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.

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.

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 concentration-dependent activity of DPPH inhibition and ABTS scavenging assay with an IC₅₀ value of 24.7 ± 2.70 and 15.8 ± 0.87 µg/mL, respectively, which is similar to standard.

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 (HC₅₀ values) was estimated to be 2838 µg/mL, which is significantly (p < 0.001) different from HC₅₀ 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%).

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 IC₅₀ 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.

The tested concentration (50–300 μg/mL) of T. foenum-graecum seed extract showed a substantial decrease in the production of IL-6 (1889.92 ± 19.32, 1338.73 ± 12.14, 1330.42 ± 10.56, 1108.32 ± 20.87, 947.33 ± 16.45, 748.52 ± 9.45, respectively) (Figure 4a and TNF-α (2158.12 ± 31.87, 1715.21 ± 28.68, 1518.68 ± 26.98, 1045.01 ± 23.55, 741.33 ± 18.84, 522.99 ± 19.03, respectively) (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%, 38.91%, 38.52%, 49.10%, 56.50%, 65.62% and 44.58%, 55.95%, 61.03%, 73.16%, 80.96%, 86.57%, respectively, with IC₅₀ values of 192.7 µg/mL and 72.03 µg/mL.