Effect of simulated microgravity on the antidiabetic properties of wheatgrass (*Triticum aestivum*) in streptozotocin-induced diabetic rats

Diabetes mellitus is a prevalent metabolic disorder characterized by impaired insulin secretion, action, or both, leading to chronic hyperglycemia and serious complications. Conventional antidiabetic therapies can lose efficacy and have side effects, and often do not address dyslipidemia. Plant-based therapies rich in antioxidant phytochemicals (phenolics, flavonoids, vitamin C) have shown hypoglycemic and hypolipidemic effects, with wheatgrass outperforming nongerminated grains and peaking in antioxidant content around 7 days of germination. Growth conditions critically influence phytochemical content, and microgravity is known to alter plant growth and composition. The study asks whether wheatgrass germinated under simulated microgravity (WGM) exhibits enhanced antioxidant and antidiabetic activities compared with wheatgrass germinated under normal gravity (WGG), and evaluates these effects in streptozotocin-induced diabetic rats to explore a low-cost, low-side-effect therapeutic approach.

The paper reviews limitations of current antidiabetic drugs (efficacy loss, toxicity, limited impact on dyslipidemia) and highlights global interest in herbal medicines with antioxidant and antihyperglycemic properties. Prior studies show germination increases antioxidants (vitamin C, phenolics, flavonoids) in wheat, with benefits to glucose and lipid metabolism. Germination outcomes depend on environmental conditions (humidity, temperature, time, medium, steeping). Microgravity has been reported to affect plant cell growth, cell-wall architecture, and composition, but no prior work evaluated the therapeutic potential of wheatgrass germinated under microgravity. This gap motivated testing WGM versus WGG for antioxidant profile and antidiabetic efficacy.

Simulation of microgravity: A three-dimensional (3D) clinostat with two continuously running motors was used to randomize the gravity vector; plants were rotated in three dimensions at 4 rotations/min for 7 days. Plant material and extraction: Triticum aestivum (Ammon cultivar) seeds were surface-sterilized (1.25% sodium hypochlorite, 30 min), rinsed with cold distilled water, steeped 24 h at 21 °C, and germinated in a growth room at 21 °C for 7 days with Hoagland’s solution. WGG (gravity) and WGM (simulated microgravity) plants were harvested, washed, shade-dried 7 days (29–32 °C; RH 70–90%), powdered, and extracted with ethanol using a Soxhlet apparatus. Solvent was removed under reduced pressure; crude extracts were stored at −20 °C. Phytochemical and antioxidant assays: Total phenolics measured by Folin–Ciocalteu assay (absorbance 724 nm; expressed as mmol gallic acid equivalents/100 g fresh weight). Total flavonoids measured per Karadeniz et al. (absorbance 510 nm; mmol rutin equivalents/100 g fresh weight). Vitamin C quantified titrimetrically with 2,6-dichlorophenolindophenol and expressed as mg/dl. Antioxidant activities assessed by hydrogen peroxide scavenging (absorbance 230 nm) and nitric oxide scavenging via Griess reaction (absorbance 546 nm), calculating IC50 values; ascorbic acid served as positive control. Animals and diabetes induction: 35 male Wistar rats (150–200 g) were housed under 12 h light/dark at 21–23 °C. After 12 h fasting, diabetes was induced by intraperitoneal streptozotocin (STZ) 55 mg/kg in 0.1 M citrate buffer (pH 4.5). Rats received 5% glucose in drinking water overnight post-STZ. Fasting blood glucose was measured 3 days later; rats with >250 mg/dl were considered diabetic. Ethics approval was obtained from the American University of Madaba (5/2018). Experimental groups and treatments: Diabetic rats were randomized (n=7/group) to: (1) diabetic control (vehicle: 1% Tween-80 in water), (2) WGM extract 150 mg/kg bw in 1% Tween-80, (3) WGG extract 150 mg/kg bw, (4) metformin 100 mg/kg bw; plus a normal control group (n=7) receiving vehicle. Treatments were given orally by gavage once daily for 30 days. Glycemic assessments: Fasting blood glucose measured from tail vein at days 0, 3, 7, 14, 21, 30 using a glucometer. Oral glucose tolerance test (OGTT): after overnight fasting, glucose 2 g/kg was administered; blood glucose recorded at 0, 30, 60, 90, 120 min. Terminal biochemical assays: After 30 days, rats were fasted overnight and sacrificed. Whole blood (EDTA) was used to assay HbA1c, urea, and creatinine. Plasma (heparinized) was analyzed for insulin and C-peptide by radioimmunoassay. Lipid profile (total cholesterol, triglycerides, HDL-C, LDL-C) and liver enzymes (AST, ALT) were measured spectrophotometrically using commercial kits. Histology: Pancreata were fixed in 10% formalin, processed, paraffin-embedded, sectioned at 6 µm, and H&E-stained. β cells per islet were counted under a ×40 objective and expressed as cells per islet. Statistics: Data are mean ± SEM. Two-way ANOVA with Tukey’s multiple comparisons was used for most outcomes (P<0.05). β-cell counts were analyzed by one-way ANOVA with Bonferroni’s test (P<0.05). Analyses were done in GraphPad Prism 7.

- Phytochemical content and in vitro antioxidant activity: - Total phenolic content (mmol gallic acid equiv./100 g fresh weight): WGM 190.0 ± 2.11 vs WGG 100.4 ± 2.15 (P<0.0001). - Total flavonoid content (mmol rutin equiv./100 g fresh weight): WGM 208.1 ± 6.57 vs WGG 30.68 ± 4.01 (P<0.0001). - Vitamin C (mg/dl): WGM 20.32 ± 0.56 vs WGG 15.43 ± 0.32 (P<0.01). - H2O2 scavenging IC50 (µg/ml): WGM 13.0 ± 2.8 vs ascorbic acid 54.9 ± 1.90 and WGG 35.0 ± 1.93 (P<0.05). - Nitric oxide scavenging: WGM showed significantly lower IC50 than ascorbic acid and WGG (P<0.05). - OGTT (0–120 min): Diabetic control remained 450–580 mg/dl; WGG decreased to 172 ± 11.31 mg/dl by 120 min; WGM reached near-control by 30 min (112.0 ± 13.36 mg/dl) and maintained 105.0 ± 7.0 mg/dl at 120 min; metformin reached 74.0 ± 16.36 mg/dl at 30 min and 65.0 ± 14.36 mg/dl at 120 min. WGM significantly outperformed WGG and diabetic control. - Fasting blood glucose over 30 days: - WGM: Day 0 370.0 ± 4.81 to Day 7 234.50 ± 14.28 mg/dl; Day 30 75.50 ± 11.02 mg/dl (−79.5% vs Day 0; −84.7% vs diabetic control). - WGG: Day 0 587.0 ± 10.61 to Day 7 361.0 ± 25.92; Day 30 129.50 ± 5.30 mg/dl (−77.9% vs Day 0; −76.8% vs diabetic control). WGM 41.6% lower than WGG at Day 30. - Metformin: Day 0 259.50 ± 11.83 to Day 7 105.0 ± 4.08; Day 30 reduced by 54.8% vs Day 0 and 79.1% vs diabetic control. - HbA1c (%): Normal 5.55 ± 0.21; Diabetic 8.85 ± 0.24; WGM 5.75 ± 0.33; WGG 7.02 ± 0.23; Metformin 5.65 ± 0.14. WGM significantly lower than WGG. - C-peptide (ng/ml): Normal 0.047 ± 0.0032; Diabetic 0.039 ± 0.0015; WGM 0.053 ± 0.0022 (↑1.35-fold vs diabetic); WGG 0.040 ± 0.0021 (ns vs diabetic/normal); Metformin 0.045 ± 0.0010 (ns vs diabetic). - Insulin (µIU/ml): Normal 2.6 ± 0.20; Diabetic 1.2 ± 0.30; WGM 3.04 ± 0.35 (↑2.53-fold vs diabetic); WGG 1.9 ± 0.12; Metformin 2.34 ± 0.21. WGM significantly higher than WGG. - Kidney function (mg/dl): Creatinine—Normal 34.40 ± 1.51; Diabetic 100.60 ± 3.50; WGM 37.80 ± 2.03; WGG 53.20 ± 1.43; Metformin 34.10 ± 1.08. Urea—Diabetic 25.8 ± 2.30; WGM 9.10 ± 1.20; WGG 12.63 ± 2.10; Metformin 8.20 ± 1.70 (all near normal vs diabetic). - Lipid profile (mg/dl): Diabetic TC 2.18 ± 0.14, TG 0.85 ± 0.17, LDL-C 2.06 ± 0.15, HDL-C 1.47 ± 0.05 (HDL reduced vs normal). WGM: TC 1.75 ± 0.04, TG 0.56 ± 0.05, LDL-C 1.11 ± 0.9 (all ↓), HDL-C 1.64 ± 0.12 (↑). WGG: LDL-C 1.46 ± 0.12 (↓), TC 1.96 ± 0.05, HDL-C 1.14 ± 0.10, TG 0.75 ± 0.20 (ns). Metformin: TC 1.64 ± 0.14, LDL-C 1.31 ± 0.13 (↓), TG 0.60 ± 0.10 (ns), HDL-C 0.97 ± 0.04 (ns). HDL-C significantly higher in WGM vs WGG. - Liver enzymes (mg/dl): AST—Normal 129.0 ± 10.20; Diabetic 373.50 ± 16.30; WGM 45.20 ± 3.60; Metformin 62.30 ± 7.20. ALT—Normal 63.0 ± 4.20; Diabetic 210.70 ± 14.10; WGM 132.40 ± 11.60; Metformin 158.0 ± 14.30. WGM significantly reduced AST/ALT vs diabetic and gravity groups. - Histology and β-cell counts (cells per islet): Normal architecture in control; diabetic showed degeneration/necrosis and islet shrinkage. WGG improved islet morphology; WGM restored near-normal islet structure; metformin showed no pathological changes. β-cell counts: Normal 51 ± 5.19; Diabetic 15 ± 3.46 (↓, P<0.001); Gravity 31 ± 2.88 (↓ vs normal); Microgravity 114 ± 7.50 (↑ vs normal, P<0.0001); Metformin 50 ± 4.04 (ns vs normal).

Simulated microgravity during germination markedly enhanced wheatgrass antioxidant composition (phenolics, flavonoids, vitamin C) and in vitro radical-scavenging capacity, which translated into superior in vivo antidiabetic efficacy versus gravity-grown wheatgrass. In STZ-induced diabetic rats, WGM improved glucose homeostasis (rapid OGTT normalization and sustained fasting glucose reduction), decreased HbA1c, and increased both C-peptide and insulin, suggesting β-cell functional recovery and/or insulinotropic effects. Histology corroborated a strong protective/regenerative effect on pancreatic islets, with β-cell counts exceeding normal levels after WGM treatment. Systemically, WGM ameliorated diabetic dyslipidemia (lower TC, TG, LDL-C; higher HDL-C), mitigated liver injury (reduced AST/ALT), and improved renal markers (urea, creatinine), indicating broad metabolic and organ-protective benefits. The findings address the study question by showing that microgravity-enhanced phytochemical profiles are associated with improved glycemic control and tissue protection, likely via antioxidant-mediated β-cell preservation/regeneration and enhanced insulin secretion/sensitivity. These results underscore the relevance of growth environment manipulation (microgravity simulation) to optimize medicinal plant efficacy.

Germinating Triticum aestivum under simulated microgravity using a 3D clinostat produced extracts with substantially higher antioxidant content and activity than gravity-grown counterparts and delivered superior antidiabetic effects in STZ-diabetic rats. WGM normalized glucose handling, lowered HbA1c, increased insulin and C-peptide, improved lipid profiles, and reduced liver and kidney dysfunction, while restoring pancreatic islet structure and β-cell numbers. The approach offers a promising, low-cost, plant-based antidiabetic intervention with minimal side effects. The study suggests leveraging microgravity simulation to enhance therapeutic phytochemical yields and proposes exploring WGM for other disease contexts.