Medicinal herbs and their constituents are of crucial importance to human health. Fenugreek (Trigonella foenum-graecum), a one-year herb belonging to the Leguminosae (Fabaceae) family, is used both as a spice and vegetable traditionally and for medicinal purposes 1, 2. Its seeds contain fibre, 3 phospholipids, oleic acid, glycolipids, 4 choline, vitamins (A, B1, B2, C, niacin, nicotinic acid), 5 linoleic and linolenic acids, and other bioactive components 6. Fenugreek has been reported to have various pharmacological activities, including anti-inflammatory, antioxidant, antimicrobial, anticholesterolemic, antidiabetic, anticancer, antiviral, demulcent, and hepatoprotective 3, 7. It also controls enzymatic functions, decreases inflammation and pain, reduces fat, enhances appetite and hormone secretion, and increases lactation. Diabetes mellitus (DM), a common metabolic syndrome, leads to both microvascular and macrovascular complications, thereby causing high morbidity and mortality 8. Globally, the prevalence of diabetes was 6.4% in 2010 and is estimated to reach 7.7% (439 million adults) by 2030 9. Type-2 diabetes mellitus (T2DM) accounts for 90–95% of DM cases. Chronic hyperglycaemia in diabetes increases the risk of myocardial infarction and other complications 10. A healthy diet plays a key role in managing diabetes, with fenugreek offering potential benefits in blood glucose regulation 7.

Herbal remedies are becoming more popular owing to their low side effects; however, when they are mixed with pharmaceuticals, there are herb-drug interactions. Research estimates that 15–20% of prescription drug users also consume herbal supplements, but fewer than 40% report this to their doctors 11. Physicians are often unaware of such interactions, but more research into their safety and efficacy is needed 12. Oral hypoglycemic drugs (OHAs) like pioglitazone (P), glipizide (G), and acarbose (A) can produce gastrointestinal side effects 13, 14, 15. This study investigated the pharmacodynamic interaction between the polyphenolic-rich fenugreek seed extract Trigonella foenum-graecum (T. foenum-graecum) and OHAs in diabetes-induced gastropathy.

Chemicals and equipment: Chemicals and equipment from well-known sources were used in this study.

Extraction of polyphenolic-rich T. foenum-graecum) Seeds: Polyphenolic-rich extract of T. foenum-graecum (PEFS) (Batch no FEN/RD/01) was procured from Green Chem Industry, Bangalore. For extraction, 100 g of fenugreek seeds was finely powdered and macerated with 80% methanol at room temperature for five days. The solution was filtered, and the solvent was evaporated. The residual residue was dissolved in water and the aqueous phase was washed repeatedly with petroleum ether until a transparent upper layer was formed. The lower aqueous phase was subsequently treated with glacial acetic acid-containing ethyl acetate (10 ml/L) and extraction was performed for 36 h at room temperature. The ethyl acetate layer was concentrated and the residue was lyophilised and stored at 70°C. The yield was approximately 6–8 g per 100 g of seed powder. Methanolic extract was used in the experiments 16.

Experimental animals: Male Sprague Dawley rats weighing 200–300 g were kept in a well-ventilated animal facility at 25 ± 5°C under a 12:12 h light-dark cycle. The experimental protocols were approved by the Institutional Animal Ethics Committee (KCP/IAEC-27/2008-09), and all procedures adhered to the guidelines of the Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA). The animals were maintained under standard conditions, and all handling was performed in accordance with Good Laboratory Practice (GLP).

Oral Glucose Tolerance Test (OGTT): OGTT was conducted in overnight-fasted (18 h) normal and diabetic rats, which were distributed into ten groups for different treatments. Glucose (2 g/kg, p.o.) was administered 30 min post-treatment, and blood samples were collected from the tail vein under light ether anaesthesia at 30,60 and 120-min. Blood glucose levels were estimated using Accu-Chek active glucose strips and a glucometer (Roche). A daily fresh fructose diet was composed of 660 g fructose, 100 g protein, 80 g fat, 0.04 g of zinc carbonate, 5 g of vitamin mixture, 5 g of mineral mixture and 150 g of cellulose all of commercial grade was prepared. For type 2 diabetes induction, rats received a daily high-fat diet (HFD) for two weeks, followed by intraperitoneal injection (i.p.) of streptozotocin (35 mg/kg) in 0.5 M citrate buffer (pH 4.4). After STZ administration, the rats were treated with a 5% glucose solution to avoid hypoglycaemia. Blood samples were collected from the tail vein, and blood glucose levels were recorded using a glucose diagnostic kit (Accu-Chek). Rats with non-fasting blood glucose ≥300 mg/dl were regarded as diabetic 17, 18.

The rats were divided into six groups as follows:

Group NC: Normal control (saline/vehicle)

Group DC: Diabetic control

Group N + PEFG: Normal rats + Polyphenol-rich T. foenum graecum seed extract (200mg/kg)

Group N + P: Normal rats + Pioglitazone (10 mg/kg) 19   

Group N + G: Normal rats + Glipizide (5 mg/kg) 20

Group N + A: Normal rats + Acarbose (5 mg/kg)21

Single-dose study: In the single-dose study, seven groups of rats were treated as follows: distilled water, extract (200 mg/kg), pioglitazone (10 mg/kg), glipizide (5 mg/kg), and acarbose (5 mg/kg). Blood was drawn 0, 1, 2, and 4 h after treatment 22.

Multidose study: In the multidose study, the same treatments were administered once a day for 10 days, and body weight and blood glucose were observed on days 1, 3, 7, and 10 22.

Impact of PEFS and Control Drugs on OGTT in Normal Rats: OGTT was performed on normal rats to compare the antihyperglycemic activity of PEFS with that of control oral hypoglycaemic drugs, Pioglitazone, Glipizide, and Acarbose, individually and in combination. The blood glucose levels of all groups decreased significantly two hours after glucose challenge (2 g/kg) compared with the control group. The combination groups utilised 75% of the normal dose of OHAs as a low dose when used with PEFS.

Effect of PEFS and OHAs on OGTT in Diabetic Rats: In STZ-induced diabetic rats, there was a marked decrease in blood glucose levels in groups receiving PEFS alone and in combination with Glipizide, Pioglitazone, or Acarbose, compared to the diabetic control group. Although single treatment with standard OHAs significantly decreased glucose levels, OHAs in combination had hypoglycaemic effects, reflecting an increased response to combination therapy.

Effect of PEFS and OHAs on Body Weight: Body weights at more than one time point showed normal rats to have a steady weight increase. However, diabetic control rats sustained weight loss throughout the experiment. PEFS and OHAs combined treatment groups showed non-significant body weight reduction compared to diabetic controls, whereas marked loss of weight was seen in OHAs alone-treated rats. Interestingly, the PEFS group and combinations of Pioglitazone + Glipizide (P+G) and Glipizide + Acarbose (G+A) showed significant improvement in body weight.

Effect of PEFS and OHAs on Blood Glucose Level: Fasting serum glucose levels were measured. The normal control group had stable glucose levels (122.5 ± 0.5 mg/dl to 125 ± 0.8 mg/dl by day 60). The diabetic control group had high glucose levels without spontaneous control. However, the groups treated with a combination of PEFS and regular OHAs had significantly lower serum glucose levels by day 60 than those receiving regular OHAs alone. The PEFS monotherapy also showed good glucose control by day 30. The OHA combination therapy caused hypoglycaemia by

day 60.

The effects of PEFS and OHAs on HDL: HDL ratios were drastically reduced in the diabetic control group. Significant increments were observed in the groups receiving PEFS along with conventional OHAs (P+PEFS, G+PEFS, and A+PEFS). Moderate increases were observed in the PEFS alone, P+G, and G+A groups. Single OHAs treatments (P, G, and A) did not show any significant alteration in HDL levels. (Table 1)

Effect of PEFS and OHAs on Total Cholesterol and Triglycerides (TC and TG): Levels of total cholesterol and triglycerides were high in diabetic controls versus normal controls. Both TC and TG levels were significantly decreased in the PEFS combination groups (P+PEFS, G+PEFS, and A+PEFS). Groups treated with PEFS alone, P+G, and G+A showed a moderate decrease. No statistically significant changes were induced by individual treatments with Pioglitazone, Glipizide, or Acarbose. (Table 2 and Table 3)

Table 3

Groups	0th day	15th day	30th day	45th day	60th day	75th day
NC	58.2±0.8	58.8±0.4	62.6±0.8	61±0.5	64.8±0.2	64.8±0.12
DC	60±0.4	110.7±0.6	116.6±0.8	117±2.5	119±0.7	128.5±0.2
D + PEFS	61.1±0.6	101.6±1.7b	95.6±2.8b	81±0.5b	79±0.7b	82.4±0.8 b
D + P	62.4±0.5	104.6±1.2c	95.6±2.4 c	90.3±1.3 c	86±1.4 c	95±0.5c
D + G	62.5±1.2	102.7±0.4 c	96±1.7 c	98±1.6 c	92±0.4 c	91±0.6 c
D + A	59.4±0.8	100.6±1.4b	91±3.4b	86±1.6 c	82±9.6 c	87±0.2 c
D + P+G	58.2±0.5	67.3±1.7 a	76.6±0.8 a	72±1.1b	71±0.9b	-
D + G+A	59.9±0.6	102.3±1.4b	85±1.7b	78.6±0.8b	72±0.8b	-
D + P+PEFS	60.8±1.1	96.3±2.6 b	75.3±0.8 a	70±0.5 a	67±0.2 a	77.8±0.5b
D + G+PEFS	61.5±1.5	68.66±2.2 a	71.66±1.2 a	72±2.4 a	70±2.5 a	74±0.4 a
D + A+PEFS	62.8±0.5	69.06±1.4 a	73.6±1.2 a	70±0.5 a	67±0.2 a	72.7±0.5 b

All values are mean ± SEM, n=6, ^a p<0.001, ^bp<0.01, ^cp<0.05 when compared to Diabetic control group

TBARS (Thiobarbituric Acid Reactive Substances)

PEFS (p < 0.001), PEFS + G (p<0.001), PEFS+A (p < 0.001), and PEFS + P significantly lowered TBARS levels in stomach homogenates compared to diabetic controls. Comparable reductions were observed in the serum TBARS levels in the PEFS + G, PEFS + A, and PEFS + P groups. (Table 4 )

Catalase activity in the stomach homogenate increased substantially in the PEFS + G, PEFS + A, and PEFS + P groups (p < 0.001). All the other treatment groups also exhibited a significant increase (p < 0.01). In the serum, catalase activity was significantly higher in the PEFS + G and P + G groups than in the diabetic controls. (Table 4 )

PEFS + G, PEFS + P, and PEFS + A treatments significantly increased SOD content in stomach homogenates. Other groups, such as P, G, A, A+G, and P+G, also exhibited significant (p < 0.01) increases in kidney homogenate SOD content compared to diabetic controls. (Table 4 )

Influence of PEFS on Colonic Contractility, Small Intestinal Transit, Gastric Fundus and Gastric Emptying Time Models: Diabetic control contractility of the colon and gastric fundus response were reduced. Functional restoration was considerable in the P + PEFS, G + PEFS, and A + PEFS groups. Moderate restorations were found in the PEFS, P+G, and G+A groups. Individual OHAs had no marked effect on any of the groups. (Table 5 & Table 6 )

Small intestinal transit was slowed in diabetic controls, but markedly improved in the P + PEFS, G + PEFS, and A + PEFS groups. Gastric fundus contractility, challenged by bethanechol-induced contractions, was markedly impaired in diabetic rats and normalised by PEFS combination treatment. Pyloric contractility was abnormally elevated in diabetic rats and decreased significantly with the PEFS combination therapy. Antral contractility was not significantly different between the groups. PEFS treatment successfully reversed the fundic hypersensitivity observed in diabetes. (Table 5 & Table 6 )

Table 5

Groups	Contractile response of acetylcholine ED 50 (µg)	Charcoal meal colon to exogenous transit in small intestine (%)
NC	1.18 ± 0.03	80.5 ± 3.01
DC	15.83 ± 2.37	59 ± 4.91
D + PEFS	6.9±0.82b	68±2.56b
D + P	4.4 ± 0.49c	70.5 ± 3.51b
D + G	6.1 ± 0.73b	73.16 ± 3.31b
D + A	6.5±0.54b	71±0.9 c
D + P+G	5.8±0.68b	64±1.53b
D + G+A	6±0.91c	62±0.98c
D + P+ PEFS	5.2±0.6a	61±2.1a
D + G+ PEFS	13.05 ± 0.82a	67.5 ± 1.23a
D + A+ PEFS	6.1±0.5a	73±1.4a

p value: ^ap < 0.001 when compared with control. ^bp < 0.001 when compared with diabetic control. ^c p < 0.01 when compared with diabetic control

Table 6

Groups	Contractile response of Bethanechol ED50 (µg)			Contractile response of Carbachol ED50 (µg)
	Fundus	Antrum	Pylorus
NC	0.45±0.1	0.06±0.9	0.2±0.8	0.82±0.018
DC	0.15±0.2	0.03±0.8	0.1±0.6	0.64±0.01
D + PEFS	0.04±0.5 b	0.03±0.2 b	0.3±0.8 c	0.52±0.05c
D + P	0.18±0.6 c	0.04±0.8 C	0.25±0.7 b	0.49±0.03b
D + G	0.25±0.9c	0.04±0.8 b	0.12±0.4 c	0.51±0.03b
D + A	0.23±0.4 c	0.05±0.5a	0.18±0.8 c	0.55±0.02c
D + P+G	0.34±0.9 b	0.046±0.8 b	0.19±0.4 b	0.50±0.01b
D + G+A	0.54±0.8a	0.038±0.3 b	0.29±0.5 a	0.60±0.02c
D + P+ PEFS	0.38±0.4c	0.02±0.5 b	0.3±0.4a	0.61±0.03a
D + G+ PEFS	0.32±0.9 a	0.03±0.5b	0.5±0.4a	0.6±0.02a
D + A+ PEFS	0.37±0.4a	0.036±0.4 a	0.4±0.8a	0.63±0.01b

p value: ^ap < 0.001 when compared with control. ^bp < 0.001 when compared with diabetic control. ^c p < 0.01 when compared with diabetic control

The current study assessed the antihyperglycemic and metabolic actions of T. foenum-graecum seed extract (PEFS) and conventional oral hypoglycaemic agents Pioglitazone, Glipizide, and Acarbose in normal and diabetic rat models. These results verified the considerable therapeutic potential of PEFS, supporting and extending previously published findings.

In the OGTT performed on healthy rats, PEFS reduced the blood glucose level two hours post-administration after glucose administration, and this effect was further potentiated when PEFS was administered together with OHAs at 75% of their usual doses. This finding is in agreement with Broca et al., who found that 4-hydroxyisoleucine, a bioactive constituent in fenugreek, released insulin in a dose-dependent manner 32. Concurrently, Bnouham et al. showed the potential of fenugreek to lower blood glucose levels through mechanisms such as delaying glucose absorption and modulating insulin 33.

In diabetic rats, PEFS single administration and combined with OHAs caused significant reductions in blood glucose levels compared to diabetic controls. Single-agent OHA treatment was efficacious; however, its combination with PEFS demonstrated improved glycaemic control and induced hypoglycaemia in certain instances. This is in agreement with the synergistic hypothesis of Jayaraj et al., who found that the combination of herbal extracts, such as fenugreek, with conventional drugs improved therapeutic effects while reducing the dosages of drugs needed 34.

Body weight is an indicator of metabolic well-being in diabetes models. In the present study, diabetic control rats showed consistent weight loss, whereas the combination therapy groups (PEFS + Pioglitazone or Glipizide) showed improved weight gain. This is likely due to the metabolic and nutrition-supportive action of fenugreek's soluble fibres, as explained by Basch et al., who reported weight control and enhanced glycaemic management of fenugreek's mucilaginous fibre content and slow gastric emptying 35.

Fasting glucose levels were lowered considerably by PEFS alone by day 30, and even more drastically when combined with OHAs by day 60. Hannan et al., support these observations with their study that fenugreek seed fiber considerably enhanced glucose tolerance in diabetic rats 36. The combination regimens caused hypoglycaemia in some cases, reproducing the intensified insulinotropic and glucose-lowering effects reported by Jayaraj et al. and Gupta et al 34, 37.

As for lipid profiles, HDL was significantly lowered in diabetic controls but increased considerably in groups treated with PEFS and OHAs. The dual OHA- and PEFS-only groups also showed a moderate increase in HDL levels. This aligns with the findings of Raghuram et al.. and Sowmya and Rajyalakshmi, who both indicated that supplementation with fenugreek resulted in increased levels of HDL and decreased serum cholesterol levels in diabetic patients and hyperlipidaemic individuals 38, 39.

These findings validate PEFS's multifaceted therapeutic potential in glycaemic control as well as in enhancing lipid metabolism and maintaining body weight in diabetic models. With combined use of adjunct therapy to traditional OHAs, PEFS seems to augment antidiabetic effectiveness while potentially reducing side effects, thus representing a compelling argument for its adoption as an adjunct treatment in the management of diabetes.

In conclusion, this study demonstrated the efficacy of fenugreek, particularly PEFS, in improving glucose homeostasis and lipid profiles in normal and diabetic rats. The interaction of PEFS with conventional OHAs resulted in greater antihyperglycemic activity, validating the hypothesis of synergistic therapy for diabetes treatment. In addition, PEFS alone was found to have encouraging outcomes, particularly in managing blood glucose and body weight improvement in diabetic animals. These findings indicate the therapeutic value of fenugreek in the management of diabetes.