Sida rhombifolia samples ( Figure 1(a) ) were collected in April 2022 in the central region of Cameroon, near the University of Yaoundé 1 in the Ngoa-ékélé district of the city of Yaoundé. The collected specimens were identified at the National Herbarium of Cameroon by Mr. MOUNMEMI KPOUMIE Hubert, who, by comparing them with patent material n˚169 of the specimen from the herbarium collection n˚9984 SRF Cam, issued a certificate of identification for the botanical reference sample n˚170/IRAD/DG/CRAM/SSRG-HNSF-PV/04/2022. The collected samples were dried at room temperature, protected from sunlight, in the Multidisciplinary Galenic Pharmacy Laboratory of the Faculty of Medicine and Biomedical Sciences of the University of Yaoundé I (LMDPG/FMSB). The plant raw material used in this study consists of the leaves ( Figure 1(b) ), stems ( Figure 1(c) ), and roots ( Figure 1(d) ) of Sida rhombifolia plant ( Figure 1(a) ).

In all our tests, the Wistar strain of Rattus norvegicus was exclusively used. For acute oral toxicity testing, we used randomly selected female rats with weights between 100 and 150 g; for the anti-inflammatory test, both male and female rats weighed between 180 and 210 g. These animals were raised in the animal facility of the Multidisciplinary Galenic Pharmacy Laboratory and the Faculty of Medicine and Biomedical Sciences (LMDPG/FMSB) of the University of Yaoundé I under natural lighting and temperature conditions. They had unlimited access to tap water and standard food. Animals were anesthetized with a mixture of ketamine, xylazine, and acepromazine at doses of 60, 7.5, and 2 mg/kg, respectively, intraperitoneally [26] . The animals were properly ventilated, and vital parameters were monitored throughout the experiment. At the end of the procedure, the animals were sacrificed by diethyl ether inhalation [27] . Albino Wistar rats were selected as the animal model for this study due to their many physiological and metabolic similarities to humans, making them preferred choice for pharmacological and toxicological studies. Additionally, these rats are commonly used in biomedical research due to their availability, ease of handling, and low genetic variability, ensuring better reproducibility of results.

Carrageenan was purchased from Sigma-Aldrich (Germany). The reference standard for anti-inflammatory activity, diclofenac (Voltaren®50), and physiological serum were obtained from a local pharmacy in Yaoundé. Dimethyl sulfoxide (DMSO) was obtained from LMDPG/FMSB (University of Yaoundé I), and distilled water was obtained from the Chemistry Laboratory of École Normale Supérieure de Yaoundé (University of Yaoundé I).

The extraction of plant material was performed by maceration, a method commonly used to preserve the integrity of bioactive compounds [12] [28] . After harvesting, the different parts of Sida rhombifolia (stem barks, leaves, and roots) were carefully washed with distilled water to remove impurities and then dried in the shade for two weeks at a controlled room temperature (27˚C - 35˚C) to avoid any thermal degradation of secondary metabolites. Once dried, the plant biomass was ground into fine particles using an electric grinder and then sieved to obtain a homogeneous powder. For extraction, 500 g of each powder were subjected to maceration in appropriate solvents (water and ethanol). After the solvent was evaporated using a rotary evaporator under reduced pressure, the dry extracts were stored in airtight glass containers, completely covered with aluminum foil to protect them from light and prevent any photochemical degradation. These measures ensure the stability and quality of the extracts before their use in biological analyses.

A total of 500 g of powder (roots, leaves, and stem barks) of Sida rhombifolia were individually macerated s in 5000 ml of distilled water for 48 hours, with stirring every 6 hours. After double filtration through Whatman no. 1 paper, the filtrates were evaporated in an oven at 50˚C until dry crude extracts were obtained. The extracts were coded as follows: EET (ethanolic extract of the stem barks), EAT (aqueous extract of the stem barks), EEF (ethanolic extract of the leaves), EAF (aqueous extract of the leaves), EER (ethanolic extract of the roots), and EAR (aqueous extract of the roots).

A total of 500 g of powdered roots, leaves, and stem barks of Sida rhombifolia were each macerated separately in 5000 ml of 96% absolute ethanol for 72 hours, with stirring every 6 hours. After double filtration through on Whatman no. 1 paper, the filtrates were concentrated under a Heidolph® Hei-VAP Core rotary evaporator under reduced pressure at 50˚C until the crude extracts were obtained.

To evaluate the yield of dry extracts obtained, the empty flask and tray were weighed before use and again after solvent evaporation. The difference between the two weights was then calculated [29] .

The phytochemical analysis was conducted following the standard protocols of references [10] [30] for qualitative studies. The targeted compound families included saponins, flavonoids, tannins, anthocyanins, alkaloids, phenols, polyphenols, triterpenes, steroids, and anthraquinones. The quantification of these secondary metabolites was carried out using the protocols described in the work of Odoh dan Okoro [31] for quantitative studies.

In this study, a limit test is carried out with a dose of 2000 and then 5000 mg/kg body weight of the extracts administered orally as a single dose to a group of six animals (three per stage) [32] . A control group of three rats received distilled water at the same time, also administered orally. Based on the chosen test dose, the corresponding weight concentration to be administered was calculated, and the test substance was administered orally to the animals. The animals were weighed individually one hour before extract administration and daily throughout the 14-day study period. From the first to the fourteenth day, weight variations (PV) were calculated using Equation (1), and the results were compared with previous measurements. For this study, 27 female rats were used. This choice is based on the recommendation of the OECD (Organization for Economic Co-Operation and Development), which advocates using females for acute toxicity tests because they are often more sensitive to the toxic effects of substances than males.

$VP=\frac{P_n-P\left(n-1\right)}{P\left(n-100\right)}\times 100$ (1)

where: VP = Percentage weight variation, P_n = nth measurement of body weight, P (n − 1) = (n − 1)th measurement of body weight, n = natural whole number.

On the 14^th day, the animals were fasted for 12 hours before being sacrificed, followed by necropsy. Organs such as the liver, spleen, lungs, heart, and kidneys were removed for relative weight calculations. Additionally, arteriovenous blood samples were collected in 5 ml dry tubes. Blood samples were centrifuged at 3000 rpm 15 minutes. The resulting serum was used for biochemical assessments, including transaminases, creatinine, and uric acid levels, measured using UV spectrophotometry. Histological analysis of the organs involved observation, fixation, trimming, dehydration, staining, mounting, embedding, and sectioning [33] .

The anti-inflammatory effect was evaluated in two stages: first, the in vivo evaluation of anti-inflammatory activity through the inhibition of edema of the right hind paw of rats induced by carrageenan [34] - [36] ; and second, the in vitro evaluation of anti-inflammatory activity using the method described by [37] . For the in vitro evaluation, a test solution (0.5 mL) was prepared, consisting of 0.45 ml of a 5% aqueous solution of bovine albumin serum (BSA) and 0.05 mL of aqueous extract at concentrations of 5; 6.25; 7.5; 8.75, and 10 μg/mL. A control test solution (0.5 ml) was also prepared, containing 0.45 ml of a 5% BSA aqueous solution and 0.05 ml of distilled water. Additionally, a blank control solution (0.5 mL) was prepared, containing 0.45 ml of distilled water and 0.05 ml of the aqueous extract at the same concentrations. The standard test solution (0.5 mL) contained 0.45 ml of the 5% BSA aqueous solution and 0.05 ml of an indomethacin standard solution at concentrations of 0.1 and 1 mg/mL. All solutions were adjusted to pH 6.3 using 1 N HCl solution. The samples were incubated at 37˚C for 20 minutes, after which the temperature was increased to 57˚C for 3 minutes. After cooling the tubes, 2.5 mL of the phosphate buffer saline solution (PBS) at pH = 6.3 was added to each solution. Absorbance was measured using a UV-visible spectrophotometer at 416 nm, and the percentage inhibition of protein denaturation was calculated using equation (2). In vivo anti-inflammatory activity was conducted on a total of 70 Witars rats, both males and females. Including both sexes allows for the assessment of potential differences in the inflammatory response and ensures that the observed effects are not biased by hormonal factors.

$\%\text{d}’\text{inhibition}=\frac{100-\left(\text{Optical density of test solution}-\text{Control optical density}\right)}{\text{Control optical density}}\times 100$ (2)

The control group was considered to have 100% denatured proteins, and the results were compared to those obtained with indomethacin (0.1 and 1 mg/mL). The in vivo evaluation of anti-inflammatory activity required 70 male and female rats, divided into 14 groups of 5 rats each. The rats were fasted for 24 hours before the experiment, then weighed and marked one hour before the start of the test [36] [38] [39] . The initial volume (V₀) of the right hind paw of each rat was measured using a plethysmometer before oral administration of the extract via orally a gastric tube. The extract doses administered were 200 and 400 mg/kg of body weight.

The rats were grouped as follows, receiving the respective solutions orally at a dose of 1 mL/100g of body weight:

One hour after the different treatments, 1% carrageenan was injected under the footpad at a dose of 0.05 mL.

The volume of paw edema was measured at 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, and 6 hours after carrageenan injection using a plethysmometer [39] . The average percentage increase in paw volume (PA) and edema inhibition percentages of edema (PI) were calculated using Equations (3) and (4).

$\%PA=\frac{V_t-V_0}{V_0}\times 100$ (3)

$\%\text{d}’\text{Inhibition}\left(\text{PI}\right)=\frac{\left(\text{PATe}-\text{PATr}\right)}{\text{PATe}}\times 100$ (4)

where: V₀ = Initial paw volume before edema induction; V_t = Paw volume after treatment administration and carrageenan injection; PATe: Percentage increase in paw volume of the control group; PATr: percentage increase in paw volume of the treated group.

Figure 2 below illustrates the steps involved in the anti-inflammatory test conducted on rats.

To evaluate the antioxidant potential of Sida rhombifolia, two activities were measured: antihemolytic activity and antilipoperoxidative activity.

1) Anti-hemolytic activity

This test is based on the reaction of red blood cells subjected to oxidative stress under strictly controlled and standardized conditions. Intra- and extracellular antioxidant defenses act to resist the oxidative attack, maintaining membrane integrity and cellular functions until the membrane is compromised, leading to the leakage of cellular contents. Hemoglobin released into the supernatant is measured by spectrophotometry at 532 nm.

Isolation and preservation of red blood cells

Blood was collected from a rat in a tube containing ethylenediaminetetraacetic (EDTA). The red blood cells were separated from the plasma and white blood cell layer by centrifugation at 1500 rpm for 15 minutes. The obtained red blood cells were washed three times with physiological saline (0.9% NaCl). The final red blood cell suspension (hematocrit at 33%) was gently agitated and stored at 4˚C. The reagents used were CuSO₄ (0.1 M) and NaCl (0.09%) are the reagents used.

Operating mode

A volume of 0.2 ml of extract (5, 6.25, 7.5, 8.75, and 10 µg/ml) prepared in water was mixed with 1 ml of NaCl (0.9%). Then, 0.1 ml of the red blood cell suspension was added. The mixture was incubated at room temperature for 30 minutes before adding 0.1 ml of CuSO₄ (0.1 M) to induce hemolysis. After 30 minutes incubation, absorbance was measured at 532 nm against a blank consisting of 1.3 ml of NaCl and 0.1 ml of the red blood cell suspension. Total hemolysis was achieved by adding 1.2 ml of NaCl (0.9%) and 0.1 ml of the suspension, incubating for 30 minutes, and then adding of 0.1 ml of CuSO₄ (0.1 M). The hemolysis rate was calculated using Equation (5).

$\text{Hemolysis rate}\left(\text{\%}\right)=\frac{\left(\text{OD total hemolysis}-\text{OD test}\right)}{\text{OD total hemolysis}}\times 100$ (5)

where: OD total hemolysis: Optical density of the total hemolysis (the positive control); OD test: Optical density of the test sample.

2) Anti-lipoperoxidative activity

Principle of the method

At physiological pH, FeSO₄ induces lipid peroxidation in polyunsaturated fatty acids, leading to the production of malondialdehydes (MDA), which absorbs at 532 nm. In the presence of antioxidants, FeSO₄’s effect on polyunsaturated fatty acids is inhibited, reducing MDA production.

Presentation of the preparation of liver homogenate:

The liver of a rat was dissected, washed in NaCl solution (0.9%), drained, weighed, and ground in a mortar. The ground material was homogenized in 10% (weight/volume) phosphate buffer (0.1 M, pH = 7.4) and centrifuged at 1500 rpm for 10 min. The supernatant (homogenate) was collected for testing. The reagents used included NaCl (0.9%), phosphate buffer (0.1 M, pH = 7.4), FeSO₄ (15 mM), TBA (1%) (Thiobarbiturique), HCl (10%), and ascorbic acid (6 mM).

Presentation of the operating mode

A volume of 300 μL of extract (5, 6.25, 7.5, 8.75 and 10 μg/mL) was added to 500 μL of liver homogenate. Lipid peroxidation was induced by adding 100 µl of iron sulfate. After incubation at 37˚C for 30 minutes, 1000 μL of TBA (Thiobarbituric) (1%) HCl (10%) were added, followed by 1000 μL of ascorbic acid. The mixture was heated to 80˚C for 20 minutes, cooled, and centrifuged at 1500 rpm for 10 min. The OD (Optical Density) was measured at 532 nm. The formula commonly used to calculate the percentage of lipoperoxidation inhibition is given by Equation (6):

$\text{\%Inhibition}=\frac{\left(\text{OD control}-\text{OD test}\right)}{\text{OD control}}\times 100$ (6)

where: OD control is the optical density of the control (without the tested extract or compound), OD test is the optical density in the presence of tested extract or compound.

In the present study, data were expressed as mean ± standard error of the mean (SEM). Statistical significance between groups was assessed using one-way analysis of variance (ANOVA), followed by Dunnett’s pos hoc test for multiple comparisons. Graph Pad Prism 8.0.1 software was used for statistical analysis, and differences were considered statistically significant when p values < 0.05.

The extraction yields of aqueous and ethanolic extracts from different parts of Sida rhombifolia are presented in Table 1 .

The results show a significant variation in extraction yields depending on the solvent used and the plant part extracted. The yields of aqueous extracts follow a decreasing order: 15.08% for leaves, 8.53% for stem barks, and 7.36% for roots. Regarding ethanolic extracts, the yields obtained are 10.73%, 6.62%, and 5.07% for leaves, stem barks, and roots, respectively. These findings indicate that aqueous extracts are obtained in greater quantities than ethanolic extracts, suggesting that water (H₂O), due to its polarity, enhances the solubilization of compounds present in the plant. These results are comparable to those reported in the literature. In India, Dhalwal, Deshpande, and Purohit [19] obtained yields of 4.28%, 5.15%, and 6.16% for ethanolic extracts of roots, stem barks, and leaves, respectively. Regarding leaves, Chaves et al. [13] in Brazil found a nearly similar yield (10.3613%) for the ethanolic extract. However, these values remain lower than those reported by Youssouf Loutfi, Vavitiana Dhalia Xavier, Rama Michela Stéphanie, and Razafindramanana Jeenath Francelline [40] , who recorded an extraction yield of 13% for a hydroalcoholic extract of Sida rhombifolia leaves. This difference could be attributed to the hydroalcoholic nature of the extract, as water contributes to more efficient extraction due to its higher polarity. The yields from leaves are consistently higher than those from stems and roots, regardless of the solvent used. This observation could be related to the presence of chlorophyll and other polar compounds in the leaves, which dissolve more easily in water. Ferro et al. [14] demonstrated that the extraction yield of secondary metabolites, particularly flavonoids and phenols, depends on the extraction method and the polarity of the solvents used. Thus, extraction efficiency is influenced not only by the chemical nature of the solvent but also by the biochemical composition of each plant part. Using water as an extraction solvent results in higher yields, particularly for leaves. This trend could be explained by the increased solubility of bioactive compounds in water compared to ethanol. However, ethanol, although less efficient in terms of overall yield, may facilitate the extraction of specific compounds that are less soluble in water, potentially influencing the composition and bioactivity of the extracts obtained.

The qualitative phytochemical Screening of aqueous and ethanolic extracts of Sida rhombifolia was carried out in triplicate. To assess the secondary metabolite content, the following codes were adopted: high content: (+++), medium content: (++), low content: (+), and absence of content: (−) [41] - [44] . The results are presented in Table 2 .

The analysis confirms the richness of aqueous and ethanolic extracts of Sida rhombifolia, harvested in the central region of Cameroon, in secondary metabolites. Identified compounds include phenolic compounds (phenols, polyphenols, tannins, flavonoids, and anthraquinones), terpenes (steroids, triterpenes, saponins), and nitrogen compounds (alkaloids). Qualitative phytochemical screening confirms the richness of Sida rhombifolia in secondary metabolites. This chemical diversity highlights the bioactive potential of the studied species. To assess the consistency of these results, a comparison with previous studies was conducted. Table 3 presents a summary of the main similarities and differences observed.

These findings align with previous studies on the chemical composition of Sida rhombifolia. According to [13] isolated steroids, alkaloids, and flavonoids from the aerial parts of the plant, confirming the presence of these metabolite classes. Similarly, [22] identified tannins, flavonoids, saponins, alkaloids, and steroids in Sida rhombifolia leaves. However, anthraquinones were absent in their study, whereas in the present research, this metabolite was detected only in ethanolic extracts. Furthermore, the work of [40] reported the presence of tannins, flavonoids, alkaloids, and anthocyanins in Sida rhombifolia leaves. However, in this study, anthocyanins were absent. This difference may be due to environmental variations influencing secondary metabolite biosynthesis. Factors such as climate, soil composition, altitude, and growth conditions can significantly impact the chemical composition of plants [14] . The observed variations in the presence or absence of certain secondary metabolites suggest an influence of environmental conditions on the biosynthesis of bioactive compounds. The detection of anthraquinones exclusively in ethanolic extracts in this study, contrary to some previous reports, could be attributed to differences in abiotic and biotic factors. Similarly, the absence of anthocyanins, which were identified in other studies, highlights the possible impact of local environmental conditions on Sida rhombifolia’s chemical profile.

The test was carried out in triplicate, and the values were expressed as mean ± standard deviation. The results of the quantitative phytochemical screening of the aqueous and ethanolic extracts of Sida rhombifolia are presented in Table 4 .

These findings align with previous studies, such as those by Youssouf Loutfi, Vavitiana Dhalia Xavier, Rama Michela Stéphanie, and Razafindramanana Jeenath Francelline [40] , who reported that the aerial parts of Sida rhombifolia contained significant levels of tannins, with average amounts of alkaloids and leucoanthocyanins, but lower levels of flavonoids, anthocyanins, and polysaccharides. Although most studies in the literature do not specify the exact quantities of each secondary metabolite family, they generally confirm that the composition of Sida rhombifolia extracts follows similar trends. Furthermore, a study by Assam et al. [22] found that the leaves of Sida rhombifolia contained significant amounts of tannins, flavonoids, saponins, alkaloids, and steroids. However, they did not report the presence of saponins in the roots, which contrasts with our results where saponins were found in the ethanolic extracts of the roots. This discrepancy could be due to differences in extraction techniques, solvent types, or environmental conditions. The extraction of secondary metabolites is highly influenced by the solvent used and the part of the plant analyzed. In the present study, alkaloids, tannins, saponins, and flavonoids were found in higher quantities compared to other metabolites. Specifically, the ethanolic extract of the roots exhibited the highest levels, with 161.42 μg/mg of alkaloids, 157.06 μg/mg of tannins, 161.63 μg/mg of saponins, and 80.26 μg/mg of flavonoids. These results suggest that ethanol is a more efficient solvent for extracting these metabolites from the roots, likely due to its ability to dissolve a wide range of bioactive compounds. This is consistent with findings from several studies. For instance, Chaves et al. [13] found that ethanol extracts of Sida rhombifolia also yielded higher concentrations of tannins and alkaloids compared to aqueous extracts. The decreasing order of secondary metabolite concentrations in this study was as follows: roots > stem barks > leaves. This distribution could be linked to the physiological role of these compounds in the plant. The roots, in direct contact with the soil and exposed to various microbial and environmental stresses, may produce and accumulate higher amounts of secondary metabolites for protective purposes. Similarly, studies reported that roots tend to accumulate more bioactive compounds compared to stems and leaves. The stems, which play an intermediary role in transporting nutrients and water, have moderate levels of secondary metabolites, while the leaves, primarily responsible for photosynthesis, generally accumulate fewer defense-related compounds. The differences observed in the content of secondary metabolites between studies could be attributed to several factors, including environmental conditions, harvest season, and the extraction methods used. The chemical composition of medicinal plants is strongly influenced by abiotic factors such as soil composition, climate, and altitude, as well as biotic factors such as stress from pests or disease and interactions with microorganisms. For instance, Dhalwal et al. [19] found that ethanol generally yields higher amounts of secondary metabolites, especially alkaloids and flavonoids, compared to aqueous extracts. The effectiveness of ethanol as an extraction solvent, due to its ability to extract both polar and non-polar compounds, may explain the higher yields found in this study compared to aqueous extraction methods. However, the specific differences in the results of various studies may be due to variations in plant material, including the geographic origin, harvest time, and plant age, all of which can significantly impact the concentration of secondary metabolites.

During this test, no deaths were recorded among the animals throughout the duration of the study. Additionally, the behavior of the animals remained unchanged. The results of the acute oral toxicity study of the aqueous and ethanolic extracts of Sida rhombifolia are presented through the kinetics of the weight evolution of the treated rats, as well as the analysis of their liver parameters.

It was observed that the extracts of Sida rhombifolia, at doses of 2000 and 5000 mg/kg body weight, had no significant effect on the weight of the animals. Table 5 presents the kinetics of the weight changes in the rats during the test.

Statistical analysis showed no significant difference in the weight change between the treated rats and the control group. Figure 3 illustrates the kinetic curves of the weight changes in the rats throughout the 14-day duration of the test.

The administration of the different Sida rhombifolia extracts at the maximum doses of 2000 mg/kg and 5000 mg/kg of body weight showed no visible sign of toxicity in the treated animals during the 14 days of observation and monitoring following treatment. Additionally, no animal deaths were recorded. It appears from this study that the animals continued their normal growth after the administration of Sida rhombifolia extracts. For acute oral toxicity, the administration of the different extracts at the maximum doses of 2000 mg/kg and 5000 mg/kg body weight showed no visible signs of toxicity in the treated animals during the 14 days of observation and monitoring after treatment. No deaths were recorded in any of the animals. These results are consistent with those in the literature, which suggest that Sida rhombifolia extracts are not toxic to the body [22] [45] . In this study, the histological sections of the animals’ organs were normal, with no signs of organ toxicity observed in the treated rats. An increase in the weight of the rats and their organs was observed in all groups, with no significant difference. In terms of blood parameters, no significant differences were recorded between the data from the control groups and those from the treated animals. Tumanggor, Bintang, and Priosoeryanto [45] demonstrated in their studies that Sida rhombifolia leaf extracts are safe for normal cells in the body.

Table 6 presents the relative mass of the organs.

** = significant difference at p < 0.01; *** = very significant difference at p < 0.001.

The results of analysis of liver parameters for the aqueous and ethanolic extracts of Sida rhombifolia are presented in Table 7 .

[22] reported that the blood and liver parameters of rats that received extracts at a dose of 8000 mg/kg showed a significant increase (p < 0.05) compared to the control group. In the present study, no significant differences were observed between the control group and the treated animals. This discrepancy may be attributed to the higher dose of 8000 mg/kg used in the study by [22] studies. Therefore, based on the present study, the LD₅₀ (lethal dose) of Sida rhombifolia extracts is greater than 5000 mg/kg of body weight. The analysis of the relative organ mass ( Table 6 ) reveals that most organs do not show significant variations compared to the control group, except for the liver. A significant increase in relative liver mass is observed with 5000 mg/kg of EER (4.069 ± 0.820; p < 0.01) and 5000 mg/kg of EET (4.174 ± 1.094; p < 0.001), suggesting hepatic hypertrophy. This increase could be related to the accumulation of metabolites or an inflammatory reaction induced by the extracts. In contrast, the relative masses of the kidneys, heart, and lungs do not show significant variations, indicating that these organs do not undergo notable effects from the extracts, even at high doses. The biochemical parameters ( Table 7 ) reveal an increase in hepatic transaminases (ASAT and ALAT) for certain extract doses. The highest values are recorded for EER at 5000 mg/kg (ASAT: 109.81 U/L; ALAT: 58.52 U/L) and EET at 5000 mg/kg (ASAT: 91.87 U/L; ALAT: 39.43 U/L). This elevation in transaminases indicates hepatic cytolysis, confirming the signs of liver damage already observed in the increased relative liver mass. Regarding renal parameters, creatinine and uric acid levels do not show significant variations between treated groups and the control group, suggesting the absence of notable renal impairment. The results suggest that the tested extracts have a dose-dependent effect on liver function, with signs of toxicity appearing mainly at high doses (5000 mg/kg). The increase in relative liver mass, accompanied by elevated transaminases, indicates hepatic distress, which may be attributed to an increased metabolic load or cellular damage. In contrast, the kidneys and other organs appear less affected by the extracts, even at high doses, indicating a selective toxic effect on the live.

The results of the in vitro and in vivo evaluation of the anti-inflammatory potential of Sida rhombifolia extracts are presented in Table 8 and Table 9 .

The protein denaturation test (BSA) results of Sida rhombifolia leaf, root and stem extracts are shown in Table 8 .

Protein denaturation plays a key role in inflammation, as denatured proteins can trigger immune responses and excessive production of inflammatory mediators. The results suggest that ethanolic extracts exhibit greater activity than aqueous extracts, indicating that bioactive anti-inflammatory compounds are more effectively extracted in ethanol than in water. The high inhibition rate observed in EET suggests that the stem bark of Sida rhombifolia is particularly rich in bioactive compounds such as flavonoids and polyphenols, which are known for their anti-inflammatory properties. These findings are consistent with previous studies showing that ethanolic extracts of medicinal plants often have superior anti-inflammatory activity due to their ability to extract non-polar bioactive compounds. The dose-dependent effect observed in this study suggests that increasing extract concentrations enhances anti-inflammatory potential, which could be relevant for the development of natural anti-inflammatory treatments. However, while these in vitro results are promising, further in vivo studies and clinical trials are needed to confirm the efficacy and safety of Sida rhombifolia extracts for therapeutic applications. The strong inhibitory activity observed, particularly in EET, highlights the potential of this plant as a valuable source of natural anti-inflammatory agents.

The in vivo test conducted on a sample of 70 rats showed almost similar significant activity for all extracts. The results of the test on the evolution of edema in the right paw of the rats that received different treatments are presented in Table 9 .

* = difference not very significant at p < 0.05; ** = significant difference at p < 0.01; *** = very significant difference at p < 0.001; **** = extremely significant difference at p < 0.0001, compared to the negative control.

The anti-inflammatory potential was evaluated by calculating the percentage reduction in paw edema of the treated rats compared to the negative control group. Figure 4 presents the curves depicting the kinetics of rat paw edema evolution over time.

The presence of statistical annotations (p < 0.05 to p < 0.0001) reinforces the reliability of the results. The very significant (*) and extremely significant (**) differences between treated groups and the negative control confirm the effectiveness of the tested extracts. This eliminates the possibility of a placebo effect and validates the hypothesis that Sida rhombifolia possesses genuine anti-inflammatory properties. The in vivo anti-inflammatory test demonstrates that the aqueous and ethanolic extracts of Sida rhombifolia significantly reduced rat paw edema. By the 3^rd hour, inhibition was already very significant at the dose of 200 mg/kg, with 87.56% for the ethanolic extract of the roots (EER) and 83.67% for the aqueous extract of the roots (EAR). The highest inhibition percentage reached 95.56% for the ethanolic extract of the twigs (EET) at 400 mg/kg in the 6th hour. At this same time point, EER at 200 mg/kg exhibited an inhibition rate of 95.31%, while diclofenac, a standard anti-inflammatory drug, showed 94.51%. Notably, from the 3rd hour onward, inhibition results exceeded 70%, indicating strong anti-inflammatory activity. In vitro anti-inflammatory activity, assessed through the protein denaturation test (BSA), revealed that the ethanolic extract of the stem exhibited the highest inhibition percentage, reaching 84.18% at a concentration of 10 mg/mL. These results align with findings from Mah, Teh, and Ee [9] , which highlighted the anti-inflammatory potential of this plant. Their study showed that the hexane extract exhibited nitric oxide (NO) inhibition with respective IC50 values of 52.16 and 58.57 µg/mL, as well as inhibition of protein denaturation at 146.03 µg/mL [9] [12] [40] . These findings collectively support the strong anti-inflammatory potential of Sida rhombifolia, reinforcing its effectiveness as a natural anti-inflammatory agent.

The results of the anti-hemolytic activity of the Sida rhombifolia extracts are presented in Table 10 .

Table 10 presents the effect of Sida rhombifolia extracts on anti-hemolytic activity at different concentrations (5 to 10 mg/mL). The results show that hemolysis inhibition increases with extract concentration, indicating a dose-dependent effect. Ethanolic extracts (EAT, EER, EEF) are generally more effective than aqueous extracts (EAF, EAR), with maximum inhibition reaching 63.08% ± 0.69% at 10 mg/mL for the ethanolic extract of aerial parts (EAT). The ethanolic extract of twigs (EET) shows relatively low inhibition at lower concentrations (6.37% ± 3.87% at 5 mg/mL) but significantly increases at 10 mg/mL (37.07% ± 2.89%). These findings suggest that Sida rhombifolia possesses anti-hemolytic properties, likely due to the presence of bioactive compounds such as flavonoids and polyphenols, known for their ability to stabilize cell membranes and reduce oxidative stress. These observations align with literature findings, particularly those of Mah, Teh, and Ee [9] , who demonstrated that the hexane extract of Sida rhombifolia inhibits NO production with IC50 values of 52.16 and 58.57 µg/mL, confirming an anti-inflammatory potential linked to membrane stabilization. Similarly, Tanumihardja et al. [12] reported protein denaturation inhibition at 146.03 µg/mL, supporting the hypothesis that this plant’s extracts protect cell membranes from oxidative damage. Compared to other medicinal plants such as Curcuma longa and Moringa oleifera, whose hemolysis inhibition often exceeds 60% at 10 mg/mL, Sida rhombifolia ranks among the plants with strong anti-hemolytic activity. Thus, these results reinforce the idea that Sida rhombifolia has genuine therapeutic potential as an anti-inflammatory and membrane-protective agent. Identifying the specific active compounds responsible for this activity and comparing these extracts to reference anti-inflammatory drugs such as diclofenac would be valuable. Furthermore, validation using in vivo oxidative stress models would provide a deeper understanding of the mechanism of action of these extracts.

The results of the anti-lipoperoxidative activity of extracts from the roots and stem of Sida rhombifolia are presented in Table 11 .

At a concentration of 10 mg/mL, the anti-lipoperoxidative test showed an inhibition of 63.41% for EAF and 51.54% for EEF, compared to 47.97% ± 1.98% for EAR and 41.58% ± 4.58% for EER. These results confirm the antioxidant potential of Sida rhombifolia. In the anti-hemolytic test, the ethanolic extract of aerial parts (EAT) at the same concentration exhibited an inhibition of 63.08%, compared to 59.26% for EEF and 58.67% for EER. Furthermore, at this concentration, the anti-lipoperoxidative test showed an inhibition of 61.41% for EAF, 47.97% for EAR, and 35.70% for EET. These findings further support the antioxidant potential of Sida rhombifolia, as demonstrated in previous studies. Mah, Teh, and Ee [9] demonstrated in their studies that the ethyl acetate extract of Sida rhombifolia exhibited antioxidant activity by scavenging DPPH (2,2-Diphenyl-1-picrylhydrazyl) radicals and ferrous ions, with EC50 (effective concentration) values of 380.5 µg/mL and 263.4 µg/mL, respectively. Similarly, research conducted in India determined that ethanolic extracts from the roots of Sida rhombifolia exhibit significantly higher antioxidant activity compared to extracts from stems or leaves [19] .