Chemical Fingerprinting, Antioxidant, and Anti-Inflammatory Potential of Hydroethanolic Extract of Trigonella foenum-graecum

In the current study, the antioxidant and anti-inflammatory potential of hydroethanolic extract of T. foenum-graecum seeds was evaluated. Phenolic profiling of T. foenum-graecum was conducted through high-performance liquid chromatography-photodiode array (HPLC-PDA) as well as through the mass spectrometry technique to characterize compounds responsible for bioactivity, which confirmed almost 18 compounds, 13 of which were quantified through a chromatographic assay. In vitro antioxidant analysis of the extract exhibited substantial antioxidant activities with the lowest IC50 value of both DPPH (2,2-diphenyl-1-picrylhydrazyl) and ABTS (2,2′-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid) inhibition assays. The extract was found to be non-toxic against human RBCs and murine macrophage RAW 264.7 cells. Moreover, the extract significantly (p < 0.001) reduced the lipopolysaccharide (LPS)-induced tumor necrosis factor alpha (TNF-α), intrlukin-6 (IL-6), prostaglandin E2 (PGE2), and nitric oxide (NO) in RAW 264.7 cells in a concentration-dependent manner. The hydroethanolic extract of T. foenum-graecum exhibited considerable anti-inflammatory potential by decreasing the cellular infiltration to the inflammatory site in both carrageenan-induced peritonitis and an air pouch model of inflammation. Pretreatment with T. foenum-graecum extract caused significant improvement in antioxidants such as superoxide dismutase (SOD), CAT (catalase), malondialdehyde (MDA), and myeloperoxidase (MPO) against oxidative stress induced by carrageenan. Based on our results of in vivo and in vitro experimentation, we concluded that hydroethanolic extract of T. foenum-graecum is a potential source of phenolic compounds with antioxidant and anti-inflammatory potential.


Introduction
Inflammation is the protective response of the body to noxious stimuli, microbes, and chemicals or irritants [1]. It causes change in vascular permeability, blood flow alteration and increased migration of leucocytes to the inflammatory area, and results in pain, heat, redness, swelling and functional failure of the affected tissue [2]. Several pathological processes, including arthritis, diabetes, cancer, and other severe inflammatory conditions, are usually characterized by pain and inflammation [3]. Although several antioxidant, antinociceptive, and anti-inflammatory medicines are available, these drugs are arguably

Chemical Fingerprinting of T. foenum-graecum Seed Extract 2.2.1. HPLC-PDA Analysis
The analytical and instrumental parameters, i.e., mobile phase composition, flow rate, and temperature, were optimized to achieve good separation among the phenolic profile of T. foenum-graecum extract by following the protocol of Hasany et al. [22]. The highefficiency reverse-phase octadecyl column Spherisorb ODS-2 (Waters Corporation, Milford, MA, USA) bearing a particle size of 10 µm and dimensions (length × internal diameter) of 300 mm × 4.6 mm was used under gradient mode of elution on high-performance liquid chromatography equipped with photo diode array detector (HPLC-PDA) (waters alliance 2998). For the 0.5% acetic acid in water (A), when mixed with organic solvent methanol (B) in the sequence 80A:20B (0-3 min), 70A:30B (3-6 min), 65A:35B (6-9 min), and 55A:45B (10-20 min) and run in the mobile phase at 1 mL/min, the well-resolved peaks were observed within 40 min analysis time.

LC-MS/MS Analysis
UHPCL equipped with mass detector (QTOF-MS/MS) Agilent 6520 was used for the analysis of sample. UHPLC chromatographic conditions were used as mobile phase in gradient mode of elution: (A) 0.1% formic acid in water and (B) 0.1% formic acid in methanol and gradient flow with 10-20% A at 1-10 min, 20-30% A at 10.1-20 min, 30-50% A at 20.1-30 min, and 50-10% A at 30.1-40 min while flow rate was set at 0.5 mL/min. Column used was octadesylsilane waters (4.6 × 100 mm, 2.5 µm) at ambient temperature. Ionization source was ESI (electrospray ionization) source operating at both positive and negative ion modes. In the ionization source, pure nitrogen gas was used as collision as well as drying gas. The capillary temperature was adjusted to 350 • C and nebulizer pressure was set to 35 psi. Ion source parameters, including flow rate of drying gas was maintained to 10 L/min, while VCap, octapole RF peak voltages, fragmentor, and skimmer were maintained to 3500, 740, 150, and 65 V, respectively, and mass range was 150-1200 Da. MS/MS fragmentation was acquired at selected precursor ions of each peak [23,24].

Determination of Total Phenolic and Total Flavonoid Contents
Total phenolic contents in T. foenum-graecum seed extract were examined by the spectrophotometer method adopted by Liu et al. [25]. Briefly, T. foenum-graecum extract (20 µL) was mixed with Folin-Ciocalteu reagent (100 µL), distilled water (1.16 mL), and sodium carbonate solution (200 µL, 20%) and incubated for 30 min at 40 • C. Absorbances of reaction mixture were measured at 700 nm. TPCs in plant extract were calculated from calibration curve prepared by different concentration of gallic acid, and results are given as mg of gallic acid equivalents (GAE)/gram of plant dry weight. For TFC, plant extract (100 µL), deionized water (200 µL), sodium nitrite (250 µL, 5%), and aluminum chloride (250 µL, 10%) were mixed and reaction mixtures were incubated for 6 min. After incubation, sodium hydroxide solution (2.5 mL, 1 M) was added and incubated for another 15 min. The reaction mixture was then diluted with deionized water (2.5 mL) and read the absorbance at 500 nm. TFC in seed extracts were calculated from the calibration curve of catechin as CE (catechin equivalents)/microgram of dry weight of plant extract.

Measurement of Total Antioxidant Capacity (TAC)
For quantification of TAC, ammonium molybdate reagent (0.004 M ammonium molybdate, 0.028 M sodium phosphate, 0.6 M sulphuric acid) was prepared. T. foenum-graecum seed extract (0.1 mL) was mixed with 1 mL of ammonium molybdate reagent and 28 µL of sodium nitrite (5%). The reaction mixtures were heated in water bath at 95 • C for 90 min. The reaction mixtures were cooled and absorbances of reaction mixtures were noted at 765 nm against blank containing ammonium molybdate reagent only. TAC in plant extract was calculated in milligrams of ascorbic acid equivalents per gram of plant dry weight (mg AAEg−1DW) [26].

DPPH Inhibition Assay
The DPPH free radicals were formed by mixing DPPH (8.87 mM) with methanol (99.9%). After which 10 µL of plant extract and 190 µL of DPPH solution was mixed and incubated at 30 • C for 15 min. Absorbances were noted at 520 nm [28].
2.6. In Vitro Cytotoxicity of T. foenum-graecum Seed Extract 2.6.1. Hemolytic Assay Hemolytic activity of T. foenum-graecum seed extract was performed against human RBCs [29]. For this, fresh human blood (5 mL) was centrifuged for 5 min at 4000 rpm. Next, supernatant was drained, and cells were washed three times with phosphate buffer saline and 10% RBC suspension was prepared. T. foenum-graecum seed extract (20 µL) was mixed with 180 µL of RBC suspension in sterile tubes and mixed with agitation. The reaction mixtures were centrifuged (5000 rpm, 5 min) after incubation of 30 min at room temperature. Supernatant (100 µL) was mixed with PBS (900 µL). Triton-X (0.1%) and PBS were used as controls. Absorbances of reaction mixtures were noted at 576 nm.

Cell Culture
The RAW 264.7 cells were provided by the Department of Cellular and Molecular Medicine, University of California, San Diego, CA, USA, and grown in DMEM (Dulbecco's Modified Eagle Medium), 10% of FBS (fetal bovine serum) and streptomycin (100 µg/mL), and penicillin (100 units/mL) solution and incubated at 37 • C and 5% CO 2 under humified conditions. Cells were counted by Trypan blue exclusion method [31].

Animals
Healthy albino rats (150-200 g) were kept at 24 • C in polypropylene cages provided with softwood shavings as bedding material, with standard conditions and free access to standard rodent diet with water ad libitum. Prior to dosing, all rats were adapted to the laboratory setting for a time period of one week [37].

Acute and Subacute Toxicity Analysis
The rats were assigned into two groups, Group I labelled as placebo control receiving saline solution and Group II as the treatment group received dose of 2000 mg/kg BW T. foenum-graecum extract at single. The treatment was given only once on starting day of experimentation and rats were observed for behavioral motor and neuronal activities, including sleep, salivation, eye color, convulsions, lethargy, skin and fur appearance, tremors, and diarrhea. All activities were monitored closely and recorded at different time intervals. In the absence of symptoms of toxicity or mortality during acute toxicity period, subacute toxicity analysis was performed on new set of rats randomly assigned into four different groups and human equivalent doses (250, 500 and 1000 mg/kg BW, respectively) were given daily for 28 days. All experiments were carried out by using OECD (Organization of Economic Co-operation and Development) guidelines and rats were monitored in the same way as in acute toxicity. Animals were euthanized after 28 days, and blood samples, and parts of different organs were stored for hematological, biochemical, and histopathological profiling. The animals were properly disposed of in accordance with the established procedures [38].
2.9. Anti-Inflammatory Potential 2.9.1. Air Pouch Inflammation Air pouch model of inflammation was used for assessment of in vivo anti-inflammatory potential of T. foenum-graecum seed extract [39]. Animals were randomly assigned into six groups. Air pouch on intracapsular region of rats was generated by injecting 5 mL of sterile air into dorsal side. An additional 3 mL of air was injected to air pouch after three days. After seven days of first injection, a carrageenan solution (0.5 mL, 1.5%) was given into the air cavity directly to execute inflammatory response. T. foenum-graecum seed extract treatment (100, 200 and 400 mg/kg) was also given along with carrageenan into the air cavity directly. Animals were sacrificed at different time points (6, 12, 24 h) and through cervical dislocation and pouch tissue were dissected precisely to collect the inflammatory exudate. Cellular infiltration in inflammatory exudate was measured to assess the anti-inflammatory response of plant extract. Morphological changes in the pouch tissues were also observed through histopathological examination.

Carrageenan-Induced Peritonitis
Animals (randomly assigned into six groups) were pretreated orally with T. foenumgraecum seed extract (100, 200, 400 mg/kg BW), saline solution (0.9%, placebo control) and dexamethasone (20 mg/kg BW, standard group) before 30 min of intraperitoneal injection of carrageenan. Then, animals were slaughtered after 4 h of carrageenan injection, and peritoneal cavities of animals were washed with normal saline solution. Cellular count was performed in peritoneal fluid by dissolving 20 µL of peritoneal fluid in Turk's solution (0.38 mL). The collected peritoneal fluid was centrifuged at 10,000 rpm for 10 min and stored at −8 • C for analysis of oxidative stress and lipid per-oxidation parameters [40].

Statistical Analysis
To determine statistical significance, one-way ANOVA was performed followed by multiple comparison tests through Tukey's test. Obtained data were presented as mean ± standard deviation of the mean. IC 50 values were also calculated using regression analysis. All analysis were performed using GraphPad Prism version 8 software (Graphpad Software Inc., San Diego, CA, USA) [46].

Screening of Phytochemicals through HPLC-DAD
T. foenum extract was characterized for its bioactive constituents through HPLC-DAD using operating conditions previously discussed in the materials and method section ( Figure 1). During the analysis, 13 compounds were identified in plant extract that were mostly phenolic acids and flavones. Peak 1 was identified as gallic acid having a response intensity of 0.17 AU (absorption unit), provided in Figure 1. When this peak was extracted for the PDA (photodiode array detector) spectrum, it showed lambda maximum (λ max nm) at 271.2 and 214.6 nm, which corresponds to a standard spectrum as well as the NIST library. The purity of the peak was also assured through the 3D spectrum as well as measuring the purity angle and purity threshold of the peak, which are given in Figure 2 as well as Table 1. The concentration of gallic acid was measured by comparing the area under the peak in comparison to the area under the peak of standard and determined 117.6 ± 1.5 mg/100 g DW. The other peaks that appeared in the chromatogram of the sample were investigated for their identification and quantification by comparison with the standards run as well as the NIST library, and the results are summarized in Table 1. The most abundant antioxidant and antimicrobial compound found was p-coumaric acid with a concentration of 256.7 ± 6.8 g/100 g of DE followed by ferulic acid 168.4 ± 1.8 g/100 g of DE. The results were in agreement with previous reports by [47,48], who reported the abundance of p-coumaric acid in plant extracts. The identification of phytochemicals indicates that antioxidant and other biological properties of extract could be due to the presence of these bioactive compounds. Almost all the phenolic acids identified have a hydroxyl group that could be responsible for the scavenging potential of these compounds [49]. Peaks of the chromatogram were identified through comparison with standards as well as the match index of the UV-visible spectrum of each peak using the NIST library. The identified compounds were presented in order of their elution on the reverse phase column.

UHPLC-Q-TOF Chromatogram of T. foenum
HPLC analysis of plant extract led to the identification and quantification of 13 phenolics, and samples were further characterized through LC-MS/MS (Q-TOF) profile (a detailed description is provided in Table 2, Figure 3). A total ion current (TIC)-based chromatogram of plant extract of T. foenum is presented in Figure 2, and the precursor ion of each peak was further processed for fragmentation pattern (MS/MS) and compared with the NIST library as well as literature reported for the identification of phytochemicals present in the sample. The results are summarized in Table 2, which described each component with retention time in order of their elution order and fragmentation pattern. Although numerous peaks were recorded in the chromatogram, we reported almost 18 compounds, which were identified through the NIST library or literature. MS profiling of peak 1 (RT 2.34 min) produced a parent ion peak at [M − H]+ m/z 441.0840 Da and daughter ions 251.0365, 233.0297 and 124.9848 Da, which corresponds to catechin gallate, and its fragmentation pattern revealed it would be catechin 3-O-gallate. MS spectra are provided in Supplementary Materials Figure S1. The other peaks appeared in chromatogram were also processed for MS1 as well as MS2 at both positive and negative ion mode, and the results were compared with literature and are described in Table 2. Almost 18 compounds were confirmed through literature and their detailed description is provided in Supplementary Materials Figures S1-S17.

Determination of TPC, TFC and In Vitro Antioxidant Activities
The results of the in vitro antioxidant profiling of T. foenum-graecum seed extract are presented in Table 3. The results reveal that T. foenum-graecum seed extract displayed excellent antioxidant potential with total phenolic contents of 454.93 ± 3.57 mg GAE/g, total flavonoid contents (TFC) of 135.04 ± 2.12 µg/CE and total antioxidant capacity (TAC) of 162.51 ± 3.81 per gram of dry plant extract. The extract represented a concentrationdependent activity of DPPH inhibition and ABTS scavenging assay with an IC 50 value of 24.7 ± 2.70 and 15.8 ± 0.87 µg/mL, respectively, which is similar to standard.

Toxicological Analysis of T. foenum-graecum Seed Extract
The results of hemolytic activity show that T. foenum-graecum seed extract has negligible toxicity in comparison to Triton-X (positive control), as shown in Figure 4. Further, the extract exhibits dose-dependent activity, and the hemolysis of erythrocytes increased with an increase in concentration of T. foenum-graecum seed extract. The concentration required for hemolysis of 50% RBCs (HC 50 values) was estimated to be 2838 µg/mL, which is significantly (p < 0.001) different from HC 50 of Triton-X-100 (64.98 µg/mL). Similarly, T. foenum-graecum extract showed a dose-dependent increase in clot lysis activity. Standard streptokinase (0.5 mL) and T. foenum-graecum extract at a high concentration (300 µg/mL) showed significant (p < 0.001) clot lysis activity of 81.82% and 63.01%, respectively comparing with the negative control (1.36%).

Cytotoxicity Determination
The results of cytotoxicity of T. foenum-graecum seed extract against RAW 264.7 cells are presented in Figure 5. Increased concentration of T. foenum-graecum seed extract has a negative impact on cell viability. The IC 50 values for T. foenum-graecum seed extract and standard doxorubicin against RAW 264.7 cells were 1055 and 614.9 µg/mL, respectively. The results exhibit that plant extract showed low-to-moderate cytotoxicity in a concentration-dependent manner. Cell viability percentage was decreased with an increase in the concentration of plant extract. Cell viabilities in murine macrophages incubated with a different concentration (100-1000 µg/mL) of T. foenum-graecum seed extract were 91.53%, 87.64%, 76.79%, 73.67%, 71.06%, 68.25%, 62.47%, 56.87%, 55.60% and 47.78%, respectively. At concentrations (100-300 µg/mL), little cytotoxic effects were observed, and these concentrations were adopted further to examine the anti-inflammatory activity of T. foenum-graecum seed extract.

Effect of T. foenum-graecum Extract on TNF-α and IL-6
The tested concentration (50-300 µg/mL) of T. foenum-graecum seed extract showed a substantial decrease in the production of IL- 6 (Figure 4b) in comparison to LPS-stimulated macrophages (2177.83 ± 37.56 µg/mL for TNF-α and 3894.42 ± 49.73 pg/mL for IL-6), suggesting significant in vitro anti-inflammatory potential. Figure 4a,b, demonstrates the effect of T. foenum-graecum seed extract on the production of TNF-α and IL-6 in RAW 264.7 cells after stimulation with LPS at various concentrations. The results indicate that the plant extract inhibited both TNF-α and IL-6 production significantly (p < 0.001) at different concentrations of 50-300 µg/mL, with inhibition rates of 13.21%, 38

NO and PGE2 Quantification
The results of NO and PGE2 inhibition by T. foenum-graecum seed extract are presented in Figure 6C,D. The findings show that both NO and PGE2 are significantly (p < 0.001) inhibited by T. foenum-graecum extract in culture of RAW 264.7 cells, reaching to the level of the control (without LPS) at higher concentrations of plant extract in a dose-dependent manner ( Figure 6C,D). Moreover, a significant (p < 0.0001) increase in NO and PGE2 concentration was recorded after treatment with LPS, which was markedly (p < 0.001) restored to the normal level after treatment with standard and plant extract with IC 50 values of 11.40 and 122.0 µg/mL. The results from the acute toxicity analysis show that T. foenum-graecum seed extract did not show any harmful effects in the treated animals, as compared with the control group at a dosage of 2000 mg/kg BW. No morbidity or mortality was noticed in experimental animals during acute toxicity analysis. Consequently, the LD 50 value of T. foenum-graecum seed extract examined was calculated to be >2000 mg/kg BW. During acute toxicity analysis, none of the animals exhibited any changes in behavior or symptoms related to the circulatory, respiratory, central, and autonomic nervous system ( Table 4). As T. foenumgraecum extract treatment did not exhibit any adverse effects in the acute study, a human equivalent dose was selected for the long-term subacute toxicity study of 28 days. In subacute toxicity analysis, the effect of plant treatment on weight, relative organ weight, liver and kidney function indices, and hematological and histopathological parameters was evaluated. Table 5 shows the variation in body weight of rats after treatment with different doses of T. foenum-graecum seed extract. The body weight of animals treated with plant extract at different dosages (250, 500, and 1000 mg/kg) did not differ significantly (p > 0.05) from those in the control group. Similarly, in comparison to the control group, there was no significant (p > 0.05) difference in the absolute weights of the kidney and liver of treated animals ( Table 6).

Hematological and Biochemical Indices
The results of the subacute toxicity of T. foenum-graecum seed extract treatment on hematological parameters are presented in Table 7, and findings reveal that hematological parameters of treatment animals were not affected after treatment with plant extract for 28 days. Similarly, treatment of T. foenum-graecum seed extract in subacute toxicity did not cause any significant change in liver and kidney function indices (Table 8). No significant alteration in AST, ALT, ALP, γ-GT, total proteins, bilirubin, glucose, creatinine, total cholesterol, total glycerides, or blood urea nitrogen was detected in experimental animals in comparison to the control group. The little variation represented has no clinical significance, because values recorded are within the normal range of rats. Histopathological examinations of liver and kidney tissues did not exhibit any morphological alterations or abnormalities under the light microscope.

Air Pouch Model of Inflammation
To evaluate the anti-inflammatory potential of T. foenum-graecum seed extract, inflammation of air pouch model was utilized, and inflammatory exudate was analyzed for different blood cells associated with inflammation ( Figure 7). In inflammatory exudate of rats treated with carrageenan only, white blood cell (15 × 10 3 cells/mL) count was about 20-fold higher compared to the control group (0.83 × 10 3 cells/mL). Dexamethasone (10 mg/kg BW) treatment caused an almost 10-fold reduction (8.13 × 10 3 cells/mL) in WBC count. T. foenum-graecum seed extract suppressed the WBC population dose-dependently. For example, WBC count in the 200 mg/kg BW treatment group was equal to dexamethasone, and in the 400 mg/kg BW treatment group the effect was more significant than dexamethasone. As shown in Figure 5, injection of carrageenan caused a noticeable increase in monocytes as compared to the carrageenan control group. However, T. foenum-graecum extract by doses of 200 and 400 mg/kg BW led to significant (p < 0.001) reductions in monocytes (1.37 × 10 3 and 1.31 × 10 3 , respectively) as compared to the carrageenan control (3.21 × 10 3 ). Moreover, T. foenum-graecum extract significantly reduced the carrageenaninduced proliferation of eosinophils and neutrophil in treatment groups, as compared to carrageenan-treated animals, in a dose-dependent manner, and this effect is comparable to dexamethasone (20 mg/kg BW), particularly at 400 mg/kg BW of T. foenum-graecum. Similarly, histopathological examination of air pouch tissue also exhibited a change in the thickness of the air pouch membrane and marked cellular influx to the inflammatory site with increased inflammatory response. The treatment with T. foenum-graecum extract and dexamethasone caused an increase in membrane thickness as compared to the carrageenantreated group, in which the membrane was narrow and condensed and cellular influx was also reduced (Figure 8).

Carrageenan-Induced Peritonitis
Treatment of T. foenum-graecum seed extract before 30 min of intraperitoneal injection of carrageenan significantly (p < 0.001) reduced total WBC and neutrophil count in peritoneal exudate (Figure 9). Injection of carrageenan caused a 16-fold increase in WBC count (11.12 × 10 3 ) as compared to the control (0.66 × 10 3 ), and dexamethasone and T. foenumgraecum (200 and 400 mg/kg dose) decreased it to 6.97 × 10 3 . Moreover, T. foenum-graecum extract significantly reduced the neutrophil count in peritoneal exudate of treated rats as compared to the control group.  Figure 9. Anti-inflammatory effect A (Neutrophils) and B (Total WBCs) of hydroethanolic extract of T. foenum-graecum on carrageenan-induced peritonitis. Rats were pretreated orally with different concentrations of T. foenum-graecum extract and dexamethasone (20 mg/kg body weight). Data are expressed as mean ± SD. ***, significant at p < 0.001.

Effect of T. foenum-graecum on Antioxidant Enzymes and Stress Markers
The levels of enzymatic antioxidants, lipid peroxidation and oxidative stress markers in air pouch exudates and peritoneal fluid were significantly (p < 0.001) altered in the carrageenan-treated group as compared to the control group. However, treatment with dexamethasone and T. foenum-graecum significantly reversed the carrageenan-induced changes in these parameters. MPO and MDA levels in the carrageenan-treated group were significantly higher as compared to those of the control group (Tables 9 and 10). Dexamethasone and T. foenum-graecum extract effectively reduced the MDA and MPO levels in the air pouch and peritoneal exudate, and the effect was dose dependent, with 400 mg/kg BW recorded as most effective dose. Similarly, carrageenan treatment increased the level of superoxide dismutase and catalase and hence TAS significantly (p < 0.001) at the inflammatory site, while dexamethasone significantly reduced the level of these enzymes, and this effect was similar to the T. foenum-graecum group at 400 mg/kg BW dose treatment group. Furthermore, total oxidant level (TOS) was also significantly higher among the carrageenan treatment group in comparison to that of the control group, which is significantly (p < 0.001) decreased in both dexamethasone and T. foenum-graecum treatment groups. The effect at higher doses of T. foenum-graecum (400 mg/kg) was similar to the dexamethasone-treated group.  Each value is mean ± SD of three replicates; values with superscripts a, b and c are significant at 0.05, 0.01 and 0.001. Eq: Equivalent.

Discussion
The in vitro and in vivo antioxidant and anti-inflammatory properties of the hydroethanolic extract of T. foenum-graecum were studied using different methods, and the overall findings have been corroborated. T. foenum-graecum is an old medicinal herb that dates back to Egyptian times and has significant antipyretic, anti-inflammatory and anti-nociceptic properties. In the current study, the phytochemical screening of T. foenumgraecum extract exhibited a considerable amount of phenolics and flavonoids and showed significant antioxidant activity. The results of our antioxidant and polyphenolic analysis are largely in agreement with Kenny et al. [56], who reported total phenolic contents (106.316 ± 0.377 mg GAE/g), FRAP (77.352 ± 0.627) and DPPH (35.338 ± 0.908) mg of Trolox equivalents per gram of Fenugreek ethyl acetate extract. Previously, Akbari et al. [57] also reported the ABTS and DPPH assays with IC 50 values of 161.3 ± 2.21 and 172.6 ± 3.1 µg/mL. Similarly, TFC and TPC, as well as the fenugreek, were also reported (14.417 ± 0.23 mg QE/g and 38.97 ± 0.34 mg GAE, respectively).
Evaluating the toxicity profile of medicinal plants and plant-based products is typically a preliminary step for the screening of the therapeutic potential of plant-derived products [19,57]. Another approach for the evaluation of cytotoxicity is hemolytic assay, which is described by the subsequent release of hemoglobin after erythrocytes lysis [58].
The polyunsaturated fatty acids and hemoglobin mostly attack RBCs because of their redoxactive oxygen transportation property [59]. Consequently, the oxidative process damages the proteins and lipids in the erythrocyte membrane during hemolysis [60]. This damage is associated with several other factors, including oxidative drugs, a high quantity of transition metals, radiation, insufficient antioxidant defense system and hemoglobinopathies [61]. Hemolysis of erythrocytes occurs when they are exposed to toxic natural or synthetic compounds. The HC 50 of T. foenum-graecum extract is very high compared to Triton-X-100 and thus supports its application as a successful pharmaceutical drug in practice. The thrombolytic potential of T. foenum-graecum extract is an important finding, which may have implications in cardiovascular health, especially in atherothrombotic patients. These results are in agreement with Ktari et al. [62], which also reported cytotoxicity of T. foenum-graecum extract, and no hemolytic activity was reported against bovine RBCs.
Many of the classic experiments in the field of inflammation have been performed using murine macrophages [4,5]. The hydroethanolic extract of T. foenum-graecum demonstrated a noticeable inhibition of pro-inflammatory cytokines, NO and PGE2 after LPS stimulation. The activation of pro-inflammatory cytokines is among the most fundamental processes that occur during inflammatory pathways [63]. TNF-α is a potent pro-inflammatory cytokine produced by a variety of immunocompetent cells, such as neutrophils, dendritic cells, macrophages, and T helper cells, and is capable of attracting immune cells to the inflammatory site to initiate the inflammatory process [64]. The hydroethanolic extract of T. foenum-graecum (50-300 µg/mL) presented dose-response anti-inflammatory activity, since it showed greater TNF-α and IL-6 inhibition in the culture of RAW 264.7 cells after stimulation with LPS. TNF-α and IL-6 are measured as basic markers of pro-inflammatory processes produced by macrophages and have the ability to activate T cells [65]. As a result, inhibiting these cytokines is thought to be an efficient way to prevent and cure a variety of inflammatory disorders [66]. The hydroethanolic extract of T. foenum-graecum also inhibited NO and PGE2 production by RAW 264.7 cells and showed similar results to those of TNF-α and IL-6 in relation to the positive control dexamethasone, evidencing its antioxidant and anti-inflammatory action [67]. NO is a powerful radical that regulates the growth, function, and death of a variety of cell types involved in inflammatory and immunological responses. Excessive NO generation has been linked to the pathophysiology of oxidative damage and inflammation [68]. Previous inhibition of pro-inflammatory cytokines by T. foenum-graecum extract has also been reported in some studies [35]. The anti-inflammatory attributes of T. foenum-graecum may be due to the large number of polyphenolic substances that have been reported good anti-inflammatory agents in previous studies [19,20].
Oral administration of extracts is the most appropriate and economical method of drug delivery in animal models during toxicity analysis [69]. Moreover, acute oral toxicity analysis in the rat model can effectively predict human acute lethal dosages in clinical setups [70]. The body weight of animals is an important index for the determination of the toxicity of synthetic or natural compounds [71]. In the current study, there was no abnormal change in the weight of animals among the treatment group and control group up to a dose of 1000 mg/kg. Likewise, variation in organ weight is also a good indication for plant-induced abnormalities, which are commonly linked with treatmentrelated effects. There were no significant variations in the body or organ weight of animals after 28 days of treatment with hydroethanolic extracts of T. foenum-graecum seed extract. All of the animals showed normal weight gain, with no significant differences among control and treatment groups. The hematopoietic system in both animals and humans is extremely sensitive to toxic substances and acts as a key indicator of pathological and physiological condition [72]. In toxicological assessment, biochemical parameters are of prime significance due to their extreme sensitivity and ability to respond against changes induced by toxicological substances [29]. These biochemical markers have a significant role in the evaluation of toxicological changes induced by natural or chemical substances [73]. In the current study, none of the biochemical parameters showed significant (p > 0.05) changes from the untreated control group. Similarly, the histopathological examination of both liver and kidney tissues was also found to be morphologically normal. Previously, some studies also reported the acute and subacute toxicities of T. foenum-graecum extract, and no toxicological effects were reported on biochemical and hematological markers [74,75].
Carrageenan-induced inflammation in rats is a useful approach for testing natural products with potential anti-inflammatory activity and also for further elucidating their mechanism of action [76]. Carrageenan injection initiates an acute inflammatory response linked with hyperalgesia, usually classified by edema and increased response to mechanical and thermal stimuli [40]. The carrageenan-induced inflammation is linked with increased leukocytes migration, mainly neutrophils, and enhanced myeloperoxidase (MPO) activity [77]. Pro-inflammatory mediators such as MPO and NOx and proinflammatory cytokine levels (TNF-α and IL-6) can modulate the inflammatory response and can significantly alter the amplitude of leukocyte activation and migration [78]. The air pouch model is a useful in vivo model for studying localized inflammation with no systemic effects. The injection of air subcutaneously into the thoracic area induces morphological alterations in the pouch's cellular lining that lasts several days [39]. This structural change develops in pouch lining, similar to that which occurs in the synovial cavity. Carrageenan injection directly into the air cavity produces an inflammatory response in patients with rheumatoid arthritis and several other chronic inflammatory diseases. Therefore, this model can be employed to screen antiinflammatory compounds [79]. One of the major benefits of air pouch inflammation over carrageenan injection directly into the knee joint is the increased volume of pouch exudate, allowing for the measurement of several parameters from each animal [80,81]. Peritonitis induced by carrageenan is a well-established model of acute inflammation frequently used for testing novel anti-inflammatory drugs focusing on analysis or quantification of cellular migration, vascular permeability, and measurement of inflammatory parameters [82]. In the current study, both the air pouch and carrageenan-induced peritonitis showed significant infiltration of leukocytes, neutrophils, and monocytes to inflammatory exudates. The lining of the air pouch was intensely invaded with inflammatory cells. Neutrophil migration to the joints of rheumatoid arthritis patients causes the destruction of synovial tissue, cartilage structure and bones through the release of different proteases and harmful oxygen metabolites. The administration of T. foenum-graecum extract efficiently abrogated the cellular influx to the pouch exudates and reduced the morphological changes in the lining of the pouch tissues.
The production of free radicals at the inflammatory site is one of the major mechanisms of tissue damage produced by several inflammatory disorders [11,12]. Activated neutrophil infiltration to the inflammatory sites is a significant source of proinflammatory mediators and oxygen-derived free radicals, which induces inflammatory reactions [83]. It has been shown that, after the injection of carrageenan, the level of free radicals, and thus total oxidant status (TOS), rises in both the air pouch and peritoneal exudates [84]. These free radicals may target the plasma membrane causing malondialdehyde (MDA) to accumulate. MDA is a basic marker of oxidative stress. Myeloperoxidase is an enzyme found in leucocytes, involved in the formation of a wide range of reactive oxygen species [85]. MPOderived oxidants have been shown to lead to tissue damage during inflammation [42,43]. Tissue damage related to oxidative stress can be reversed via the CAT and SOD enzyme. The activity of these enzymes controls the cytotoxic properties of toxic free radicals [41,44]. In this study, there were significant increases in catalase and SOD enzyme activity in both the air pouch and peritoneal exudate; hence, there was an increase in TAS level after treatment with T. foenum-graecum extract. Furthermore, there was a substantial decrease in MDA and MPO levels after treatment with T. foenum-graecum extract. Oxidative stress may, therefore, be inhibited by hydroethanolic extract of T. foenum-graecum. A decrease in MDA and MPO activity is linked to reduced inflammatory progression, and these effects could be attributed to polyphenols, quercetin, and gallic acid, among other compounds found in the plant extract.

Conclusions
In conclusion, hydroethanolic extract of T. foenum-graecum contains a significant number of polyphenolic compounds, which decreases cellular infiltration, lipid peroxidation, and the level of pro-inflammatory cytokines and inflammatory mediators. Pre-treatment of animals with T. foenum-graecum extract significantly improves tissue antioxidant status, which ameliorates oxidative stress and inflammatory processes induced by carrageenan. Moreover, the T. foenum-graecum extract was also characterized through ESI-Q-TOF MS/MS as well as HPLC-PDA to investigate the phenolic profile likely responsible for bioactivity. However, further studies are essential and need to be conducted to investigate the mechanisms of action, bioaccessibility, and bioavailability of these compounds for their proper nutritional and medicinal properties.