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Fatima, H. Antioxidant, and Anti-Inflammatory Potential of Trigonella foenum-graecum. Encyclopedia. Available online: https://encyclopedia.pub/entry/20081 (accessed on 17 November 2024).
Fatima H. Antioxidant, and Anti-Inflammatory Potential of Trigonella foenum-graecum. Encyclopedia. Available at: https://encyclopedia.pub/entry/20081. Accessed November 17, 2024.
Fatima, Hina. "Antioxidant, and Anti-Inflammatory Potential of Trigonella foenum-graecum" Encyclopedia, https://encyclopedia.pub/entry/20081 (accessed November 17, 2024).
Fatima, H. (2022, March 02). Antioxidant, and Anti-Inflammatory Potential of Trigonella foenum-graecum. In Encyclopedia. https://encyclopedia.pub/entry/20081
Fatima, Hina. "Antioxidant, and Anti-Inflammatory Potential of Trigonella foenum-graecum." Encyclopedia. Web. 02 March, 2022.
Antioxidant, and Anti-Inflammatory Potential of Trigonella foenum-graecum
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This entry is adapted from the peer-reviewed paper 10.3390/antiox11020364

Trigonella foenum-graecum (Fenugreek) is a valuable medicinal plant belonging to the Fabaceae family. Plant seeds are mostly used in Asian, African, and Mediterranean countries as major ingredients of daily diets and in domains such as cosmetics, fragrances, beverages, nutrition, medicine and industry. The major pharmacological attributes of fenugreek are hypotensive, antioxidant, antiviral, anticarcinogenic, galactagogue, laxative, febrifuge, carminative, anticholesterolemic, antimicrobial, etc. 

T. foenum-graecum air pouch inflammation antioxidants oxidative stress markers cellular infiltration lipid peroxidation peritonitis

1. 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 inaccessible, costly, less effective, and have multiple side effects [4]. Non-conventional medicines occupy a significant place in healthcare systems, as more than 80% of people worldwide depend on them for their daily healthcare requirements, particularly in Asia and Africa [5].
Trigonella foenum-graecum (Fenugreek) is a valuable medicinal plant belonging to the Fabaceae family [6]. Plant seeds are mostly used in Asian, African, and Mediterranean countries as major ingredients of daily diets and in domains such as cosmetics, fragrances, beverages, nutrition, medicine and industry [7]. The major pharmacological attributes of fenugreek are hypotensive, antioxidant, antiviral, anticarcinogenic, galactagogue, laxative, febrifuge, carminative, anticholesterolemic, antimicrobial, etc. [8][9]. Overproduction of nitrogen and reactive oxygen species, as well as insufficient quenching/stabilization in the body, causes oxidative stress, which destroys important biomolecules (nucleic acids, lipids, and proteins) [10]. Several human diseases, including cardiac disorders, diabetes, cancer, inflammation, and neurodegenerative diseases, are exacerbated or triggered by oxidative damage to these biological molecules [11]. Plant-derived natural products, particularly polyphenolics, and related antioxidant phytochemicals have a diverse range of bioactivities, including anti-inflammation, owing to their ability to quench and alleviate oxidative stress in biological systems, thus restoring health [12]. As a result, the latest research focus has switched towards plant-based natural products as one of the most encouraging sources of curative agents of inflammation and pain [13].
Plants are an abundant source of bioactive compounds, including phenolics, phenolic acids, simple phenolics, flavonoids, derivatives of hydroxycinnamic acid, and anthocyanins [14]. Based upon their physiological properties, including scavenging of free radicals, as well as antimutagenic, anti-inflammatory, and anti-carcinogenic effects, phenolic compounds of different classes continue to attract much scientific attention [15]. Previously, anti-inflammatory properties of T. foenum-graecum have been reported against the carrageenan-induced paw edema model of inflammation, but there is no report available against carrageenan-induced air pouch inflammation and carrageenan-induced peritonitis. The antioxidant effects of polyphenolics are mainly due to their redox potential, serving as hydrogen donors, potent reducing agents, metal chelators, along with singlet oxygen quenchers [16][17][18]. Several phenolics and flavonoids have been demonstrated to have significant effects on the functioning of the immune system, including inflammatory processes [19]. Quercetin, luteolin, hesperidin, and apigenin are flavonoids containing potential anti-inflammatory activities [20]. 

2. 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 (λmax 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 [21][22], 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 [23]. 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.
Figure 1. (A) Chromatogram of high-pressure liquid chromatography equipped with diode array detector (HPLC-DAD) at 280 nm and (B) extracted spectrum (200–400 nm) of each peak.
Figure 2. Purity plots of representative peaks, (A) gallic acid, and (B) trihydroxybenzoic acid.
Table 1. High-pressure liquid chromatography equipped with diode array detector (HPLC-DAD) summary results of each peak.
Peak No. Compound Name Retention Time ʎmax (nm) Concentration
mg/100 g DW
1 Gallic acid (GA) 4.4 271.2, 214.6 117.6 ± 1.5
2 Trihydroxybenzoic (TBHA) acid 7.3 260.5, 221.4 112.6 ± 5.6
3 Protochateuic acid (PTA) 13.4 291.4, 260.5, 214.4 103.8 ± 2.4
4 Ferulic acid (FA) 14.1 323.7, 236.8 168.4 ± 1.8
5 Protocatechualdehyde (PTCA) 15.0 308.2, 279.5, 229.7 98.4 ± 2.3
6 p-coumaric acid (PCA) 18.4 309.4, 229.7 256.7 ± 6.8
7 Vanillic acid (VN) 19.7 279.5, 246.4 57.9 ± 2.4
8 Protocatechualdehyde (PhA) 22.2 280.6, 224.3 116.8 ± 1.9
9 Vaniline (VNA) 23.6   87.5 ± 2.4
10 Apigenic acid (APG) 26.9 294.6, 261.7, 219.8 117.7 ± 3.6
13 Syringic acid (peak 13) 27.2 310.5, 222.5 95.6 ± 11.3
14 p-hydroxybenzoic acid (PHBA) 28.1 328.5, 234.4 113.7 ± 3.8

3. 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, researchers 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 (supplementary could be found in https://www.mdpi.com/2076-3921/11/2/364#supplementary). 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.
Figure 3. UHPLC-Q-TOF chromatogram of T. foenum.
Table 2. Ultra-high-pressure liquid chromatography equipped with electrospray ionization-quadrupole time-of-flight mass detector (UPLC-ESI-Q-TOF-MS/MS) characterization of phytochemicals of T. foenum.
Serial# Compound Detected Retention Time (min) MS M − H (Predicted) M − H (Found) MS2 Formula Reference
1 Catechin 3-O-gallate 2.34 442.09 441.0827 441.0840 251.0365,
233.0297,
124.9848		 C22H18O10 [23][24]
2 4-O-methylepigallocatechin 11.20 320.0896 321.1569 321.1468 303.1364, 241.1007 C16H16O7 [24][25][26]
3 Sativanone 12.84 300.1398 299.1425 299.1474 283.1462, 223.0883, 179.0974 C17H16O5 [24][26]
4 Gallic acid 13.36 170.12 169.1345 169.1342 125.4622 C7H6O5 [24][26]
5 Kampferol 18.34 286.23 285.1487 285.1425 245.1357
189.1109		 C15H10O6 [24][27]
6 Methylviolanone 18.57 330.1103 329.1103 329.1539 311.1424, 279.1167 C18H18O6 [20][24]
7 homovanilic acid-hexoside 19.23 344.138 343.1135 343.1762 279.1162, 255.1146, 181.0796, 163.0396 C15H20O9 [24][25]
8 5-O-Feruloylquinic acid 20.99 368.1568 367.1448 367.1648 337.1599, 293.1295, 191.0640 C17H20O9 [23][24][26]
9 Cyanidin 25.17 288.2 287.2 287.1768 287.1768, 253.095, 167.1364, 149.1272 C15H11O6 [27][28]
10 Coumaroylquinic acid 26.09 338.1002 337.1927 337.1900 265.1338,
173.1554		 C16H18O8 [25][26][27]
11 Quercetin 27.15 302.236 301.1915 301.1950 295.1747
291.1551, 163.1428		 C15H10O7 [20][26]
12 Schisandrin C 27.49 384.1573 385.1646 385.1393 339.1704, 137.0888, 123.0786 C22H24O6 [20][26]
13 p-coumaric acid O-hexoside 27.95 326.1455 325.1529 325.1552 295.1080, 165.0856, 123.0763 C15H18O8 [20][26]
14 Hydroxytyrosol-4-o-glycoside 29.29 316.2158 315.2085 315.2140 251.1190, 237.1404,
237.1042
177.1555		 C14H20O8 [24][29]
15 3-Methoxysinensetin 31.42 402.2315 403.2388 403.2755 399.2602,
383.2710, 199.0671		 C21H22O8 [26][28]
16 Pinoresinol 31.74 358.1416 357.1343 357.2531 313.2297, C20H22O6 [26][28]
17 Glycitin 32.45 446.3213 447.3286 447.3361 403.3065, 368.4185 C22H22O10 [20][26]
18 Dihydroferulic acid 4-O-glucuronide 33.52 372.1156 371.1054 371.1674 291.1162, 266.9912,
73.0421		 C6H20O10 [24][29]

4. 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 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.
Table 3. In vitro antioxidant activities and total phenolic and flavonoid contents of T. foenum-graecum seed extract.
Antioxidant Assay T. foenum-graecum Ascorbic Acid
Total phenolic contents (GAE mg/g) 454.93 ± 3.57 -
Total flavonoid contents (CE µg/g) 135.04 ± 2.12 -
Total antioxidant capacity (AAE mg/g) 162.51 ± 3.81 -
DPPH inhibition assay (IC50) (µg/mL) 24.7 ± 2.70 a 25.05 ± 1.45 a
ABTS cation inhibition (IC50) (µg/mL) 15.8 ± 0.87 a -
Each value is mean ± SD of three replicates. Alphabets a shows significant difference at (p < 0.05). -, not determined; AAE, ascorbic acid equivalents; CE, catechin equivalents; GAE, gallic acid equivalents; (µg/mL), microgram per milliliter.

5. 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 (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%).
Figure 4. Hemolytic (a) and thrombolytic activity (b) of different doses (50–300 µg/mL) of hydroethanolic extract of T. foenum-graecum seeds through hemolytic and thrombolytic assays. (***, significant at p < 0.001).

6. 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 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.
Antioxidants 11 00364 g005 550
Figure 5. Cytotoxic activity (cell viability) of hydroethanolic extract of T. foenum-graecum and doxorubicin (standard) against RAW 264.7 macrophage through MTT assay.

7. 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 (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.

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