Phytochemical Elucidation and Effect of Maesa indica (Roxb.) Sweet on Alleviation of Potassium Dichromate-Induced Pulmonary Damage in Rats

Maesa indica (Roxb.) Sweet is one of the well-known traditionally-used Indian plants. This plant is rich in secondary metabolites like phenolic acids, flavonoids, alkaloids, glycosides, saponins, and carbohydrates. It contains numerous therapeutically active compounds like palmitic acid, chrysophanol, glyceryl palmitate, stigmasterol, β-sitosterol, dodecane, maesaquinone, quercetin 3-rhaminoside, rutin, chlorogenic acid, catechin, quercetin, nitrendipine, 2,3-dihydroxypropyl octadeca-9,12-dienoate, kiritiquinon, and β-thujone. The Maesa indica plant has been reported to have many biological properties including antidiabetic, anticancer, anti-angiogenic, anti-leishmanial, antioxidant, radical scavenging, antibacterial, antiviral, and anti-coronavirus effects. One purpose of the current study was to investigate the leaves’ metabolome via Triple-Time-of-Flight-Liquid-Chromatography-Mass Spectrometry (T-TOF LC/MS/MS) to identify the chemical constituents of the Maesa indica ethanolic extract (ME). Another purpose of this study was to explore the protective effect of ME against potassium dichromate (PD)-induced pulmonary damage in rats. Rats were assigned randomly into four experimental groups. Two different doses of the plant extract, (25 and 50 mg/kg), were administered orally for seven consecutive days before PD instillation injection. Results of our study revealed that ME enhanced cellular redox status as it decreased lipid peroxidation marker, MDA and elevated reduced glutathione (GSH). In addition, ME upregulated the cytoprotective signaling pathway PI3K/AKT. Moreover, ME administration ameliorated histopathological anomalies induced by PD. Several identified metabolites, such as chlorogenic acid, quercetin, apigenin, kaempferol, luteolin, and rutin, had previously indicated lung-protective effects, possibly through an antioxidant effect and inhibition of oxidative stress and inflammatory mediators. In conclusion, our results indicated that ME possesses lung-protective effects, which may be the result of its antioxidant and anti-inflammatory properties.

The LC-MS method is a strong analytical tool for profiling plant metabolites.Plants biochemistry is moderately complex and includes several semi-polar components, including important secondary metabolite classes, which may be effectively separated and identified using LC-MS techniques [6].Electrospray ionization (ESI), both in positive and negative ion modes, has quickly become the method of choice for secondary metabolites analysis, particularly when used in conjunction with MS-MS techniques.The method makes it possible to analyze pure and mixed samples quickly and without the requirement for derivatization by using small amounts of ingredients (less than 1 mg).
Natural polyphenols are a broad class of compounds present in plant-based food that offer protection against neurological, metabolic, cardiovascular, and cancerous diseases [7].Due to well-established evidence of their antioxidant activities both in vitro and in-vivo, polyphenols have garnered significant interest as potential therapeutic agents in lung degenerative disorders, particularly in acute lung injury [7,8].
Acute lung injury (ALI), also known as acute respiratory distress syndrome (ARDS), is a severe form of acute inflammatory lung injury linked to increased morbidity and mortality as well as the development of multiple organ failure [8].ALI affects more than 3 million patients a year, accounting for up to 10% of intensive care unit (ICU) patients [9].Diffuse alveolar epithelial destruction, pulmonary vascular endothelial cells, neutrophil infiltration, and the flow of protein-rich fluid into the alveolar gaps are among the primary pathological characteristics of acute lung injury.Inflammatory responses, apoptosis, redox imbalance, and goblet cell hyperplasia (GCH) are a few possible ALI causes [10].
Large amounts of hazardous chromium compounds are released into the environment as a result of the widespread usage of chromium in industry.While the hexavalent form of chromium is typically toxic, the trivalent form of chromium is less toxic and soluble [11].Reactive oxygen species (ROS) are produced when hexavalent chromium is reduced, and these ROS have the potential to harm DNA, lipids, and proteins in cells [12].In both experimental and clinical studies, the involvement of oxidants and oxidative damage in the etiology of ALI/ARDS has been extensively described [12].Oxidative stress can result from an imbalance in the production and elimination of ROS by the antioxidant defense system [10].Many inflammatory diseases, including idiopathic pulmonary fibrosis (IPF), acute respiratory distress syndrome, cystic fibrosis, and human immunodeficiency diseases, have been linked to changes in glutathione (GSH) and malondialdehyde (MDA) levels [13].According to [14], the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT) is thought to be connected to cell division, death, metabolism, and proliferation.Many recent research investigations have focused on the relationship between PI3K/AKT and pulmonary protection as it is believed that PI3K/Akt promotes the survival of pulmonary cells [15].
The objective of this study was to investigate the lung-protective effect of ME in potassium dichromate-induced pulmonary injury through measuring the levels of GSH, MDA, AKt, and PI3K as well as exploring the prevention of histopathological changes associated with exposure to PD.In addition, the chemical profile of ME was analyzed using Triple-Time-of-Flight-Liquid-Chromatography-Mass Spectrometry (T-TOF LC/MS/MS) to identify constituents that may be relevant to the plant's pulmonary protective action.

Metabolic Profiling Using T-TOF LC/MS/MS
In this study, we sought to provide a metabolite profile of ME using UPLC-ESI-TOF-MS and correlate the expected anti-pulmonary damage activity to its phytochemical Plants 2024, 13, 338 3 of 21 composition.Fifty-six metabolites were detected in ME.The metabolites were classified according to their chemical structure.We found 26 flavonoids, 17 amino acids, 8 phenolics, 3 sugars, and 2 alkaloids.For flavonoid identification, differentiating between O and C-linked flavonoid and phenolic conjugates required tandem mass-spectrometric analyses (MS 2 ).While the fragmentation pattern of the C-glycosyl conjugates shows major fragment ions as a result of neutral losses of 90 and 120 amu of pentose and hexose sugars, respectively, the O-glycosyl attachment typically can be easily identified in MS 2 spectra by the neutral losses of 162, 146, and 132 amu indicative of hexose, deoxyhexose, or pentose moieties, respectively [16].The base peak chromatograms of ME in the negative and positive ESI modes are depicted in Figure 1.The details of the detected and assigned major metabolites are listed in Table 1.

Metabolic Profiling Using T-TOF LC/MS/MS
In this study, we sought to provide a metabolite profile of ME using UPLC-ESI-TOF-MS and correlate the expected anti-pulmonary damage activity to its phytochemical composition.Fifty-six metabolites were detected in ME.The metabolites were classified according to their chemical structure.We found 26 flavonoids, 17 amino acids, 8 phenolics, 3 sugars, and 2 alkaloids.For flavonoid identification, differentiating between O and Clinked flavonoid and phenolic conjugates required tandem mass-spectrometric analyses (MS 2 ).While the fragmentation pattern of the C-glycosyl conjugates shows major fragment ions as a result of neutral losses of 90 and 120 amu of pentose and hexose sugars, respectively, the O-glycosyl attachment typically can be easily identified in MS 2 spectra by the neutral losses of 162, 146, and 132 amu indicative of hexose, deoxyhexose, or pentose moieties, respectively [16].The base peak chromatograms of ME in the negative and positive ESI modes are depicted in Figure 1.The details of the detected and assigned major metabolites are listed in Table 1.S1).Quercetin   [34] (Figure S23).
Many compounds could only be identified in only one ionization mode during the LC-MS analysis, because the chemical stability of these compounds is higher in one mode.We used both the negative and the positive ionization LC-MS mode to enhance the chemical profiling of the plant extract by studying the fragmentation pathway in the negative and the positive ion modes.The study confirmed the presence of phenolic acids, flavonoids, alkaloids, nitrogenous compounds, amino acids, and carbohydrates.The major classes were flavonoids and phenolics, which are characterized by having many therapeutic effects, such as anti-inflammatory, hepatoprotective, antioxidant, antiaging, lung protection, cardioprotective, antidiabetic, anxiolytic, neuroprotective, immunomodulatory, gastroprotective, hormone synthesis regulation, and anti-Alzheimer's [41,42].
To our knowledge, only a few studies have been done to study the chemical composition of the Maesa indica plant.This was the first screening and analysis of ME using LC/MS/MS, and it resulted in the identification of fifty-six compounds.Only five of the identified compounds were reported to be present in ME in previous studies.The previously reported compounds were chlorogenic acid, quercetin, quercetrin, catechin, and rutin.The remaining fifty-one compounds were identified for the first time in this study.

In-Vivo Study 2.2.1. Acute Toxicity
Male and female mice treated with a single dose of 0.5 g/kg of ME did not exhibit any toxicity signs such as death, hair loss, gastrointestinal disturbances, yellow spots, or abnormal behavior.As a result, the acute fatal toxicity test confirmed that Measa indica extract could be administered orally without a significant risk.

Investigation of the Lung Protection Activity of ME Effect of ME on GSH and MDA Lung Contents
PD, containing hexavalent chromium, generates ROS inducing acute lung injury [43].In the current study, PD instillation significantly (p-value < 0.05) reduced GSH lung tissue concentration from 0.0666 ± 0.0023 mmol/L (in the control group) to 0.0494 ± 0.00152 mmol/L (Figure 2A), which means that PD instillation induced a decrease of GSH levels of 26%.PD instillation also significantly (p-value < 0.05) increased MDA lung tissue concentration from 5.07771 ± 0.8338 322 nmol/g to 7.9637 ± 0.54515 nmol/g, compared to the control group (Figure 2B), which means that PD instillation induced an increase of MDA levels of 57%.levels, which attenuates brain injury [46].
In another study, Maesa indica, rich in polyphenols, proved to be a radical scavenger with antioxidant properties [2].Maesa indica contains the naturally bioactive compound quercetin, which reduces oxidative damage and chronic diseases such as diabetes, cardiovascular diseases, and hepatotoxicity by reducing amounts of the inflammatory cytokines TNF-α and NF-κ B [47,48].In addition, Maesa indica is a favorite plant of practitioners of folk medicine for its ability to inhibit oxidative hemolysis and its nitric oxide radical and DPPH radical scavenging effects [49].The highly potent antioxidant compounds quercetin and gallic acid, which were previously identified in ME, have been reported to enhance lung SOD activity and GSH levels and decrease NO and IL-6 levels [50].On the other hand, the phenolic compounds of M. lanceolate, another species of genus Maesa, were found to be responsible for its excellent antioxidant activity [51].Data were expressed as mean ± SD.Statistical analysis was carried out by one-way ANOVA followed by Tukey's multiple comparisons test.* Significant difference at p < 0.05 when compared to the normal group.# Significant difference at p < 0.05 when compared to the PD group.

Effect of ME on AKt and PI3K Lung Contents
Intracellular phosphatidylinositol-3 kinase (PI3K) regulates oxidative damage through phosphorylation of AKt, which is a serine/threonine kinase that controls cell survival and stimulates Nrf2 gene expression and nuclear translocation, leading to the Interestingly, administration of ME 25 mg/kg significantly (p-value < 0.05) increased GSH by 9% and decreased MDA contents by 17% compared to the PD group.As shown in Figure 2, the levels of GSH increased to 0.054 ± 0.00071 mmol/L (vs.0.0494 ± 0.00152 mmol/L) (A), and MDA contents were decreased to 6.6248 ± 0.140640 nmol/L (vs.7.9637 ± 0.54515 nmol/g) (B).More effective results were achieved with the high dose of the extract (50 mg/kg).We measured a GSH increase of 16% and an MDA decrease of 32%.Specifically, GSH measured 0.0576 ± 0.00089 mmol/L and MDS measured 5.4268 ± 0.5308 325 nmol/L (Figures 2A and 2B, respectively).
These results indicated that ME, via its antioxidant effect, significantly decreased lung injury caused by the administration of PD.Phenolics scavenge initial free radicals such as hydroxyl radicals and bind metal ion catalysts, decomposing primary oxidation products into non-radical species [44,45].Phenolic compounds have antioxidant potential; they work by elevating GSH-PX and SOD enzyme levels and decreasing MDA radical levels, which attenuates brain injury [46].
In another study, Maesa indica, rich in polyphenols, proved to be a radical scavenger with antioxidant properties [2].Maesa indica contains the naturally bioactive compound quercetin, which reduces oxidative damage and chronic diseases such as diabetes, cardiovascular diseases, and hepatotoxicity by reducing amounts of the inflammatory cytokines TNF-α and NF-κ B [47,48].In addition, Maesa indica is a favorite plant of practitioners of folk medicine for its ability to inhibit oxidative hemolysis and its nitric oxide radical and DPPH radical scavenging effects [49].The highly potent antioxidant compounds quercetin and gallic acid, which were previously identified in ME, have been reported to enhance lung SOD activity and GSH levels and decrease NO and IL-6 levels [50].On the other hand, the phenolic compounds of M. lanceolate, another species of genus Maesa, were found to be responsible for its excellent antioxidant activity [51].

Effect of ME on AKt and PI3K Lung Contents
Intracellular phosphatidylinositol-3 kinase (PI3K) regulates oxidative damage through phosphorylation of AKt, which is a serine/threonine kinase that controls cell survival and stimulates Nrf2 gene expression and nuclear translocation, leading to the inhibition of oxidative stress and inflammation [10].Our results indicated that the concentration of PI3K in lung tissue was significantly reduced (p-value < 0.05) in the PD group as compared to the control group.The concentration fell from 732.5 ± 14.773 pg/g to 413.9 ± 10.2311 pg/g (Figure 3A).This indicates that PD instillation induces a decrease of PI3K lung level with 43%.PD instillation also significantly decreased AKt lung contents (p-value < 0.05) in the PD group as compared to the control group.The levels fell from 644 ± 65.761 pg/g to 290.13 ± 4.652 pg/g (Figure 3B), which indicates that PD instillation induces a 55% decrease in AKt levels.
These results strongly suggest that ME possesses antioxidant and anti-inflammatory properties.In line with our results, a previous study showed that phytochemicals of Maesa indica have anti-arthritic properties in vitro [52].In addition, an extract of Maesa mesozygia, another species of genus Maesa, is three times more effective than diclofenac sodium as an anti-inflammatory and anti-arthritic drug [51].

Histopathological Findings
No histopathological alterations were detected, and the normal histological structure of the bronchiole (b) with the surrounding air alveoli (a) were recorded in group of control rats (Figure 4A).The peribronchiolar tissue showed focal lymphoid cells aggregation (L) with dilatation of the blood vessels (b.v) associated with fibroblastic cells (f), and aggregation surrounding the bronchioles as well as in between the obliterated air alveoli in the group of rats experimentally-inducted with PD (Figure 4B,C).The peribronchiolar tissue showed moderate inflammatory cell (i) aggregation in the group of experimentallyinducted rats treated with ME (25 mg/kg) (Figure 4D).However, mild inflammatory cell aggregation was observed in the peribronchiolar tissue, and we found a few lymphoid cell aggregations in the group of experimentally-inducted rats treated with ME (50 mg/kg) Figure 4E.From these histopathological findings, we can conclude that the use of ME at a higher concentration (50 mg/kg) provides better lung protection than that produced by the lower concentration (25 mg/kg).ME reduces the histopathological changes caused by However, administration of ME 25 mg/kg significantly (p-value < 0.05) elevated AKt by 35% and PI3K contents by 43% compared to the PD group.As shown in Figure 3, the level of AKt increased to 390.53 ± 34.857 pg/g (vs.290.13 ± 4.652 pg/g) (A) and PI3K contents increased to 592.2 ± 24.191 pg/g (vs.413.9 ± 10.2311 pg/g) (B).Larger effects were achieved with the higher dose of the plant extract (50 mg/kg).We measured an AKt elevation of 82% and a PI3K elevation of 58%.Particularly, 529.067 ± 2.891 pg/g were measured for AKt and 654.5 ± 13.233 pg/g for PI3K (Figure 3A,B).
These results strongly suggest that ME possesses antioxidant and anti-inflammatory properties.In line with our results, a previous study showed that phytochemicals of Maesa indica have anti-arthritic properties in vitro [52].In addition, an extract of Maesa mesozygia, another species of genus Maesa, is three times more effective than diclofenac sodium as an anti-inflammatory and anti-arthritic drug [51].

Histopathological Findings
No histopathological alterations were detected, and the normal histological structure of the bronchiole (b) with the surrounding air alveoli (a) were recorded in group of control rats (Figure 4A).The peribronchiolar tissue showed focal lymphoid cells aggregation (L) with dilatation of the blood vessels (b.v) associated with fibroblastic cells (f), and aggregation surrounding the bronchioles as well as in between the obliterated air alveoli in the group of rats experimentally-inducted with PD (Figure 4B,C).The peribronchiolar tissue showed moderate inflammatory cell (i) aggregation in the group of experimentallyinducted rats treated with ME (25 mg/kg) (Figure 4D).However, mild inflammatory cell aggregation was observed in the peribronchiolar tissue, and we found a few lymphoid cell aggregations in the group of experimentally-inducted rats treated with ME (50 mg/kg) Figure 4E.From these histopathological findings, we can conclude that the use of ME at a higher concentration (50 mg/kg) provides better lung protection than that produced by the lower concentration (25 mg/kg).ME reduces the histopathological changes caused by the exposure to PD which might be due to the high content of phenolics and flavonoids.Phenolic and flavonoid compounds have been reported to be able to markedly reduce lung histopathological changes [50].
Plants 2024, 13, x FOR PEER REVIEW 11 of 19 the exposure to PD which might be due to the high content of phenolics and flavonoids.Phenolic and flavonoid compounds have been reported to be able to markedly reduce lung histopathological changes [50].All of these results confirmed the lung-protecting effect of the ethanolic extract of Maesa indica.This plant is rich in secondary metabolites, especially those which have antioxidant effects, such as phenolic and flavonoid compounds [5].In this study, we identified 26 flavonoid compounds and 8 phenolic compounds.Most of these identified compounds have confirmed antioxidant capabilities and can prevent lung injury.Chlorogenic acid and phenolic acid were confirmed to have a potential protective effect against various toxicities like metals, pesticides, natural toxins, and pharmaceuticals.Mechanistic evaluations showed that inhibition of oxidative stress, free radical scavenging, apoptosis pathways, and decreasing the inflammatory responses are among the beneficial mechanisms mediated by chlorogenic acid [53].Another study concluded that chlorogenic acid elevates the activity of SOD and the level of GSH and decreases the production of ROS and the accumulation of MDA [54].Quercetin has been said to suppress lung inflammation in mice and to have an antioxidant effect, because the intratracheal administration of quercetin decreases the wet lung-to-body weight ratio (an index of pulmonary edema that is correlated with the severity of a lung injury) [55].Apigenin administration reduces the biochemical parameters of oxidative stress and inflammation, improves oxygenation, and decreases lung edema in acute lung injuries in mice by inhibiting inflammation and oxidative stress [56].According to [57], apigenin, quercetin, and luteolin were confirmed to have antioxidant effects.Kaempferol has proved to be a strong antioxidant, and it could significantly reduce the damage caused by lung ischemia-reperfusion injuries by inhibiting the release of inflammatory factors including interleukin lung ischemia-reperfusion injury and tumor necrosis factor α into All of these results confirmed the lung-protecting effect of the ethanolic extract of Maesa indica.This plant is rich in secondary metabolites, especially those which have antioxidant effects, such as phenolic and flavonoid compounds [5].In this study, we identified 26 flavonoid compounds and 8 phenolic compounds.Most of these identified compounds have confirmed antioxidant capabilities and can prevent lung injury.Chlorogenic acid and phenolic acid were confirmed to have a potential protective effect against various toxicities like metals, pesticides, natural toxins, and pharmaceuticals.Mechanistic evaluations showed that inhibition of oxidative stress, free radical scavenging, apoptosis pathways, and decreasing the inflammatory responses are among the beneficial mechanisms mediated by chlorogenic acid [53].Another study concluded that chlorogenic acid elevates the activity of SOD and the level of GSH and decreases the production of ROS and the accumulation of MDA [54].Quercetin has been said to suppress lung inflammation in mice and to have an antioxidant effect, because the intratracheal administration of quercetin decreases the wet lung-to-body weight ratio (an index of pulmonary edema that is correlated with the severity of a lung injury) [55].Apigenin administration reduces the biochemical parameters of oxidative stress and inflammation, improves oxygenation, and decreases lung edema in acute lung injuries in mice by inhibiting inflammation and oxidative stress [56].According to [57], apigenin, quercetin, and luteolin were confirmed to have antioxidant effects.Kaempferol has proved to be a strong antioxidant, and it could significantly reduce the damage caused by lung ischemia-reperfusion injuries by inhibiting the release of inflammatory factors including interleukin lung ischemia-reperfusion injury and tumor necrosis factor α into the broncho-alveolar lavage fluid and reducing oxidative stress reactions [58].
In a lipopolysaccharide (LPS)-induced acute lung injury, rutin pretreatment prevented polymorphonuclear granulocyte infiltration into broncho-alveolar lavage fluid in addition to histological alterations in lung tissues.Furthermore, rutin reduced LPS-induced inflammatory responses in a concentration-dependent manner, including enhanced proinflammatory cytokine release and lipid peroxidation.Moreover, rutin reduces the effects of LPS on the activity of antioxidative enzymes such as catalase, glutathione peroxidase, hemeoxygenase-1, and superoxide dismutase [59].The lung-protection effect of catechin and quercetin was detected during an assay of their effects on chlorpyrifos-induced lung toxicity in male rats.As a result, glutathione-S-transferase and glutathione peroxidase activities significantly increased, while MDA and SOD activities significantly decreased.Light microscopic studies revealed that histopathological changes were milder in animals treated with catechin or quercetin than in a control group [60].Ref. [61] Explored the effect of naringenin on lipopolysaccharide (LPS)-induced acute lung injury in mice and the supposed mechanism.The results proved that naringenin pretreatment increased the rate of survival, enhanced histopathologic changes, attenuated pulmonary edemas and lung vascular leaks, and downregulated ROS level.In addition, naringenin pretreatment reduced the total and phosphorylated protein levels of phosphatidylinositol 3-hydroxy kinase (PI3K) and AKt.Pretreatment with rosmarinic acid strongly decreases lung injury caused by diesel exhaust particles (DEP) through its antioxidative and anti-inflammatory activities.Oral treatment of mice with rosmarinic acid inhibited DEP-caused lung injuries and edemas [62].Ref. [63] Investigated the protective effect of caffeic acid phenethyl ester on acetylsalicylic-acid-induced lung injury in rats.The study concluded that caffeic acid phenethyl ester administration ameliorated the inflammation and eosinophil accumulation in the pulmonary interstitium in a histopathological manner, and it provided its lung-protecting effect by reducing oxidative damage and inflammation.Cinnamic acid was confirmed as a lung-protective compound effective against histopathological damage induced by methotrexate in rats.This was associated with parallel improvements in measured oxidative, inflammatory, and fibrotic parameters [64].
As mentioned before, alkaloids are strong therapeutic compounds, and they have many reported biological properties, including antimitotic, anticancer, anti-inflammatory, analgesic, antibacterial, antifungal, local analgesic, pain-relieving, antioxidant, antiparasitic, anti-plasmodic, antibacterial, anti-HIV, and as a local anesthetic [39].The total alkaloids of the Aconitum tanguticum plant were found to have protective effects on acute lung injury induced by lipopolysaccharide in rats.The alkaloids significantly reduced the lung wet/dry ratio, reduced lung histopathological changes, decreased the nuclear factor kappa B (NF-κB) activation, downregulated inflammatory cell infiltration, and reduced vascular leakage and inflammatory cytokine release [65].In lipopolysaccharides-induced acute lung injury in rats, total alkaloids decreased the levels of neutrophils, the number of WBCs, the levels of albumin, alkaline phosphate, and lactate dehydrogenase in broncho-alveolar lavage fluid (BALF), and increased the content of albumin in serum.It also improved SOD activity in lung, serum, and BALF, increased nitric oxide levels, and decreased MDA levels in lungs.Total alkaloids also inhibited the production of the inflammatory cytokines TNF-α and IL-8 in BALF and in the lung tissues.Histopathological examination showed that alkaloids suppressed histopathological changes caused by acute lung injury [66].The two identified alkaloids, caffeine and trigonelline, were proven to have antioxidant properties, increase SOD activity and GSH levels, and decrease ROS production and MDA accumulation [67].
A variety of natural compounds can target cell-signaling pathways that promote beneficial activity against respiratory diseases.Natural products, including flavonoids, phenolics, alkaloids, and terpenes are at the same time a treasure trove of essential chemotherapeutics that produce desirable effects against respiratory diseases and lung injury.They also facilitate the development of novel drug systems by providing pharmacophores suitable for optimal effects on target pathways associated with the development of respiratory diseases [68].This concept suggests the synergistic effect of ME phenolics, flavonoids, and alkaloid effects in lung protection, side by side with the approved lung protection of many of the compounds in ME identified in this study.

Plant Materials
M. indica Roxb.Sweet aerial parts before blooming were collected from the EL-MAZHAR botanical garden, El-Barageel, Giza, Egypt.The plant was verified by Therese Labib, a senior botanist at El-Orman Botanical Gardens and a plant taxonomy consultant at the Ministry of Agriculture, Egypt.The specimen voucher with the number 19.06.2022 was kept in the Pharmacognosy Department herbarium of the Faculty of Pharmacy, Cairo University.The plant material was allowed to air dry before being pounded into a coarse powder, put in an appropriate amber glass, sealed in an airtight container, and kept at room temperature.

Extraction of Plant Materials
Five hundred grams of the powdered MI were extracted using one liter of 70% ethanol (three times until complete extraction) at room temperature.The ethanol was evaporated using rotavapor until dryness giving about 6 gm of thick extract (ME).Methanol and formic acid (LC grade) were supplied from Fisher Scientific (Hampton, UK).Acetonitrile and ammonium formate (LC grade) were purchased from Sigma-Aldrich (Darmstadt, Germany).Millipore water was used (Burlington, MA, USA).

Instrument
LC-MS/MS was performed using an ExionLC™ AC system coupled with an AB Sciex TripleTOF 5600+ mass spectrometer (SCIEX, Toronto, ON, Canada).Non-targeted screening was applied using electron spray ionization (ESI) as the ionization probe.Data processing and peak identification were performed using an MS-DIAL 4.6 with the Respect library database.

Chromatographic Conditions
The separation was performed using an X select HSS T3 Column (2.1 × 150 mm, 2.5 µm).The mobile phases consisted of two eluents A: 5 mM ammonium formate buffer pH 8 containing 1% methanol; B: acetonitrile.The mobile phase elution was programmed as follows: 10% B from 0-1 min, 10-90% B from 1-21 min, 90% B from 21-25 min, 90-10% B from 25-25.01 min, and finally holds for three minutes at 10% B. The flow rate was 0.3 mL/min and the injection volume was 10 µL.Negative ionization mode was applied with a workflow EMS-IDAEPI and a scan from 50 to 1000 Da.For MS1, the following parameters were adjusted: curtain gas: 25 psi; IonSpray voltage: −4500, positive mode IonSpray voltage was 4500; source temperature: 500 • C; ion source gas 1 & 2 were 45 psi while in the case of MS2, a scan from 50 to 1000 Da was also adjusted with a declustering potential: −80; collision energy: −35; collision energy spread: 15.

Sample Preparation
Prepare a reconstitution solvent composed of Water: Methanol: Acetonitrile (50:25:25) v/v.50 mg of the sample is dissolved in 1 mL of the reconstitution solvent.Vortex for 2 min followed by ultra-sonication for 10 min.Centrifuge for 10 min at 10,000 rpm.Dilute 50 µL of stock solution to 1000 µL by reconstitution solvent.The injected concentration is 2.5 µg/µL.Inject 10 µL on negative mode.Inject 10 µL reconstitution solvent as a blank sample.

Acute Toxicity Study
For this investigation, male and female Swiss mice weighing 20-30 gm were procured from the animal house laboratory at the National Research Centre (NRC), Cairo, Egypt.Mice were kept in a hygienic laboratory environment for seven days before the start of the biological experiment (adaptation period), held in a well-ventilated box at 22 ± 2 • C for a 12-h lighting and darkness cycle.A natural baseline diet was given to the mice.Diets and water were provided freely.They were managed according to the animal testing guidelines approved by the Ethical Committee of Medical Research, NRC, Cairo, Egypt.Acute toxicity was carried out in compliance with the guidelines of the World Health Organization (WHO) for assessing the safety and efficacy of herbal remedies.Forty Swiss mice were divided into four groups (ten for each group).Groups one and two: control male and female mice were orally administered with saline.Groups three and four: male and female mice were orally administered with a single dose of ME in graded doses up to 5 g/kg.The animals were observed daily for signs of behavioral changes for two weeks [69].

Investigation of Pulmonary Protection Activity Animals
Adult male Wister albino rats (150-200 g) were provided by the Animal House of the National Research Centre (Cairo, Egypt).The rats were group-housed under temperatureand light-controlled conditions ( 22 Drugs, Chemicals and Kits PD was purchased from (Santa Cruz, CA, USA).MDA and GSH were determined using a Biodiagnostic kit, Giza, Egypt.AKt and PI3K were determined using ELISA kits procured from (Sunlong Biotech Co., Ltd., Hangzhou, China).

Experimental Design of Pulmonary Damage
Male Wister albino rats were randomly allocated into four groups (n = 8) as follows: Control group: Rats were injected with a single intranasal (i.n) instillation of normal saline and received normal saline orally for ten consecutive days.PD group: Rats were injected with a single i.n instillation of PD (2 mg/kg in a volume of 500 µL) [10].ME groups: rats were administered with ME (25 & 50 mg/kg) once daily for seven consecutive days before the intranasal (i.n) instillation of PD.

Biochemical Analysis
At the end of the experimental period, the animals were anesthetized with pentobarbital sodium and sacrificed by decapitation.One lung from each rat was immediately dissected out, washed with ice-cooled physiological saline, and homogenized in phosphatebuffered saline (PBS) (pH 7.4) as 20% (w/v) for the biochemical measurements [70].The other lung was kept for histopathological assessment.

Estimation of MDA and GSH
Serum was used for the estimation of reduced glutathione (GSH) and malondialdehyde (MDA) levels.The GSH estimation method is based on the reduction of 5,5 dithiobis (2-nitrobenzoic acid) (DTNB) with reduced GSH to produce a yellow compound.The reduced chromogen is directly proportional to GSH concentration, and its absorbance can be measured at 405 nm [71].MDA measuring depends on the formation of MDA as an end product of lipid peroxidation.The MDA reacts with thiobarbituric acid, producing a thiobarbituric acid reactive substance (TBARS), a pink chromogen, which can be measured spectrophotometrically at 532 nm.An MDA standard was used to construct a standard curve against which readings of the samples were plotted [10].

Estimation of AKt and PI3K
The contents of AKt and PI3K in each lung were determined using ELISA kits.Standards and samples (50 µL) were pipetted into wells with immobilized antibodies specific for rat AKt and PI3K and were then incubated for 30 min at 37 • C.After incubation and washing, horseradish peroxidase-conjugated streptavidin was pipetted into the wells and incubated for 30 min at 37 • C, which were washed once again.Tetramethylbenzidine (TMB) substrate solution was added to the wells and incubated for 15 min at 37 • C; a color developed proportionally to the amount of AKt and PI3K bound.Color development was discontinued (stop solution) and after 10 min the color intensity was measured at 450 nm [72].
Then the concentration of each parameter (pg/g) and the % change in concentration between groups are calculated as follows: % change = 100 − (conc of treatment/mean conc of diseased) × 100

Histological Examination
The dissected lungs of different groups were fixed in 10% formalin.Fixation for one or two days was followed by dehydration in ascending grades of alcohol (70%, 90%, and three changes in absolute alcohol), clearance with xylene, impregnation in three successive changes in soft paraffin at 50 • C, and finally embedded in paraffin wax to obtain solid blocks containing the tissue.Serial transverse sections of 7 µm thick were cut.Paraffin sections were mounted on glass slides covered by albumin glycerin and then stained with Haematoxylin and Eosin.Hematoxylin and Eosin sections were evaluated qualitatively under light microscopy.

Statistical Analysis
All the values are presented as means ± SD.Data of this study were evaluated by one-way analysis of variance followed by Tukey's multiple comparisons test.Graphpad Prism software, version 5 (Iglesia Ni Cristo., San Diego, CA, USA) was used to carry out these statistical tests.The difference was considered significant when p < 0.05.

Conclusions
The present study investigated the chemical profile of 70% Maesa indica ethanolic extract and identified 56 compounds via UPLC-ESI-TOF-MS in both negative and positive ESI ionization modes.The identified compounds include 26 flavonoids, 17 amino acids, 8 phenolics, 3 sugars, and 2 alkaloids.The study also investigated the lung-protective capacity of the plant ethanolic extract against potassium dichromate-induced pulmonary damage in rats.The results confirmed the lung-protection capacity of ME.The ME inhibited the histopathological changes caused by potassium dichromate.Furthermore, the administration of ME caused a significant increase in GSH, AKt, and PI3K levels and significant decrease in MDA level.Considering these results, it can be inferred that Maesa indica is a potent lung-protective plant and can alleviate potassium dichromate-induced pulmonary damage.Further investigation and clinical trials are needed to assess the exact mechanism of action of ME and to determine if these results can be applied to human diseases.

Figure 2 .
Figure 2. Effect of ME on the lung content of (A) GSH in mmol/L.(B) MDA in nmol/g.

Figure 2 .
Figure 2. Effect of ME on the lung content of (A) GSH in mmol/L.(B) MDA in nmol/g.Data were expressed as mean ± SD.Statistical analysis was carried out by one-way ANOVA followed by Tukey's multiple comparisons test.* Significant difference at p < 0.05 when compared to the normal group.# Significant difference at p < 0.05 when compared to the PD group.

Figure 3 .
Figure 3.Effect of ME on: (A) Lung content of PI3K in pg/g.(B) Lung content of AKt in pg/g.Data were expressed as mean ± SD.Statistical analysis was carried out by one-way ANOVA followed by Tukey's multiple comparisons test.* Significant difference at p < 0.05 when compared to the normal group.# Significant difference at p < 0.05 when compared to the PD group.

Figure 3 .
Figure 3.Effect of ME on: (A) Lung content of PI3K in pg/g.(B) Lung content of AKt in pg/g.Data were expressed as mean ± SD.Statistical analysis was carried out by one-way ANOVA followed by Tukey's multiple comparisons test.* Significant difference at p < 0.05 when compared to the normal group.# Significant difference at p < 0.05 when compared to the PD group.

Figure 4 .
Figure 4. Effect of treatment with ME on PD-induced lung histopathological anomalies.(A) Lung sections from normal control rats showed average bronchioles with normal histological features, (B,C) Lung sections from the PD-treated group showing severe focal lymphoid aggregation, blood vessel dilatation and congestion as well as fibrotic changes (arrows).(D) Lung sections from rats treated with PD and ME 25 mg/kg, showing moderate infiltration of inflammatory cells (arrows).(E) Lung sections from rats treated with PD and ME 50 mg/kg, showing mild inflammatory infiltration (arrows), indicating dose-dependent protection against PD-induced lung injury.

Figure 4 .
Figure 4. Effect of treatment with ME on PD-induced lung histopathological anomalies.(A) Lung sections from normal control rats showed average bronchioles with normal histological features, (B,C) Lung sections from the PD-treated group showing severe focal lymphoid aggregation, blood vessel dilatation and congestion as well as fibrotic changes (arrows).(D) Lung sections from rats treated with PD and ME 25 mg/kg, showing moderate infiltration of inflammatory cells (arrows).(E) Lung sections from rats treated with PD and ME 50 mg/kg, showing mild inflammatory infiltration (arrows), indicating dose-dependent protection against PD-induced lung injury.

3. 3 .
Metabolic Profiling Using T-TOF LC/MS/MS 3.3.1.Chemicals ± 2 • C under a 12 h light/dark cycle) and had free access to standard laboratory rodent chow and water.The animal experiments were performed according to NRC and recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (NIH No. 85:23 revised 1985).This study was approved by the Ethics Committee of the Faculty of Pharmacy, Cairo University, Egypt [Serial number of the protocol: MP (2992)].

Table 1 .
Metabolites tentatively identified in the 70% ethanol extract of (Maesa indica) via UPLC ESI-TOF-MS in both negative/positive ESI ionization modes.