Ayurveda-based phytochemical composition attenuates lung inflammation and precipitates pharmacokinetic interaction with favipiravir: an in vivo investigation using disease-state of acute lung injury

Abstract Acute respiratory distress syndrome (ARDS) is a critical form of acute lung injury (ALI). Here, we investigated the effect of a defined combination of ten pure phytochemicals in equal proportions of weight (NPM) from plants, recommended by Ayurveda for any protective action against lipopolysaccharide (LPS)-induced ALI. Results indicate that NPM markedly improved protein and neutrophil contents, myeloperoxidase and hydroxyproline levels, oxidative stress markers (glutathione and malonaldehyde), inflammatory cytokines, and genes (IL-6, TNF-α, TGF-β, and NF-κB/IκBα) in BALF/lung tissue. The histopathological examination of the lung revealed the shielding effect of NPM against ALI. NPM exhibited a protective effect on the lung by reducing oxidative stress and inhibiting inflammation. A substantial drop in favipiravir’s oral exposure was observed in ALI-state compared to normal-state, but oral exposure upon NPM treatment in ALI-state followed similar behaviour of favipiravir alike normal-state without NPM treatment. Overall, results offer potential insight into Ayurvedic recommendations for immunity boosting during ALI situations. Graphical abstract


Introduction
Acute respiratory distress syndrome (ARDS) is a life-threatening condition with a high mortality rate (Mason et al. 2016).It is characterized by inflammatory conditions associated with the increased weight of the lung, elevated alveolar-capillary permeability, and damage to alveolar tissues leading to the development of the hypoxic condition and respiratory failure (Ashbaugh et al. 1967).Despite understanding the pathophysiology of the diseased state, their laboratory diagnosis and therapeutic interventions are still not progressed to save the life of ARDS patients (Boyle et al. 2013).The importance of ARDS treatment has again been instigated since the outbreak of SARS-CoV-2/COVID-19 infection, where the mortality is due to the severity of the ARDS condition (Gorain et al. 2020).Generation of cytokine storm is the key pathological feature during this COVID-19 disease progression, which leads to lung injury followed by ARDS and failures of major organs (Gour et al. 2021).Research progress in treating this ARDS condition can't bring any beneficial compound yet to the bedside of the patients, which is compensated by thousands of lives every day (Gorain et al. 2020).Under this circumstance, it has been suggested to boost the immunity for combating the pathogen, thereby diminishing the chances of infection and disease severity.
In this pursuit, phytochemicals have shown immense potential to develop new therapeutics; therefore, a substantial portion of marketed drugs are directly or indirectly discovered from plant products (Veeresham 2012).Moreover, the experimental phytochemicals may be beneficial to prevent the likelihood of infection and may be able to combat the cytokine release storm.It is due to their helpful pharmacological actions like immunomodulatory, anti-inflammatory, antiviral activities, and many more (Gour et al. 2021).In addition, The Ministry of AYUSH in India recommends boosting immunity during the COVID-19 crisis (Sathya et al. 2020).Several plant materials are suggested for maintaining respiratory health; however, scientific evidence on those recommendations is yet to be established.
Favipiravir is turned out to be a useful drug for managing mild to moderate infection of COVID-19 (Udwadia et al. 2021).However, there is a lack of information on its pharmacokinetic behaviour during ALI and the possibility of any herb-drug interaction (Kotwal et al. 2020).Therefore, further investigations were done to evaluate the effect of disease-state (ALI-state) in comparison to normal-state on the pharmacokinetics of favipiravir alone.Further NPM was also explored to assess any pharmacokinetic interaction of favipiravir in ALI-state using a rat model.

NPM reduced protein level and neutrophil count
In the quest to explore the effect of NPM against LPS-induced ALI in the rat model, a battery of studies was carried out using BALF or lung tissue.Treatment of LPS significantly enhanced the protein content in the BALF compared to the control group.Treatment of dexamethasone (DEX) as a standard notably declined the protein content in LPS-treated animals.NPM treatment reduced the protein level substantially in the BALF at all the dose levels compared to the disease control group (Figure S1A).Similarly, the neutrophil count was also considerably elevated due to the treatment of LPS and decreased upon treatment with DEX in a significant manner.Treatment with NPM (20 to 40 mg/kg) notably lessened the neutrophil count compared to the disease control group (Figure S1B).It is evident from the literature that an increase in the total protein and extracellular traps of neutrophils is associated with ALI conditions (Liu et al. 2016).Therefore, results indicate that NPM could lower the protein content and neutrophil count to exert the shielding effect against ALI.

NPM decreased MPO activity and hydroxyproline content
MPO is considered a marker of inflammatory conditions in the lungs.The migrated polymorphonuclear neutrophils secrete this marker at the lung site, which is the risk factor for long-term mortality (Lei et al. 2020).Moreover, hydroxyproline is associated with collagen deposition leading to lung damage (Woessner 1961).LPS treatment caused a significant increase in MPO activity and hydroxyproline content in the lung tissue compared to the control group.Treatment with DEX considerably decreased the MPO activity and hydroxyproline content compared to the diseased control group.Treatment of NPM (10 to 20 mg/kg) exhibited a noteworthy improvement in both MPO activity (Figure S1C) and hydroxyproline content (Figure S1D) compared to the diseased control group.Thus, results suggest that NPM could restrict the LPS treatment linked to leakage of lung fluid.

NPM improved MDA content and GSH level
Malondialdehyde (MDA) is generated due to lipid peroxidation and can serve as a marker of oxidative stress.Glutathione (GSH) neutralizes reactive oxygen species and protects cells from cellular damage linked to oxidative stress (Chow et al. 2003).MDA content and GSH level were considerably augmented and declined upon LPS treatment, respectively in the lung tissue.DEX displayed a significant MDA content reduction and GSH level enhancement compared to the diseased control group.NPM (10 to 40 mg/kg) markedly decreased MDA content (Figure S1E).NPM (20 mg/kg) exhibited a prominent effect against LPS-induced depletion in GSH levels (Figure S1F).Hence, results reveal that NPM could limit the LPS-induced oxidative damage in the lung tissue.

NPM inhibited inflammatory cytokines: IL-6, TNF-a, and TGF-b
Excessive infiltration of inflammatory cells in the lungs is the main pathological hallmark of ALI (Cross and Matthay 2011).Administration of LPS caused significant enhancement in IL-6, TNF-a, IL-1b, and TGF-b levels (Figure S2A-2D) in the lung tissue compared to the control group.DEX exhibited a pronounced effect on each inflammatory cytokine up to various extents.NPM (10 to 40 mg/kg) displayed a substantial effect (IL-6 > TGF-b > TNF-a) against LPS-induced elevation of inflammatory cytokine levels in comparison to the disease control group.However, NPM treatment did not show any notable effect on IL-1b.So, results demonstrate that the action of NPM in ALI conditions could decelerate the release of inflammation mediators leading to control of lung inflammation.

NPM downregulated NF-jB/IjBa protein expression
During ALI conditions, alveolar macrophages release inflammatory cytokines and play a key role in activating inflammatory cascades leading to severe lung inflammation (Cross and Matthay 2011).Western blotting was done to evaluate the altered protein expression of p-NF-jB (p65) and p-IjBa in the lung tissue.A significant elevation in p-NF-jB (p65) and p-IjBa was observed in the diseased control group due to the treatment with LPS in comparison to the control group.DEX treatment displayed a considerable drop in both the experimental protein expression levels.NPM significantly downregulated the expression of both test proteins at all dose levels, except NPM at 40 mg/kg compared to the diseased control group.Results suggest that NPM could be more effective at the dose level of 20 mg/kg in decreasing the protein expression of NF-jB and IjBa (Figure S3).Therefore, results advocate a substantial effect of NPM on inflammatory gene regulation toward reducing lung inflammation.

NPM restricted alteration in lung histopathology
To get better insight into the pathological changes of the lung tissue, histopathological examination was done (Figure S4).Treatment of LPS caused lung injury, which was evaluated based on the following parameters: inflammatory infiltration, collagen deposition, and thickening of alveolar walls (Figure S5).Based on these parameters, lung architecture was significantly improved for all the treatment groups of DEX and NPM at all dose levels.NPM at the dose level of 20 mg/kg showed a more pronounced action in preventing lung histopathological features.Hence, overall results illustrate that the impact of NPM on oxidative stress and inflammation would possibly contribute to the protection against LPS-induced lung damage.

NPM increased the oral exposure of favipiravir in ALI-state
Favipiravir is one of the repurposed drugs to treat COVID-19 infection.Disease-states like inflammatory conditions can modulate the protein expression for transporters and drug-metabolism enzymes, leading to drug interaction (Morgan 2009, Wu andLin 2019).On the contrary, the simultaneous administration of phytochemicals with the prescribed therapy may cause drug interaction by elevating or reducing oral exposure of the drug (Kotwal et al. 2020).The pharmacokinetics of favipiravir was assessed under the ALI-state and normal-state using the rat model.LPS was used to induce ALI, which was confirmed by including an additional set of animals (Figure S6).Favipiravir was administrated orally at 20 mg/kg to rats in both normal-state and ALI-state (Figure S7 and Table S1).C max of favipiravir was significantly dropped (# 61%) in ALI-state compared to the normal-state.Although decrease in AUC for favipiravir was observed in ALI-state, it lacks statistical significance compared to the normal-state.So, results indicate that oral exposure of favipiravir was severely reduced in ALI-state in comparison to the normal-state, which could lead to therapeutic failure.
When oral exposure levels of favipiravir under ALI-state in the absence or presence of NPM (20 mg/kg) were compared, a substantial enhancement in C max of favipiravir (2.6-fold) was observed due to NPM treatment.Although the remaining pharmacokinetic parameters were not altered, the difference in AUC 0-t showed statistical significance.Co-administration of favipiravir with NPM in the ALI-state showed similar pharmacokinetic behaviour to favipiravir alone in the normal-state.Further exploration should be performed to clarify this type of drug interaction is friend or foe.On the other hand, NPM containing experimental phytochemicals like piperine (Choudhary et al. 2020), curcumin (Rocha and de Assis 2020), thymoquinone (Xu et al. 2021), linalool (Roviello and Roviello 2022), ursolic acid (Matondo et al. 2021), cinnamaldehyde (Lucas et al. 2021), 6-gingerol (Rathinavel et al. 2020), menthol (Sarker et al. 2021), eugenol (Paidi et al. 2021), and hesperidin (Agrawal et al. 2021) are reported in the literature for anti-viral activity against COVID-19 infection using in silico/in vitro models.
Hence, further efficacy studies along with underlying mechanisms should be designed for the combination of favipiravir with NPM in the specific animal model of COVID-19 to elucidate any potentiating role of NPM in the improvement of the anti-viral efficacy of favipiravir.

Conclusion
The current study findings revealed that NPM could provide a protective effect against LPS-induced ALI by inhibiting neutrophil infiltration, oxidative stress, the release of inflammatory cytokines, and the down-regulation of protein activation linked to inflammation.These pathophysiological changes contributed to reduce edema formation and lung inflammation, which are beneficial for protection against lung injury.Further, results of pharmacokinetic interaction of favipiravir with NPM in ALI-state should be clinically validated.It can be helpful to ensure any requirement of dose adjustment for favipiravir towards desired efficacy and safety.The present study data is beneficial for exploring NPM as a defensive weapon to combat cytokine storms leading to ARDS due to COVID-19 infection.