Potential Surviving Effect of Cleome droserifolia Extract against Systemic Staphylococcus aureus Infection: Investigation of the Chemical Content of the Plant

The increasing rates of morbidity and mortality owing to bacterial infections, particularly Staphylococcus aureus have necessitated finding solutions to face this issue. Thus, we elucidated the phytochemical constituents and antibacterial potential of Cleome droserifolia extract (CDE). Using LC-ESI-MS/MS, the main phytoconstituents of CDE were explored, which were kaempferol-3,7-O-bis-alpha-L-rhamnoside, isorhamnetin, cyanidin-3-glucoside, kaempferide, kaempferol-3-O-alpha-L-rhamnoside, caffeic acid, isoquercitrin, quinic acid, isocitrate, mannitol, apigenin, acacetin, and naringenin. The CDE exerted an antibacterial action on S. aureus isolates with minimum inhibitory concentrations ranging from 128 to 512 µg/mL. Also, CDE exhibited antibiofilm action using a crystal violet assay. A scanning electron microscope was employed to illuminate the effect of CDE on biofilm formation, and it considerably diminished S. aureus cell number in the biofilm. Moreover, qRT-PCR was performed to study the effect of CDE on biofilm gene expression (cna, fnbA, and icaA). The CDE revealed a downregulating effect on the studied biofilm genes in 43.48% of S. aureus isolates. Regarding the in vivo model, CDE significantly decreased the S. aureus burden in the liver and spleen of CDE-treated mice. Also, it significantly improved the mice’s survival and substantially decreased the inflammatory markers (interleukin one beta and interleukin six) in the studied tissues. Furthermore, CDE has improved the histology and tumor necrosis factor alpha immunohistochemistry in the liver and spleen of the CDE-treated group. Thus, CDE could be considered a promising candidate for future antimicrobial drug discovery studies.

Staphylococcus aureus is a common species of Staphylococci highly associated with multidrug resistance [15].Such bacterial species can adapt to various environments and possess frequent virulence factors [16].In addition, it is a common nosocomial pathogen that can trigger various diseases that range from mild severity to life-threatening ailments.The infections triggered by S. aureus are mild skin and soft tissue infections, bacteremia, osteomyelitis, endocarditis, and pneumonia [17].
S. aureus can exhibit resistance to antibiotics by various mechanisms, including decreasing the bacterial membrane permeability to the antibiotics, efflux, and excessive production of resistance enzymes like β-lactamases [18].Multi-drug resistance (MDR) is a worldwide issue that has a deleterious effect on health care.S. aureus acquires resistance to antibiotics owing to persistent exposure to various antimicrobials.MDR S. aureus is resistant to multiple chemotherapeutic agents.Such MDR isolates have led to increasing global rates of mortality as well as morbidity in S. aureus-infected patients [19].
Biofilm is an important virulence factor of S. aureus, and it is defined as an extracellular complex structure that compromises a population of bacterial cells anchored to living or nonliving surfaces [20].The cells are surrounded by an extracellular polymer matrix formed by themselves as a protective tactic for bacterial survival to adapt to their surrounding environments [21].
Traditional antibiotics are excessively losing their potential to combat bacterial infections, particularly S. aureus [22].Thus, novel treatment approaches should be elucidated to face such global concerns.Natural sources, like plants, are rich in many bioactive phytochemicals with various therapeutic activities [23].Many studies have elucidated the potential antimicrobial action of plants and their bioactive constituents against viruses, fungi, and bacteria [24][25][26][27][28].
Here, we aimed to explore CDE's potential antibacterial action in vitro and in vivo.Also, the phytoconstituents of this plant will be elucidated using liquid chromatographyelectrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) to explore the active principles with different chemical entities which may have a role in developing new antibacterial agents against the MDR S. aureus isolates.

Bacterial Isolates and Antibiotic Resistance
The clinical specimens from which the bacterial isolates were recovered include blood, wounds, sputum, and urine (Figure 1).The antibiotic susceptibility of the tested isolates is revealed in Figure 2.

Susceptibility of S. aureus to CDE
The susceptibility of S. aureus to CDE was elucidated using the agar well diffusion method as a preliminary method to reveal whether CDE possesses antibacterial action (Figure 3 and Table S2).Then, the MICs were determined, as shown in Table S3.

Susceptibility of S. aureus to CDE
The susceptibility of S. aureus to CDE was elucidated using the agar well diffusion method as a preliminary method to reveal whether CDE possesses antibacterial action (Figure 3 and Table S2).Then, the MICs were determined, as shown in Table S3.

Susceptibility of S. aureus to CDE
The susceptibility of S. aureus to CDE was elucidated using the agar well diffusion method as a preliminary method to reveal whether CDE possesses antibacterial action (Figure 3 and Table S2).Then, the MICs were determined, as shown in Table S3.

Determination of Antibiofilm Potential by Crystal Violet Assay, SEM, and qRT-PCR
CDE revealed antibiofilm potential through the semiquantitative method, crystal violet (Table 2), as it decreased the percentage of strong and moderate biofilm-forming isolates from 73.91% to 30.43%.Then, SEM was employed to elucidate the effect of CDE on the morphology of the biofilm (Figure 4).CDE has significantly decreased the number of cells in the formed biofilm.

Determination of Antibiofilm Potential by Crystal Violet Assay, SEM, and qRT-PCR
CDE revealed antibiofilm potential through the semiquantitative method, crystal violet (Table 2), as it decreased the percentage of strong and moderate biofilm-forming isolates from 73.91% to 30.43%.Then, SEM was employed to elucidate the effect of CDE on the morphology of the biofilm (Figure 4).CDE has significantly decreased the number of cells in the formed biofilm.qRT-PCR was utilized to reveal the potential of CDE on the expression level of biofilm genes.CDE was found to downregulate the biofilm-encoding genes in 43.48% of the isolates, as revealed in  qRT-PCR was utilized to reveal the potential of CDE on the expression level of biofilm genes.CDE was found to downregulate the biofilm-encoding genes in 43.48% of the isolates, as revealed in Figure 5.  qRT-PCR was utilized to reveal the potential of CDE on the expression level of biofilm genes.CDE was found to downregulate the biofilm-encoding genes in 43.48% of the isolates, as revealed in Figure 5.

In Vivo Infection Model in Mice
A systemic infection was induced in mice, and the bacterial burden was detected in the liver and spleen in the experimental groups (Figure 6).

In Vivo Infection Model in Mice
A systemic infection was induced in mice, and the bacterial burden was detected in the liver and spleen in the experimental groups (Figure 6).The survival curve was constructed as shown in Figure 7.No mice died in group in group II, two died after three days, one after five days, and another after one we Also, two mice died after nine days.Regarding groups III and IV, one mouse died on eighth and sixth days.The survival curve was constructed as shown in Figure 7.No mice died in group I; in group II, two died after three days, one after five days, and another after one week.Also, two mice died after nine days.Regarding groups III and IV, one mouse died on the eighth and sixth days.The survival curve was constructed as shown in Figure 7.No mice died in group I; in group II, two died after three days, one after five days, and another after one week.Also, two mice died after nine days.Regarding groups III and IV, one mouse died on the eighth and sixth days.

Histopathological and Immunohistochemical Investigations
The effect of CDE on the histological features of the liver and spleen is shown in Figures 8 and 9. Also, the TNF-α immunohistochemical staining of the liver and spleen of the different experimental groups is shown in Figures 10 and 11

Histopathological and Immunohistochemical Investigations
The effect of CDE on the histological features of the liver and spleen is shown in Figures 8 and 9. Also, the TNF-α immunohistochemical staining of the liver and spleen of the different experimental groups is shown in Figures 10 and 11

ELISA
Levels of IL-1β and IL-6 were detected in the liver and spleen tissues of the different groups (Table 3).

ELISA
Levels of IL-1β and IL-6 were detected in the liver and spleen tissues of the different groups (Table 3).

Collection, Drying, and Extraction of the Plant Material
The stems, flowers, and leaves of C. droserifolia shrubs were collected in March 2022 from the wadis around Sharm El-Sheikh and Dahab in South Sinai Governorate.The plant was dried and powdered to obtain 1.2 kg of dry weight.It was recognized by Prof. Dr. Hanafey Farouk Maswada, Professor of Plant Physiology, Agricultural Botany Department, Faculty of Agriculture, Tanta University, and depositing a voucher specimen (PG-A-00124) in the herbarium of the Department of Pharmacognosy, Faculty of Pharmacy, Tanta University.Methanol (Sigma Chemical Co., St. Louis, MO, USA) was used as an extracting solvent and was mixed with the powder.The extraction was performed thrice (4 L × 3 times), and then the concentration of the solvent was performed by a rotary evaporator under vacuum to obtain 45.3 g of dry residue from the plant's extract.

Exploration of the Plant's Phytoconstituents by LC-ESI-MS/MS
Different compounds in the extract were identified by the Proteomics and Metabolomics Unit, Children's Cancer Hospital (57357), Basic Research Department, Cairo, Egypt.The crude extract was reconstituted in DI-Water:Methanol:Acetonitrile-50:25:25, and HPLC separation was accomplished by using in-line filter disks (0.5 µm × 3.0 mm, Phenomenex ® , Torrance, CA, USA) and X select HSS T3 (2.5 µm, 2.1 × 150 mm, Waters ® , Milford, MA, USA, 40 • C).The first is a pre-column, and the second is an analytical column.The mobile phases were composed of mobile phase A, which is composed of 5 mM ammonium formate buffer pH 8 with 1% methanol.Mobile phase B is 100% acetonitrile.Isocratic elution was done using 90% of solvent A and 10% of solvent B for one minute.After that a gradient elution from 90 to 10% solvent A and 10 to 90% of solvent B in twenty minutes was done, then elution with 90% of solvent B for four minutes was done then return to the initial condition (10% of acetonitrile) for three minutes.The flow rate was 0.3 mL/min.The instrument was coupled with Triple TOF 5600+ (Sciex ® , Framingham, MA, USA) for IDA acquisition and Analyst TF 1.7.1 (Sciex ® ) for LC-Triple TOF control.Raw data files were loaded into MS-DIAL 3.52 for data-independent MS/MS deconvolution [69].Compounds were recognized with >70% probability using an MS1 and MS2 tolerance of 0.2 mass units to be accepted as positive identifications.The ReSpect negative (1573 records) database was used as a reference database.PeakView 2.2 with the MasterView 1.1 package (AB SCIEX, Framingham, MA, USA) were used for feature or peaks extraction from the total ion chromatogram (TIC) based on that the signal-to-noise of features is more than ten, as well as their intensities of the sample-to blank should be more than three [37].

Bacteria
Twenty-three S. aureus isolates were from clinical specimens, including blood, wounds, sputum, and urine.They were identified by standard biochemical tests.

Antibacterial Action of C. droserifolia Methanol Extract
Antibacterial action was revealed by agar well diffusion in MHA plates [71].The bacterial suspension (0.5 McFarland) was dispersed on the surface of the MHA plates.Three wells were performed.The first well-received CDE (2 mg/mL), the second received linezolid (positive control), and the third received dimethyl sulfoxide (DMSO, negative control).The appearance of inhibition zones revealed CDE's antibacterial activity after incubating the plates at 37 • C for 24 h [72].

Determination of the Minimum Inhibitory Concentration (MIC) of CDE
The broth microdilution assay in MH broth was employed to estimate the MIC values of CDE against S. aureus isolates, as previously reported [72].The MIC had the lowest concentration of CDE, and no growth was detected visually after overnight incubation at 37 • C [73].

Biofilm Inhibition
The effect of CDE on biofilm formation was investigated at 0.5 MIC values [74].A tryptone soy broth (TSB) suspension was prepared from an over-night bacterial culture, and was adjusted to 10 6 CFU/mL in freshly prepared TSB.Then, 200 µL of the bacterial suspension was added to the microtitration plates and wells in the presence and absence of sub-MIC (0.5 MIC) of SAM and incubated at 37 • C for 48 h.The TSB was gently removed, and the wells were washed to remove any planktonic cells and subsequently left for air drying.Add 200 µL of 99% methanol for 20 min, and then the formed biofilm was stained with 200 µL of 1% crystal violet (CV) solution for 15 min.After washing the plate, 33% glacial acetic acid was utilized as a solvent for CV.Using a microtitration plate reader (Sunrise, Männedorf, Switzerland), the absorbance of the solubilized dye was measured at 570 nm [72].

Scanning Electron Microscope (SEM)
The antibiofilm action of CDE on S. aureus bacteria was visualized under SEM, as previously explained (JEOL, Tokyo, Japan) [75].

Gene Expression Measurement Using qRT-PCR
The influence of CDE on the expression levels of the biofilm genes (cna, fnbA, and icaA) was elucidated using qRT-PCR.After growing the isolates in TSB in the presence and absence of sub-MICs of SAM, they were incubated overnight at 37 • C.After the incubation period, cells were harvested by centrifugation and immediately stored at −80 • C. The total RNA from S. aureus isolates was extracted and purified using TRIzol ® reagent (Life Technologies, Carlsbad, CA, USA) following the manufacturer protocol.Reverse transcription was employed using the QuantiTect Reverse Transcription kit (Qiagen, Hilden, Germany).Then, the formed cDNA was amplified using Maximas SYBR Green/Fluorescein qPCR master mix (Thermo Fisher Scientific, Waltham, MA, USA).The average threshold cycle (CT) values were normalized to the housekeeping gene (16s rRNA).The relative gene expression of the treated isolates was compared to that of the untreated ones according to the 2 −∆∆Ct method [76].Primers are exposed in Table S1 [77,78].

In Vivo Assay
Forty male mice weighing 25-30 g and aged 6-8 weeks were obtained from the faculty of pharmacy at Tanta University, Egypt.They were grouped into four groups, each with ten mice.The first group was a normal control.The residual three groups were infected with 0.1 mL via intravenous injection of 1.5 × 10 7 colony-forming units (CFUs) of S. aureus [79].The second group served as a positive control group (placebo), and the third group administered linezolid (160 mg/kg/24 h) orally as a standard drug.The fourth group administered CDE orally (200 mg/kg/24 h) [12].The experimental procedures were approved by the ethics committee at the faculty of pharmacy, Tanta University (TP/RE/3/24 p-03).
After two weeks, mice from the diverse groups were euthanized.Liver and spleen samples were obtained from each group.The bacterial burden was determined in the liver and spleen after homogenization.A 1:10 serial dilution of the tissue homogenates in phosphate buffered saline was performed.Then, 100 µL of undiluted and each subsequent dilution were spread onto tryptic soya agar plates in duplicate using a glass spreader.The plates were then incubated at 37 • C overnight.
The number of colonies was counted on each plate, and the count of CFU/mL was determined as follows: CFU/mL = number of colonies × dilution factor/0.1 mL.CFU/g = CFU/mL × number of mL/g [80].
On the other hand, tissue samples (2 × 3 mm) were excised, fixed in buffered formalin (10%), treated as previously described [81], and finally stained with hematoxylin and eosin (H&E) and photographed using a light microscope.Also, tumor necrosis factor-alpha (TNF-α) monoclonal antibodies were utilized for staining the tissues.The immunostained tissues were then checked by a light microscope.

ELISA
The anti-inflammatory potential of CDE was illuminated by determining the levels of the inflammatory mediators, interleukin IL-1β and IL-6 in pg/mg protein in the liver and spleen tissues by an ELISA kit from Abcam Co., Waltham, MA, USA, following the manufacturer's instructions.

Histopathological Examination
Formalin-fixed hepatic and splenic tissues were processed, and 5-µm-thick paraffin sections were stained with hematoxylin and eosin (H&E).Photomicrographs were taken at different magnification powers using a light microscope (Olympus, Tokyo, Japan) to assess the morphological changes [82].Histopathological evaluation of the hepatic and splenic tissue damage was performed.A score of zero indicates the absence of tissue necrosis; a score of one indicates mild damage, 10-20% liver cell degeneration, necrosis, and 10-20% red blood cell depletion.A score of two indicates moderate degeneration in the form of 20-40% liver cell degeneration, necrosis, and 20-40% red blood cell depletion in the spleen.A score of three indicates severe degeneration in the form of >40% liver cell degeneration and necrosis and >40% red blood cell depletion in the spleen.

Immunohistochemistry
Six-micrometer tissue sections from the liver and spleen were subjected to immunohistochemical staining; they were first dewaxed, rehydrated by a diminishing alcohol series, and treated with 10% hydrogen peroxide in methanol for ten minutes.Following this, the sections were microwaved for ten minutes in 0.01 M sodium citrate buffer (pH 6.0), allowed to cool at room temperature, and then repeatedly washed with PBS for five minutes.After washing, antigens were recovered by autoclaving in citrate buffer for 11 min.Next, slices were incubated with primary antibodies for a whole night at 4 • C.Then, the tissues were treated for 30 min at room temperature with 3, 3-diaminobenzidine and a rabbit polyclonal TNF-α antibody.The tissue sections were mounted for visibility, washed in xylene, and subtly counterstained with hematoxylin.Slides were examined under a light microscope at a magnification of ×400 [83].Using image analysis tools (Image J, 1.46a, NIH, Bethesda, MD, USA), morphometric analysis was carried out.At ×400 magnification, the mean area percentage of TNF-α protein expression for each of the experimental groups was evaluated in ten non-overlapping fields within each region.

Statistics
The assays were carried out three times and exposed as mean ± standard deviation (SD).ANOVA was utilized to reveal the significance of differences among the experimental groups by GraphPad software version 8.0 (GraphPad Software, LLC, Boston, MA, USA).Results of the histopathological scores were analyzed using Kruskal-Wallis test followed by Dunn's multiple comparison test.

Discussion
Medicinal plants can act as a natural source of numerous therapeutic compounds that can be utilized safely to treat various diseases in humans and animals [84].In recent decades, increasing attention has been paid to elucidating plants as an important source for numerous drugs, principally antimicrobials, to combat multidrug-resistant bacteria [85].
CDE revealed antibacterial action on S. aureus isolates with MIC values of 128-512 µg/mL.An earlier study described the antibacterial action of CDE on S. aureus NCTC 10788, Salmonella senftenberg ATCC 8400, Escherichia coli BA 12296, and Candida albicans ATCC MAY-2876 [12].In addition, the antibacterial action of the essential oil obtained from Cleome species was previously reported on Gram-positive and Gram-negative bacterial species [92].
As biofilm is an important virulence factor for S. aureus, it enables it to resist multiple antibiotics by transferring the genes of resistance among the bacterial cells embedded in the biofilms and by hindering the penetration of the antibiotics across the biofilm [99,100].Thus, antibiofilm agents are beneficial for managing MDR S. aureus infections [101].Herein, CDE revealed antibiofilm action by crystal violet and SEM.Also, it revealed a downregulating effect on the biofilm-encoding genes (cna, fnbA, and icaA) using qRT-PCR in 43.48% of the isolates.Such genes encode intracellular adhesion molecules (icaA) and microbial surface components recognizing adhesive matrix molecules (fnbA and cna), which have a great role in biofilm formation [102].
Regarding the in vivo model, which was employed to simulate the human body [103], CDE revealed a promising effect in the studied infection model as it significantly decreased the bacterial count in the liver and spleen, indicating its antibacterial action in vivo.Also, it improved the histological features of the liver and spleen, manifested by regaining the normal hepatic structure and the splenic architecture.
As bacterial infections are among the causes that often trigger inflammation as a body response [104], we elucidated the consequence of CDE on the inflammatory markers in the liver and spleen using immunohistochemistry and ELISA.TNF-α is an inflammatory cytokine produced by macrophages as a response to acute inflammation [105,106].Also, IL-1β is a proinflammatory cytokine that mediates numerous physiological responses, such as fever and lymphocyte activation [107,108].IL-6 is an important pleiotropic cytokine in the inflammatory response [109][110][111].Remarkably, CDE has significantly diminished these inflammatory mediators, which could have a role in its antibacterial potential.The experiment demonstrated a notable enhancement in the survival rate of rats infected with S. aureus when treated with CDE compared to the positive control group.CDE efficacy was comparable to that of the standard drug, linezolid.These results highlight the potential therapeutic value of CDE as an alternative treatment for S. aureus infections, warranting further investigation into the clinical relevance of these findings.

Conclusions
LC-ESI-MS/MS revealed that C. droserifolia methanol extract had variable phytochemicals with valuable biological activities.Flavonoid glycosides, anthocyanins, and other phenolic compounds were believed to cause multiple effects, such as antimicrobial, antibiofilm, and anti-inflammatory properties.The predominant compounds in CDE were kaempferol-3,7-O-bis-alpha-L-rhamnoside, isorhamnetin, cyanidin-3-glucoside, 3, 5, 7trihydroxy-4 ′ -methoxyflavone (kaempferide), kaempferol-3-O-alpha-L-rhamnoside, caffeic acid, isoquercitrin, quinic acid, isocitrate, mannitol, apigenin, acacetin, and naringenin.CDE exhibited a potent antibacterial action on S. aureus isolates with MICs that ranged from 128 to 512 µg/mL.It also revealed antibiofilm action using a crystal violet assay and SEM.This antibiofilm potential was further studied at the molecular level using qRT-PCR on the biofilm-encoding genes, and it revealed a downregulating action on the studied genes in 43.48% of the isolates.In the in vivo aspect, using ELISA and immunohistochemical studies, the CDE-treated group showed a significant improvement in the histological features, with a significant lessening in the inflammatory markers in the liver and spleen.Our work has shown that treatment with CDE can significantly improve the survival rate of S. aureus-infected rats, with efficacy similar to that of the standard drug linezolid, suggesting potential therapeutic value and prompting further exploration of its clinical relevance.Thus, it is important to perform future studies on CDE to reveal its potential activity on other bacterial species and to elucidate its action in clinical practice.

Supplementary Materials:
The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/antibiotics13050450/s1,Table S1.Sequences of the utilized primers.Table S2.Inhibition zone diameter of SAM, linezolid (positive control), and DMSA (negative control).Table S3.Minimum inhibitory concentrations (MICs) of SAM.Author Contributions: J.A.: data curation, funding acquisition, software, validation, and writing-review and editing.W.A.N.: data curation, formal analysis, methodology, writing-original draft, and writing-review and editing.E.E.: data curation, formal analysis, methodology, writing-original draft, and writing-review and editing.I.A.H.: funding acquisition, investigation, resources, software, and writing-review and editing.H.S.H.: resources, software, validation, and writing-original draft.A.R.A.: funding acquisition, investigation, resources, and software.E.M.: data curation, project administration, resources, validation, and writing-review and editing.R.A.: data curation, investigation, software, validation, and writing-review and editing.S.I.: methodology, software, and writing-original draft.S.A.E.-S.: data curation, formal analysis, methodology, writing-original draft, and writing-review and editing.All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement:
The experimental procedures were approved by the ethics committee at the faculty of pharmacy, Tanta University (TP/RE/3/24 p-03).
Informed Consent Statement: Not applicable.

Antibiotics 2024 , 22 Figure 1 .
Figure 1.Pie chart revealing the percentages of the clinical specimens.

Figure 2 .
Figure 2. Heat map representing the susceptibility of S. aureus isolates to different antibiotics.

Figure 1 . 22 Figure 1 .
Figure 1.Pie chart revealing the percentages of the clinical specimens.

Figure 2 .
Figure 2. Heat map representing the susceptibility of S. aureus isolates to different antibiotics.

Figure 2 .
Figure 2. Heat map representing the susceptibility of S. aureus isolates to different antibiotics.

Figure 3 .
Figure 3.The inhibition zone diameters of CDE against the tested isolates.

Figure 3 .
Figure 3.The inhibition zone diameters of CDE against the tested isolates.

Figure 4 .
Figure 4. SEM micrograph revealing the morphology of S. aureus biofilm formed on the surfaces of cover glass: (A) without (untreated isolates) and (B) with CDE (treated isolates).

Figure 4 .
Figure 4. SEM micrograph revealing the morphology of S. aureus biofilm formed on the surfaces of cover glass: (A) without (untreated isolates) and (B) with CDE (treated isolates).

Figure 4 .
Figure 4. SEM micrograph revealing the morphology of S. aureus biofilm formed on the surfaces of cover glass: (A) without (untreated isolates) and (B) with CDE (treated isolates).

Figure 5 .
Figure 5. Impact of CDE on the gene expression levels of the biofilm.

Figure 5 .
Figure 5. Impact of CDE on the gene expression levels of the biofilm.

Figure 7 .
Figure 7. Survival curve constructed for the experimental groups. .

Figure 7 .
Figure 7. Survival curve constructed for the experimental groups. .

Figure 8 .
Figure 8.Light microscopic H&E-stained images of hepatic sections of adult albino rats of all studied groups.(A) Group I shows normal hepatic architecture in the form of hepatic cords (c) radiating from the central vein (CV) and separated by the hepatic sinusoids (S), with pericentral zone and midzone hepatocytes.Polyhedral hepatocytes appear with rounded vesicular nuclei and granular eosinophilic cytoplasm (black arrow) separated by sinusoids (S) lined with endothelial cells and Kupffer cells.(B) Group II shows disturbed hepatic architecture, compressed hepatic sinusoids, multiple pyknotic nuclei (black arrow), multiple karyolitic nuclei (blue arrow), and diffuse vacuolar degeneration of hepatocytes with multiple large vacuoles (V) as well as ballooned hepatocytes.(C) Group III shows organized hepatic cords (C) around the central vein, resembling the normal hepatic structure of Group I. (D) Group IV shows marked improvement and a regaining of the normal hepatic structure.However, with this enhancement, inflammatory cells infiltrate the pericentral zone (green arrow).(E) Histopathological score of the hepatic tissue cross sections in all studied groups.A box plot was used to express the data.The bottom of the plot represents 25%, the middle represents the median, and the top represents 75% of the data.Significant difference at p ≤ 0.05, where (a) in comparison with group I, (b) in comparison with group III, and (c) in comparison with group IV using the Kruskal-Wallis test followed by Dunn's pairwise comparison post-hoc test.(H&E × 400, scale bar = 50 μm).

Figure 8 . 22 Figure 9 .
Figure 8.Light microscopic H&E-stained images of hepatic sections of adult albino rats of all studied groups.(A) Group I shows normal hepatic architecture in the form of hepatic cords (c) radiating from the central vein (CV) and separated by the hepatic sinusoids (S), with pericentral zone and midzone hepatocytes.Polyhedral hepatocytes appear with rounded vesicular nuclei and granular eosinophilic cytoplasm (black arrow) separated by sinusoids (S) lined with endothelial cells and Kupffer cells.(B) Group II shows disturbed hepatic architecture, compressed hepatic sinusoids, multiple pyknotic nuclei (black arrow), multiple karyolitic nuclei (blue arrow), and diffuse vacuolar degeneration of hepatocytes with multiple large vacuoles (V) as well as ballooned hepatocytes.(C) Group III shows

Figure 9 .
Figure 9. Photomicrographs of H&E-stained sections of spleen in all studied groups.(A) Group I shows the normal histological organization of white pulp (WP) and red pulp (RP) surrounded by a capsule (arrowhead).The WP presents the central arteriole (dashed arrow) and lymphoid follicles (LF), with germinal centers (GC) and mantle regions (M), surrounded by a loosely distributed marginal zone (MZ).The RP presents lymphocytes, trabeculae, and sinusoids.(B) Group II shows a loss of normal architecture, shrunken WP, and broadened RP.Note congested, dilated splenic sinuses (S) in the RP.Many cells in the WP appear vacuolated.Thick fibrous trabeculae are observed (asterisk).(C) Group III shows a nearly normal appearance of the WP and RP splenic architecture.(D) Group IV shows a nearly normal outline of splenic architecture, but congested splenic sinuses are also seen.(E) Histopathological score of the splenic tissue cross sections in all studied groups.A box plot was used to express the data.The bottom of the plot represents 25%, the middle represents the median, and the top represents 75% of the data.Significant difference at p ≤ 0.05 (a) in comparison with group I, (b) in comparison with group III, and (c) in comparison with group IV using the Kruskal-Wallis test followed by Dunn's pairwise comparison post-hoc test.(H&E × 100, scale bar = 200 µm).

Figure 10 .Figure 10 .
Figure 10.Light microscopic TNF-α-stained pictures of a liver section in all studied groups.(A) Group I shows a negative TNF-α expression within the hepatocytes' cytoplasm.(B) Group II shows a strong positive expression of TNF-α, which appears as brownish cytoplasm in hepato-Figure 10.Light microscopic TNF-α-stained pictures of a liver section in all studied groups.(A) Group I shows a negative TNF-α expression within the hepatocytes' cytoplasm.(B) Group II shows a strong positive expression of TNF-α, which appears as brownish cytoplasm in hepatocytes (arrows).(C) Group III shows a negative expression of TNF-α.(D) Group IV shows a mild positive expression of TNF-α in a few hepatocytes (arrow) and a negative expression in most of the cells.(E) The area percentage of TNF-α in each group.Mean ± SD was used to represent the data.One-way ANOVA was used for the statistical comparison, and Tukey's post-hoc test was used for multiple comparisons.The single asterisk indicates a significant change, and the abbreviation NS denotes a non-significant change (p < 0.05).(TNF-α × 400, scale bar = 50 µm).
cytes (arrows).(C) Group III shows a negative expression of TNF-α.(D) Group IV shows a mild positive expression of TNF-α in a few hepatocytes (arrow) and a negative expression in most of the cells.(E) The area percentage of TNF-α in each group.Mean ± SD was used to represent the data.One-way ANOVA was used for the statistical comparison, and Tukey's post-hoc test was used for multiple comparisons.The single asterisk indicates a significant change, and the abbreviation NS denotes a non-significant change (p < 0.05).(TNF-α × 400, scale bar = 50 μm).

Figure 11 .
Figure 11.Light microscopic TNF-α-stained pictures of the spleen in all studied groups.(A) Group I shows a negative expression of TNF-α within the cytoplasm of the cells.(B) Group II shows a strong positive expression of TNF-α, which appears as brownish cytoplasm in most cells (arrows).(C) Group III shows a negative expression of TNF-α.(D) Group IV shows few TNF-α-positive cells (arrow) and a negative expression in most cells.(E) The area percentage of TNF-α in each group.Mean ± SD is used to represent the data.One-way ANOVA was used for the statistical comparison, and Tukey's post-hoc test was used for multiple comparisons.The single asterisk indicates a significant change, and the abbreviation NS denotes a non-significant change (p < 0.05).(TNF-α × 400, scale bar = 50 μm).

Figure 11 .
Figure 11.Light microscopic TNF-α-stained pictures of the spleen in all studied groups.(A) Group I shows a negative expression of TNF-α within the cytoplasm of the cells.(B) Group II shows a strong positive expression of TNF-α, which appears as brownish cytoplasm in most cells (arrows).(C) Group III shows a negative expression of TNF-α.(D) Group IV shows few TNF-α-positive cells (arrow) and a negative expression in most cells.(E) The area percentage of TNF-α in each group.Mean ± SD is used to represent the data.One-way ANOVA was used for the statistical comparison, and Tukey's post-hoc test was used for multiple comparisons.The single asterisk indicates a significant change, and the abbreviation NS denotes a non-significant change (p < 0.05).(TNF-α × 400, scale bar = 50 µm).
Figure S1.Negative mode total ion chromatogram of LC-ESI-MS/MS of Cleome droserifolia methanol extract.

Table 1 .
The phytoconstituents of C. droserifolia methanol extract recognized by LC-ESI-MS/ MS analysis.

Table 3 .
Effect of CDE on IL-1β and IL-6 levels in the liver and spleen of the experimental groups.

Table 3 .
Effect of CDE on IL-1β and IL-6 levels in the liver and spleen of the experimental groups.