Bronchom (Internal batch no: CHIH/BROA/0421/1128) was sourced from Divya Pharmacy, Haridwar, India. The phyto- and mineral components of the test article are mentioned in Table 1. Dexamethasone (D4902), Geimsa stain (48900), gallic acid (91215; potency-97.3%), eugenol (35995; potency-99.6%), piperine (P49007; potency-97.0%), rosmarinic acid (CFN99103; potency-98.0%) and anti-Actin, α-Smooth Muscle antibody (A5228) were purchased from Sigma-Aldrich, USA. Methyl gallate (G0017; potency-99.9%), methacholine (M0073) and succinylcholine (S0149) were procured Tokyo Chemical Industries, India. Protocatechuic acid (P006; potency-99.5%) and glycyrrhizin (G2137; potency-93.0%) were bought from Natural Remedies), whereas 6-gingerol (11707; potency-99.3%) was procured from Cayman Chemical. House dust mite, comprised of the crushed whole bodies of Dermatophagoides pteronyssinus (XPB70D3A2.5) was purchased from Stallergenes Greer, USA. Thiopental sodium was bought from Neon Laboratories Limited, India whereas isoflurane was purchased from Baxter, India. The powder for preparing Hank’s Balanced Salt Solution (HBSS, TS1098) was obtained from HiMedia, India. Th2 cytokines and chemokines in the BALF were evaluated by employing multiplexing kit (MCYTOMAG-70K-08) and was purchased from Merck. TRIzol reagent (15596018), Verso cDNA synthesis kit (AB-1453/B), Alexa Fluor Plus 488 (A32723) and DAPI (D1306, 4’,6-Diamidino-2-Phenylindole, dihydrochloride) were purchased from Thermo Fisher Scientific, USA.

To 250 mg of Bronchom powder, 5 mL of water: methanol (20:80) was added. It was then sonicated for 30 min, centrifuged at 10000 rpm for 5 min and finally filtered using a 0.45 µm nylon filter. This solution was used for the further analysis. The stock solutions of gallic acid, methyl gallate, protocatechuic acid, eugenol, 6-gingerol, piperine, rosmarinic acid and glycyrrhizin were dissolved in methanol to prepare standard solutions of concentration 1000 µg/mL, individually. Subsequently, standard mix working solution of 50 µg/mL concentration were prepared for each of the standards. Analysis was performed on a Prominence-XR UHPLC system (Shimadzu, Japan), which was equipped with Quaternary pump (NexeraXR LC-20AD XR), PDA detector (SPD-M20 A), autosampler (Nexera XR SIL-20 AC XR), degassing unit (DGU-20A 5R) and column oven (CTO-10 AS VP). Separation by UHPLC was achieved using a Shodex C18-4E (5µm, 4.6 × 250 mm) column subjected to binary gradient elution. The two solvents used for the analysis consisted of water containing 0.1% orthophosphoric acid; adjusted to pH 2.5 with diethyl amine (solvent A) and acetonitrile (solvent B). Gradient programming of the solvent system was initially at 5% B for 0-5 min, 5-25% B from 5-30 min, 25-45% B from 30-40 min, 45-70% B from 40-50 min, 70% B from 50-55 min, 70-85% B from 55-65 min, 85-5% B from 65-66 min, 5% B from 66-70 min with a flow rate of 1.0 ml/min. 10 µl of standard and sample solution were injected and column temperature was maintain at 35°C. Wavelengths were set 270 nm (for gallic acid, methyl gallate, protocatechuic acid, eugenol and 6-gingerol), 250 nm (for glycyrrhizin) and 325 nm (for rosmarinic acid and piperine).

Female, specific pathogen free BALB/c mice (6-7 weeks of age) were purchased from Hylasco Biotechnology (India) Pvt. Ltd., Telangana, India, a Charles River Laboratories-accredited domestic animal breeder. The current study is reported according to the ARRIVE guidelines (9) and all the experimental procedures performed were in strict accordance with the recommendations specified by Committee for Control and Supervision of Experiments on Animals, Department of Animal Husbandry and Dairying, Ministry of Fisheries, Animal Husbandry and Dairying, Government of India. Prior to the initiation of the experiments, the experimental protocol was thoroughly reviewed and subsequently ratified by the Institutional Animal Ethics Committee of Patanjali Research Foundation, vide protocol number PRIAS/LAF/IAEC-103. Post-receipt, the mice were quarantined for a week and were then shifted to an experimental room, earmarked for the study. Here the animals were housed in polypropylene cages of standard dimensions at room temperature ranging from of 21-25 °C and relative humidity between 60-70%, along with a 12-hour light/dark cycle. Animals had unrestricted supply of gamma-irradiated standard pelleted laboratory animal diet (Purina 5L79 Rodent Lab diet, USA), which was purchased from Hylasco Biotechnology (India) Pvt. Ltd., Telangana, India. Similarly, reverse osmosis (Make: MERCK) water was also provided ad libitum in steam sterilized polypropylene bottles. Due care was taken to minimize animal suffering during the entire course of the study. Intranasal instillation of HDM was performed under transient isoflurane anesthesia. Further, lung function assessment was performed under thiopental sodium anesthesia (50 mg/kg), administered intraperitoneally and at the end of the experiment the mice were humanely sacrificed under overdose of thiopental sodium anesthesia (150 mg/kg), which was also administered by the intraperitoneal route.

The doses of Bronchom tested in the present study were computed on the basis of the variances in the body surface areas of humans and mice. The clinically recommended therapeutic dose of Bronchom is 2000 mg/day, in two divided doses of 1000 mg, respectively. Accordingly, the therapeutic dose for a human weighing 60 kg will be 2000/60 i.e. 33.33 mg/kg/day. The equivalent dose (in mg/kg) for mice was computed by multiplying the human equivalent dose (in mg/kg) by factor of 12.3 (10). The mouse equivalent dose was consequently calculated to be 409.99 mg/kg/day. Rounding off to the nearest hundreds, 400 mg/kg/day or 200 mg/kg, b.i.d. was adjudged to be the mouse equivalent dose. With an objective of capturing a dose-response relationship in the studied parameters, the remaining doses chosen were from 1/10^th to 3 times the recommended therapeutic dose i.e. 40, 120 and 1200 mg/kg/day. This corresponds to twice daily doses of 20, 60 and 600 mg/kg, respectively.

After completion of quarantine, mice were randomized on the basis of their respective body weights and subsequently allocated into seven groups as outlined in Figure 1.

Bronchom was daily administered to the mice, prophylactically as a gavage, for 15-consecutive days prior to initiation of disease induction, whereas dexamethasone was administered 3 weeks after commencement of disease induction, orally. The dose volume was fixed at 10 mL/kg and 0.5% methylcellulose (MC) solution was used as the vehicle for formulating the compounds.

To induce allergic asthma, 25 µg of HDM protein dissolved in 20 µL of normal saline was instilled into the nares of the mice, 5 days a week for 5-consecutive weeks, under transient isoflurane anesthesia. Animals allocated to Group 1 were administered 20 µL of normal saline, intranasally Further, the animals continued to receive their respective treatments, as outlined above, for the entire duration of the experiment, concurrent with HDM instillation.

Twenty-four hours after the last HDM instillation, the mice were anaesthetized by intraperitoneal administration of thiopental sodium (50 mg/kg). They were then tracheostomized, followed by the insertion of an 18-gauge stainless steel cannula in the trachea. Subsequently, respiratory paralysis was induced by intraperitoneal administration of succinylcholine-7.5 mg/kg following which, the tracheal cannula was attached to a flexiVent system (Emka-Scireq, Canada). The intubated mice were ventilated with a tidal volume of 10 mL/kg at 150 breaths per minute, with a positive end expiratory pressure of 3 cm of H₂O. The animals were then subjected to challenge with escalating doses of aerosolized methacholine, ranging from 0 (saline) to 50 mg/mL. By employing the low frequency forced oscillation technique, total respiratory system resistance (Rrs) and total respiratory system elastance (Ers) was derived from the single compartment model. The Rrs and Ers values obtained subsequent to challenging the animals allocated to groups 2 to group 7, at the concentration of 50 mg/mL of methacholine, was then normalized with the response obtained in the normal-control group (Group 1) at 50 mg/mL. Average value of Rrs or Ers obtained at MCh-50 mg/mL in Group 2 (disease-control group) was utilized for the calculation of % protection from AHR. The percentage protection from airway hyperresponsiveness (AHR) for Groups 3-7 was then computed by employing the following formula:

After the measurement of lung mechanics, the animals were euthanized with thiopentone administered intraperitoneally at the dose of 150 mg/kg. Then 1 ml of ice cold HBSS was slowly introduced into the lungs via the tracheal cannula. Subsequently, the thorax of the animal was gently massaged and the BALF was collected in a centrifuge tube. This exercise was repeated for four additional times. The first BALF retrieved from the animals was centrifuged at 2800 × g for 10 minutes at 4°C by employing a refrigerated centrifuge (Sorvall Legend Micro 21R; Thermo Fisher Scientific Inc., USA). Subsequently, the supernatant was separated and immediately stored at -80 °C for cytokine estimation. The cell pellet was then re-suspended with 1 ml of HBSS. The remaining lung washes were similarly centrifuged using a refrigerated centrifuge (Sorvall ST 8R; Thermo Fisher Scientific Inc.; USA) and the retrieved cell pellet was then pooled with the one obtained from the first wash. Total leukocytes in the re-suspended cell pellet were enumerated by employing a hematology analyzer (BC5000 Vet, Mindray, China). Further, the differential leukocyte counts were enumerated in smears obtained by employing a cytocentrifuge (Medspin5; Medilabsolutions; India), set at 500 rpm for 10 min. The obtained smears were then stained with Giemsa stain. A total of three hundred leukocytes were counted by using a microscope (Olympus BX43, Perkin Elmer; USA) and the following cells were identified in the stained smears according to their morphology: macrophages, eosinophils, lymphocytes, neutrophils and basophils. Subsequently, the counted cells were expressed as a percentage and the absolute counts of each of the identified leukocytes were computed by employing the formula.

After completion of BALF collection, lungs were excised from the animals. The left lung was fixed in 10% neutral buffered formalin and the right lung was flash frozen in liquid nitrogen and stored at -80 °C for biochemical and molecular estimations. The left lung was processed for histopathological evaluation using standard procedures and subsequently 3-5 µm sections were stained with hematoxylin and eosin (H&E) stain to detect the manifestation of infiltration of inflammatory cells, Periodic Acid-Schiff (PAS) stain for evidence of goblet cell metaplasia and Masson’s Trichrome (MT) stain for the presence of sub-epithelial fibrosis, as described previously (5). In order to assess the severity of peribronchiolar and perivascular inflammation as well as sub-epithelial fibrosis, the following semi-quantitative scoring system was employed: 0 = Nil; 1 = Mild; 2 = Moderate and 3 = Severe. Further, to assess the extent of goblet cell metaplasia, the following scoring criteria was used, based on the number of goblet cells present per bronchiole: 0 = 0-75 cells; 1 = 76-150 cells; 2 = 151-225 cells; 3 = 226-300 cells; 4 = 301-375 cells; 5 = 376-450 cells; 6 = 451-525 cells; 7 = 526-600 cells; 8 = 601-675 cells and 9 = 676-750 cells.

Additionally, the increase in airway smooth muscle mass was determined by morphometric analysis subsequent to immunofluorescence staining of alpha-smooth muscle actin (α-SMA). For this procedure, processed lung tissues were cut in 2-2.5 micron sections and mounted on poly-l-lysine coated slides. Then the sections were deparaffinized in xylene and rehydrated in descending grades of alcohol. Finally, these sections were washed in deionized water twice. For antigen retrieval, sections were subjected to heat-induced epitope retrieval using sodium citrate buffer (pH 6.0). The slides were cooled followed by washing in deionized water and blocking of non-specific binding using blocking buffer (Vitro Master Diagnostica Master polymer plus detection system). Then the sections were immunolabelled with primary antibody (Anti-Actin, α-Smooth Muscle antibody, 1:2000 dilution) and incubated at room temperature for 1 hour followed by staining with secondary antibody (Alexa Fluor 488, 1:500 dilution) at room temperature for 1 hour. The sections were washed in PBS followed by staining with DAPI with anti-fade mountant and mounted with coverslip. Then fluorescent evaluation was performed using an Olympus BX43 fluorescent microscope and images were obtained using Mantra Imaging System (PerkinElmer). The thickness of alpha smooth muscle actin around the bronchioles was measured by employing Zeiss Axioscope microscope, using AxioVision software, in six randomly chosen areas per animal. The average of the measured thickness was subsequently computed for each animal.

The cytokines (IL-4 and IL-5) and chemokines (Eotaxin and IP-10) were measured by multiplexing that employed a MILLIPLEX^®MAP Mouse Cytokine/Chemokine Magnetic Bead Panel based on the Luminex^® xMAP^® technology (Merck).

The total RNA was extracted from lung tissue using TRIzol reagent and was used to synthesize the cDNA. 1μg of the total RNA was used with Verso cDNA synthesis kit. The qRT-PCR analysis was performed as described previously (11), using GAPDH as housekeeping gene. Primers used are mentioned in Supplementary Table 1.

Levels of reduced glutathione (GSH) and oxidized Glutathione (GSSG) were estimated as per previously reported methods (12). Superoxide dismutase (SOD) activity was determined as per the method of Beauchamp and Fridovich, 1971 (13), whereas malondialdehye (MDA) activity was measured according to the method of Heath and Packer, 1968 (14). For nitrosative stress, nitrite levels were measured as previously reported (15). The values were normalized to the protein content of the lung, which was additionally estimated in the lung homogenate by Bicinchoninic acid (BCA) assay using a commercially available kit (Thermo Fisher), as per the manufacturer’s instructions.

All the data for the studied parameters were compiled from each of the study groups and expressed as mean ± standard error of mean (SEM). Statistical analysis was performed using GraphPad Prism version 7.04 software (GraphPad Software, San Diego, CA, USA). A one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison post-hoc test was employed to calculate the statistical differences between the mean values for all the evaluated parameters except AHR measurement at different concentration of MCh, for which, two-way ANOVA followed by Tukey’s multiple comparison test was employed. A p value < 0.05 was considered to be statistically significant.

UHPLC-PDA analysis of Bronchom revealed the presence of eight bioactive phyto-metabolites, subsequent to comparison with the obtained chromatographs of pure standard compounds (Table 2 and Figure 2). Each milligram of Bronchom powder contained gallic acid (3.407 µg), protocatechuic acid (0.092 µg), methyl gallate (1.393 µg), rosmarinic acid (0.793 µg), glycyrrhizin (8.281 µg), eugenol (2.332 µg), 6-gingerol (0.308 µg) and piperine (2.411 µg).

Repeated intranasal exposure of HDM to mice for five weeks resulted in an increased bronchoconstrictor response to aerosolized methacholine, reflected by an increase in the Rrs and Ers values, when compared to the normal-control group (Figures 3A–D). A significant difference between the airway responsiveness of disease control vs. normal control animals was evident when they were challenged with the highest concentration of methacholine-50 mg/mL (Figures 3A, C). Hence, we normalized the increased Rrs and Ers observed in disease control animal with that noted in the normal-control animals and accordingly computed the percentage protection from AHR for all the treated groups. Reference control dexamethasone administered at the dose of 1 mg/kg, p.o., significantly reversed HDM-induced AHR (Figures 3B, D), thereby validating the model. Bronchom administered orally at the doses of 20, 60, 200 and 600 mg/kg, b.i.d. reduced the development of AHR in a dose-related manner (Figures 3B, D). Significant reduction of AHR was evident at the highest tested dose of Bronchom.

Similar, to the development AHR, repeated HDM-challenge in untreated animals, elicited a robust increase in BALF cellularity as evident by a significant elevation in the number of total leukocytes, eosinophils, lymphocytes and neutrophils, as compared to the normal-control animals (Figures 4A–D). Dexamethasone, completely abrogated the HDM-induced infiltration of these inflammatory cells in the BALF. Oral administration of Bronchom at the tested doses, exhibited a dose-related inhibition of HDM-induced influx of inflammatory cells in the BALF (Figures 4A–D). When compared to the disease-control group, Bronchom-600 mg/kg, b.i.d. significantly inhibited the infiltration of the total inflammatory cells and neutrophils. Further, the significant inhibitory effects of Bronchom on eosinophil influx were evident at the doses of 200 and 600 mg/kg, b.i.d., respectively. The most pronounced effect of Bronchom was observed in the inhibition of absolute lymphocyte influx, wherein, significant differences were noted at the doses of 60, 200 and 600 mg/kg, b.i.d., respectively.

When compared to the saline-challenged animals, all the features of airway remodeling were clearly evident in mice that were subjected to repeated HDM exposure. These included peri-bronchiolar and peri-vascular inflammation (Figure 5), goblet cell metaplasia (Figure 6), sub-epithelial fibrosis (Figure 7), and increased alpha-smooth muscle actin around the airways (Figure 8). The reference control drug, dexamethasone ameliorated the airway remodeling changes. Oral administration of Bronchom inhibited inflammatory cell infiltration in the peri-bronchial and peri-vascular areas in a dose-related manner (Figures 5A, B) with a significant inhibition observed at the dose of 600 mg/kg, b.i.d. Further, the highest tested dose of Bronchom was able to significantly limit the development of goblet cell metaplasia (Figures 6A, B). Additionally, Bronchom reduced the emergence of HDM-induced sub-epithelial fibrosis (Figures 7A, B), wherein, significant effects were evident at all the tested doses of Bronchom. When compared to the disease-control group, the total histopathological lesion score was significantly lesser in Bronchom-60 and 600 mg/kg, b.i.d. treated groups, respectively.

Immunofluorescence staining of alpha-smooth muscle actin in the sections of the lung additionally revealed that Bronchom was able to dose-dependently reduce the HDM-induced increase in the airway smooth muscle mass (Figures 8A, B) and the effects were significant at all the tested doses.

HDM exposure in mice for 5-weeks resulted in significant elevation of Th2-cytokines, IL-4 and IL-5 as well as chemokines (Eotaxin and IP-10) in BALF (Figures 9A–D). Dexamethasone-1 mg/kg, p.o. significantly decreased (Figures 9A–D) the induction of the evaluated cytokines and chemokines. Oral administration of Bronchom exhibited a dose-related inhibition of HDM-induced increase in the levels of the tested cytokines and chemokines (Figures 9A–D). For IL-4 and IL-5 the effect was significant at 200 and 600 mg/kg, b.i.d. as compared to the disease-control group. Further, for Eotaxin the effect was statistically significant at 60, 200 and 600 mg/kg, b.i.d., respectively. Furthermore, in the case of IP-10, the effect was statistically significant, when compared to the disease-control group at 200 and 600 mg/kg, b.i.d.

In the current study, we also evaluated the effect of Bronchom on the mRNA expression of pro-inflammatory cytokines (TNF-α, IL-6 and IFN-γ), one Th2 cytokine: IL-13 and in addition, IL-33, another pro-inflammatory cytokine, which is regarded to be central for the generation of Th2-type cytokine-related immune responses. Since, Bronchom at the dose of 20 mg/kg, b.i.d. did not demonstrate significant efficacy in most of the tested parameters, the mRNA expression analysis was conducted in the lungs of the animals that received Bronchom at the doses 60, 200 and 600 mg/kg, b.i.d. HDM-challenge in vehicle-treated animals significantly augmented the TNF-α expression (Figure 10A), when compared to the normal-control group. This effect was significantly inhibited by Dexamethasone and Bronchom at the doses of 60, 200 and 600 mg/kg, b.i.d, respectively (Figure 10A).

The expression of IL-6 was significantly increased in HDM-challenged animals, as compared to the saline-challenged animals (Figure 10B). Dexamethasone significantly inhibited the observed increase (Figure 10B). Similarly, Bronchom also significantly attenuated the increased expression of IL-6 at 60, 200 and 600 mg/kg, b.i.d., respectively, Figure 10B).

Further, the repeated exposure to HDM in disease-control animals, significantly increased the expression of IFN-γ (Figure 10C). This observed elevation was significantly reduced by dexamethasone (Figure 10C) as well as by Bronchom at 60, 200 and 600 mg/kg, b.i.d. respectively (Figure 10C).

Additionally, the expression of IL-13, another key Th2 cytokine was significantly elevated by HDM-challenge, when compared to saline-challenged group (Figure 10D). This increase was significantly suppressed by Dexamethasone (Figure 10D) and Bronchom at 60, 200 and 600 mg/kg, b.i.d. respectively, Figure 10D).

The mRNA level of IL-33 was also significantly elevated in the disease-control group, as compared to the normal-control group (Figure 10E). Dexamethasone significantly reduced the expression of IL-33 (Figure 10E). Likewise, Bronchom also attenuated the increase in the expression of the evaluated cytokine at 60, 200 and 600 mg/kg, b.i.d., respectively (Figure 10E)

When compared to the normal-control group, a significant decrease was noted in the anti-oxidant biomarker, GSH in the HDM-challenged animals (Figure 11A) with a concomitant elevation of its oxidized form, GSSG (Figure 11B). Further, the ratio of the reduced to oxidized glutathione was also significantly reduced (Figure 11C). Reference control drug, dexamethasone significantly restored the levels of GSH (Figure 11A) and suppressed the level of GSSG (Figure 11B). Additionally, it also reinstated the altered GSH/GSSG ratio (Figure 11C). Similar to the dexamethasone, oral administration of Bronchom, significantly restored the levels of GSH at all the tested doses (Figure 11A); decreased the level of GSSG at all the tested doses (Figure 11B) and furthermore, restored the decrease in GSH/GSSG ratio that was noted in the vehicle-treated, HDM-challenged group (Figure 11C).

Furthermore, we also evaluated the effect of Bronchom on MDA activity, a marker of lipid peroxidation in the lung homogenate. The MDA activity was significantly elevated in HDM-challenged animals, when compared to the normal-control group (Figure 11D). Both dexamethasone and Bronchom were able to inhibit the HDM-induced increase in MDA activity. The effects were significant for dexamethasone-1 mg/kg, q.d., whereas Bronchom demonstrated significant inhibition at the doses of 60, 200 and 600 b.i.d., respectively (Figure 11D).

HDM-challenged animals also exhibited a decrease in SOD activity, which was however not significant when compared to saline-challenged animals (Figure 11E). Dexamethasone (1 mg/kg, q.d.) and Bronchom (20, 60 and 200 mg/kg, b.i.d.) both tended to restore the reduced SOD activity. However, Bronchom-600 mg/kg, b.i.d. showed a significant elevation of SOD activity (Figure 11E).

Finally, we additionally assessed the nitrosative stress parameter, nitrite in the lung homogenates. HDM-challenge tended to result in an increase in the nitrite levels when compared to the saline-challenged animals (Figure 11F). This increase was significantly attenuated by Bronchom at all the tested doses as compared to the disease-control group (Figure 11F). Dexamethasone also tended to lower the HDM-induced increase in lung nitrite levels (Figure 11F).