briefly describes the identification and quantification of the specified peak number. The peak number 1 in the enlarged chromatogram refers to cinnamaldehyde. Abbreviations: DHP, Divya-Herbal-Peya; HPTLC, high performance thin liquid chromatography; UHPLC, ultra high performance liquid chromatography; GC-MS, gas chromatography-mass spectrometry; RA, rosmarinic acid; CA, cinnamic acid; PE, Piperine; GA, Glycyrrhizin.

Table 3 The weight percentage of herbal ingredients used to formulate DHP

Figure 3 Identification and quantification of phytochemicals in DHP based on HPTLC, UHPLC and GC-MS. (A) (Left) The digital fingerprint of the TLC plate captured at 254 nm shows the bands corresponding to GA and glycyrrhizin in the DHP sample. (Right) The digital fingerprint of the TLC plate captured at 254 nm shows that the bands identified in the DHP sample correspond to RA, CA, and PE, where RA is rosmarinic acid, CA is cinnamic acid, and PE is piperine. (B) A graph showing the content (in μg/mg) of designated plant metabolites identified by HPTLC. (C) The 3-D superimposed density chart recorded at 280 nm shows the peaks of RA, CA and PE in the DHP sample, reference standard. (D) The 3-D superimposed density plot monitored at 280 nm shows the peak of GA, which is the glycyrrhizin in the DHP sample and standard. (E) The chromatogram generated at 250 nm shows the superposition of the DHP sample (pink) and the standard (blue). The number in the chromatogram indicates the number of peaks determined by the retention time of the reference standard. Peak numbers Figures 5 and 7 have been enlarged to provide a clear illustration of overlapping peaks in DHP samples and standards. (F) The bar graph shows the quantification (in μg/mg) of phytochemicals identified by UHPLC. (G) The GC-MS chromatogram shows the superposition of the DHP sample (green) and the cinnamaldehyde standard (blue). Table 5 briefly describes the identification and quantification of the specified peak number. The peak number 1 in the enlarged chromatogram refers to cinnamaldehyde.

Abbreviations: DHP, Divya-Herbal-Peya; HPTLC, high performance thin liquid chromatography; UHPLC, ultra high performance liquid chromatography; GC-MS, gas chromatography-mass spectrometry; RA, rosmarinic acid; CA, cinnamic acid; PE, Piperine; GA, Glycyrrhizin.

We identified and quantified the plant components in DHP through HPTLC and UHPLC-based methods, and identified and quantified the volatile components through GC-MS-based analysis. The DHP extract separated on the TLC plate is exposed at 254 nm, and the Rf value of the reference standard is used to identify the phytochemicals. The TLC plate showed that the mobile phase (A) promoted the separation of rosmarinic acid (RA), cinnamic acid (CA) and piperine (PE), with Rf values ​​of 0.27, 0.62 and 0.55, respectively, while the mobile phase (B) made licorice For the separation of sodium cyclamate (GA), the Rf value was 0.27 (Figure 3A). TLC panel analysis of different wavelengths can quantify the identified phytochemicals (Figure 3B). In addition, a 3-D overlay density map at 280 nm was generated to further confirm the presence of RA, CA, PE, and GA in DHP (Figure 3C and D). UHPLC chromatograms of the DHP extract and standard mixture were generated at 250 nm and then superimposed to identify and quantify plant metabolites (Figure 3E). The chromatogram showed the presence of a large amount of gallic acid (24.46 µg/mg), protocatechuic acid (0.24 µg/mg), ellagic acid (4.02 µg/mg), rosmarinic acid (0.65 µg/mg), cinnamic acid (0.11) µg/mg) mg), glycyrrhizin (2.93 µg/mg), 6-gingerol (0.39 µg/mg) and piperine (0.03 µg/mg) in DHP (Figure 3F, Table 4). In addition, when analyzing the hexane extract of DHP by GC-MS, the presence of cinnamaldehyde, widdrol, gingerol, n-hexadecanoic acid, octaethylene glycol monolauryl ether and octaethylene glycol was described (Figure 3G ,table 5). The quantification of cinnamaldehyde is carried out according to the standard. Table 4 Using UHPLC to identify and quantify the phytochemicals present in DHP . (A) The graph shows the average speed of each group (n = 24 for each group). The circles in the figure represent the mean±SEM speed value of each group. (B) A box-and-whisker plot describing the average number of turns (revolutions per minute) for each zebrafish is calculated (n = 24 per group). The average value of each group is represented by a + sign. (C) Record the number of operculum beats for 3 minutes in each zebrafish. On this basis, we calculated the rate of opercular beating per minute. The bar graph shows the average opercular frequency of each group (n = 24 per group). Error bars represent ±SEM; the significance of the data is expressed as **p<0.005, ***p<0.0005, ****p<0.00005, if p>0.05, it is not significant (ns). Abbreviations: DHP, Divya-Herbal-Peya; DEX, dexamethasone; LPS, lipopolysaccharide; NC, normal control.

Table 4 UHPLC was used to identify and quantify the phytochemicals present in DHP

Table 5 GC-MS identification and quantification of volatile compounds present in DHP

Figure 4. Abnormal behavioral activity stimulated by normalized LPS with pre-administration of DHP in a zebrafish model. (A) The graph shows the average speed of each group (n = 24 for each group). The circles in the figure represent the mean±SEM speed value of each group. (B) A box-and-whisker plot describing the average number of turns (revolutions per minute) for each zebrafish is calculated (n = 24 per group). The average value of each group is represented by a + sign. (C) Record the number of operculum beats for 3 minutes in each zebrafish. On this basis, we calculated the rate of opercular beating per minute. The bar graph shows the average opercular frequency of each group (n = 24 per group). Error bars represent ±SEM; the significance of the data is expressed as **p<0.005, ***p<0.0005, ****p<0.00005, if p>0.05, it is not significant (ns).

Abbreviations: DHP, Divya-Herbal-Peya; DEX, dexamethasone; LPS, lipopolysaccharide; NC, normal control.

We evaluated the modulation of zebrafish movement by calculating the average swimming speed (mm/sec) and turning rate of each test group. The LPS challenge significantly reduced swimming speed, indicating that the zebrafish’s motor activity is impaired. However, zebrafish pretreated with DHP showed a dose-dependent recovery of average swimming speed. Zebrafish managed by DEX showed similar results to DHP. In fact, the highest dose of DHP pretreatment significantly eliminated swimming speed disturbances because the difference between the NC and DHP (142 µg/kg) treatment groups was not statistically significant (Figure 4A, Supplementary Figure 1S). Figure 5 The zebrafish model pretreated with DHP is not affected by behavioral fever stimulated by LPS. (A) An illustration of an experimental device for studying zebrafish's behavioral fever. Adjacent perforated tanks of 18°C ​​(low) and 40°C (high) adjust the indicated temperature of the interconnected hot tank. The temperature preference of zebrafish was studied by introducing zebrafish alone in a hot tank at 29°C. (B) The horizontal bar graph depicts the average time (s) spent independently by the six groups at 23°C, 29°C, and 37°C (n = 24 per group). Significance only shows the difference in the average time spent in the 6 groups at 37 °C. (C) The scatter plot shows the time spent by each zebrafish at 37°C (n = 24). Each fish symbol represents the zebrafish subject participating in the experimental setup described in (A). The colors of the fish symbols represent their respective groups. (Four). The fold change of CRP mRNA expression level (n = 3 per group). The error bars in (B and D) represent ±SEM; the significance of the data is expressed as *p<0.05, **p<0.005, ****p<0.00005. Abbreviations: DHP, Divya-Herbal-Peya; DEX, dexamethasone; LPS, lipopolysaccharide; NC, normal control; CRP, C-reactive protein.

Figure 5 The zebrafish model pretreated with DHP is not affected by behavioral fever stimulated by LPS. (A) An illustration of an experimental device for studying zebrafish's behavioral fever. Adjacent perforated tanks of 18°C ​​(low) and 40°C (high) adjust the indicated temperature of the interconnected hot tank. The temperature preference of zebrafish was studied by introducing zebrafish alone in a hot tank at 29°C. (B) The horizontal bar graph depicts the average time (s) spent independently by the six groups at 23°C, 29°C, and 37°C (n = 24 per group). Significance only shows the difference in the average time spent in the 6 groups at 37 °C. (C) The scatter plot shows the time spent by each zebrafish at 37°C (n = 24). Each fish symbol represents the zebrafish subject participating in the experimental setup described in (A). The colors of the fish symbols represent their respective groups. (Four). The fold change of CRP mRNA expression level (n = 3 per group). The error bars in (B and D) represent ±SEM; the significance of the data is expressed as *p<0.05, **p<0.005, ****p<0.00005.

Abbreviations: DHP, Divya-Herbal-Peya; DEX, dexamethasone; LPS, lipopolysaccharide; NC, normal control; CRP, C-reactive protein.

In addition to speed, the turn rate of the zebrafish was also evaluated. LPS induction greatly reduced the turning rate of zebrafish, while the reduction in zebrafish pre-administered DEX or DHP (142 µg/kg) was negligible (Figure 4B, Supplementary Figure 1S). The dose-dependent effect of DHP confirmed the specificity of herbal preparations in restoring physical activity in LPS-treated zebrafish. The administration of LPS triggers several systemic inflammatory cascades, leading to respiratory physiology. 18 In zebrafish, opercular movement represents respiratory physiology. Higher opercular pulsation was observed after LPS induction, while the DEX or DHP pre-administration group showed no abnormality in opercular pulsation frequency. In fact, as the dose of DHP increased, the frequency of opercular pulsation was observed to decrease (Figure 4C, Supplementary Figure 1S). Taken together, these data indicate that zebrafish fed with conventional DHP doses greatly delayed LPS-mediated behavioral abnormalities.

Behavioral fever is an important parameter to solve the activation of zebrafish immune response. 19 We determined that compared with the control, zebrafish injected with LPS had a longer average time at 37 °C, indicating that the zebrafish developed behavioral fever (the setup described in Figure 5A of the experiment). On the other hand, zebrafish in the normal control group and the DEX treatment group showed a preference for water at the optimal temperature of 29 °C. Interestingly, the zebrafish group pre-administered DHP showed a significant reduction in heat preference in a dose-dependent manner (Figure 5B). The LPS treatment group had the largest number of fish staying at higher temperatures, while the number of fish in the control group and the DHP-142 µg/kg treatment group was significantly reduced. This indicates that DHP pre-administration protects zebrafish from behavioral fever caused by LPS (Figure 5C). Elevated C-reactive protein (CRP) in tissues is considered to be a key marker for enhancing the immune response to microbial infections. 20 Consistent with this, we determined that the tissue CRP level of the zebrafish treated with LPS increased by 10 times, while the zebrafish fed with DHP-142 µg/kg in the feed showed a CRP level comparable to that of the control group (Figure 5D) . DHP feed did regulate the CRP levels in zebrafish attacked by LPS in a dose-dependent manner.

Caudal fin injury is an important parameter to evaluate the severity of inflammation, reflecting the acute inflammatory response. 21 Phenotypic screening of the caudal fin showed that LPS treatment damaged the caudal fin and caused mild discoloration. However, compared with the normal control group, DEX and DHP pretreatment maintained the anatomical structure of the caudal fin (Figure 6). In conclusion, we recommend DHP as an effective herbal immunomodulator to eliminate LPS-mediated pathophysiology. Figure 6 DHP rescued the zebrafish caudal fin damage induced by LPS. Representative images of caudal fin injury in each group (Group I-NC; Group II-LPS; Group III-DEX; Group IV-6 µg/kg DHP; Group V-28 µg/kg DHP; Group IV-142 µg/kg) Captured under the microscope. Abbreviations: DHP, Divya-Herbal-Peya; DEX, dexamethasone; LPS, lipopolysaccharide; NC, normal control.

Figure 6 DHP rescued zebrafish caudal fin damage caused by LPS. Representative images of caudal fin injury in each group (Group I-NC; Group II-LPS; Group III-DEX; Group IV-6 µg/kg DHP; Group V-28 µg/kg DHP; Group IV-142 µg/kg) Captured under the microscope.

Abbreviations: DHP, Divya-Herbal-Peya; DEX, dexamethasone; LPS, lipopolysaccharide; NC, normal control.

It is known that LPS stimulation stimulates the immune response and releases inflammatory cytokines. 22 To determine the same situation in LPS-treated zebrafish, we evaluated the expression levels of marker genes that were up-regulated during the inflammatory response. Compared with the saltwater-treated zebrafish (NC), the expression levels of TNFα, IL-6, and IL-1β in the zebrafish treated with LPS were 6, 14 and 3 times higher, respectively. Interestingly, the relative mRNA expression levels of TNFα, IL-6, and IL-1β in zebrafish fed on DHP-142 µg/kg were similar to those in the control group (Figure 7). Here, we conclude that DHP is an effective immunomodulator and can alleviate the exacerbated inflammatory response. Figure 7 DHP feed coordinated the LPS-stimulated cytokine surge in zebrafish. The fold change of the relative mRNA expression levels of IL-6, TNFα, and IL-1β (n = 3 per group). Error bars represent ±SEM; the significance of the data is expressed as **p<0.005, ***p<0.0005, ****p<0.00005, if p>0.05, it is not significant (ns). Abbreviations: DHP, Divya-Herbal-Peya; DEX, dexamethasone; LPS, lipopolysaccharide; NC, normal control; IL-6, interleukin-6; TNFα, tumor necrosis factor α; IL-1β, interleukin-1β.

Figure 7 DHP feed coordinated the LPS-stimulated cytokine surge in zebrafish. The fold change of the relative mRNA expression levels of IL-6, TNFα, and IL-1β (n = 3 per group). Error bars represent ±SEM; the significance of the data is expressed as **p<0.005, ***p<0.0005, ****p<0.00005, if p>0.05, it is not significant (ns).

Abbreviations: DHP, Divya-Herbal-Peya; DEX, dexamethasone; LPS, lipopolysaccharide; NC, normal control; IL-6, interleukin-6; TNFα, tumor necrosis factor α; IL-1β, interleukin-1β.

Our body is susceptible to multiple infections; the different immune responses of individuals determine the degree, severity, and related clinical manifestations of the infection. The activation of the inflammatory immune response is a natural host defense mechanism against pathogens. However, in the case of repeated infections, the chronic activation of the inflammatory response can cause severe tissue damage. Therefore, it is important to obtain effective immunity so that pathogens with short-term inflammation can be quickly controlled. There are several nutrients, vitamins and food supplements available over the counter to boost immunity, but most of them are either synthetic or lack scientific evidence.

Practitioners in Ayurveda recommend using DHP to combat the inflammatory symptoms that occur during mild recurrent infections, fever, colds, and coughs. Our research investigated the basic principles behind the use of DHP through experiments. Thorough component analysis of DHP proved the existence of herbs with proven anti-inflammatory properties (Figure 2). We identified some marker plant metabolites with therapeutic potential in DHP through HPTLC and UHPLC (Figure 3A-D); the most important one is gallic acid. In vitro and in vivo studies have demonstrated the anti-inflammatory and antimicrobial properties of gallic acid. 23,24 Ellagic acid is another important plant metabolite, which belongs to polyphenol tannins and also has scientifically proven antioxidant, antimicrobial and antimicrobial effects. -Inflammatory activity. 25-27

The root of Glycyrrhiza glabra in DHP is a rich source of glycyrrhizin, which has also been identified by HPTLC and UHPLC techniques. Various in vitro and in vivo models have determined that glycyrrhizin can treat asthma by inhibiting the inflammatory response enhanced by the PI3K/Akt/GSK3β pathway. 28,29 Another study showed that glycyrrhizin can also inhibit the transactivation of NFκB, such as dexamethasone. 30 Rosmarinic acid is an important polyphenol derived from members of the Lamiaceae family, accounting for 11% of the total components of DHP. Rosmarinic acid is used as a food supplement because of its many health benefits and scientific indicators involving anti-inflammatory and antioxidant properties. 31-33 Protocatechuic acid and 6-gingerol have also been identified as active ingredients in DHP preparations. Scientific reports show that these two plant metabolites have free radical scavenging activity and immunomodulatory properties. 34-38

The potent antimicrobial activity of cinnamic acid has been used to develop new drugs with improved potency, solubility and bioavailability to fight countless bacterial infections. 39–41 Long pepper and black pepper, a metabolite of alkaloids in piperine from DHP, passed ultra-high performance liquid chromatography. Piperine has anti-inflammatory, antimicrobial, analgesic, antipyretic, and antioxidant properties, and can also improve the bioavailability of certain drugs. 42 We use GC-MS to identify and quantify cinnamaldehyde, which also has proven antimicrobial and anti-inflammatory activities. 43

In conclusion, most of the plant metabolites identified in DHP extracts are fully supported by in vitro and in vivo studies because of their anti-inflammatory and antimicrobial properties. Therefore, we hypothesize that DHP has immunomodulatory properties and may be able to re-regulate the immune response. This may be the reason why DHP is recommended to reduce infection-related symptoms. It is well known that LPS induction activates inflammation and stress responses, which may also lead to abnormal behaviors. Adult zebrafish exhibit countless mature behaviors related to anxiety, exercise, learning, social interaction, and temperature preference, which encourages us to think that they are suitable for this study. Therefore, we outline the study of LPS-induced zebrafish, which is already an established model of inflammation. In our preliminary experiments for model optimization, we also observed behavioral diseases and abnormal movement patterns when challenged by LPS, which encouraged us to consider zebrafish as an experimental model higher than higher vertebrates. Our study aimed to determine the preventive effect of DHP on the abnormal behavioral responses of zebrafish induced by LPS. By abnormal behavior response, we refer to the performance observed in quantifiable behavior, which is the result of the biochemical and physiological changes that occur after LPS induction. Our results indicate that the average swimming speed and turning rate are significantly reduced after LPS induction in zebrafish. However, in the group pretreated with DEX or DHP, normal swimming dynamics and turning rate were observed (Figure 4A-D). Similarly, in the zebrafish treated with LPS, the opercular pulsation rate (which is an established parameter for assessing pressure) was significantly higher. An increase in opercular frequency also indicates an increase in respiratory rate and oxygen demand. In addition, a previous report also showed that the opercular rate is directly proportional to the heartbeat. 44 Therefore, these studies indicate that the unstable rate of the gill cover means abnormal respiratory and cardiovascular function of the fish.

The feed added with DHP showed that the effect of LPS on the frequency of the operculum was negligible, which confirmed our claim that DHP has an immunomodulatory effect and can maintain normal pathophysiology during infection (Figure 4E and F). Similarly, even after LPS stimulation, DHP-pretreated zebrafish showed negligible behavioral fever (Figure 5A-D). The response of endothermic animals to infection is physiological fever. On the other hand, the temperature-changing animals respond by moving in higher temperature water. 45 These thermal preferences are called behavioral fevers. Fish regulate their body temperature by relocating themselves to a higher temperature to enhance the immune response. Zebrafish pretreated with DHP or DEX prefer to stay in water maintained at the optimal temperature (29 °C), indicating that DHP treatment counteracts the LPS-mediated immune response. Due to LPS-mediated cytokine production, inflammatory tissue damage is usually observed. We also observed damage to the caudal fin of zebrafish treated with LPS, despite zero mortality. However, DHP pretreatment did not show significant caudal fin damage, which suggests that DHP may modulate the inflammatory response induced by LPS. LPS is an effective inducer of the immune system and can trigger TNFα, IL-6 and IL-1β, which are also known to be secreted during common infections such as colds, coughs, and fevers. We determined that LPS stimulates the inflammatory response in zebrafish by increasing the gene expression levels of TNFα, IL-6, and IL-1β. On the other hand, preventive treatment of DHP mediated a dose-dependent reduction in the expression levels of TNFα, IL-6 and IL-1β genes, indicating that DHP has potential immunomodulatory properties (Figures 6 and 7). This may be an important mechanism of action. Even after LPS treatment, DHP-treated zebrafish will not be ineffective due to abnormal behavioral reactions.

DHP may have nutritional and health care significance, in which herbal decoctions can be consumed by healthy people to prevent recurrent infections. Overall, these results encourage us to further decipher the mechanism of DHP through in vitro and in vivo experimental models. However, our research has not yet investigated the therapeutic potential of DHP as an herbal beverage.

DHP prevents the inflammatory response induced by LPS. It is administered preventively to healthy subjects, which may endow the zebrafish model with health benefits of nutritional supplements against LPS. Our research shows that DHP has a scientific basis and can be used to relieve symptoms associated with mild infections.

DHP, Divya-Herbal-Peya; HPTLC, high performance thin liquid chromatography; UHPLC, ultra high performance liquid chromatography; GC-MS, gas chromatography-mass spectrometry; LPS, lipopolysaccharide; TNF, tumor necrosis factor; IL-6, interleukin 6; IL-1β, interleukin 1β.

We thank our CRO partner Pentagrit Labs, the zebrafish testing facility and experiment in Chennai, India. We thank Ms. Meenu Tomer, Mr. Yash Varshney and Mr. Sudeep Verma for their phytochemical analysis. We thank Mr. Shoor Singh, Mr. Arun Raturi, and Dr. Preeti Raj for their excellent support in generating video graphics. We thank Ms. Priyanka Kandpal, Mr. Tarun Rajput, Mr. Gagan Kumar and Mr. Lalit Mohan for their prompt administrative support. The test article (DHP) was from Divya Pharmacy, Haridwar, Uttarakhand, India. In addition, Divya Pharmacy provided test items and did not participate in any aspect of this research. Divya Pharmacy, Haridwar India, produces and sells many herbal products, including DHP.

All authors have made significant contributions to design concepts, data collection, data analysis and interpretation. Assist/cooperate/participate in the drafting work or critically modify important knowledge content; agree to the final approval of the version to be released. All authors agree to be responsible for all aspects of the work to ensure that issues related to the accuracy or completeness of any part of the work are properly investigated and resolved.

The work presented was carried out using internal research funds from the non-commercial and non-profit Patanjali Research Foundation Trust Fund of Haridwar, India.

Acharya Balkrishna is the honorary trustee of the Divya Yog Mandir Trust, which manages the Divya Pharmacy in Haridwar. In addition, Acharya Balkrishna holds an honorary management position at Patanjali Ayurved Ltd. in Haridwar, Uttarakhand, India. Savita Lochab, Monali Joshi, Jyotish Srivastava and Anurag Varshney are employed by the Patanjali Research Institute, which is managed by the Patanjali Research Foundation (PRFT) in Haridwar, Uttarakhand, India. A non-profit organization. In addition, Anurag Varshney is an adjunct professor in the Department of Joint and Applied Sciences at Patanjali University, NH-58, Haridwar-249405, Uttarakhand, India; and Special Center for Systems Medicine, Jawaha Lal Nehru University, New Delhi-110067, India. The authors report no other conflicts of interest in this work.

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