Antidiabetic, antihyperlipidemic and immunomodulatory effects of Trigonella foenum-graecum extract in streptozotocin-induced diabetic rats

Study design and ethical approval

The study was carried out in the Department of Pharmacology and Therapeutics, King George’s Medical University, Lucknow, with the objective of assessing the antidiabetic, lipid-lowering and immunomodulatory effects of TFE extract. Ethical clearance was obtained from the Institutional Animal Ethics Committee (IAEC No. 183/IAEC/2023), and all experimental procedures complied with CCSEA regulations.

Experimental animals

Adult male Wistar rats (160–200 g) were used for the experiment. Thirty-six animals were procured from CDRI (Old Campus), Lucknow, and housed in the Institutional Animal Facility under standard laboratory conditions: Ambient temperature 24–27°C, humidity 60–65% and a 12 h light/dark cycle. Food and water were provided ad libitum. Two diets were used – a normal pellet diet (NPD) and a high-fat diet (HFD). The HFD, sourced from Bharat Science Solution Company, Unnao (Uttar Pradesh), delivered 58% calories from fat, 25% from protein and 17% from carbohydrate. All rats were acclimatised for 1 week before starting the protocol.

Drugs and chemicals

STZ was obtained from SRL Diagnostics (India) and dissolved in freshly prepared cold 0.1 M citrate buffer (pH 4.5) immediately before intraperitoneal use. Metformin, purchased from Abhilasha Pharma Pvt. Ltd. (Ankleshwar, Gujarat), was dissolved in distilled water and administered orally. TFE extract in powdered form was procured from Tokyo Chemical Industry Co., Ltd. (TCI Chemicals), Tokyo, Japan, and suspended in distilled water for oral administration at doses of 200, 500 and 1000 mg/kg body weight (BW). All other chemicals used were of analytical grade.

Induction of T2DM

All groups except Group I (Normal Control) were placed on an HFD for 28 days to promote insulin resistance. On Day 29, following an overnight fast, these rats received a single intraperitoneal injection of STZ (40 mg/kg BW), prepared in freshly made cold citrate buffer (0.1 M, pH 4.5) and administered within 15–20 min.^[12] Group I animals were given citrate buffer alone. After 1 week, fasting blood glucose (FBG) was measured by tail vein sampling using a glucometer (Accu-Chek). Animals with glucose values above 250 mg/dL were confirmed diabetic and enrolled for further treatment.^[13]

Experimental design

A total of 36 rats were enrolled and, after 1 week of acclimatisation, randomly allocated into six groups of six animals each. Group I served as Normal Control (NPD-fed + citrate buffer injection). Group II represented the Diabetic Control (HFD-fed + STZ 40 mg/kg + distilled water). Groups III, IV and V consisted of diabetic rats receiving TFE at 200, 500 and 1000 mg/kg/day, respectively. Group VI was treated with metformin (100 mg/kg/day).^[14,15] STZ was administered on Day 29, and treatment with TFE or metformin was initiated thereafter for 21 days, following confirmation of hyperglycaemia (FBG >250 mg/dL) [Figure 1].

Close

Figure 1:

Flowchart showing experimental design, animal grouping, induction of diabetes using HFD and STZ and subsequent treatment with TFE or metformin. HFD: High-fat diet, NPD: Normal pellet diet, STZL: Streptozotocin, TFE: Trigonella foenum-graecum extract, BW: Body weight, FBS: Fasting blood sugar.

Export to PPT

BW, FBG, lipid profile and serum resistin were evaluated at 3 time points: Baseline (Day 0), week 5 (post-STZ) and week 8 (after treatment). Blood was collected through tail vein puncture and, at the terminal stage, through the retro-orbital plexus under anaesthesia.^[16,17] These assessments were aimed at determining the antidiabetic, lipid-regulating and immunomodulatory potential of TFE in STZ-induced diabetic rats on HFD.

Blood collection

Blood was obtained at baseline, week 5 and week 8 for analysis of fasting blood sugar (FBS), body weight, lipid profile and serum resistin. For tail vein collection, animals were gently restrained, and tails were briefly immersed in lukewarm water to dilate the veins. After cleaning with alcohol, blood was drawn from the lateral tail vein using a fine needle inserted nearly parallel to the vessel. Correct entry was confirmed by flashback, and samples were aspirated slowly.^[16] At study termination, retro-orbital blood was collected under light anaesthesia using a capillary tube.^[17] Serum was separated and preserved for subsequent biochemical and immunological analyses.

Parameters measured

Statistical analysis

Data entry and tabulation were performed using Microsoft Excel (Office 365), and statistical analysis was carried out with the Statistical Package for the Social Sciences version 24.0. Results were expressed as mean ± standard deviation. Paired t-test was used for within-group comparisons across time points, while intergroup variations were assessed using one-way analysis of variance followed by Tukey’s honest significant difference post hoc test. Significance was defined at P < 0.05, with P < 0.01 and P < 0.001 indicating stronger levels of significance.

Intergroup comparison of BW at different time intervals

At baseline (Day 0), mean BWs were similar across groups (P = 0.853), ranging from 164.00 ± 6.75 g in Group I to 168.32 ± 4.05 g in Group II. By the 5^th week, all diabetic groups (II–VI) recorded significantly greater weights compared with Group I (P < 0.001), with the maximum increase observed in Group VI (263.01 ± 3.46 g). At the 8^th week, Group II continued to show the highest weight (262.82 ± 5.84 g), while Groups III–V demonstrated reductions, most pronounced in Group IV (196.24 ± 8.61 g). The metformin-treated group (VI) showed partial weight control (170.48 ± 5.92 g). Intergroup variations remained strongly significant (P < 0.001), underscoring the therapeutic impact of both TFE and metformin [Figure 2].

Close

Figure 2:

Bar graph showing intergroup comparison of body weight (g) at baseline (Day 0), 5^th week and 8^th week among control, diabetic and treatment groups. Values are expressed as mean ± standard deviation (n = 6). SD: Standard deviation.

Export to PPT

Intergroup comparison of blood glucose (mg/dL) at different time intervals

At baseline, no significant differences in blood glucose were observed among groups (P > 0.05), with values ranging from 72.83 ± 8.7 mg/dL in Group I to 79.33 ± 8.7 mg/dL in Group IV. By the 5^th week, glucose levels in Group I remained much lower (76.17 ± 5.38 mg/dL) compared with all diabetic groups (P < 0.001), confirming successful diabetes induction. At the 8^th week, Group II continued to exhibit markedly high glucose (304 ± 10.43 mg/dL), whereas Groups V (128 ± 7.85 mg/dL) and VI (95.17 ± 6.88 mg/dL) recorded the greatest reductions. Groups III and IV showed moderate declines. Overall, intergroup variation was strongly significant (P < 0.001), with Group VI providing the best glycaemic control [Figure 3].

Close

Figure 3:

Bar graph showing intergroup comparison of fasting blood glucose (mg/dL) at baseline, 5^th week and 8^th week among control, diabetic and treatment groups. Values are expressed as mean ± standard deviation (n = 6). SD: Standard deviation.

Export to PPT

Intergroup comparison of lipid levels at 5 weeks and 8 weeks

At both the 5^th and 8^th weeks, lipid parameters showed significant variation across groups (P < 0.001). By week 8, Group II displayed the most adverse profile, with the highest TC (151.9 ± 6.6 mg/dL), TG (122.4 ± 7.7 mg/dL), LDL (96.4 ± 6.2 mg/dL) and VLDL (24.5 ± 1.6 mg/dL), accompanied by the lowest HDL (33.2 ± 2.4 mg/dL). In contrast, Group I demonstrated the healthiest pattern, characterised by an HDL of 45.1 ± 3.3 mg/dL and LDL of 48.5 ± 4.3 mg/dL. Among the treatment arms, Group VI (metformin) achieved values closest to normal (TC: 112.5 ± 4.5 mg/dL; HDL: 46 ± 1.4 mg/dL), while Group V (TFE 1000 mg/kg) also showed marked improvement. These findings reinforce the lipid-lowering efficacy of both the test extract and the reference drug [Figure 4a and b].

Close

Figure 4:

(a) Intergroup comparison of lipid levels at the end of the 5^th week among control, diabetic and treatment groups. Parameters include TC, TG, LDL, VLDL and HDL. Values are expressed as mean ± SD (n = 6). (b) Intergroup comparison of lipid levels at the end of the 8^th week among control, diabetic and treatment groups. Parameters include TC, TG, LDL, VLDL and HDL. Values are expressed as mean ± SD (n = 6). TC: Total cholesterol, TG: Triglycerides, VLDL: Very low-density lipoprotein, HDL: High-density lipoprotein, SD: Standard deviation.

Export to PPT

Intergroup comparison of serum resistin levels at 5 weeks and 8 weeks

At the 5^th week, Group II recorded the highest serum resistin (18.17 ± 0.50), while Group I showed the lowest (10.14 ± 0.30). By the 8^th week, marked intergroup differences persisted (P < 0.001). Group II continued to exhibit the highest concentration (19.64 ± 0.48), whereas the lowest value was noted in Group VI (10.65 ± 0.28), followed by Group V (11.83 ± 0.30) and Group I (10.32 ± 0.28). Groups III and IV displayed moderate declines. The overall ranking at 8 weeks was: VI < I < V < IV < III < II, underscoring the most pronounced immunomodulatory action of metformin and high-dose TFE [Figure 5].

Close

Figure 5:

Bar graph showing intergroup comparison of serum resistin (pg/mL) at the 8^th week among control, diabetic and treatment groups. Values are expressed as mean ± standard deviation (n = 6). SD: Standard deviation.

Export to PPT

Comparison of blood glucose levels among therapeutic groups and controls

At week 5, both the diabetic control (Group II) and treatment groups (III–VI) demonstrated a marked elevation in mean FBS relative to the normal control (Group I) (P < 0.001), confirming effective induction of diabetes. Group I maintained the lowest FBS (76.17 ± 5.38 mg/dL), while values in diabetic groups ranged from 276.33 ± 25.50 mg/dL to 279.33 ± 30.51 mg/dL. At this point, the treated groups did not differ significantly from the diabetic control.

By the 8^th week, all treatment groups showed a significant fall in FBG compared with the diabetic control (P < 0.001). Group II maintained the highest glucose concentration (304.00 ± 10.43 mg/dL), while Group VI (metformin: 95.17 ± 6.88 mg/dL) recorded the lowest, followed by Group V (TFE 1000 mg/kg: 128.00 ± 7.85 mg/dL), indicating a dose-related antihyperglycaemic effect of TFE. Groups III and IV showed moderate improvements. These results reaffirm the glucose-lowering efficacy of both metformin and TFE, with greater benefit at higher TFE doses.

These observations are consistent with Alchalabi et al. (2019), who demonstrated that TFE significantly decreased fasting glucose and enhanced glucose tolerance in diabetic models, most effectively at 1000 mg/kg.^[18] Similarly, Tewari et al. (2024) noted that fenugreek promotes insulin release and hepatic glycogen storage, mechanisms that may underlie the glycaemic improvements seen in our study.^[10]

Comparison of BW among therapeutic groups and controls

At baseline, average BWs were similar across groups (P = 0.853), ranging from 164.00 ± 6.75 g in Group I to 168.32 ± 4.05 g in Group II, confirming uniform selection. By week 5, diabetic groups (II–VI) showed significantly greater weight than Group I (P < 0.001), with Group VI (metformin) displaying the highest mean (263.01 ± 3.46 g), indicative of HFD-driven metabolic alterations.

At 8 weeks, Group II displayed a pronounced fall in BW (139.89 ± 5.84 g), reflecting a 16.89% decline from baseline, probably due to severe metabolic disturbances. Conversely, treatment groups showed significant recovery in a dose-related fashion. The highest restoration occurred in Group VI (metformin: 190.02 ± 5.92 g), followed by Group V (TFE 1000 mg/kg: 184.18 ± 4.15 g), Group IV (TFE 500 mg/kg: 178.24 ± 8.61 g) and Group III (TFE 200 mg/kg: 175.48 ± 5.83 g). These findings corroborate Tewari et al. (2024), who demonstrated that trigonelline, a major bioactive compound of TFE, improved weight gain and glycaemic control in diabetic rats in a dose-dependent pattern.^[10]

Weight reduction in diabetic rats primarily arises from insulin deficiency, increased protein breakdown and defective energy metabolism. The bioactive components of TFE appeared to correct feeding patterns and energy balance, leading to weight improvement. Metformin, known for its weight-regulating effects, also supported weight recovery, most likely through better glycaemic control and efficient energy use. These observations are consistent with earlier studies showing metformin’s combined action on glucose regulation and BW improvement in diabetic models.^[19]

Comparison of lipid profile among therapeutic groups and controls

By the 8^th week, the diabetic control group (Group II) exhibited pronounced dyslipidaemia, characterised by raised TC, TG, LDL and VLDL, together with decreased HDL. In contrast, treatment with TFE extract and metformin produced significant improvements in lipid parameters. The most pronounced reductions in TC (112.5 ± 4.5 mg/dL) and LDL (49.5 ± 3.6 mg/dL) were achieved in Group VI (metformin 100 mg/kg), followed sequentially by Groups V (TFE 1000 mg/kg), IV (TFE 500 mg/kg) and III (TFE 200 mg/kg). A comparable pattern was noted for TG and VLDL, with Group VI again showing the strongest effect, whereas Group III showed a non-significant declining trend.

HDL levels rose significantly in Groups IV, V and VI, with the greatest increase noted in Group VI (46.0 ± 1.4 mg/dL), reflecting restoration of anti-atherogenic potential. Group III showed only a slight, non-significant elevation. These results are consistent with Alchalabi et al. (2019), who demonstrated that fenugreek seed extract lowered TG, TC and LDL while raising HDL.^[18] Similar lipid-regulating actions were described by Geberemeskel et al. (2019) and Bafadam et al. (2021), reinforcing the cardioprotective and antihyperlipidaemic role of TFE.^[20,21]

The lipid-lowering effect of metformin in this study is consistent with previous findings by Solymar et al. (2018), which attributed its benefits to the modulation of hepatic lipid metabolism and enhanced insulin sensitivity.^[22] The hypolipidaemic properties of TFE are most likely linked to trigonelline and associated alkaloids that suppress lipogenic enzymes such as peroxisome proliferator-activated receptor-γ, fatty acid synthase and acetyl-CoA carboxylase. In addition, trigonelline may stimulate bile acid production and improve hepatic lipid clearance, leading to better lipid regulation and a reduced cardiovascular risk in diabetes.

Comparison of serum resistin levels among therapeutic groups and controls

By the 8^th week, serum resistin levels were markedly higher in the diabetic control group (Group II: 55.67 ± 3.5 pg/mL) compared with the normal control (Group I: 25.5 ± 3.02 pg/mL), reflecting increased inflammatory activity and insulin resistance. Administration of TFE produced a dose-related reduction, with Group V (1000 mg/kg: 37.17 ± 2.79 pg/mL) and Group IV (500 mg/kg: 47.17 ± 2.48 pg/mL) showing significant decreases relative to Group II. Group III (200 mg/kg: 51.00 ± 3.16 pg/mL) exhibited only a mild, non-significant decline. Among the treated groups, the lowest resistin was observed in Group VI (metformin 100 mg/kg: 30.00 ± 3.16 pg/mL), approaching values seen in normal controls.

Our findings are consistent with those of Visuvanathan et al., who demonstrated that trigonelline – an active principle of fenugreek – improves insulin signalling by activating PPAR-γ and GLUT4, leading to reduced resistin expression.^[23] Similarly, Sfar et al. highlighted trigonelline’s antioxidant and anti-inflammatory effects, noting its ability to influence cytokine profiles, including resistin, thereby mitigating metabolic inflammation in T2DM.^[24]

The immunomodulatory action of TFE is likely mediated through suppression of the nuclear factor-kappa B (NF-κB) pathway, a central regulator of pro-inflammatory cytokine expression. Inhibiting NF-κB decreases resistin and other inflammatory mediators, thereby enhancing insulin sensitivity. In addition, fenugreek bioactives may activate adenosine monophosphate-activated protein kinase (AMPK), further strengthening insulin signalling and attenuating resistin-associated inflammation. In support, Pandey et al. (2024) observed that metformin and sodium-glucose cotransporter 2 inhibitors lowered leptin while elevating adiponectin in diabetic rats, suggesting a shared mechanism of immunometabolic regulation in T2DM.^[25]