MTT assay: Performed on HCT116, HT29, and SW480 cells (treatment duration: 48 h).

DAPI/PI staining: Conducted on HCT116 and HT29 cells (treatment: 24 h).

Western blot analysis: Conducted on HCT116 and HT29 cells (treatment: 24 h).

Protein denaturation, HRBC membrane stabilization, and NO assays: Conducted across all extracts at 100, 300, and 600 μg/mL. The 25–50 mg/kg doses used in mice correspond to human equivalent doses of approximately 2–4 mg/kg, based on body surface area conversion, ensuring translational relevance and safety.

Extract administration: AR-EA and AR-Met at 25 mg/kg and 50 mg/kg (oral gavage).

Control and reference groups: Normal saline (Group I), LPS-only (Group II), and dexamethasone (Group VII).

Assessment time points: Behavioural monitoring (0–13 days), histopathology (end of study), and blood sample collection (day 14).

With few adjustments, this assay was carried out in accordance with Djuichou (Djuichou Nguemnang et al., 2019). We prepared a reaction mixture through a component combination of varying concentrations of different extracts of Alcea rosea (100, 300, and 600 μg/mL) with 5% bovine serum albumin. The reaction mixtures were heated at 37°C in an incubator for 20 min, followed by 15 min at 70°C to induce the denaturation of protein. The absorbance of the reaction mixture was measured at 660 nm. The standard drug aspirin, an acetylsalicylic acid, was selected as the affirmative control.

With slight modifications, we carried out this assay following the protocol described by Leelaprakash and Dass (Leelaprakash and Dass, 2011). The reaction mixture was prepared by mixing 0.06 mg of trypsin, 1 mL of 20 mM Tris HCl buffer (pH 7.4), and 1 mL of test samples or extracts of Alcea rosea at varying concentrations (100, 300, and 600 μg/mL). The reaction mixtures were heated at 37°C in an incubator. After 5 min, 0.8% casein (1 mL) was added to the mixture. This combination of reactants was heated at 37°C in an incubator for 20 min. The reaction was completed by introducing 2 mL of 70% perchloric acid to the reaction mixture. The turbid mixture produced at the conclusion underwent centrifugation, and the resulting sediment was discarded. This experiment involved quantifying the absorbance of the supernatant at 210 nm.

This analysis was conducted in compliance with the procedure mentioned earlier (Tantary et al., 2017). Several solutions were prepared with citric acid, sodium citrate, sodium chloride, and dextrose in water at concentrations of 0.05%, 0.8%, 0.42%, and 2%, respectively. Mice blood was mixed with Alsever solution, centrifuged at 3000 rpm for 10 min, and washed with isosaline solution. The final cell volume was reconstituted to 10% v/v with isosaline. Hypotonic solution (50 mM NaCl) triggered mice red blood cell lysis. The experimental mixture included 0.50 mL of initial erythrocyte suspension in 10 mM sodium phosphate-buffered saline (pH 7.4) and 5 mL of hypotonic solution spiked with Alcea rosea extracts at 100, 300, and 600 μg/mL in separate test tubes. The standard drug diclofenac sodium served as a positive control.

We conducted a nitric oxide assay in accordance with the protocol outlined by Boora (Boora et al., 2014) with minor alterations. Different concentrations (600, 300, and 100 μg/mL) of all five Alcea rosea extracts were prepared alongside gallic acid as a standard. Griess reagent was freshly prepared. Equal volumes of 0.1% N-1-naphthyl ethylene-diamine and 1% SAA in 2.5% phosphoric acid were mixed. A mixture of 10 mM sodium nitroprusside in 1× PBS (pH 7.3) with Alcea rosea extracts underwent 3-h incubation. Griess reagent was then added. Control samples contained only 1× PBS. The reaction mixture was pipetted into a 96-well plate, and absorbance was measured at 546 nm using an ELISA microplate reader. Gallic acid served as a positive control for nitric oxide scavenging.

The MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide] assay was conducted following Yang’s protocol with minor adjustments (Yang et al., 2023). Human colon cancer cell lines (HCT116, HT29, SW480) from NCCS Pune were cultured in DMEM supplemented with penicillin, FBS, and streptomycin. Optimal growth conditions were maintained in a CO₂ incubator. Alcea rosea extracts were obtained via Soxhlet extraction and solubilised in DMSO. Cells were plated in 96-well plates and treated with various extract concentrations. After 48 h, MTT assay was performed, and absorbance was measured at 570 nm using an ELISA microplate reader. IC₅₀ values were determined by nonlinear regression analysis using the log[inhibitor] vs. normalized response (variable slope) model with GraphPad Prism version 10.1.0 software. Extract stock solutions were prepared at 100 mg/mL in DMSO and diluted directly in culture medium to achieve a target concentration of 200 μg/mL. At this concentration, 2 µL of stock was added to 1 mL of medium, ensuring that the final DMSO concentration did not exceed 0.2% (v/v), thereby minimizing any DMSO-related cytotoxicity.

HCT116 and HT29 cells were treated with AR-EA and AR-Met for 24 h. After treatment, the cells were fixed with 4% paraformaldehyde and washed twice with PBS. Nuclear staining was performed using DAPI, while propidium iodide (PI) was used to identify dead cells. Next, a Floid™ Cell Imaging System (Thermo Scientific, USA) was used to visualize the stained cells (Parry et al., 2025).

For Western blotting, HCT116 and HT29 cells were seeded in 100 mm cell culture dishes at 2.2 × 106 density. After 24 h, cells at 70% confluency were treated with ethyl acetate and methanol extracts for 24 h under standard conditions. Post-treatment, cells were harvested, lysed in NP-40 buffer, and stored at −80°C. Protein quantification (30–100 μg) was done by the Bradford method, followed by SDS-PAGE (10%–12%) and transfer onto PVDF membranes. Membranes were blocked with ODYSSEY LICOR buffer, incubated with primary antibodies overnight, washed thrice, and then incubated with secondary antibodies. Signal detection utilized LI-COR equipment (Guesmi et al., 2024).

Agilent technologies GC systems with MassHunter Qual 10.0 (GC-7000D_MS_QQQ model) (Santa Clora, CA, USA) equipped with HP-5 MS column were used to identify chemical components in Alcea rosea ethyl acetate (AR-EA), and methanol (AR-Met) extracts using GC-MS analysis (Gomathi et al., 2015). High-energy electrons (70 eV) were used to ionize the sample components. Pure helium gas served as the carrier gas. The temperature was gradually raised from 50-150°C at constant addition of 5°C per minute until it reached 250°C and was kept isothermally for 10 min. In a splitless mode, 4 μL of sample (AR extracts diluted with respective solvent) was injected. The metabolites retention time (t_R) and their mass spectra were then compared to those of previously recognized metabolites in the NIST collection (NIST17.L library, 2023).

BALB/c mice (25–30 g) aged 8 ± 1 weeks, were procured from the CSIR-Indian institute of integrative medicine (IIIM Jammu). Animals were well maintained at the Kashmir University animal house with regular 12-h light/dark cycle, relative humidity of 50% ± 20%, temperature of 23 ± 2°C and ventilation of 10–15 air changes/hour. Mice were properly fed with food and water and libitum. Each experimental animal group consisted of 6 BALB/c mice divided into seven groups: Group-I (normal saline only), Group-II (LPS only), Group-III (LPS + 25 mg/kg AR-EA), Group-IV (LPS + 50 mg/kg AR-EA), Group-V (LPS + 25 mg/kg AR-Met), Group-VI (LPS + 50 mg/kg AR-Met) and Group-VII (LPS + 0.5 mg/kg Dexamethasone). Effect of AR-EA and AR-Met extracts on general behaviour and safety of BALB/c mice was investigated. A single oral dose of 1500 mg/kg of AR-EA and AR-Met was administered to the experimental animals via oral gavage. Following the administration, the animals were closely monitored for potential toxic effects over the next 4 hours. After 24 h, no mortality was observed, and they remained under continuous observation for 13 days. At the end of the study, the mice were euthanized through cervical dislocation, and the weights of key organs were measured to assess any changes. All in vivo experiments were carried in accordance with the guidelines of Materials and methods of the “committee for purpose of control and supervision of experiments on animals” (CPCSEA) norms (Pandey and Sharma, 2011).

For histopathological analysis, paw tissues from mice were preserved in 10% formalin. The fixed tissues underwent a graded alcohol series to ensure complete dehydration. Subsequently, the dehydrated tissues were embedded in paraffin blocks. Thin sections, 3–5 μm in thickness, were sliced using an automated microtome. These tissue sections were then stained with hematoxylin and eosin. Finally, the stained sections were mounted with disterene phthalate xylene (DPX) and observed under Carl Zeiss Primovert (415510-1101-000) microscope (Ali et al., 2024).

Blood samples were obtained from mice via the retro-orbital plexus using fine glass capillaries and collected in properly labelled red-top vacutainers. To separate the serum, the vacutainers were centrifuged at 3000 rpm. The serum from each sample was then transferred into pyrogen-free Eppendorf tubes and stored at −20°C for subsequent analysis.

All the in Vitro and in vivo experiments were repeated three times to ensure statistical validity, and the results were presented as the mean ± standard deviation. One-way ANOVA was performed, followed by Dunnett’s post-hoc test for comparison of treatment groups versus the LPS-only group. This approach was selected to emphasize the anti-inflammatory and protective effects of extracts relative to the inflammatory control. Statistical analysis was conducted using GraphPad Prism, version 10.1.0 (316) software. Results with a p-value lower than 0.05 were deemed statistically significant.

Among the five tested extracts of Alcea rosea, the ethyl acetate extract (AR-EA) was the most potent at suppressing protein denaturation, with an inhibition percentage of 80.72 ± 2.93 (p < 0.001), followed by the methanolic extract of Alcea rosea (AR-Met), with an inhibition percentage of 71.21 ± 3.68 (p < 0.001) at a concentration of 600 μg/mL. The ethanolic extract (AR-E) had an inhibitory effect of 58.93 ± 3.06 (p < 0.01), and the aqueous extract (AR-Aq) had an inhibitory effect of 49.41 ± 2.95 (p < 0.05) at 600 μg/mL. The hexane extract (AR-H) showed the least inhibition (38.51% ± 1.89%) (p < 0.05) at 600 μg/mL. Aspirin, used as a positive control, demonstrated maximum inhibition (87.54% ± 4.02%) (p < 0.001) at 600 μg/mL. AR-EA and AR-Met exhibited IC₅₀ values of 220.27 ± 7.69 and 268.46 ± 9.87 μg/mL, respectively, while the IC₅₀ values of the other extracts ranged between 779.57 ± 39.02 and 460.35 ± 23.14 μg/mL. Aspirin had an IC₅₀ value of 83.08 ± 3.98 μg/mL (Table 1).

While investigating the anti-proteinase capacity of various extracts of Alcea rosea, it was observed that there was a marked dose-dependent inhibition of proteinase activity due to treatment with the ethyl acetate extract (AR-EA), attaining a maximum inhibition of 78.65% ± 3.24% at 600 μg/mL (Table 1). The methanol extract (AR-Met) inhibited 70.46% ± 3.27%, while the percentage inhibition of the other extracts ranged between 42.21% ± 2.10% and 57.30% ± 2.65% at the maximum concentration. Aspirin, which was used as a positive control, showed an inhibition of 81.1% ± 4.055% at 600 μg/mL. AR-EA and AR-Met exhibited very low IC₅₀ values, i.e., 86.38 ± 5.28 μg/mL and 190.92 ± 8.69 μg/mL, respectively, while in the other extracts, the IC₅₀ values ranged between 779.69 ± 36.54 and 360.96 ± 19.12 μg/mL. Aspirin had an IC₅₀ value of 68.49 ± 3.73 μg/mL.

All the tested extracts of Alcea rosea inhibited red blood cell (RBC) membrane haemolysis in a dose-dependent manner, with the ethyl acetate extract (AR-EA) exhibiting the greatest prevention of RBC haemolysis (81.29% ± 3.99%), followed by the methanolic extract (AR-Met) (79.85% ± 4.09%) at a concentration of 600 μg/mL, while the rest of the extracts showed an inhibition ranging between 48.97% ± 2.48% and 63.97% ± 3.31% at the maximum concentration. Diclofenac sodium, which served as a positive control, showed an inhibition of 86.98% ± 4.37% at 600 μg/mL. The efficacy of the AR-EA and AR-Met extracts was also evident by their IC₅₀ values, which were 47.46 ± 2.17 μg/mL in AR-EA and 61.33 ± 2.84 μg/mL in AR-Met, while in the other extracts, the IC₅₀ values ranged between 612.75 ± 29.14 and 169.30 ± 8.59 μg/mL. Diclofenac sodium had the lowest IC₅₀ value of 38.55 ± 1.69 μg/mL (Table 1).

The maximum nitrite radical scavenging potential was found for the ethyl acetate extract (AR-EA), followed by the methanol extract (AR-Met), while the hexane extract showed the least scavenging ability. The ethyl acetate extract (AR-EA) showed the greatest nitrite scavenging activity (79.89% ± 3.90%) at 600 μg/mL, followed by the methanolic extract (AR-Met), which inhibited (72.92% ± 3.66%) at 600 μg/mL (Table 1). The percentage of NO inhibition exhibited by the other extracts ranged between 53.24% ± 2.65% and 67.13% ± 3.48%. The gallic acid used as a positive control exhibited an inhibition of 89.41% ± 4.07% at the maximum concentration. AR-EA and AR-Met had IC₅₀ values of 78.20 ± 3.49 and152.05 ± 5.90 μg/mL, respectively, while the IC₅₀ values of the other extracts ranged between 513.26 ± 26.29 and 223.25 ± 11.70 μg/mL. Gallic acid had the lowest IC₅₀ value of 47.79 ± 1.94 μg/mL.

MTT assay was performed on the HCT116 cell line using extracts (25, 50, 75, 100, 125, 150, 175, and 200 μg/mL). We found that Alcea rosea ethyl acetate extract significantly affected the viability of HCT116 cells (IC₅₀ = 30.94 μg/mL), followed by Alcea rosea methanol extract (IC₅₀ = 55.62 μg/mL) and ethanolic extract, while hexane and aqueous extracts had less inhibitory effects (Figures 1A–E). Next, we evaluated the effects of ethyl acetate extract and methanol extract on HT29 and SW480 cell lines. The ethyl acetate extract showed IC₅₀ = 46.89 μg/mL in HT29 (Figure 1F) and IC₅₀ = 63.40 μg/mL in SW480; Figure 1H, while the methanol extract showed IC₅₀ = 58.15 μg/mL in HT29 (Figure 1G) and IC₅₀ = 75.37 μg/mL in SW480; (Figure 1I).

We next investigated the effect of these two extracts on key inflammatory proteins, such as COX-2, NFκB, and PPARγ, in HCT116 and HT29 cells. As shown in Figures 2A,E, the protein levels of all the probed genes decreased in both cell lines upon treatment with AR-EA and AR-Met. In HCT116 cells, AR-EA treatment decreased COX-2 levels by 1.86-fold (Figure 2B), NFκB by 3.12-fold (Figure 2C), and PPARγ by 2.6-fold (Figure 2D), and AR-Met treatment decreased COX-2 levels by 1.7-fold (Figure 2B), NFκB by 1.81-fold (Figure 2C), and PPARγ by 2.00-fold (Figure 2D). However, in HT29 cells, AR-EA treatment decreased COX-2 expression by 2.08-fold (Figure 2F), NFκB by 3.32-fold (Figure 2G), and PPARγ by 2.62-fold (Figure 2H), and AR-Met treatment decreased COX-2 expression by 1.88-fold (Figure 2F), NFκB by 1.83-fold (Figure 2G), and PPARγ by 1.76-fold (Figure 2H).

We next investigated the effect of AR-EA and AR-Met on the induction of apoptosis in HCT116 and HT29 cells. For this purpose, we exposed HCT116 and HT29 cells to AR-EA and AR-Met for 24 h. After treatment, we probed the induction of cell death in the cell lines by using DAPI(4′,6-diamidino-2-phenylindole)-PI staining, as shown in (Figure 3A) HCT116 and (Figure 3E) HT29. The exposure of HCT116 and HT29 cells to AR-EA and AR-Met for 24 h led to a significant increase in the number of apoptotic cells. These results were further validated by probing the extent of PARP and Caspase 3 cleavage. As shown in (Figures 3B,F), the protein levels of cleaved PARP and cleaved Caspase 3 were significantly increased in both cell lines upon treatment with AR-EA and AR-Met, respectively. In HCT116 cells, AR-EA treatment increased C-PARP levels 2.70-fold (Figure 3C) and C-Caspase 3 levels 3.59-fold (Figure 3D) and AR-Met treatment increased C-PARP levels 2.36-fold (Figure 3C) and C-Caspase 3 levels 2.19-fold (Figure 3D). However, in HT29 cells, AR-EA treatment increased C-PARP levels 3.06-fold (Figure 3G), C-Caspase 3 treatment 2.77-fold (Figure 3H), and AR-Met treatment 2.29-fold (Figure 3G) and C-Caspase 3 treatment 2.29-fold (Figure 3H). It can be inferred from these experimental findings that the AR-EA and AR-Met extracts of A. rosea display strong anticancer potential in HCT116 and HT29 colorectal cancer cell lines by mediating PARP and Caspase 3 cleavage.

Chromatograms of metabolites identified in the Alcea rosea ethyl acetate and methanol extracts are shown in (Figure 4). Some of the major metabolites identified in the aforementioned extracts are Nonanoic Acid, N-Hexadecenoic Acid, Eicosanoic Acid, Octadecanoic Acid, Propionic Acid, Hentriacontane, Kaempferol 3-O-(6″-Galloyl)-Beta-D-Glucopyranoside, Phytol, α-Tocopherol, 1-Beta-D-Ribofuranosyl-3-[5-Tetraazolyl]-1,2,4-Triazole, Ergosterol and Stigmasterol etc. (Supplementary Table 1). While the present study employed GC-MS for qualitative metabolite profiling, quantitative analysis of key compounds such as kaempferol and phytol using HPLC-DAD/LC-MS, along with batch-to-batch reproducibility and bioactivity correlation, are part of our ongoing and future research.

Administering lipopolysaccharide (LPS) into the sub-plantar area of the right hind paw in mice triggers a rapid inflammatory reaction. The histopathological analysis of the control group showed intact dermis and epidermis with no signs of lesion development, cellular infiltration, or edema. Nevertheless, the group that received only LPS had significant vasodilation and swelling, accompanied with neutrophil migration into the gaps between cells, collagen fibers, and blood vessels (Figure 5). On the other hand, mice who received ethyl acetate extract and methanolic extract at dosages of 25 and 50 mg/kg body weight exhibited a decrease in swelling and a decrease in the presence of polymorphonuclear (PMN) cells infiltrating the affected area. This reduction occurred in a way that was dependent on the dosage administered. Furthermore, these groups had reduced vascular congestion and epidermal thickening. The anti-inflammatory effects of the highest dosages of AR-EA and AR-Met were almost like those noted in the Dexamethasone-treated group, which was used as a positive control (Figure 5). While representative histological images were included to illustrate tissue-level changes, quantitative histopathological scoring was not performed, as the study prioritized molecular and biochemical assessments to evaluate inflammation.

This study investigated the impact of LPS-induced inflammation on a comprehensive panel of blood markers. We measured total and direct bilirubin, liver enzyme activities (AST and ALT), alkaline phosphatase, total protein profile (including albumin and globulin), kidney function markers (blood urea, blood urea nitrogen, and creatinine), uric acid, serum appearance, cholesterol, triglycerides, and C-reactive protein across all groups. These markers are known to be sensitive to changes caused by inflammation. Compared to the normal control group (receiving only saline), animals in the LPS group displayed a significant increase (p < 0.001) in all measured parameters except serum uric acid, which decreased. Conversely, animals in the AR-EA and AR-Met groups showed a significant improvement (p < 0.001) as evidenced by a decrease in all measured parameters and an increase in serum uric acid, as detailed in (Table 2).