Chenopodium murale Juice Shows Anti-Fungal Efficacy in Experimental Oral Candidiasis in Immunosuppressed Rats in Relation to Its Chemical Profile

Chenopodium murale (Syn. Chenopodiastrum murale) (amaranthaceae) is used in the rural Egypt to treat oral ulcers in newborn children. The current study aimed to discover new natural products suitable for treating candidiasis disease with minimal side effects. Characterization of bioactive compounds by LC-QTOF-HR-MS/MS from Chenopodium murale fresh leaves’ juice (CMJ) was carried out in order to elucidate their potential anti-fungal and immunomodulatory effects in oral candidiasis in immunosuppressed rats. An oral ulcer candidiasis model was created in three stages: (i) immunosuppression by drinking dexamethasone (0.5 mg/L) for two weeks; (ii) Candida albicans infection (3.00 × 106 viable cell/mL) for one week; and (iii) treatment with CMJ (0.5 and 1.0 g/kg orally) or nystatin (1,000,000 U/L orally) for one week. Two doses of CMJ exhibited antifungal effects, for example, through a significant reduction in CFU/Petri (236.67 ± 37.86 and 4.33 ± 0.58 CFU/Petri), compared to the Candida control (5.86 × 104 ± 1.21 CFU/Petri), p ≤ 0.001. In addition, CMJ significantly induced neutrophil production (32.92% ± 1.29 and 35.68% ± 1.77) compared to the Candida control level of 26.50% ± 2.44. An immunomodulatory effect of CMJ at two doses appeared, with a considerable elevation in INF-γ (103.88 and 115.91%), IL-2 (143.50, 182.33%), and IL-17 (83.97 and 141.95% Pg/mL) compared with the Candida group. LC-MS/MS analysis operated in negative mode was used for tentative identification of secondary (SM) metabolites based on their retention times and fragment ions. A total of 42 phytoconstituents were tentatively identified. Finally, CMJ exhibited a potent antifungal effect. CMJ fought Candida through four strategies: (i) promotion of classical phagocytosis of neutrophils; (ii) activation of T cells that activate IFN-γ, IL-2, and IL-17; (iii) increasing the production of cytotoxic NO and H2O2 that can kill Candida; and (iv) activation of SOD, which converts superoxide to antimicrobial materials. These activities could be due to its active constituents, which are documented as anti-fungal, or due to its richness in flavonoids, especially the active compounds of kaempferol glycosides and aglycone, which have been documented as antifungal. After repetition on another type of small experimental animal, their offspring, and an experimental large animal, this study may lead to clinical trials.

. Microbiological study of therapeutic efficacy of Chenopodium murale juice (CMJ) against oral candidiasis in immunosuppressed rats.

Strength CFU/Petri
Negative control +ve 11.33 ± 1.53 C. murale (0.5 g/kg) ve+ −ve 1 Data are presented as mean ± SE. Data were analyzed using a one-way ANOVA followed by post hoc analysis for multiple comparisons, and p < 0.001.

Immunomodulatory Effect of CMJ
Compared to the negative control, hemoglobin and red blood cell count were not affected either by Candida infection or CMJ administration. However, dexamethasone-induced immunosuppressed rats experienced significant thrombocytopenia. The platelet count of the ulcer control group significantly decreased by about 55.38% compared to the negative control (p ≤ 0.001). On the contrary, CMJ administration exhibited significant thrombocytosis, while the platelet count of CMJ (0.5 g/kg)-treated and CMJ (1.0 g/kg)-treated groups was significantly elevated to 234.99 × 10 9 ± 5.06 and 321.98 × 10 9 ± 8.33 platelet/L, compared to the ulcer control group (194.75 × 10 9 ± 5.41 platelet/L). The same trend was noticed in the positive groups, in which platelet counts significantly increased compared with the negative control (Table 2). Data are presented as mean ± SE. Data were analyzed using a one-way ANOVA followed by post hoc analysis for multiple comparisons, and p < 0.001. The means followed by the same letter in each column are not significantly different at the 0.1% probability level (Duncan's multiple range test).
Candida infection caused a condition of inflammation, in which ulcer group animals produced a large number of leukocyte cells (13.30 × 19 9 ± 1.50 cell/L); this number was larger than the negative group by about 54.65% (p ≤ 0.001). Additionally, the leukocyte differential of the ulcer group recorded remarkable alternation, where the lymphocyte% significantly reduced (59.07% ± 4.22). Meanwhile, the monocyte% was significantly induced (9.56% ± 0.79), with no significant change in neutrophil, eosinophil, and basophil percentages compared with the corresponding values in the negative group. CMJ force-feeding significantly depleted the leukocyte count and remarkably modulated its differentiation, where neutrophil% increased at the expense of the lymphocyte, monocyte, and basophil%. The leucocyte counts of the CMJ (0.5 g/kg)-treated and CMJ (1.0 g/kg)-treated groups were significantly depleted compared with that of the ulcer group, by about 66.17 and 64.14%, respectively. Additionally, more than in the ulcer group, the neutrophil percentages were significantly elevated by about 24.23 and 34.65%. Meanwhile, the last two groups' lymphocyte, monocyte, eosinophil, and basophil% were remarkably reduced compared with those of the ulcer group (p ≤ 0.001). CMJ administration decreased leukocyte count and increased neutrophil% more effectively than nystatin, compared to the ulcer group. The leucocyte count of the positive groups was also considerably reduced. The leucocyte differential was remarkably ameliorated toward the level of the neutrophil%, compared to the negative group. CMJ (0.5 g/kg)-treated and CMJ (1.0 g/kg)-treated groups appeared with a leucocyte (6.97 × 10 9 ± 0.18 and 7.23 × 10 9 ± 0.44 cells/L) and a lym-phocyte% (59.17% ± 3.67 and 54.94% ± 0.81) which were lesser than the negative group, and a neutrophil% (30.73% ± 2.99 and 35.07% ± 1.01) and monocyte% (6.99% ± 0.78 and 6.88% ± 0.51) larger than those of the negative group. There were no significant differences in the eosinophil and basophil percentages between the positive and negative groups.
In Figure 1A, the oral ulcer control group appeared with a low INF-γ concentration (140.90 ± 2.25 Pg/mL), which is about 32.62% less than that of the negative group (209.11 ± 2.32 Pg/mL), p ≤ 0.001. CMJ caused significant immune enhancement, where the INF-γ concentration was significantly elevated in the CMJ (0.5 g/kg)-treated and CMJ (1.0 g/kg)-treated groups, at a level of 287.27 ± 4.19 and 304.22 ± 3.76 Pg/mL, respectively, compared to the ulcer group (140.90 ± 2.25 Pg/mL). The INF-γ concentration of the two groups was higher than that of the nystatin group. The same trend was recorded in the INFγ concentration of CMJ (0.5 g/kg) and CMJ (0.5 g/kg)-positive groups, being significantly increased to 279.18 ± 4.91 and 291.74 ± 2.95 Pg/mL, respectively, compared to the negative control (209.11 ± 2.32 Pg/mL). Compared to the negative control, the INF-γ concentration of all groups that were administered CMJ was higher. g/kg)-treated groups was significantly elevated by about 143.50 and 182.33%, respectively. CMJ at 0.5 and 1.0 g/kg significantly increased IL-2, more than nystatin (110.94 ± 1.18, 128.63 ± 1.02, 94.49 ± 0.75 Pg/mL, respectively). In addition, CMJ significantly raised IL-2 in the positive groups, where the IL-2 of CMJ (0.5 g/kg)-positive and CMJ (1.0 g/kg)-positive groups increased more than the IL-2 of the negative group (96.64 ± 1.35, 114.86 ± 1.16, and 72.34 ± 1.08 Pg/mL). The IL-2 of all groups administered CMJ was higher than the IL-2 of the negative control. A B C Figure 1. The effect of Chenopodium murale juice (CMJ) on the INF-gamma (A), IL-17 (B), and IL-2 (C) of oral candidiasis in immunosuppressed rats. Data are presented as mean ± SE. Data were analyzed using a one-way ANOVA followed by post hoc analysis for multiple comparisons, and p ≤ 0.001. Value with the different superscript letters means significance at a probability level of 0.1%.
The data in Figure 1C show that the IL-17 of the ulcer group was remarkably less than that of the negative group, by about 38.06% (p ≤ 0.001). On the contrary, CMJ significantly increased the IL-17 of the CMJ (0.5 g/kg)-treated and CMJ (1.0 g/kg)-treated groups (210. 19   , and IL-2 (C) of oral candidiasis in immunosuppressed rats. Data are presented as mean ± SE. Data were analyzed using a one-way ANOVA followed by post hoc analysis for multiple comparisons, and p ≤ 0.001. Value with the different superscript letters means significance at a probability level of 0.1%.
The data in Figure 1B illustrate that Candida infection significantly reduced IL-2 from 72.34 ± 1.08 Pg/mL in the negative control to 45.56 ± 0.87 Pg/mL in the ulcer control (p ≤ 0.001). Compared to the ulcer control, the IL-2 of CMJ (0.5 g/kg)-treated and CMJ (1.0 g/kg)-treated groups was significantly elevated by about 143.50 and 182.33%, respectively. CMJ at 0.5 and 1.0 g/kg significantly increased IL-2, more than nystatin (110.94 ± 1.18, 128.63 ± 1.02, 94.49 ± 0.75 Pg/mL, respectively). In addition, CMJ significantly raised IL-2 in the positive groups, where the IL-2 of CMJ (0.5 g/kg)-positive and CMJ (1.0 g/kg)-positive groups increased more than the IL-2 of the negative group (96.64 ± 1.35, 114.86 ± 1.16, and 72.34 ± 1.08 Pg/mL). The IL-2 of all groups administered CMJ was higher than the IL-2 of the negative control.
The data in Figure 1C show that the IL-17 of the ulcer group was remarkably less than that of the negative group, by about 38.06% (p ≤ 0.001). On the contrary, CMJ significantly increased the IL-17 of the CMJ (0.5 g/kg)-treated and CMJ (1.0 g/kg)-treated groups (210. 19

Antioxidant Impact of CMJ
The results in Table 3 show that oral ulcer induction caused significant depletion in the CAT activity of the serum and oral mucosa, but not the spleen, compared to the negative control (p ≤ 0.001). Data are presented as mean ± SE. Data were analyzed using a one-way ANOVA followed by post hoc analysis for multiple comparisons, and p ≤ 0.001. The means followed by the same letter in each column are not significantly different at the 0.1% probability level (Duncan's multiple range test).
CMJ showed an antioxidant effect, and significantly increased CAT activity. The CMJ (0.5 g/kg)-treated and CMJ (1.0 g/kg)-treated groups recorded CAT activity higher than that of the ulcer group by about 146.25 and 172.12% for the serum, 123.73 and 135.85% for oral mucosa, 24.09 and 63.81% for the spleen, respectively. Nystatin significantly elevated the CAT activity of the serum, oral mucosa, and spleen, compared to the ulcer group, but this elevation was less than that of CMJ. Additionally, CMJ significantly increased the CAT activity of positive groups, where the CAT activity of the serum, oral mucosa, and spleen in CMJ (0.5 g/kg-positive and CMJ (1.0 g/kg)-positive groups was higher than that of the negative control (p ≤ 0.001). CMJ augmented CAT activity in all groups the administered it, more so than the negative control.
The data in Table 3 demonstrate that Candida infection significantly reduced the SOD activity of the serum (226.52 ± 3.94 U/mL), oral mucosa (6.00 ± 0.05 U/mg protein), and spleen (5.01 ± 0.05 U/mL), compared to the negative control (272.08 ± 3.43 U/mL, 8.00 ± 0.08 U/mg protein, and 5.72 ± 0.16 U/mg protein, respectively). The SOD enzymes of CMJ (0.5 g/kg)-treated groups remarkably activated to 289.16 ± 1.57 U/mL for the serum, 9.23 ± 0.07 U/mg protein for the oral mucosa, and 6.29 ± 0.06 U/mg protein for the spleen, compared to ulcer group. Increasing the CMJ dose to 1.0 g/kg increased the SOD activity of the serum and oral mucosa of the CMJ (1.0 g/kg)-treated groups. Meanwhile, the SOD of the spleen did not significantly change. The SOD of the serum and oral mucosa of the two positive groups significantly increased, but the spleen SOD activity was not significantly affected. Oral ulcer induction caused a significant oxidative stress condition that represented as a significant rise in the H 2 O 2 concentration of serum (35.15 ± 0.59 mM/L), oral mucosa (3.82 ± 0.13 mM/g), and spleen (1.80 ± 0.03 mM/g) in comparison to the corresponding values in the negative control (30.80 ± 0.32 mM/L, 1.42 ± 0.03 mM/g, and 0.655 ± 0.02 mM/g, respectively (p ≤ 0.001)), Table 4. Data are presented as mean ± SE. Data were analyzed using a one-way ANOVA followed by post hoc analysis for multiple comparisons, and p ≤ 0.001. The means followed by the same letter in each column are not significantly different at the 0.1% probability level (Duncan's multiple range test).
The H 2 O 2 concentration of serum was significantly boosted as a response to CMJ force-feeding. Therefore, the H 2 O 2 concentration of the serum of the CMJ (0.5 g/kg)-treated and CMJ (1.0 g/kg)-treated groups was higher than the H 2 O 2 of the ulcer group by about 20.51 and 80.11%, respectively. An opposite trend was recorded in the H 2 O 2 concentration of the oral mucosa and spleen of CMJ (0.5 g/kg)-treated and CMJ (1.0 g/kg)-treated groups; these were significantly reduced (about 35.86 and 29.85% for the oral mucosa and 73.89 and 77.78% for the spleen, respectively). Nystatin treatment caused a significant reduction in the H 2 O 2 concentration of the serum, oral mucosa, and spleen compared to the ulcer group. CMJ force-feeding caused a substantial decrease in the H 2 O 2 concentration of the serum, oral mucosa, and spleen of the positive groups compared to the negative group. Interestingly, CMJ behavior towards serum H 2 O 2 production differed in healthy and infected animals. It significantly reduced the serum H 2 O 2 in positive groups and also significantly raised the serum H 2 O 2 in treated groups.
Candida infection significantly raised MDA levels, a lipid peroxidation biomarker, in the serum (6.32 ± 0.11 nmol/mL), oral mucosa (4.85 ± 0.08 nmol/g tissue), and spleen (3.53 ± 0.14 nmol/g tissue), compared to the negative group (p ≤ 0.001), Table 4. CMJ significantly reduced the MDA level of the serum, oral mucosa, and spleen in CMJ (0.5 g/kg)treated and CMJ (1.0 g/kg)-treated groups compared to the ulcer control. No significant difference was noticed between the MDA levels (serum, oral mucosa, and spleen) of infected animals administered CMJ and the others administered nystatin. However, CMJ significantly decreased the MDA of the positive groups to levels lower than that of the negative group.
Only a high dose of CMJ significantly raised the NO concentration in the mucosal membrane (5.94 ± 0.21 µmol/g) and spleen (1.41 ± 0.04 µmol/g), compared to Candida control (2.91 ± 0.24 and 0.87 ± 0.06 µmol/g, respectively) (p ≤ 0.001). CMJ at a high dose increased NO concentration in a manner similar to nystatin. In positive groups, CMJ did not affect NO concentration in the mucosal membrane and spleen, compared to the negative control.

Chemical Composition of CMJ
The CMJ (10.00 g extract/100 g powder) used had powder-like characteristics, with spinach-like odor and a light green color ( Table 5). The total assessments carried out for CMJ exhibited that it contains polyphenols (66.33 ± 0.88 mg gallic acid/g extract), flavonoids (44.45 ± 1.45 mg quercetin /g extract), and tannins (14.49 ± 0.70 mg tannic acid/g extract); finally, alkaloids represented 200.43 ± 8.60 mg alkaloid/g extract. Table 5. Physical examination and organoleptic characters of the Chenopodium murale juice (CMJ).

Yield and Physical Characters Ethanol 70%
Yield (g, %) 10.00 g extract/100 g dried powder Color Light

Characterization of Phytoconstituents in Chenopodium murale Juice (CMJ) by HPLC/QTOF-HR-MS/MS Analysis
The valuable biological effects of CMJ prompted us to identify its phytochemical profile through a non-targeted profiling method using high-performance liquid chromatography (HPLC) coupled with a high-resolution quadrupole time-of-flight mass spectrometer (QTOF-MS) operated in both negative (−ve) and positive (+ve) ionization modes. CMJ was analyzed in (−ve) ionization mode. Some 42 compounds belonging to different natural product classes are listed in Table 6; these were tentatively identified using HPLC-QTOF-HR-MS/MS, based on their retention times (R t ), detected accurate mass, (−ve) ionization mode, and molecular formula; the error in ppm (between the observed mass and the real mass) of each phytochemical and the MS/MS fragment ions were used to determine the limit of detection for each peak of the compounds, by comparing the reference compounds' spectra and reported data. The total ion current (TIC) and base peak (BPC) MS-chromatograms ( Figure 2) revealed that the CMJ is rich in polyphenols such as phenolic acids, anthocyanines and flavonoids, especially kaempferol derivatives, aurone, flavones, flavonols and their glycosides.
The chemical structures of the individual polyphenolics were determined by analysis of fragment patterns, in which glycosides of flavonoids such as glucose, rhamnose, glucuronic acid and neohesperidoside (m/z 162, 146, 176 and 308) were cut out from their structures [17]. In this work, all of the separated compounds were tentatively identified and detected for the first time in CMJ. Some 13 compounds out of a total of 42 were already reported in C. species [18][19][20].
The relative concentration percentage (Rel. Conc.%) of the identified compounds was calculated, as shown in Table 6. It was found that the compounds of kaempferol derivatives, aurone, phenolics and fatty acids, anthocyanides, flavones, flavonols and their glycosides are the most concentrated compounds in the plant, compared to the rest of the separated compounds. It was noted specifically that the compound Kaempferol3-O-(6-pcoumaroyl)-glucoside has a high concentration that doubles compared to the rest of the high-concentration compounds.
LC-MS coupled with Q-TOF-MS also achieved the detection of chemical compounds with high sensitivity. The peaks of the most concentrated compounds were responsible for anti-candidal effect of CMJ, especially Kaempferol3-O-(6-p-coumaroyl)-glucoside, which is present in a concentration that significantly exceeds the rest of the Kaempferol derivatives. Different types of flavonoid glycosides in such abundance contribute to antifungal activity [21].     Relative concentration (%) calculated based on the individual peak areas for identified components, * highly concentrated compounds; ** The most concentrated compounds.

Characterization of the First Detected Kaempferol Derivatives
Flavonoid glycosides have a variety of isomers with the same molecular weight but distinct aglycone and sugar components at various places on the aglycone ring. It is difficult to determine the identification of the sugars and how they are connected using only mass spectrometry. For instance, the loss of 162 amu implies the presence of a hexose sugar, but it is unclear whether it is glucose or galactose. The locations and types of sugars were identified, as we compared the R t values, mass spectra and chromatography of the main compounds, such as Kaempferol3-O-(6-p-coumaroyl)-glucoside, Kaempferol

Characterization of Flavone Derivatives
Observing HPLC-MS/MS spectral data, peaks 11, 13, 15 and 16 are mono-O-flavone glycosides; this could be determined from the fragmentation pattern of sugar units. Fragmentation due to the loss of 14 amu was detected for the methyl, the loss of 18 amu for the hydroxyl and the loss of 30 amu for the methoxy groups ( Figure 3). Baicalein-7-Oglucuronide was detected for the first time, proposed for peak 11, with a molecular ion peak at m/z 445.0777, R t 5.856 min, and a molecular formula of C 21 H 18 O 11 [28].   [25]. The ion at m/z 446.0835 also proved that the deoxyhexose was in a terminal position. The loss of (−308 amu) showed the two sugars were linked at the same position. Compound 24, with a molecular ion at m/z 447.0932, exhibited a fragmentation pattern similar to that of Maritimetin-6-O-glucoside, at m/z 285.0400, 151.0000 and 133.0000 [30].

Characterization of Phenolics and Fatty Acids
Phenolic acids are a class of 2 ry metabolites (SM) that have a variety of interesting biochemical pathways. They usually form a pseudomolecular ion [M − H] corresponding to a deprotonated molecule and characteristic fragment ion [M − H-44] related to CO 2 loss from the carboxylic acid group. In this work, six free phenolic acids were tentatively identified, including chlorogenic acid, quinic acid, caffeic acid, salicylic acid, P -hydroxybenzoic acid and 3,4-Dihydroxybenzoic acid [18,31], the highest concentration of which was found to be chlorogenic acid. Three highly concentrated fatty acids were detected as lactic acid, citraconic acid and D-3-Phenyllactic acid, respectively.
Candida yeasts are generally present in healthy humans, and are frequently part of the human body's normal oral and intestinal flora, particularly on the skin; however, their growth is usually limited by the human immune system and by competition of other microorganisms. Candida albicans is not considered a pathogen in healthy individuals. However, in immunocompromised patients, it can cause severe systemic candidiasis [34]. In this study, a candidiasis model in rats was created as follows: (i) the rats were administered dexamethasone solution for 15 days to cause immunosuppression; (ii) the rats were infected with Candida for seven days; and (iii) the rats were treated with CMJ or nystatin for seven days. Candida control rats appeared to have a weak immune system, in which IL-17, IL-2, and INF-γ levels were statistically reduced. Additionally, altered hematological parameters, wherein rats recorded leukocytosis but the neutrophil% did not change significantly. Furthermore, Candida control rats suffered from oxidative stress, where antioxidant enzymes and CAT and SOD activity significantly decreased alongside significant elevation of oxidative stress biomarkers (i.e., H 2 O 2 and MDA concentrations). Our results are in accordance with the immunosuppressive effect of glucocorticoid medications such as dexamethasone.
Dexamethasone is an immunosuppressive and anti-inflammatory glucocorticoid medication for treating many diseases, including (i) rheumatic problems, (ii) skin diseases, (iii) severe allergies, (iv) asthma, (v) chronic obstructive lung diseases, (vi) croup, (vii) brain swelling, (viii) eye pain following eye surgery, (ix) superior vena cava syndrome, and (x) antibiotics in tuberculosis. Unfortunately, dexamethasone has many side effects, including candidiasis and leukocytosis. Glucocorticoids work through inhibiting genes that code for the cytokines IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, and TNF-α, reducing T-cell proliferation. Glucocorticoids suppress hormonal immunity, causing B-cells to express fewer IL-2 and IL-2 receptors, diminishing both B cells' clone expansion and antibody synthesis. In addition, glucocorticoids induce the resolution of inflammation by increasing the secretion of anti-inflammatory factors (IL-10 and tumor growth factor (TGF)-β). They also affect the adaptive immune system, suppressing CD4+ T cell activation by modulating dendritic cell function and promoting the polarization of T helper (Th) cells, with the preferential differentiation of Th2 and T regulatory (Treg) cells and the inhibition of Th1 and Th17 cells. In addition, glucocorticoids can alter patients' microbiome and promote M2 macrophage polarization [35].
On the contrary, Candida-infected rats who received CMJ with two doses showed an opposite trend. CMJ caused an immunomodulatory action that manifested as a significant increase in INF-γ, IL-17, and IL-2. CMJ ameliorated hematological biomarkers toward phagocytosis, in which state the neutrophil% statistically increased. Meanwhile, the other percentages (lymphocyte, monocyte, basophil, and eosinophil) reduced considerably. CMJ activated antioxidant enzymes, CAT and SOD, and inhibited the oxidative stress biomarkers (i.e., H 2 O 2 and NO) and MDA of the serum, mucosa, and spleen. CMJ showed a promising anti-fungal effect, immunomodulatory action, and antioxidant properties compared to nystatin treatment. CMJ showed the same trend as nystatin, an anti-fungal medication.
Both innate and adaptive immunity play a role in protection against fungal infections. Fungi are recognized by the innate immune system (e.g., dendritic cells and macrophages), resulting in phagocytosis and the initiation of killing mechanisms (e.g., production of reactive oxygen species) and helping to drive the development of adaptive immunity. In adaptive immunity, CD4+ T-cells cause IFN-γ (Th1) or IL-17 (Th17) to provide the best protection during fungal infections. This helps to drive effective killing by innate effector cells such as neutrophils and macrophages [36]. CMJ affected both innate and adaptive immunity, wherein it activated phagocytosis, which manifested as a significant elevation in neutrophils; it returned the INF-γ, IL-17, and IL-2 of the infected rats to a level close to that of the negative control.
Firstly, macrophages and neutrophils are the prototypical innate immune cell, essential for host defense against candidiasis. Neutrophils' depletion in neutropenic patients leads to an increased susceptibility to mucosal candidiasis [37]. Neutrophils are highly phagocytic granulocytic polymorphonuclear cells that are important in antimicrobial immunity. A reduction in circulating neutrophils increases the risk of candidiasis [38]. Additionally, chronic granulomatous disease patients, whose neutrophils cannot make ROS, suffer from invasive aspergillosis [39]. The neutrophil's classical killing mechanism works through the production of ROS and hydrolytic enzymes, which kill phagocytosed microbes when the granules that contain them fuse with the phagosome. The respiratory burst provides neutrophils with oxygen to generate ROS. The respiratory burst activates the NADPH oxidase, releasing large quantities of superoxide, a ROS [40]. Superoxide rapidly breaks down to H 2 O 2 through SOD. Then, H 2 O 2 is converted to hypochlorous acid (HClO) by myeloperoxidase. HClO is bactericidal enough to kill the bacteria phagocytosed by the neutrophil [41]. CMJ significantly elevated neutrophils and H 2 O 2 and SOD activity, which suggests that CMJ promotes the classical phagocytosis of neutrophils by increasing ROS production.
Secondly, CMJ enhanced adaptive immunity, wherein it statistically elevated the levels of INF-γ, IL-17, and IL-2 that were significantly depleted in the Candida control.
INF-γ is a cytokine called the macrophage activation factor. It is produced by natural killer cells, dendritic cells, killer T cells, CD4 Th1, CD8 cytotoxic T lymphocytes, and effector T cells. INF-γ alters transcription in up 30 genes, producing a variety of physiological and cellular responses, including (i) promotion of natural killer cells, which indirectly fight Candida by promoting the production of TNF-α and INF-γ [37]; (ii) increasing antigen presentation and the lysosome effect of macrophages, causing them to engulf and digest the Candida cells (they are also essential for wound healing); (iii) activation of inducible NO synthase that mediates the cytotoxic effect of the pathogen; (iv) activation of B cells to produce IgG2a and IgG3 antibodies that enhance the neutrophil-mediated killing of Candida, opsonophagocytosis, and induce complement activation [36,42,43]; (v) induction of the expression of defense factors against retroviruses that have directly antiviral effects; and (vi) promotion of the adhesion and binding required for leukocyte migration [44,45].
IL-17 is a pro-inflammatory interleukin produced by CD4+ T cells. IL-17 is essential for protecting against mucosal candidiasis. The anti-candidal defense action of IL-17 was confirmed by the high susceptibility of −/− mice to mucosal candidiasis, which correlates with defects in neutrophil recruitment and reduced antimicrobial peptide (AMP) production. In addition, host recognition of C. albicans via immunoreceptors is required to mount an appropriate immune response, including activation of IL-17 [46]. Additionally, IL-17 inhibitory drugs used to treat psoriasis have been shown statistically to increase the risk of fungal and bacterial infections [47]. Finally, in humans, the candida-specific memory T cells are predominantly Th-17 cells [48]. IL-17 fights Candida infection through several mechanisms, including up-regulation of hematopoietic cells such as phagocytes, adaptive Th-17 cells, neutral Th-17 cells, dendritic cells, non-major histocompatibility complex-restricted T cells, and subsets γ and δ T cells. Additionally, IL-17 activates non-hematopoietic cells, including mucosal epithelial cells, which are responsible for expression of IL-17 receptors (IL-17RA/RC). In addition, IL-17 is involved in fungal clearance via the production of various pro-inflammatory cytokines and antimicrobial peptides during infection [33]. Th-17 produces cytokine IL-22, which is also vital for anti-fungal immune responses; however, in experimental OPC, IL-22 −/− mice are only mildly susceptible to Candida infection compared to IL-17RA −/− mice [48]. IL-17 up-regulated many antimicrobial peptides' excretion, including defensins (β-defensins), calprotectin (S100A8/9) and mucin [49]. IL-17 promotes expression of the mucin gene MUC5B that has implications for the inhibition of virulence factors, including genes of adhesion, filamentation and biofilm formation, which lead to Candida clearance [50]. IL-17 may be proven to affect immune modulation through the recruitment of additional IL-17+ lymphocytes to the site of infection. Signaling through the IL-17 receptor results in a neutrophil influx, which assists fungal control. Finally, Murine β-defensin-3, which is involved in protection against Candida, is strongly IL-17-dependent [51].
IL-2 is produced by activated CD4+ T cells and activated CD8+ T cells. IL-2 has several immunity functions, including (i) a direct effect on the antibody-producing clones of B lymphoblasts, and on activated T-lymphocyte proliferation; (ii) induction of the phagocytic capacity of polymorphonuclear leukocytes, and the destruction of target cells by natural killer cells and cytotoxic T lymphocytes; (iii) activation of non-major histocompatibility complex-restricted cytotoxic cells, resulting in the generation of lymphokine-activated killer cells with the capacity to kill tumor and fungal targets; (iv) lectin-like properties, which have specificity for high-mannose groups. Despite the previous essential functions of IL-2, it has mannose-binding properties, whereby IL-2 binds to Candida via its surface mannose groups. Cuneyt et al. [52] reported that Candida mannan has immunosuppressive properties. Lilic et al. [53] demonstrated that IL-2 and IL-10 cytokines are deregulated and Candida-specific. Incubation of Candida albicans with IL-2 inhibited its growth [54].
Finally, we suggest that the anti-fungal properties of CMJ may be due to the activation of CD4+ T cells, which causes significant increase in INF-γ, IL-2, and IL-17 production. Additionally, CMJ activated natural killer cells, dendritic cells, killer T cells, CD4+ Th1, CD8+ cytotoxic T lymphocyte, and effector T cells, which produce INF-γ.
Collectively, we may conclude that CMJ treated oral candidiasis in immunosuppressed rats via several strategies, including (i) promotion of classical phagocytosis of neutrophils; (ii) activation of T cells that activate IFN-γ, IL-2, and IL-17 production; (iii) increasing the production of cytotoxic NO and H 2 O 2 , which can kill Candida; (iv) activation of the SOD enzyme that converts superoxide to antimicrobial materials; and (v) induction of the repair process of damaged tissues. CMJ exhibited these characteristics due to its chemical composition; CMJ contains considerable quantities of polyphenols, flavonoids, and alkaloids. Additionally, LC-Mass identified several active constituents recognized in the CMJ, with anti-fungal and immunomodulatory effects.
For example, Kaempferol and kaempferol-bound gylcosides were the major components in the CMJ that were determined to be anti-fungal when tested alone or with the extract. Kaempferol-containing extracts such as Scabiosa hymettia, Allium ursinum, and Bryophyllum pinnatum exhibited antifungal activity against Candida types [55][56][57]. Pure kaempferol has a strong effect against oral and vaginal candidiasis [58][59][60]. Gallic acid suppressed the protein synthesis of C. albicans, reducing the number of hyphal cells and germ tubes [59]. Chlorogenic acid exhibited an in vitro anti-candidal effect, resulting in (i) cell viability reduction; (ii) the elevated possibility of mitochondria depolarization and the release of ROS; (iii) DNA fragmentation and phosphatidylserine liberation; and (iv) the induction of apoptosis. Additionally, chlorogenic acid exhibited considered interactions with the ALS3 active site residues of C. albicans, which enable it to adhere to and resist fluconazole [61]. Cinnamic acid has an in vitro immunoregulatory effect on monocytes against C. albicans. Caffeic acid inhibited C. albicans through an isocitrate lyase enzyme effect.
Quercetin and naringenin are good suppressors of C. albicans; they halted biofilm synthases and induced membrane disorders, thereby reducing cell size and infiltration of intracellular components. Quercetin activated phosphatidylserine, which inhibited fatty acid synthase, a main component of the cell wall. Quercetin regulated mitochondrial functions by (i) inhibiting oxidative phosphorylation; (ii) alternating the ROS production in mitochondria; and (iii) modulating the transcription factors that control mitochondrial proteins' expression. The previous functions cause pro-apoptotic functions by discharging cytochrome C from the mitochondria; they can also do so indirectly by inducing the expression of the pro-apoptotic proteins of Bcl-2, and by reducing anti-apoptotic proteins. In vitro treatment with apigenin and quercitrin downregulated genes encoding efflux pumps (CDR1). Apigenin recorded antifungal activity against C. albicans through reducing the fungal virulence and expression of antifungal resistance-linked genes [62]. It also induces membrane disorders, leading to cell shrinkage and the loss of intracellular constituents [63,64]. Additionally, it causes mitochondrial disorders through stimulating mitochondrial calcium uptake, resulting in (i) membrane disruption; (ii) increased mitochondrial mass; and (iii) an elevation in ROS production. Furthermore, apigenin activated apoptosis via (i) phosphatidylserin exposure; (ii) DNA fragmentation; and (iii) caspase activation. Baicalein has potent antifungal activity against C. albicans, C. tropicalis and C. parapsilosis [65], wherein exposure of Candida cells to baicalin disrupted their biofilms' components [66]. Myricetin has an antifungal effect against C. albicans via (i) injuring the cell wall's integrity; (ii) increasing membrane permeability; (iii) causing DNA and protein loss; and (iv) causing alternations in the lipid components or order of the cell membrane [67].

Collection of C. murale and Preparation of the Juice
Leaves of C. murale were collected from fields in Sharkia Governorate, Egypt, in March 2021. Washed fresh leaves (500 g) were crushed using a blender (Toshiba, Egypt) and were filtrated by filter paper (Whatman No. 1). The filtrate was lyophilized using a lyophilizer. The residue extract (50 g) was kept at −20 • C until it was incorporated into the bioassay. The acute toxicity assay of the CMJ was performed according to according to per OECD guideline 423 (2001) for acute oral toxicity -Up-and-Down-Procedure (UDP) [68]. The dosing pattern started at 500 to 6000 mg/kg body weight. Mice were force-fed the extract by gastric tube (5 mice), and control mice received saline only. All groups were kept under observation and were assessed for any changes and mortality for 48 h. The live animals were observed for 14 days. Using mortality numbers in each concentration during the first 48 h and the BioStat program (BioStat 2009 Build 5.8.4.3 © 2023 analystSoft Inc., Alexandria, VA, USA), the CMJ LD 50 was determined to be 5000 mg/kg.

Microbial Strains and Culture Conditions
The C. albicans strain was isolated from the sputum culture of a patient suffering from oral candidiasis and was used for all experimental assays in this study. This strain was stored as frozen stocks in 30% glycerol at −80 • C, subcultured on Sabouraud Dextrose agar plates, and routinely grown in Sabouraud liquid medium at 37 • C. A single colony from the Sabouraud Dextrose agar plate was produced in a yeast extract-peptone glucose medium YPG: (yeast extract, 2%; bacto peptone, 1%; glucose, 2%) for 18 h at 30 • C in a shaker. The culture was harvested by centrifugation at 2500 rpm, and then cells were washed three times in phosphate-buffered saline (PBS) and adjusted to a final concentration of 5 × 10 6 CFU/mL. Therefore, samples from the entire oral cavity or only from a specific site, e.g., the tongue's surface, were collected with a swab, for posterior CFU counting, onto Sabouraud dextrose agar (SDA). After plating, the Petri dishes were incubated at 35-37 • C for 24 or 48 h (using a hemocytometer chamber for counting cells) [69].

Animal Preparation and Oral Infection
Some 70 Wistar male albino rats (60 days old), weighing 180-200, were obtained from the Central Animal House of the National Research Centre, Giza, Egypt. The rats were maintained in cages at the animal care facility (20-25 • C, 55-65% humidity, 10-12 h light/dark cycle). The rats were fed the standard chow diet obtained from the Central Animal House. Water and food were available ad libitum over the experimental period.

Oral Ulcer Induction
A model of oral candidiasis in immunosuppressed rats reported by Martinez et al. [4] was used. The experiment was performed in three stages: decreasing the immunity of the rats, infecting the rats with Candida, and treating them with CMJ. In the first stage, rats were immunosuppressed with dexamethasone and treated with tetracycline to enhance the infection rate. Before infection, for a week, the rats drank dexamethasone (0.5 mg/L) and tetracycline (0.1% to prevent bacterial infections) in water. On the day of infection, the rats drank dexamethasone (1 mg/L) and tetracycline (0.01%) in water. The rats were maintained with this water until the end of the experiment. In the second stage, the rats were orally infected three times at 48 h intervals (days-7, -5, and -3) with 0.1 mL of saline suspension containing 3.00 × 10 6 viable cells of C. albicans (Table 7). Oral infection was achieved using a cotton swab rolled twice over all mouth parts. After the last inoculation (72 h), all groups were sampled using a cotton swab to confirm the infection and quantify the number of CFU (colony-forming units) in the oral cavity, before starting CMJ administration. Groups of mice were sacrificed under anesthesia and subjected to evaluation of the severity of lesions of the tongue. Macroscopic assessment of the infection was expressed by scoring lesions from 0 to 4 based on the extent and severity of whitish, curd-like patches on the tongue surface, as follows: 0, normal; 1, white patches in less than 20%; 2, white patches in less than 90% but more than 21%; 3, white patches in more than 91%; 4, thick white patches similar to pseudomembranes in more than 91%. In the third stage, rats were treated with CMJ or nystatin for seven days. Table 7. Experimental layouts.

Stages Experimental Days Protocols
Adaptation period 1st-7th days • Animals were kept in laboratory conditions. • water and food provided 'ad labium'. According to the LD 50 , the two doses of the extract were chosen to be 1.0 and 0.5 g/kg body weight, i.e., 1 /4 and 1/10 of LD 50 [70,71]. Nystatin oral suspension was used as a standard drug. Nystatin was typically applied at the recommended dose of 1 mL/kg/day (equalling 100,000 units). CMJ and nystatin were dispersed in a viscous 0.8% agar solution as an excipient for oral treatment [72]. CMJ was typically applied using a 1 mL dropper similar to that of nystatin.

Experimental Scheme
Rats were randomly divided into seven groups, every ten rats, as follows. Group I: rats received normal saline for 7 days and were maintained as a negative control. Group II and III: rats received CMJ at two doses, a high dose (1.0 g/kg/day) and a low dose (0.50 g/kg/day for 7 days), respectively, and were maintained as a positive control for these doses. Group IV: Candida-induced oral ulcer rats received normal saline for 7 days and were kept as an ulcer control. Group V and VI: Candida-induced oral ulcer rats were treated with CMJ at high doses (1.0 g/kg/day) and low doses (0.50 g/kg/day), respectively, for 7 days. Group VII: Candida-induced oral ulcer rats were treated with nystatin, the reference drug, at the recommended dose, for 7 days [72]. Some 48 h later, after the last drug administration, the rats were fasted overnight. The rats were anesthetized using an injection of ketamine 87 mg/kg of body weight and xylazine 13 mg/kg of body weight (dissolved in normal saline), and each rat received 0.2 mL/100 g body weight of this liquid [73]. Animals were sacrificed after anesthesia, and the blood samples were collected from the retro-orbital plexus and organs. Blood samples (2 mL) were collected over EDITA to CBC analysis. Blood samples were collected in a clean centrifuge tube and centrifuged at 3000 r/min for 10 min using Sigma Laborzentrifugen (Osterode am Harz, Germany). The organs were washed and weighted freshly. The oral mucosae were excised from the animals. The oral mucosae and spleens were homogenized using an ultrasonic tissue homogenizer, then centrifuged at 4000× g, at 4 • C for 15 min. Serum was used for determination of the antioxidant parameters and oxidative stress parameters. The oral mucosae and spleens were kept in 10% formalin for histopathological examination.

Microbiological Evaluation of the Progression of the Infection
The whole oral cavity, including the buccal mucosa, tongue, soft palate, and other oral mucosal surfaces, was swabbed using a cotton pad. The end of the cotton pad was then cut off and placed in a tube containing 5 mL sterile saline. After mixing on a Vortex mixer to release Candida cells from the swab into the saline, serial 100-fold dilutions of the cell suspension were incubated on a Candida GS plate at 37 • C for 20 h. The CFU (colony-forming units) of the Candida colonies were counted.

Biochemical Analysis
In accordance with the work of Dacie and Lewis [74], blood samples were introduced to determine the hematological parameters. In addition, antioxidant biomarkers and catalase (CAT) and superoxide dismutase (SOD) activities of the serum, mucosa, and spleen tissues were determined spectrophotometrically [75,76].
According to the manufacturer's instructions, using an enzyme-linked immunosorbent assay, serum interleukins, IL-17 and IL-2, and interferon-gamma (IFN-γ) were determined using ELISA kits.

Chemical Composition of the CMJ
Quantitative Analysis of the CMJ The total phenolic content was estimated using the Folin-Ciocalteu method [80]. The total flavonoid content was measured according to Lin and Tang [81], and was expressed as mg quercetin/g of extract. Crude alkaloids were gravimetrically determined according to Onwuka [82]. The tannin content was evaluated by the standard method of Broadhurst and Jones [83].

Qualitative Analysis of Phytoconstituents in CMJ by HPLC/QTOF-HR-MS/MS
Liquid chromatography-mass/mass spectrometry analysis was used to identify the chemical composition of CMJ [84,85]. LC-mass/mass analysis was carried out in the Proteomics and Metabolomics Research Program of the Basic Research Department at the Children's Cancer Hospital, Cairo, Egypt.

Sample Preparation
A stock solution of the CMJ was prepared from 50 mg of the lyophilized CMJ dissolved in 1000 µL of the solvent mixture, itself composed of water: methanol: acetonitrile (H 2 O:MeOH:ACN) in a ratio of 2:1:1. Complete solubility of the stock solution was obtained by vortexing the sample for 2 min and ultra-sonicating the sample at 30 kHz for 10 min. An aliquot, 20 µL of the stock solution, was again diluted with 1000 µL of H 2 O:MeOH:ACN (2:1:1) and centrifuged at 10,000 rpm for 10 min. Finally, 10 µL of stock with a concentration of 2.5 µg/µL was injected. Some 10 µL of reconstitution solvent was injected as a blank sample. The sample was injected in negative modes.

Instruments and Acquisition Method
The mass spectrometry (MS) was performed on a Triple TOF 5600+ system equipped with a duo-spray source operating in the ESI mode (AB SCIEX, Concord, ON, Canada). The sprayer capillary and declustering potential voltages were −4500 and −80 V in the negative mode. The source temperature was set at 600 • C, the curtain gas was 25 psi, and gas 1 and gas 2 were 40 psi. A collision energy of −35 V (negative mode), a CE spreading of 20 V and an ion tolerance of 10 ppm were used. The TripleTOF5600+ was operated using an information-dependent acquisition (IDA) protocol. Batches for MS and MS/MS data collection were created using Analyst-TF 1.7.1. The IDA method was used to simultaneously collect full-scan MS and MS/MS information. The technique consisted of high-resolution survey spectra from 50 to 1100 m/z, and the mass spectrometer was operated in a pattern, wherein a 50-ms survey scan was detected. Subsequently, after each scan, the top 15 intense ions were selected for acquisition of the MS/MS fragmentation spectra.

LC-MS Data Processing
MS-DIAL 3.70 open-source software was used for the sample's non-targeting, small molecule comprehensive analysis. ReSpect negative (1573 records) databases were used as reference databases according to the acquisition mode. The MS-DIAL output was used to run again on PeakView 2.2 with the Master View 1.1 package (AB SCIEX) for feature (peaks) confirmation, based on the criteria, using the total ion chromatogram (TIC). Aligned features had a signal-to-noise ratio greater than 5, and a sample intensity of greater than 5.

Statistical Analysis
Data were analyzed as mean ± SE for every six rats. Comparisons among groups were performed using a one-way analysis of variance ANOVA test, at p ≤ 0.001, followed by a Tukey comparison test using IBM-SPSS (version 25), followed by a post hoc test.

Conclusions
The current study demonstrated the antifungal effect of C. murale fresh juice (CMJ). CMJ treated oral candidiasis in immunosuppressed rats, where the Candida count in mucosal parts was dramatically reduced. Concurrently, CMJ exhibited significant enhancement of neutrophils, NO, H 2 O 2 production and SOD activity, meaning CMJ fought Candida through classical phagocytosis of the neutrophils by increasing ROS production. Besides, CMJ promoted adaptive immunity and significantly increased INF-γ, IL-17, and IL-2 production. The antifungal and immunomodulatory effects of CMJ may attributed to its active constituents, which were identified by LC-MS/MS. This study can be generalized to a broader study population, and may prompt a clinical trial using CMJ as an antifungal drug.