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

Effect of simulated microgravity on the antidiabetic properties of wheatgrass (Triticum aestivum)... www.nature.com/npjmgrav ARTICLE OPEN Effect of simulated microgravity on the antidiabetic properties of wheatgrass (Triticum aestivum) in streptozotocin-induced diabetic rats 1 1 1 2 Wajdy J. Al-Awaida , Ahmad S. Sharab , Hamzeh J. Al-Ameer and Nabil Y. Ayoub Microgravity affects plant growth and content. A three-dimensional clinostat was used at 4 rotations/min to rotate the seeds of Triticum aestivum cultivar (Ammon) in three dimensions for 7 days, following which the antioxidant activities of ethanolic extracts were evaluated using both nitric oxide- and hydrogen peroxide-scavenging activities. The antidiabetic activities of ethanolic extracts were evaluated by measuring the concentration of plasma glucose, insulin, C peptide, and glycated hemoglobin (HbA1c); determining the number of β cells in the pancreatic islets; and performing the glucose tolerance test. Furthermore, the effects of the ethanolic extracts on the lipid profile and liver function were estimated. After rats were sacrificed, their pancreases were isolated and used for histopathological processing. The results indicated that the antioxidant potential and antioxidant metabolite content were significantly increased under microgravity conditions in comparison to those under normal gravity conditions. Rats treated with an extract of wheatgrass (T. aestivum) germinated over a period of 6–10 days under microgravity (WGM) showed a significant reduction in the levels of serum glucose, HbA1C, urea, creatinine, aspartate aminotransferase and alanine aminotransferase, and insulin resistance compared to rats treated with an extract of wheatgrass germinated under gravity. Additionally, the total cholesterol and low-density lipoprotein cholesterol levels were significantly decreased. In contrast, high- density lipoprotein cholesterol, C-peptide, and insulin levels rose significantly after treatment with T. aestivum germinated under microgravity. WGM is a promising potential diabetic treatment without side effects with a low manufacturing cost. npj Microgravity (2020) 6:6 ; https://doi.org/10.1038/s41526-020-0096-x INTRODUCTION effects. Therefore, the current focus on diabetes treatment is on the development of an alternative solution for the treatment of Diabetes is a metabolic syndrome that arises mainly due to diabetes from natural sources, which are affordable and effective deficiencies in insulin activity, insulin secretion, or both. This and have fewer side effects than synthetic drugs. In traditional disorder can cause serious problems that affect human health. medicine, medicinal plants are used for the treatment of diabetes; Over the long term, uncontrolled diabetes can lead to several these plants are a rich source of hypolipidemic, hypoglycemic, and chronic complications, including renal failure, heart disease, and 3 antioxidant agents, such as phenols, flavonoids, gallotannins, and blindness. Statistically, 8.8% of the world’s population in 2015 11,12 other related polyphenols. exhibited the symptoms of this disease, and this percentage is Wheatgrass (T. aestivum germinated over a period of 6–10 days) predicted to rise to more than 10.4% by 2040. was found to improve metabolic and lipid profiles and to restore Despite the significant developments made in the management 14,15 the levels of plasma glucose, insulin, and liver glycogen. of diabetes over the past two decades, the effects of diabetes The nutrient value of germinated grains depends on the treatments in patients are still far from exemplary. These conditions in which they are grown. These conditions include treatments have many disadvantages, including a reduction in humidity, temperature, length of germination, culture medium, drug efficiency, toxicity, and side effects. For example, sulfonylur- and steeping protocol. The microgravity environment encoun- eas lose their efficiency after 6 years of treatment in 44% of tered during space flight has long been thought to affect plant patients. It is also believed that glucose-lowering medications are growth and content. Until now, no research on the therapeutic not capable of controlling hyperlipidemia. Recently, the use of potential of wheatgrass germinated under microgravity (WGM) herbal medications with insignificant toxicity and no side effects conditions has been carried out. Therefore, the present study was for the treatment of diabetes mellitus has been demonstrated to carried out to produce wheatgrass with high antioxidant and be of global importance. antidiabetic potential, but no side effects with a low Wheatgrass is believed to have a better nutritive value than manufacturing cost. nongerminated grains and their products. Antioxidant com- pounds, such as vitamins C, flavonoids, and phenolic compounds, RESULTS are scarcely measurable in dry grains. However, upon germination, the concentrations of these antioxidant compounds increase with Plant phenolics and flavonoids play a substantial role in increasing germination time and peak after 7 days. scavenging free radicals in the body and act as antioxidants. There are numerous hypoglycemic medications available in the Our results showed the total phenolic content (TPC) of the clinic, but these medications are associated with many side ethanolic extracts of wheatgrass germinated under gravity (WGG) 1 2 Department of Biology and Biotechnology, American University of Madaba, Madaba, Jordan. Department of Basic Sciences and Humanities, Faculty of Science, American University of Madaba (AUM), Amman 11821, Jordan. *email: w.alawaida@aum.edu.jo Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA 1234567890():,; W.J. Al-Awaida et al. Fig. 1 Triticum aestivum growth under gravity and microgravity conditions and the effect of simulated microgravity on the total phenolic content, total flavonoid content, vitamin C content, and total antioxidant activities in wheatgrass extract. a shows T. aestivum growth under gravity conditions. b shows T. aestivum growth under microgravity conditions simulated by a three-dimensional clinostat. c shows the total phenolic content of ethanolic extracts of T. aestivum under gravity and microgravity conditions. d shows the total flavonoid content of ethanolic extracts of T. aestivum under gravity and microgravity conditions. e shows the vitamin C content of ethanolic extracts of T. aestivum under gravity and microgravity conditions. f shows the hydrogen peroxide- and nitric oxide radical-scavenging activities of ethanolic extracts of T. aestivum under gravity and microgravity conditions. The values are expressed as the mean ± SEM of seven rats in each group. Data in c–e were analyzed by using two-way ANOVA, followed by Tukey’s multiple comparison test with the level of significance set at P < 0.05. *, **, ***, **** = P < 0.05, P < 0.01, P < 0.001, and P < 0.0001, respectively. Data in f were analyzed by using two-way ANOVA, followed by Tukey’s multiple comparison test with the level of significance set at P < 0.05. a refers to microgravity versus a reference compound ascorbic acid; b refers to gravity versus a reference compound ascorbic acid; and c refers to microgravity versus a gravity. and WGM conditions. The TPC is expressed as mmol equivalents metabolism. Vitamin C also helps with iron absorption and is of gallic acid/100 g fresh weight. In the ethanolic extract of WGM, important for immune system health. the TPC (190.0 ± 2.11 mmol) was found to be significantly higher The quantity of vitamin C, quantified as the total ascorbic acid (P < 0.0001; TPC of extracts by two-way analysis of variance content, was higher in the ethanolic extract of WGM (20.32 ± (ANOVA), followed by Tukey’s multiple comparison test) than that 0.56 mg/dl) (P < 0.01; vitamin C of extracts by two-way ANOVA, of the ethanolic extract of wheatgrass grown under gravity followed by Tukey’s multiple comparison test) than in the (100.4 ± 2.15 mmol), as shown in Fig. 1c. ethanolic extract of WGG (15.43 ± 0.32 mg/dl) (Fig. 1e). Our results present the total flavonoid content (TFC) of Hydrogen peroxide and nitric oxide radical-scavenging activities ethanolic extracts of WGG and WGM conditions. The TFC is were measured to determine the ability of the WGM extracts to expressed as mmol equivalents of rutin/mg fresh weight. The detoxify and scavenge free radicals. TFC of the ethanolic extract of WGM was significantly higher Our results present the hydrogen peroxide and nitric oxide (208.1 ± 6.57 mmol) (P < 0.0001; TFC of extracts by two-way radical-scavenging activities of ethanolic extracts of WGG and ANOVA followed by Tukey’s multiple comparison test) than the WGM. The ethanolic extract of WGM showed significantly greater TFC of the ethanolic extract of WGG (30.68 ± 4.01 mmol), as (P < 0.05; H O -scavenging activity of extracts by two-way ANOVA, 2 2 shown in Fig. 1d. followed by Tukey’s multiple comparison test) H O -scavenging 2 2 Vitamin C, also known as ascorbic acid, is an antioxidant activity with a lower half-maximal inhibitory concentration (IC ) compound and a component of an enzyme required for protein value (13.0 ± 2.8 µg/ml) compared to ascorbic acid (54.9 ± 1.90 µg/ npj Microgravity (2020) 6 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA 1234567890():,; W.J. Al-Awaida et al. (105.0 ± 7.0 mg/dl) (Fig. 2a). In diabetic rats treated with metfor- min, blood glucose levels began to decrease and reached the control level after 30 min (74.0 ± 16.36 mg/dl), at which they remained until 120 min (65.0 ± 14.36 mg/dl) (Fig. 2a). In the diabetic group treated with the ethanolic extract of WGM, the blood glucose levels were significantly decreased compared to those in the diabetic control group, the diabetic group treated with the ethanolic extract of WGG, and the diabetic group treated with metformin (Fig. 2a). Diabetes is characterized by a high level of glucose in the bloodstream caused by insufficient insulin secretion or insulin resistance. The diabetic group showed a significant increase (P < 0.05; blood glucose levels by two-way ANOVA, followed by Tukey’s multiple comparison test) in blood glucose levels in comparison to the control group. First, oral administration of the ethanolic extract of WGM in the diabetic group revealed a highly significant reduction (P < 0.05; blood glucose levels by two-way ANOVA, followed by Tukey’s multiple comparison test) in blood glucose levels starting on the seventh day of treatment (234.50 ± 14.28 mg/dl) compared to the levels in the same group before treatment (day 0) (370.0 ± 4.81 mg/dl) as well as the levels in the diabetic group on the corresponding days (560.0 ± 13.06 and 371.50 ± 24.11 mg/dl, respectively). This reduction was marked on the 30th day, as blood glucose levels were reduced by 79.5% compared to those at day 0 and 84.7% compared to the value in the diabetic group (Fig. 2b). Fig. 2 The effects of ethanolic extracts of T. aestivum germinated Subsequently, the group treated with the ethanolic extract of under gravity and microgravity conditions on oral glucose WGG exhibited a highly significant reduction (P < 0.05; blood tolerance tests over 2 h and fasting blood glucose levels over glucose levels by two-way ANOVA, followed by Tukey’s multiple 30 days in experimental rats. a shows the effect of ethanolic comparison test) in blood glucose levels starting on the seventh extracts of T. aestivum under gravity and microgravity conditions on the results of the oral glucose tolerance test (OGTT). b shows the day of treatment (361.0 ± 25.92 mg/dl) compared to levels on day effect of ethanolic extracts of T. aestivum under gravity and 0 (587.0 ± 10.61 mg/dl) and the levels in diabetic rats on the microgravity conditions on fasting blood glucose levels in experi- corresponding days (560.0 ± 13.06 and 371.50 ± 24.11 mg/dl, mental rats. The values are expressed as the mean ± SEM of seven respectively). This reduction was marked on the 30th day, as rats in each group. Data were analyzed by using two-way ANOVA blood glucose levels were reduced by 77.9% compared to those followed by Tukey’s multiple comparison test with the level of on day 0 and 76.8% compared to those in the diabetic group (Fig. significance set at P < 0.05. a refers to treatment versus normal 2b). The diabetic group treated with metformin showed a control; b refers to treatment versus diabetic control; c refers to significant reduction (P < 0.05; blood glucose levels by two-way microgravity versus gravity. ANOVA, followed by Tukey’s multiple comparison test) in blood glucose levels starting on the seventh day of treatment (105.0 ± ml), which was used as a positive control, and the ethanolic extract 4.08 mg/dl) compared to blood glucose levels on day 0 (259.50 ± of WGG (35.0 ± 1.93 µg/ml). In contrast, the ethanolic extract of 11.83 mg/dl) and those of the diabetic group on the correspond- WGM showed significantly greater (P < 0.05; nitric oxide- ing days (560.0 ± 13.06 and 371.50 ± 24.11 mg/dl, respectively). scavenging activity of extracts by two-way ANOVA, followed by This reduction was marked on the 30th day, as blood glucose Tukey’s multiple comparison test) nitric oxide-scavenging activity levels were reduced by 54.8% compared to those on day 0 and with a lower IC compared to the positive control, ascorbic acid, 79.1% compared to those in the diabetic group (Fig. 2b). In the and the ethanolic extract of WGG, as shown in Fig. 1f. diabetic group treated with the ethanolic extract of WGM, blood The glucose tolerance test (GTT) has been extensively used for glucose levels were significantly decreased (P < 0.05; blood the study of carbohydrate metabolism in experimental animals. glucose levels by two-way ANOVA, followed by Tukey’s multiple Time-dependent changes in blood glucose levels during the oral comparison test) compared to the group treated with the GTT (0–120 min) for all groups are shown in Fig. 2a. The rats in the ethanolic extract of WGG beginning on day 7 (234.50 ± 14.28 diabetic group had significantly higher (P < 0.05; GTT by two-way and 361.0 ± 25.92 mg/dl, respectively). This reduction was marked ANOVA, followed by Tukey’s multiple comparison test) blood on the 30th day (75.50 ± 11.02 and 129.50 ± 5.30 mg/dl), when glucose levels throughout the total experimental period (120 min) blood glucose were 41.6% lower in the group treated with the (450.0 ± 15.14 to 580.0 ± 17.07 mg/dl) compared to the normal ethanolic extract of WGM (Fig. 2b). control group (111.0 ± 13.0 to 87.0 ± 13.0 mg/dl) (Fig. 2a). In Insufficient insulin prevents the body from transporting glucose diabetic rats treated with the ethanolic extract of WGG, even after from the bloodstream into the body’s cells to use as energy. When the administration of glucose, blood glucose levels remained the this occurs, the body starts burning fat and muscle for energy, same as those at 0 min (430 ± 24.74 mg/dl) and even those at causing a decrease in overall body weight. Changes in the body 60 min (373 ± 14.14 mg/dl), following which the blood glucose weight of rats in the normal control, diabetic control, microgravity, levels began to decrease after 90 min (200.0 ± 14.84 mg/dl) and gravity, and metformin groups over a period of 30 days are shown reached the upper limit for normal glucose levels after 120 min in Fig. 3. A significant decrease (P < 0.05; body weight by two-way (172.0 ± 11.31 mg/dl) (Fig. 2a, P < 0.05; GTT by two-way ANOVA, ANOVA, followed by Tukey’s multiple comparison test. *, **, ***, followed by Tukey’s multiple comparison test). In diabetic rats **** = P < 0.05, P < 0.01, P < 0.001, and P < 0.0001, respectively) in treated with the ethanolic extract of WGM, the blood glucose body weight was observed in the diabetic and gravity groups levels began to decrease, reached the control level after 30 min compared with their body weights at the initial day of the (112.0 ± 13.36 mg/dl) and remained the same until 120 min experiment (from 190.0 ± 12.4 to 152.0 ± 7.49 g and from 201.0 ± Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2020) 6 W.J. Al-Awaida et al. Fig. 3 The effects of ethanolic extracts of T. aestivum germinated under gravity and microgravity conditions on body weight in experimental rats after 30 days. a shows changes in body weight in untreated nondiabetic rats on days 0 and 30. b shows changes in body weight in untreated diabetic rats on days 0 and 30. c shows changes in diabetic rats treated with the ethanolic extract of T. aestivum germinated under microgravity conditions on days 0 and 30. d shows changes in diabetic rats treated with the ethanolic extract of T. aestivum germinated under gravity condition group on days 0 and 30. e shows changes in diabetic rats treated with metformin on days 0 and 30. All values represent the mean ± SD. All comparisons were performed between the same group of animals on days 0 and 30. Data were analyzed by using two-way ANOVA, followed by Tukey’s multiple comparison test with the level of significance set at P < 0.05. *, ***, **** = P < 0.05, , P < 0.001, and P < 0.0001, respectively. 8.7 to 172.0 ± 9.23 g, respectively). However, there was no significant difference (P < 0.05; body weight by two-way ANOVA, followed by Tukey’s multiple comparison test. *, **, ***, **** = P < 0.05, P < 0.01, P < 0.001, and P < 0.0001, respectively) in body weight between the normal control, microgravity, and metformin groups on the initial day of the experiment (Fig. 3). Glycated hemoglobin (HbA1c), a form of hemoglobin that is chemically linked to glucose, indicates the sugar level in the bloodstream over a long period. Our results show the HbA1C levels in normal and experimental rats. In the diabetic control group, there was a significant increase (P < 0.05; HbA1C level by two-way ANOVA, followed by Tukey’s multiple comparison test) in the HbA1C level (8.85 ± 0.24%) compared to the normal control group (5.55 ± 0.21%) (Fig. 4a). In the diabetic group treated with the ethanolic extract of WGM, the HbA1C level was significantly decreased (5.75 ± 0.33%) (P < Fig. 4 Effect of treatment with ethanolic extracts of T. aestivum 0.05; HbA1C level by two-way ANOVA, followed by Tukey’s under gravity and microgravity conditions for 30 days on multiple comparison test) compared to the diabetic control group C-peptide, serum insulin, and glycated hemoglobin (HbA1c) (Fig. 4a). In the diabetic group treated with the ethanolic extract of levels in experimental rats. a shows the effect of ethanolic extracts WGG, the HbA1C levels was significantly decreased (7.02 ± 0.23%) of T. aestivum germinated under gravity and microgravity conditions (P < 0.05; HbA1C level by two-way ANOVA, followed by Tukey’s on body weight in experimental rats. b shows the effect of ethanolic multiple comparison test) compared to the diabetic group. The extracts of T. aestivum germinated under gravity and microgravity HbA1C level was higher in the diabetic gravity group. In diabetic conditions on C-peptide levels in experimental rats. c shows the effect of ethanolic extracts of T. aestivum germinated under gravity rats treated with metformin, the HbA1C levels was significantly and microgravity conditions on serum insulin levels in experimental decreased (5.65 ± 0.14%) (P < 0.05; HbA1C level by two-way rats. The values are expressed as the mean ± SEM of seven rats in ANOVA, followed by Tukey’s multiple comparison test) compared each group. Data were analyzed by using two-way ANOVA, followed to the diabetic group. A significant difference (P < 0.05; HbA1C by Tukey’s multiple comparison test with the level of significance set level by two-way ANOVA, followed by Tukey’s multiple compar- at P < 0.05. a refers to treatment versus normal control, b refers to ison test) in the HbA1C level was observed between the gravity treatment versus diabetic control, and c refers to microgravity versus and WGM groups. The HbA1C level in the diabetic group treated gravity. with WGM was significantly lower than the HbA1C level in the diabetic group treated with WGG (Fig. 4a). C-peptide of insulin, a metabolic byproduct produced during ANOVA, followed by Tukey’s multiple comparison test) compared the activation of insulin, is a useful and common method used to to the diabetic group by 1.35-fold (Fig. 4b). There was no assess pancreatic β cell function. significant difference (P < 0.05; C-peptide level by two-way The C-peptide level was reduced in the diabetic group (0.039 ± ANOVA, followed by Tukey’s multiple comparison test) in the C- 0.0015 ng/ml) compared to the normal group (0.047 ± 0.0032 ng/ peptide level between the diabetic group treated with the ethanolic ml) (Fig. 4b). In the diabetic group treated with the ethanolic extract of WGG (0.04 ± 0.0021 ng/ml) and both the diabetic and extract of WGM, the C-peptide level was significantly increased normal control groups (Fig. 4b). C-peptide levels in the diabetic (0.053 ± 0.0022 ng/ml (P < 0.05; C-peptide level by two-way group treated with metformin (0.045 ± 0.0010 ng/ml) were not npj Microgravity (2020) 6 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA W.J. Al-Awaida et al. significantly different (P < 0.05; C-peptide level by two-way ANOVA, Diabetic rats showed a significant increase (P < 0.05; TC, TG, and followed by Tukey’s multiple comparison test) compared to those in LDL-C levels by two-way ANOVA followed by Tukey’s multiple the diabetic group (Fig. 4b). In the diabetic group treated with the comparison test) in the TC (2.18 ± 0.14 mg/dl), TG (0.85 ± .17 mg/ ethanolic extract of WGG, there was a significant increase (P <0.05; dl), and LDL-C (2.06 ± 0.15 mg/dl) levels in the blood (Fig. 5b). In C-peptide level by two-way ANOVA, followed by Tukey’s multiple contrast, the high-density lipoprotein cholesterol (HDL-C) level (1.47 ± 0.05 mg/dl) was significantly reduced (P < 0.05; HDL-C level comparison test) in the C-peptide level insulin level by 1.32-fold by two-way ANOVA, followed by Tukey’s multiple comparison test) compared to the gravity group (Fig. 4b). compared to the normal control group. In diabetic rats treated The plasma insulin level was significantly reduced (P < 0.05; with the ethanolic extract of WGM, the TC (1.75 ± 0.04 mg/dl), TG insulin level by two-way ANOVA, followed by Tukey’s multiple (0.56 ± 0.05 mg/dl), and LDL-C (1.11 ± 0.9 mg/dl) levels in the comparison test) in the diabetic group (1.2 ± 0.30 µlU/ml) com- blood were significantly decreased (P < 0.05; TC, TG, and LDL-C pared to the normal control group (2.6 ± 0.20 µlU/ml) (Fig. 4c). The levels by two-way ANOVA, followed by Tukey’s multiple compar- plasma insulin level in the diabetic group treated with the ison test). However, the level of HDL-C (1.64 ± 0.12 mg/dl) was ethanolic extract of WGM was significantly increased by 2.53-fold significantly increased (P < 0.05; HDL-C level by two-way ANOVA, (3.04 ± 0.35 µlU/ml) (P < 0.05; insulin level by two-way ANOVA, followed by Tukey’s multiple comparison test) (Fig. 5b). In diabetic followed by Tukey’s multiple comparison test) compared to the rats treated with the ethanolic extract of WGG, the blood level of diabetic control group (Fig. 4c). There was no significant difference LDL-C (1.46 ± 0.12 mg/dl) was significantly decreased (P < 0.05; (P < 0.05; insulin level by two-way ANOVA, followed by Tukey’s LDL-C level by two-way ANOVA, followed by Tukey’s multiple multiple comparison test) in the plasma insulin level of the comparison test). However, TC (1.96 ± .05 mg/dl), HDL-C (1.14 ± diabetic group treated with the ethanolic extract of WGG (1.9 ± 0.10 mg/dl), and TG (0.75 ± 0.20 mg/dl) levels were not changed 0.12 µlU/ml) compared to the diabetic and normal control groups (Fig. 5b). In diabetic rats treated with metformin, the TC (1.64 ± (Fig. 4c). In diabetic rats treated with metformin, the insulin level 0.14 mg/dl) and LDL-C (1.31 ± 0.13 mg/dl) levels in the blood were was significantly increased (2.34 ± 0.21 µlU/ml) (P < 0.05; insulin significantly decreased (P < 0.05; TC and LDL-C levels by two-way level by two-way ANOVA, followed by Tukey’s multiple compar- ANOVA, followed by Tukey’s multiple comparison test). However, ison test) by 1.95-fold compared to the diabetic group (Fig. 4c). In the TG (0.60 ± 0.10 mg/dl) and HDL-C (0.97 ± 0.04 mg/dl) levels the diabetic group treated with the ethanolic extract of WGM, the were not changed (Fig. 5b). The level of HDL-C in the microgravity insulin level was significantly increased (P < 0.05; insulin level by group was significantly increased (P < 0.05; HDL-C level by two- two-way ANOVA, followed by Tukey’s multiple comparison test) by way ANOVA, followed by Tukey’s multiple comparison test) 1.6-fold compared to the gravity group (Fig. 4c). compared to the gravity group (Fig. 5b). Serum urea and creatinine are known to be increased with Increased activities of the liver enzymes aspartate aminotrans- hyperglycemia in patients with uncontrolled diabetes and usually ferase (AST) and alanine aminotransferase (ALT) are indicators of correlate with the severity of kidney damage. Our results show the liver cell injury and associated with uncontrolled diabetes. levels of creatinine and urea in normal and experimental rats. In The induction of diabetes led to significant increases (P < 0.05; diabetic rats, creatinine and urea levels were significantly AST and ALT levels by two-way ANOVA, followed by Tukey’s increased (100.60 ± 3.50 and 25.80 ± 2.30 mg/dl, respectively) multiple comparison test) in the serum activities of AST (373.50 ± (P < 0.05; creatinine and urea levels by two-way ANOVA, followed 16.30 mg/dl) and ALT (210.70 ± 14.10 mg/dl) in the diabetic group by Tukey’s multiple comparison test) compared to the normal compared to the normal group (129.0 ± 10.20 and 63.0 ± 4.20 mg/ control group (34.40 ± 1.51 and 6.0 ± 0.80 mg/dl, respectively) dl, respectively) (Fig. 5c). ALT (132.40 ± 11.60 mg/dl) and AST (Fig. 5a). The creatinine level in the diabetic group treated with the (45.20 ± 3.60 mg/dl) activities were significantly reduced (P < 0.05; ethanolic extract of WGM was significantly decreased (37.80 ± AST and ALT levels by two-way ANOVA, followed by Tukey’s 2.03 mg/dl) (P < 0.05; creatinine level by two-way ANOVA, followed multiple comparison test) in the group of diabetic rats treated by Tukey’s multiple comparison test) compared to the diabetic with the ethanolic extract of WGM compared to the diabetic control group (100.60 ± 3.50 mg/dl) (Fig. 5a). The creatinine level group. The AST and ALT levels in the diabetic gravity group were in the diabetic group treated with the ethanolic extract of WGG higher than those in the diabetic microgravity group. (Fig. 5c). was significantly decreased (53.20 ± 1.43 mg/dl) (P < 0.05; creati- Interestingly, the diabetic group treated with metformin showed a nine level by two-way ANOVA, followed by Tukey’s multiple significant reduction (P < 0.05; AST and ALT levels by two-way comparison test) compared to the diabetic control group ANOVA, followed by Tukey’s multiple comparison test) in ALT (100.60 ± 3.50 mg/dl) (Fig. 5a). The creatinine level in the diabetic (158.0 ± 14.30 mg/dl) and AST (62.30 ± 7.20 mg/dl) activities com- gravity group was higher than that in the diabetic microgravity pared to the diabetic group (Fig. 5c). group. In diabetic rats treated with metformin, the creatinine level β Cells are a type of cell found in pancreatic islets that produce and was significantly increased (34.10 ± 1.08 mg/dl) (P < 0.05; creati- secrete insulin. In diabetes, β cell mass and function are diminished, nine level by two-way ANOVA, followed by Tukey’s multiple leading to insufficient insulin secretion and hyperglycemia. comparison test) compared to the diabetic control group To elucidate the preventative effects of ethanolic extracts of (100.60 ± 3.50 mg/dl) (Fig. 5a). The blood urea level was increased WGG and WGM conditions on streptozotocin (STZ)-induced in the diabetic group (25.8 ± 2.30 mg/dl). However, the blood urea diabetes, the pancreatic tissues of the ethanol extract-treated level was significantly decreased (P < 0.05; urea level by two-way and control groups were examined. The results revealed the ANOVA, followed by Tukey’s multiple comparison test) in the following: the normal control group showed a normal pancreatic microgravity (9.10 ± 1.20 mg/dl), gravity (12.63 ± 2.10 mg/dl) and structure (Fig. 6a), whereas the diabetic group showed degenera- metformin (8.20 ± 1.70 mg/dl) groups compared to the diabetic tion, necrotic changes, and islet shrinkage in the pancreas control group. No significant differences (P < 0.05; urea level by (Fig. 6b). The pancreatic sections of rats administered the two-way ANOVA, followed by Tukey’s multiple comparison test) ethanolic extract of WGG showed improved islet morphology in blood urea level were found in the microgravity, gravity, and (Fig. 6c). The pancreatic sections of rats administered the ethanolic metformin groups in comparison to the normal control group extract of WGM showed a normal structure in the pancreatic islets (Fig. 5a). of Langerhans (Fig. 6d). There were no pathological changes in the Uncontrolled diabetes tends to raise total cholesterol (TC), total pancreatic islets of Langerhans in diabetic rats treated with triglyceride (TG), and low-density lipoprotein cholesterol (LDL-C) metformin (Fig. 6e). β Cells in the pancreatic islets was measured levels, which increases the risk of heart disease and stroke. This under a ×40 objective lens. The results revealed a significant common condition is called diabetic dyslipidemia. decrease (P < 0.001; β cells by one-way ANOVA with Bonferroni’s Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2020) 6 W.J. Al-Awaida et al. Fig. 5 Effect of treatment with ethanolic extracts of T. aestivum under gravity and microgravity conditions for 30 days on liver and kidney functions and lipid profiles in experimental rats. a shows the effect of ethanolic extracts of T. aestivum under gravity and microgravity conditions on biomarkers of kidney function in experimental rats. b shows the effect of ethanolic extracts of T. aestivum under gravity and microgravity conditions on the lipid profiles of experimental rats. c shows the effect of ethanolic extracts of T. aestivum under gravity and microgravity conditions on biomarkers of liver function in experimental rats. The values are expressed as the mean ± SEM of seven rats in each group. Data were analyzed by using two-way ANOVA, followed by Tukey’s multiple comparison test with the level of significance set at P < 0.05. a refers to treatment versus normal control, b refers to treatment versus diabetic control, and c refers to microgravity versus gravity. multiple comparisons test. *, ***, ****, ns = P < 0.05, P < 0.001, P < 0.05, P < 0.001, P < 0.0001, and nonsignificant, respectively) in the 0.0001, and nonsignificant, respectively) in the number of β cells number of β cells per islet in the microgravity group (114 ± 7.50) per islet in the diabetic control group (15 ± 3.46) compared to the compared to the normal control group. However, there was no normal control group (51 ± 5.19). In addition, there was a significant difference (ns) in the number of β cells per islet in the significant decrease (P < 0.05; β cells by one-way ANOVA with metformin group (50 ± 4.04) compared to the normal control Bonferroni’s multiple comparisons test. *, ***, ****, ns = P < 0.05, P group. < 0.001, P < 0.0001, and nonsignificant, respectively) in the number of β cells per islet in the gravity group (31 ± 2.88) DISCUSSION compared to the normal control group (15 ± 3.46). However, there was a significant increase (P < 0.0001; β cells by one-way ANOVA This study evaluated the effect of simulated microgravity on the with Bonferroni’s multiple comparisons test. *, ***, ****, ns = P < antidiabetic and antioxidant potential of wheatgrass extracts in npj Microgravity (2020) 6 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA W.J. Al-Awaida et al. Fig. 6 Photomicrographs of H&E-stained histological slides of pancreatic specimens. a Normal histological appearance of the islets of Langerhans from the pancreases of rats in the control group. b The pancreases of rats in the diabetic control group showed degeneration, necrotic changes, and islet shrinkage. c The appearance of pancreas sections from rats administered the ethanolic extract of T. aestivum under gravity conditions showed improved islet morphology. d The appearance of pancreas sections from rats administered the ethanolic extract of T. aestivum under microgravity conditions showed the normal structure of the pancreatic islets of Langerhans. e shows that there were no pathological changes in the islets of Langerhans of the pancreas in diabetic rats treated with metformin. Original magnification was ×400. f The pancreatic β cell number per islet in different treatment groups. Groups were compared to the control untreated group. As shown in the figure, microgravity extract treatment had a highly significant effect on the pancreatic β cell number per islet compared to the normal untreated control group. Data were compared against data for the normal control by using one-way ANOVA with Bonferroni’s multiple comparisons test with the level of significance at P < 0.05. *, ***, ****, ns = P < 0.05, P < 0.001, P < 0.0001, and nonsignificant, respectively. STZ-induced diabetic rats. Insulin-dependent diabetes mellitus conditions was detected in experimental animals following the 23,24 (IDDM) is a disorder initiated by progressive damage to insulin- administration of antioxidant compounds. secreting β cells. STZ is normally used to induce diabetes in Although wheatgrass germinated under normal conditions (under gravity) showed significant antioxidant potential and pancreatic rats by DNA alkylation and β cell fragmentation. STZ antihyperglycemic effects in STZ-induced diabetic rats as a result selectively devastates the β cells, which secrete insulin, decreasing 14,25 of its high TP and flavonoid concentrations, our results under the β cell efficiency and resulting in diabetes mellitus. Many microgravity conditions show even more promise in the field. The living β cells remain after treatment with low-dose STZ, and their microgravity environment created by a three-dimensional (3D) regeneration is also possible. clinostat produced wheatgrass with a higher concentration of In our study, significantly more β cells were observed in the antioxidant compounds and a higher potential for antioxidant group treated with the ethanol extract of WGM conditions, and activity than WGG. We hypothesize that the presence of a high stimulation due to insulin secretion could be responsible for most concentration of some bioactive compounds, such as phenolic of the observed metabolic effects. Bioactive compounds in compounds, flavonoids, and vitamin C, in WGM conditions medicinal plants may have insulin-like properties and have been produces an insulinotropic effect due to the antidiabetic effects shown to enhance insulin sensitivity, improve glucose-dependent of these compounds that facilitates a decrease in blood glucose insulin secretion, and activate the regeneration of the pancreatic levels. The elevated plasma glucose levels in diabetic rats were islets of Langerhans in STZ-induced diabetic rats. Currently, the decreased due to the administration of wheatgrass germinated only option for achieving normal glucose levels in diabetic extract under microgravity conditions, and the administration of patients is the stimulation of insulin secretion by medicinal plants WGM elevated C-peptide and plasma insulin levels compared to 20,21 or renewal of β cells. Increased free radical generation and those in diabetic control rats. The possible mechanism of action of oxidative stress together with hyperglycemia play a key role in the WGM conditions is its regeneration and stimulation of β cells to pathogenesis of diabetes and its late complications. β Cells are release insulin, which mediates a decrease in glucose levels, as highly sensitive to destruction by oxidative stress because of their shown in our results. low level of free radical-scavenging enzymes, which protect the Wheatgrass ethyl alcohol extract was shown to decrease cell from the damaging effects of oxidative stress. Decreased mononuclear cell infiltration in the β cells of the pancreas oxidative stress due to a reduced hyperglycemic status in diabetic compared to that in the untreated group. This effect coincides Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2020) 6 W.J. Al-Awaida et al. with the protective effect of wheatgrass extract on the diabetes blood sugar levels in experimental animal models. The antiox- parameters investigated in this study. This effect was reported idant activity of these phenolic and flavonoid compounds depends using germinated T. aestivum. on their redox properties, and these compounds can act as The induction of diabetes by STZ led to a reduction in body hydrogen donors, reducing agents, and singlet oxygen quench- weight due to structural protein degradation as a result of ers. Polyphenolics containing hydroxyl groups are essential plant disturbances in carbohydrate metabolism and thus energy ingredients that can defend the body against oxidative stress. The metabolism. Due to damage to β cells caused by STZ, a present study concluded that the microgravity environment decrease in body weight and an increase in water and food intake created by a 3D clinostat produced germinated T. aestivum with a are commonly detected in diabetes, and these effects may be high concentration of antioxidant compounds and high level of caused by metabolic changes due to the absence or deficiency of potential antioxidant activity; the produced wheatgrass has insulin. The nonsignificant changes in the body weights of potential as a diabetic treatment without any side effects and a diabetic rats treated with extracts of WGG and WGM conditions as low manufacturing cost. This method can be used to explore the well as metformin-treated diabetic rats compared to the control therapeutic potential of WGM conditions for other diseases. group revealed the effect of a normal blood glucose level, which in turn prevents the loss of body weight. HbA1c is used as an indicator to assess the degree of METHODS hemoglobin glycation in diabetes mellitus. HbA1c was found to The 3D clinostat represent the average fasting blood glucose level of diabetic Earth’s gravitational field at its surface at a certain location is a fixed vector patients over the previous 120 days. Under diabetic conditions, pointing downwards (towards the Earth’s center) whose average extra glucose in the blood is covalently bound to hemoglobin to magnitude is 9.8 m/s. Many plants and organisms contain cells that form HbA1c. Hence, HbA1c levels are increased in untreated sense gravity through sedimentation due to gravity. A procedure in which cells in plants or organisms are placed in a randomizing machine in which diabetic rats. Treatment with WGM conditions significantly the gravity vector loses its effect on these cells would simulate the reduced HbA1c levels in comparison to those in the gravity and conditions of weightlessness in outer space as the direction of the gravity diabetic control groups. vector would be lost. The rotational speed of such a machine is determined The liver plays a central role in the metabolism, storage, by the time scale of the movement of the cells inside the plant. There are excretion, and detoxification of xenobiotics and their metabolites, many such machines, and the one used in this study was a 3D clinostat. We and AST and ALT are effective markers of liver function and STZ- aimed to optimize a 3D clinostat for a life science experiment. Since a 3D induced diabetic liver injury in rats. For that reason, elevated clinostat is prepared with two motors, we fixed the angular velocity of one activities of SGOT and SGPT in the plasma might be due mostly to (primary) motor and varied for the angular velocity of the other the escape of these enzymes from the liver cell cytosol into the (secondary) motor. In this setup, each motor ran continuously and bloodstream, which is a signal of the hepatotoxic effect of STZ. constantly in one direction through the experiment; this device was used Treatment of the diabetic rats with the extract of WGM conditions to rotate plants in three dimensions at a speed of 4 rotations/min. Wheatgrass seeds were germinated under these conditions for 7 days. reduced the activities of these enzymes in plasma compared to those in the gravity and diabetic control groups and consequently alleviated liver damage caused by STZ-induced diabetes, as Preparation of plant extracts presented in our results. To limit microbial growth, all equipment and containers involved in the However, insulin deficiency deactivates the lipoprotein lipase germination process were treated with boiling water. Prior to germination, enzyme. Under conditions of insulin deficiency with low wheat grains of the same size were submerged in 1.25% sodium hypochlorite lipoprotein lipase, TG, cholesterol, and LDL levels were elevated, at room temperature for 30 min to disinfect and kill any contaminating and the HDL level was decreased. This hypertriglyceridemia was microorganisms on the grain surface. Distilled water at ~10 °C was then run over the grains for 15 min to thoroughly rinse off any remaining sodium reduced rapidly after insulin treatment therapy. In this study, the hypochlorite. The sterilized grains were steeped in tap water for 24 h at administration of WGM conditions significantly increased serum 21 °C. For germination, wheat grains were placed into a special container with insulin levels, a marked decrease in TC, TG, and LDL levels in rats drain holes at the bottom and irrigated with Hoagland’s solution. treated with WGM conditions and metformin-treated rats was Germination was carried out in a plant growth room at 21 °C for 7 days. observed in comparison to the diabetic control group. Addition- After harvest, the whole plants were washed carefully with distilled water for ally, an increased HDL level was observed compared to diabetic 16 3 min to remove any remaining perlite particles or nutrient elements. control rats. This activity of WGM conditions reduces hypertrigly- WGG and WGM conditions were dried under shade for 7 days at room ceridemia under diabetic conditions and therefore prevents temperature ranging from 29 to 32 °C with a relative humidity of 70–90% diabetic-associated complications. and then ground using a blender, while nongerminated seeds were Elevated plasma urea and creatinine levels signal the develop- discarded. An appropriate amount of the powdered plant material was extracted with ethanol using a Soxhlet extraction apparatus. The solvent ment of diabetic nephropathy in diabetic rats. The urea and was completely removed under reduced pressure in a rotary vacuum creatinine levels in rats with diabetic nephropathy are higher than evaporator to yield the crude extract. All extracts were packed in an air- those in normal rats. The observed maintenance of urea and proof container before being stored at −20 °C until use. creatinine levels closer to those in control rats using extracts of WGM and gravity conditions, as well as metformin treatment, Determination of the TP, flavonoid, and vitamin C contents suggests that germinated wheatgrass extract plays an either direct or indirect primary role in protecting against diabetic nephropathy The TP content of WGG and WGM conditions was estimated using the Folin–Ciocalteu assay. A total of 475 µl of a 5% sodium carbonate or retarding its development. solution was mixed with 50 µl of plant extract. The reaction mixture was An ethyl alcohol extract was selected because of the expected incubated for 3–5 min, and 475 µl of 50% Folin–Ciocalteu reagent was presence of phenolic and flavonoid compounds with antihypergly- added. The reaction mixture was mixed and incubated at room cemic properties. The presence of phenolic and flavonoid temperature in darkness for 1 h. The absorbance of the reaction product compounds in our extract with antioxidant potential, as seen in was measured at 724 nm using a ultraviolet–visible spectrophotometer. our results, further confirms this prediction. Phenolic and flavonoid The TP content was calculated from a standard calibration curve based on compounds were shown to have antihyperglycemic effects. gallic acid, and the results are expressed as mmol of gallic acid equivalents −1 Phytochemical analysis of germinated wheatgrass showed the (GAEs) per 100 mg of plant dry weight (mmol GAE 100 mg DW). presence of tannins, flavonoids, saponins, and sterols. Their The total flavonoid (TF) content of WGG and WGM conditions was antidiabetic effects and ability to regenerate pancreatic β cells determined according to Karadeniz et al. Fifty microliters of plant extract have already been demonstrated. Sterols were shown to decrease and 600 µl of ddH O were mixed with 40 µl of 5% potassium nitrite. The npj Microgravity (2020) 6 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA W.J. Al-Awaida et al. mixture was incubated at room temperature for 6 min, and then 70 µl of a overnight fasting on the 0th (before the start of the experiment), 3rd, 7th, 4.26% aluminum chloride solution was added. After 5 min of incubation at 14th, 21st, and 30th day, and glucose levels were measured by using an 25 °C, we added 240 µl of sodium hydroxide (1 M) to the reaction mixture, Accu-Chek active glucometer. and the solution was mixed well. The absorbance of the reaction product was measured at 510 nm, and the TF content was calculated from a Oral glucose tolerance test standard curve based on rutin. The results are expressed as mmol of rutin −1 Briefly, after overnight fasting, the rats were intragastrically administered equivalents (REs) per 100 mg of plant dry weight (mmol RE 100 mg DW). glucose (2 g/kg). Blood samples were withdrawn from the tail vein after 0, Five milliliters of dilute extracts of T. aestivum germinated under gravity 30, 60, 90, and 120 min, and blood glucose levels were determined using and microgravity conditions was added into a test tube, following which an Accu-Chek active glucometer. 1 ml of glacial acetic acid was added, the solution was titrated with a 2,6- dichlorophenolindophenol solution (0.2 g/l) until a faint permanent pink color was observed, after which the titer (T) was recorded. The titration Assay of biochemical parameters was repeated with 5 ml of water as a blank (B 1) and 5 ml of an ascorbic After 30 days of treatment, the rats were fasted overnight and sacrificed. acid standard solution (0.1 g/l). The vitamin C content of the test sample Blood was collected using EDTA as the anticoagulant, and whole blood was calculated according to the following equation: was used to measure HbA1c, urea, and creatinine levels. The plasma was Vitamin C content (mg/dl) = (T − B1/ ST − B1) × 2 × dilution factor. separated by collecting blood in a heparin-coated tube and centrifuging it at 1000 × g for 15 min at 4 °C. The plasma was used to estimate insulin, and Hydrogen peroxide- and nitric oxide-scavenging activity assays plasma C-peptide assays were performed using a radioimmunoassay (RIA) kit for rats. TC, TG, HDL-C, and LDL levels and the activities of the liver The hydrogen peroxide-scavenging activity of ethanolic extracts of WGG enzymes AST and ALT were determined spectrophotometrically using and WGM conditions was evaluated according to the method of Ruch commercial kits. et al. The extract at different concentrations (3.4 ml, pH 7.4) was mixed with a hydrogen peroxide solution (43 mM, 0.6 ml, pH 7.4). After 10 min, the absorbance of the reaction mixture was measured at 230 nm. The reaction Histological analysis mixture without sample was used as a blank. Ascorbic acid was used as a Specimens of the pancreatic tissue of the different groups were reference compound. The percentage inhibition activity was evaluated as immediately fixed in 10% formalin and then underwent standard [(Abs. of the control− Abs. of the sample)/Abs. of the control] × 100%. treatment with an alcohol gradient and xylene. For histopathological Nitric oxide was produced from sodium nitroprusside and measured by examination, 6-µm-thick pancreatic specimens were stained with hema- the Griess reaction. Nitric oxide was generated spontaneously from toxylin and eosin. The β cells were counted under a ×40 objective lens. Cell sodium nitroprusside compound dissolved in aqueous solution at densities are expressed as cells per pancreatic islet. physiological pH and interacted with oxygen to generate nitrite ions, which were measured by the use of Greiss reagent. Different concentra- tions of ethanolic extracts of WGG and WGM conditions dissolved in Statistical analysis dimethyl sulfoxide were mixed with sodium nitroprusside (5 mM) in Statistical analysis was performed using GraphPad Prism 7. Values are phosphate-buffered distilled water and incubated at 25 °C for 150 min. A expressed as the mean ± SEM, and data were analyzed by using two-way control experiment without the test compound but with the equivalent ANOVA, followed by Tukey’s multiple comparison test, with the level of amount of alcohol was conducted similarly. At intervals, samples (0.5 ml) of significance set at P < 0.05. the incubation solution were diluted with 0.5 ml of Griess reagent (1% sulfanilamide, 0.1% naphthyl ethylenediamine dihydrochloride and 2% H PO ). 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Effect of simulated microgravity on the antidiabetic properties of wheatgrass (Triticum aestivum) in streptozotocin-induced diabetic rats

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Abstract

www.nature.com/npjmgrav ARTICLE OPEN Effect of simulated microgravity on the antidiabetic properties of wheatgrass (Triticum aestivum) in streptozotocin-induced diabetic rats 1 1 1 2 Wajdy J. Al-Awaida , Ahmad S. Sharab , Hamzeh J. Al-Ameer and Nabil Y. Ayoub Microgravity affects plant growth and content. A three-dimensional clinostat was used at 4 rotations/min to rotate the seeds of Triticum aestivum cultivar (Ammon) in three dimensions for 7 days, following which the antioxidant activities of ethanolic extracts were evaluated using both nitric oxide- and hydrogen peroxide-scavenging activities. The antidiabetic activities of ethanolic extracts were evaluated by measuring the concentration of plasma glucose, insulin, C peptide, and glycated hemoglobin (HbA1c); determining the number of β cells in the pancreatic islets; and performing the glucose tolerance test. Furthermore, the effects of the ethanolic extracts on the lipid profile and liver function were estimated. After rats were sacrificed, their pancreases were isolated and used for histopathological processing. The results indicated that the antioxidant potential and antioxidant metabolite content were significantly increased under microgravity conditions in comparison to those under normal gravity conditions. Rats treated with an extract of wheatgrass (T. aestivum) germinated over a period of 6–10 days under microgravity (WGM) showed a significant reduction in the levels of serum glucose, HbA1C, urea, creatinine, aspartate aminotransferase and alanine aminotransferase, and insulin resistance compared to rats treated with an extract of wheatgrass germinated under gravity. Additionally, the total cholesterol and low-density lipoprotein cholesterol levels were significantly decreased. In contrast, high- density lipoprotein cholesterol, C-peptide, and insulin levels rose significantly after treatment with T. aestivum germinated under microgravity. WGM is a promising potential diabetic treatment without side effects with a low manufacturing cost. npj Microgravity (2020) 6:6 ; https://doi.org/10.1038/s41526-020-0096-x INTRODUCTION effects. Therefore, the current focus on diabetes treatment is on the development of an alternative solution for the treatment of Diabetes is a metabolic syndrome that arises mainly due to diabetes from natural sources, which are affordable and effective deficiencies in insulin activity, insulin secretion, or both. This and have fewer side effects than synthetic drugs. In traditional disorder can cause serious problems that affect human health. medicine, medicinal plants are used for the treatment of diabetes; Over the long term, uncontrolled diabetes can lead to several these plants are a rich source of hypolipidemic, hypoglycemic, and chronic complications, including renal failure, heart disease, and 3 antioxidant agents, such as phenols, flavonoids, gallotannins, and blindness. Statistically, 8.8% of the world’s population in 2015 11,12 other related polyphenols. exhibited the symptoms of this disease, and this percentage is Wheatgrass (T. aestivum germinated over a period of 6–10 days) predicted to rise to more than 10.4% by 2040. was found to improve metabolic and lipid profiles and to restore Despite the significant developments made in the management 14,15 the levels of plasma glucose, insulin, and liver glycogen. of diabetes over the past two decades, the effects of diabetes The nutrient value of germinated grains depends on the treatments in patients are still far from exemplary. These conditions in which they are grown. These conditions include treatments have many disadvantages, including a reduction in humidity, temperature, length of germination, culture medium, drug efficiency, toxicity, and side effects. For example, sulfonylur- and steeping protocol. The microgravity environment encoun- eas lose their efficiency after 6 years of treatment in 44% of tered during space flight has long been thought to affect plant patients. It is also believed that glucose-lowering medications are growth and content. Until now, no research on the therapeutic not capable of controlling hyperlipidemia. Recently, the use of potential of wheatgrass germinated under microgravity (WGM) herbal medications with insignificant toxicity and no side effects conditions has been carried out. Therefore, the present study was for the treatment of diabetes mellitus has been demonstrated to carried out to produce wheatgrass with high antioxidant and be of global importance. antidiabetic potential, but no side effects with a low Wheatgrass is believed to have a better nutritive value than manufacturing cost. nongerminated grains and their products. Antioxidant com- pounds, such as vitamins C, flavonoids, and phenolic compounds, RESULTS are scarcely measurable in dry grains. However, upon germination, the concentrations of these antioxidant compounds increase with Plant phenolics and flavonoids play a substantial role in increasing germination time and peak after 7 days. scavenging free radicals in the body and act as antioxidants. There are numerous hypoglycemic medications available in the Our results showed the total phenolic content (TPC) of the clinic, but these medications are associated with many side ethanolic extracts of wheatgrass germinated under gravity (WGG) 1 2 Department of Biology and Biotechnology, American University of Madaba, Madaba, Jordan. Department of Basic Sciences and Humanities, Faculty of Science, American University of Madaba (AUM), Amman 11821, Jordan. *email: w.alawaida@aum.edu.jo Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA 1234567890():,; W.J. Al-Awaida et al. Fig. 1 Triticum aestivum growth under gravity and microgravity conditions and the effect of simulated microgravity on the total phenolic content, total flavonoid content, vitamin C content, and total antioxidant activities in wheatgrass extract. a shows T. aestivum growth under gravity conditions. b shows T. aestivum growth under microgravity conditions simulated by a three-dimensional clinostat. c shows the total phenolic content of ethanolic extracts of T. aestivum under gravity and microgravity conditions. d shows the total flavonoid content of ethanolic extracts of T. aestivum under gravity and microgravity conditions. e shows the vitamin C content of ethanolic extracts of T. aestivum under gravity and microgravity conditions. f shows the hydrogen peroxide- and nitric oxide radical-scavenging activities of ethanolic extracts of T. aestivum under gravity and microgravity conditions. The values are expressed as the mean ± SEM of seven rats in each group. Data in c–e were analyzed by using two-way ANOVA, followed by Tukey’s multiple comparison test with the level of significance set at P < 0.05. *, **, ***, **** = P < 0.05, P < 0.01, P < 0.001, and P < 0.0001, respectively. Data in f were analyzed by using two-way ANOVA, followed by Tukey’s multiple comparison test with the level of significance set at P < 0.05. a refers to microgravity versus a reference compound ascorbic acid; b refers to gravity versus a reference compound ascorbic acid; and c refers to microgravity versus a gravity. and WGM conditions. The TPC is expressed as mmol equivalents metabolism. Vitamin C also helps with iron absorption and is of gallic acid/100 g fresh weight. In the ethanolic extract of WGM, important for immune system health. the TPC (190.0 ± 2.11 mmol) was found to be significantly higher The quantity of vitamin C, quantified as the total ascorbic acid (P < 0.0001; TPC of extracts by two-way analysis of variance content, was higher in the ethanolic extract of WGM (20.32 ± (ANOVA), followed by Tukey’s multiple comparison test) than that 0.56 mg/dl) (P < 0.01; vitamin C of extracts by two-way ANOVA, of the ethanolic extract of wheatgrass grown under gravity followed by Tukey’s multiple comparison test) than in the (100.4 ± 2.15 mmol), as shown in Fig. 1c. ethanolic extract of WGG (15.43 ± 0.32 mg/dl) (Fig. 1e). Our results present the total flavonoid content (TFC) of Hydrogen peroxide and nitric oxide radical-scavenging activities ethanolic extracts of WGG and WGM conditions. The TFC is were measured to determine the ability of the WGM extracts to expressed as mmol equivalents of rutin/mg fresh weight. The detoxify and scavenge free radicals. TFC of the ethanolic extract of WGM was significantly higher Our results present the hydrogen peroxide and nitric oxide (208.1 ± 6.57 mmol) (P < 0.0001; TFC of extracts by two-way radical-scavenging activities of ethanolic extracts of WGG and ANOVA followed by Tukey’s multiple comparison test) than the WGM. The ethanolic extract of WGM showed significantly greater TFC of the ethanolic extract of WGG (30.68 ± 4.01 mmol), as (P < 0.05; H O -scavenging activity of extracts by two-way ANOVA, 2 2 shown in Fig. 1d. followed by Tukey’s multiple comparison test) H O -scavenging 2 2 Vitamin C, also known as ascorbic acid, is an antioxidant activity with a lower half-maximal inhibitory concentration (IC ) compound and a component of an enzyme required for protein value (13.0 ± 2.8 µg/ml) compared to ascorbic acid (54.9 ± 1.90 µg/ npj Microgravity (2020) 6 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA 1234567890():,; W.J. Al-Awaida et al. (105.0 ± 7.0 mg/dl) (Fig. 2a). In diabetic rats treated with metfor- min, blood glucose levels began to decrease and reached the control level after 30 min (74.0 ± 16.36 mg/dl), at which they remained until 120 min (65.0 ± 14.36 mg/dl) (Fig. 2a). In the diabetic group treated with the ethanolic extract of WGM, the blood glucose levels were significantly decreased compared to those in the diabetic control group, the diabetic group treated with the ethanolic extract of WGG, and the diabetic group treated with metformin (Fig. 2a). Diabetes is characterized by a high level of glucose in the bloodstream caused by insufficient insulin secretion or insulin resistance. The diabetic group showed a significant increase (P < 0.05; blood glucose levels by two-way ANOVA, followed by Tukey’s multiple comparison test) in blood glucose levels in comparison to the control group. First, oral administration of the ethanolic extract of WGM in the diabetic group revealed a highly significant reduction (P < 0.05; blood glucose levels by two-way ANOVA, followed by Tukey’s multiple comparison test) in blood glucose levels starting on the seventh day of treatment (234.50 ± 14.28 mg/dl) compared to the levels in the same group before treatment (day 0) (370.0 ± 4.81 mg/dl) as well as the levels in the diabetic group on the corresponding days (560.0 ± 13.06 and 371.50 ± 24.11 mg/dl, respectively). This reduction was marked on the 30th day, as blood glucose levels were reduced by 79.5% compared to those at day 0 and 84.7% compared to the value in the diabetic group (Fig. 2b). Fig. 2 The effects of ethanolic extracts of T. aestivum germinated Subsequently, the group treated with the ethanolic extract of under gravity and microgravity conditions on oral glucose WGG exhibited a highly significant reduction (P < 0.05; blood tolerance tests over 2 h and fasting blood glucose levels over glucose levels by two-way ANOVA, followed by Tukey’s multiple 30 days in experimental rats. a shows the effect of ethanolic comparison test) in blood glucose levels starting on the seventh extracts of T. aestivum under gravity and microgravity conditions on the results of the oral glucose tolerance test (OGTT). b shows the day of treatment (361.0 ± 25.92 mg/dl) compared to levels on day effect of ethanolic extracts of T. aestivum under gravity and 0 (587.0 ± 10.61 mg/dl) and the levels in diabetic rats on the microgravity conditions on fasting blood glucose levels in experi- corresponding days (560.0 ± 13.06 and 371.50 ± 24.11 mg/dl, mental rats. The values are expressed as the mean ± SEM of seven respectively). This reduction was marked on the 30th day, as rats in each group. Data were analyzed by using two-way ANOVA blood glucose levels were reduced by 77.9% compared to those followed by Tukey’s multiple comparison test with the level of on day 0 and 76.8% compared to those in the diabetic group (Fig. significance set at P < 0.05. a refers to treatment versus normal 2b). The diabetic group treated with metformin showed a control; b refers to treatment versus diabetic control; c refers to significant reduction (P < 0.05; blood glucose levels by two-way microgravity versus gravity. ANOVA, followed by Tukey’s multiple comparison test) in blood glucose levels starting on the seventh day of treatment (105.0 ± ml), which was used as a positive control, and the ethanolic extract 4.08 mg/dl) compared to blood glucose levels on day 0 (259.50 ± of WGG (35.0 ± 1.93 µg/ml). In contrast, the ethanolic extract of 11.83 mg/dl) and those of the diabetic group on the correspond- WGM showed significantly greater (P < 0.05; nitric oxide- ing days (560.0 ± 13.06 and 371.50 ± 24.11 mg/dl, respectively). scavenging activity of extracts by two-way ANOVA, followed by This reduction was marked on the 30th day, as blood glucose Tukey’s multiple comparison test) nitric oxide-scavenging activity levels were reduced by 54.8% compared to those on day 0 and with a lower IC compared to the positive control, ascorbic acid, 79.1% compared to those in the diabetic group (Fig. 2b). In the and the ethanolic extract of WGG, as shown in Fig. 1f. diabetic group treated with the ethanolic extract of WGM, blood The glucose tolerance test (GTT) has been extensively used for glucose levels were significantly decreased (P < 0.05; blood the study of carbohydrate metabolism in experimental animals. glucose levels by two-way ANOVA, followed by Tukey’s multiple Time-dependent changes in blood glucose levels during the oral comparison test) compared to the group treated with the GTT (0–120 min) for all groups are shown in Fig. 2a. The rats in the ethanolic extract of WGG beginning on day 7 (234.50 ± 14.28 diabetic group had significantly higher (P < 0.05; GTT by two-way and 361.0 ± 25.92 mg/dl, respectively). This reduction was marked ANOVA, followed by Tukey’s multiple comparison test) blood on the 30th day (75.50 ± 11.02 and 129.50 ± 5.30 mg/dl), when glucose levels throughout the total experimental period (120 min) blood glucose were 41.6% lower in the group treated with the (450.0 ± 15.14 to 580.0 ± 17.07 mg/dl) compared to the normal ethanolic extract of WGM (Fig. 2b). control group (111.0 ± 13.0 to 87.0 ± 13.0 mg/dl) (Fig. 2a). In Insufficient insulin prevents the body from transporting glucose diabetic rats treated with the ethanolic extract of WGG, even after from the bloodstream into the body’s cells to use as energy. When the administration of glucose, blood glucose levels remained the this occurs, the body starts burning fat and muscle for energy, same as those at 0 min (430 ± 24.74 mg/dl) and even those at causing a decrease in overall body weight. Changes in the body 60 min (373 ± 14.14 mg/dl), following which the blood glucose weight of rats in the normal control, diabetic control, microgravity, levels began to decrease after 90 min (200.0 ± 14.84 mg/dl) and gravity, and metformin groups over a period of 30 days are shown reached the upper limit for normal glucose levels after 120 min in Fig. 3. A significant decrease (P < 0.05; body weight by two-way (172.0 ± 11.31 mg/dl) (Fig. 2a, P < 0.05; GTT by two-way ANOVA, ANOVA, followed by Tukey’s multiple comparison test. *, **, ***, followed by Tukey’s multiple comparison test). In diabetic rats **** = P < 0.05, P < 0.01, P < 0.001, and P < 0.0001, respectively) in treated with the ethanolic extract of WGM, the blood glucose body weight was observed in the diabetic and gravity groups levels began to decrease, reached the control level after 30 min compared with their body weights at the initial day of the (112.0 ± 13.36 mg/dl) and remained the same until 120 min experiment (from 190.0 ± 12.4 to 152.0 ± 7.49 g and from 201.0 ± Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2020) 6 W.J. Al-Awaida et al. Fig. 3 The effects of ethanolic extracts of T. aestivum germinated under gravity and microgravity conditions on body weight in experimental rats after 30 days. a shows changes in body weight in untreated nondiabetic rats on days 0 and 30. b shows changes in body weight in untreated diabetic rats on days 0 and 30. c shows changes in diabetic rats treated with the ethanolic extract of T. aestivum germinated under microgravity conditions on days 0 and 30. d shows changes in diabetic rats treated with the ethanolic extract of T. aestivum germinated under gravity condition group on days 0 and 30. e shows changes in diabetic rats treated with metformin on days 0 and 30. All values represent the mean ± SD. All comparisons were performed between the same group of animals on days 0 and 30. Data were analyzed by using two-way ANOVA, followed by Tukey’s multiple comparison test with the level of significance set at P < 0.05. *, ***, **** = P < 0.05, , P < 0.001, and P < 0.0001, respectively. 8.7 to 172.0 ± 9.23 g, respectively). However, there was no significant difference (P < 0.05; body weight by two-way ANOVA, followed by Tukey’s multiple comparison test. *, **, ***, **** = P < 0.05, P < 0.01, P < 0.001, and P < 0.0001, respectively) in body weight between the normal control, microgravity, and metformin groups on the initial day of the experiment (Fig. 3). Glycated hemoglobin (HbA1c), a form of hemoglobin that is chemically linked to glucose, indicates the sugar level in the bloodstream over a long period. Our results show the HbA1C levels in normal and experimental rats. In the diabetic control group, there was a significant increase (P < 0.05; HbA1C level by two-way ANOVA, followed by Tukey’s multiple comparison test) in the HbA1C level (8.85 ± 0.24%) compared to the normal control group (5.55 ± 0.21%) (Fig. 4a). In the diabetic group treated with the ethanolic extract of WGM, the HbA1C level was significantly decreased (5.75 ± 0.33%) (P < Fig. 4 Effect of treatment with ethanolic extracts of T. aestivum 0.05; HbA1C level by two-way ANOVA, followed by Tukey’s under gravity and microgravity conditions for 30 days on multiple comparison test) compared to the diabetic control group C-peptide, serum insulin, and glycated hemoglobin (HbA1c) (Fig. 4a). In the diabetic group treated with the ethanolic extract of levels in experimental rats. a shows the effect of ethanolic extracts WGG, the HbA1C levels was significantly decreased (7.02 ± 0.23%) of T. aestivum germinated under gravity and microgravity conditions (P < 0.05; HbA1C level by two-way ANOVA, followed by Tukey’s on body weight in experimental rats. b shows the effect of ethanolic multiple comparison test) compared to the diabetic group. The extracts of T. aestivum germinated under gravity and microgravity HbA1C level was higher in the diabetic gravity group. In diabetic conditions on C-peptide levels in experimental rats. c shows the effect of ethanolic extracts of T. aestivum germinated under gravity rats treated with metformin, the HbA1C levels was significantly and microgravity conditions on serum insulin levels in experimental decreased (5.65 ± 0.14%) (P < 0.05; HbA1C level by two-way rats. The values are expressed as the mean ± SEM of seven rats in ANOVA, followed by Tukey’s multiple comparison test) compared each group. Data were analyzed by using two-way ANOVA, followed to the diabetic group. A significant difference (P < 0.05; HbA1C by Tukey’s multiple comparison test with the level of significance set level by two-way ANOVA, followed by Tukey’s multiple compar- at P < 0.05. a refers to treatment versus normal control, b refers to ison test) in the HbA1C level was observed between the gravity treatment versus diabetic control, and c refers to microgravity versus and WGM groups. The HbA1C level in the diabetic group treated gravity. with WGM was significantly lower than the HbA1C level in the diabetic group treated with WGG (Fig. 4a). C-peptide of insulin, a metabolic byproduct produced during ANOVA, followed by Tukey’s multiple comparison test) compared the activation of insulin, is a useful and common method used to to the diabetic group by 1.35-fold (Fig. 4b). There was no assess pancreatic β cell function. significant difference (P < 0.05; C-peptide level by two-way The C-peptide level was reduced in the diabetic group (0.039 ± ANOVA, followed by Tukey’s multiple comparison test) in the C- 0.0015 ng/ml) compared to the normal group (0.047 ± 0.0032 ng/ peptide level between the diabetic group treated with the ethanolic ml) (Fig. 4b). In the diabetic group treated with the ethanolic extract of WGG (0.04 ± 0.0021 ng/ml) and both the diabetic and extract of WGM, the C-peptide level was significantly increased normal control groups (Fig. 4b). C-peptide levels in the diabetic (0.053 ± 0.0022 ng/ml (P < 0.05; C-peptide level by two-way group treated with metformin (0.045 ± 0.0010 ng/ml) were not npj Microgravity (2020) 6 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA W.J. Al-Awaida et al. significantly different (P < 0.05; C-peptide level by two-way ANOVA, Diabetic rats showed a significant increase (P < 0.05; TC, TG, and followed by Tukey’s multiple comparison test) compared to those in LDL-C levels by two-way ANOVA followed by Tukey’s multiple the diabetic group (Fig. 4b). In the diabetic group treated with the comparison test) in the TC (2.18 ± 0.14 mg/dl), TG (0.85 ± .17 mg/ ethanolic extract of WGG, there was a significant increase (P <0.05; dl), and LDL-C (2.06 ± 0.15 mg/dl) levels in the blood (Fig. 5b). In C-peptide level by two-way ANOVA, followed by Tukey’s multiple contrast, the high-density lipoprotein cholesterol (HDL-C) level (1.47 ± 0.05 mg/dl) was significantly reduced (P < 0.05; HDL-C level comparison test) in the C-peptide level insulin level by 1.32-fold by two-way ANOVA, followed by Tukey’s multiple comparison test) compared to the gravity group (Fig. 4b). compared to the normal control group. In diabetic rats treated The plasma insulin level was significantly reduced (P < 0.05; with the ethanolic extract of WGM, the TC (1.75 ± 0.04 mg/dl), TG insulin level by two-way ANOVA, followed by Tukey’s multiple (0.56 ± 0.05 mg/dl), and LDL-C (1.11 ± 0.9 mg/dl) levels in the comparison test) in the diabetic group (1.2 ± 0.30 µlU/ml) com- blood were significantly decreased (P < 0.05; TC, TG, and LDL-C pared to the normal control group (2.6 ± 0.20 µlU/ml) (Fig. 4c). The levels by two-way ANOVA, followed by Tukey’s multiple compar- plasma insulin level in the diabetic group treated with the ison test). However, the level of HDL-C (1.64 ± 0.12 mg/dl) was ethanolic extract of WGM was significantly increased by 2.53-fold significantly increased (P < 0.05; HDL-C level by two-way ANOVA, (3.04 ± 0.35 µlU/ml) (P < 0.05; insulin level by two-way ANOVA, followed by Tukey’s multiple comparison test) (Fig. 5b). In diabetic followed by Tukey’s multiple comparison test) compared to the rats treated with the ethanolic extract of WGG, the blood level of diabetic control group (Fig. 4c). There was no significant difference LDL-C (1.46 ± 0.12 mg/dl) was significantly decreased (P < 0.05; (P < 0.05; insulin level by two-way ANOVA, followed by Tukey’s LDL-C level by two-way ANOVA, followed by Tukey’s multiple multiple comparison test) in the plasma insulin level of the comparison test). However, TC (1.96 ± .05 mg/dl), HDL-C (1.14 ± diabetic group treated with the ethanolic extract of WGG (1.9 ± 0.10 mg/dl), and TG (0.75 ± 0.20 mg/dl) levels were not changed 0.12 µlU/ml) compared to the diabetic and normal control groups (Fig. 5b). In diabetic rats treated with metformin, the TC (1.64 ± (Fig. 4c). In diabetic rats treated with metformin, the insulin level 0.14 mg/dl) and LDL-C (1.31 ± 0.13 mg/dl) levels in the blood were was significantly increased (2.34 ± 0.21 µlU/ml) (P < 0.05; insulin significantly decreased (P < 0.05; TC and LDL-C levels by two-way level by two-way ANOVA, followed by Tukey’s multiple compar- ANOVA, followed by Tukey’s multiple comparison test). However, ison test) by 1.95-fold compared to the diabetic group (Fig. 4c). In the TG (0.60 ± 0.10 mg/dl) and HDL-C (0.97 ± 0.04 mg/dl) levels the diabetic group treated with the ethanolic extract of WGM, the were not changed (Fig. 5b). The level of HDL-C in the microgravity insulin level was significantly increased (P < 0.05; insulin level by group was significantly increased (P < 0.05; HDL-C level by two- two-way ANOVA, followed by Tukey’s multiple comparison test) by way ANOVA, followed by Tukey’s multiple comparison test) 1.6-fold compared to the gravity group (Fig. 4c). compared to the gravity group (Fig. 5b). Serum urea and creatinine are known to be increased with Increased activities of the liver enzymes aspartate aminotrans- hyperglycemia in patients with uncontrolled diabetes and usually ferase (AST) and alanine aminotransferase (ALT) are indicators of correlate with the severity of kidney damage. Our results show the liver cell injury and associated with uncontrolled diabetes. levels of creatinine and urea in normal and experimental rats. In The induction of diabetes led to significant increases (P < 0.05; diabetic rats, creatinine and urea levels were significantly AST and ALT levels by two-way ANOVA, followed by Tukey’s increased (100.60 ± 3.50 and 25.80 ± 2.30 mg/dl, respectively) multiple comparison test) in the serum activities of AST (373.50 ± (P < 0.05; creatinine and urea levels by two-way ANOVA, followed 16.30 mg/dl) and ALT (210.70 ± 14.10 mg/dl) in the diabetic group by Tukey’s multiple comparison test) compared to the normal compared to the normal group (129.0 ± 10.20 and 63.0 ± 4.20 mg/ control group (34.40 ± 1.51 and 6.0 ± 0.80 mg/dl, respectively) dl, respectively) (Fig. 5c). ALT (132.40 ± 11.60 mg/dl) and AST (Fig. 5a). The creatinine level in the diabetic group treated with the (45.20 ± 3.60 mg/dl) activities were significantly reduced (P < 0.05; ethanolic extract of WGM was significantly decreased (37.80 ± AST and ALT levels by two-way ANOVA, followed by Tukey’s 2.03 mg/dl) (P < 0.05; creatinine level by two-way ANOVA, followed multiple comparison test) in the group of diabetic rats treated by Tukey’s multiple comparison test) compared to the diabetic with the ethanolic extract of WGM compared to the diabetic control group (100.60 ± 3.50 mg/dl) (Fig. 5a). The creatinine level group. The AST and ALT levels in the diabetic gravity group were in the diabetic group treated with the ethanolic extract of WGG higher than those in the diabetic microgravity group. (Fig. 5c). was significantly decreased (53.20 ± 1.43 mg/dl) (P < 0.05; creati- Interestingly, the diabetic group treated with metformin showed a nine level by two-way ANOVA, followed by Tukey’s multiple significant reduction (P < 0.05; AST and ALT levels by two-way comparison test) compared to the diabetic control group ANOVA, followed by Tukey’s multiple comparison test) in ALT (100.60 ± 3.50 mg/dl) (Fig. 5a). The creatinine level in the diabetic (158.0 ± 14.30 mg/dl) and AST (62.30 ± 7.20 mg/dl) activities com- gravity group was higher than that in the diabetic microgravity pared to the diabetic group (Fig. 5c). group. In diabetic rats treated with metformin, the creatinine level β Cells are a type of cell found in pancreatic islets that produce and was significantly increased (34.10 ± 1.08 mg/dl) (P < 0.05; creati- secrete insulin. In diabetes, β cell mass and function are diminished, nine level by two-way ANOVA, followed by Tukey’s multiple leading to insufficient insulin secretion and hyperglycemia. comparison test) compared to the diabetic control group To elucidate the preventative effects of ethanolic extracts of (100.60 ± 3.50 mg/dl) (Fig. 5a). The blood urea level was increased WGG and WGM conditions on streptozotocin (STZ)-induced in the diabetic group (25.8 ± 2.30 mg/dl). However, the blood urea diabetes, the pancreatic tissues of the ethanol extract-treated level was significantly decreased (P < 0.05; urea level by two-way and control groups were examined. The results revealed the ANOVA, followed by Tukey’s multiple comparison test) in the following: the normal control group showed a normal pancreatic microgravity (9.10 ± 1.20 mg/dl), gravity (12.63 ± 2.10 mg/dl) and structure (Fig. 6a), whereas the diabetic group showed degenera- metformin (8.20 ± 1.70 mg/dl) groups compared to the diabetic tion, necrotic changes, and islet shrinkage in the pancreas control group. No significant differences (P < 0.05; urea level by (Fig. 6b). The pancreatic sections of rats administered the two-way ANOVA, followed by Tukey’s multiple comparison test) ethanolic extract of WGG showed improved islet morphology in blood urea level were found in the microgravity, gravity, and (Fig. 6c). The pancreatic sections of rats administered the ethanolic metformin groups in comparison to the normal control group extract of WGM showed a normal structure in the pancreatic islets (Fig. 5a). of Langerhans (Fig. 6d). There were no pathological changes in the Uncontrolled diabetes tends to raise total cholesterol (TC), total pancreatic islets of Langerhans in diabetic rats treated with triglyceride (TG), and low-density lipoprotein cholesterol (LDL-C) metformin (Fig. 6e). β Cells in the pancreatic islets was measured levels, which increases the risk of heart disease and stroke. This under a ×40 objective lens. The results revealed a significant common condition is called diabetic dyslipidemia. decrease (P < 0.001; β cells by one-way ANOVA with Bonferroni’s Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2020) 6 W.J. Al-Awaida et al. Fig. 5 Effect of treatment with ethanolic extracts of T. aestivum under gravity and microgravity conditions for 30 days on liver and kidney functions and lipid profiles in experimental rats. a shows the effect of ethanolic extracts of T. aestivum under gravity and microgravity conditions on biomarkers of kidney function in experimental rats. b shows the effect of ethanolic extracts of T. aestivum under gravity and microgravity conditions on the lipid profiles of experimental rats. c shows the effect of ethanolic extracts of T. aestivum under gravity and microgravity conditions on biomarkers of liver function in experimental rats. The values are expressed as the mean ± SEM of seven rats in each group. Data were analyzed by using two-way ANOVA, followed by Tukey’s multiple comparison test with the level of significance set at P < 0.05. a refers to treatment versus normal control, b refers to treatment versus diabetic control, and c refers to microgravity versus gravity. multiple comparisons test. *, ***, ****, ns = P < 0.05, P < 0.001, P < 0.05, P < 0.001, P < 0.0001, and nonsignificant, respectively) in the 0.0001, and nonsignificant, respectively) in the number of β cells number of β cells per islet in the microgravity group (114 ± 7.50) per islet in the diabetic control group (15 ± 3.46) compared to the compared to the normal control group. However, there was no normal control group (51 ± 5.19). In addition, there was a significant difference (ns) in the number of β cells per islet in the significant decrease (P < 0.05; β cells by one-way ANOVA with metformin group (50 ± 4.04) compared to the normal control Bonferroni’s multiple comparisons test. *, ***, ****, ns = P < 0.05, P group. < 0.001, P < 0.0001, and nonsignificant, respectively) in the number of β cells per islet in the gravity group (31 ± 2.88) DISCUSSION compared to the normal control group (15 ± 3.46). However, there was a significant increase (P < 0.0001; β cells by one-way ANOVA This study evaluated the effect of simulated microgravity on the with Bonferroni’s multiple comparisons test. *, ***, ****, ns = P < antidiabetic and antioxidant potential of wheatgrass extracts in npj Microgravity (2020) 6 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA W.J. Al-Awaida et al. Fig. 6 Photomicrographs of H&E-stained histological slides of pancreatic specimens. a Normal histological appearance of the islets of Langerhans from the pancreases of rats in the control group. b The pancreases of rats in the diabetic control group showed degeneration, necrotic changes, and islet shrinkage. c The appearance of pancreas sections from rats administered the ethanolic extract of T. aestivum under gravity conditions showed improved islet morphology. d The appearance of pancreas sections from rats administered the ethanolic extract of T. aestivum under microgravity conditions showed the normal structure of the pancreatic islets of Langerhans. e shows that there were no pathological changes in the islets of Langerhans of the pancreas in diabetic rats treated with metformin. Original magnification was ×400. f The pancreatic β cell number per islet in different treatment groups. Groups were compared to the control untreated group. As shown in the figure, microgravity extract treatment had a highly significant effect on the pancreatic β cell number per islet compared to the normal untreated control group. Data were compared against data for the normal control by using one-way ANOVA with Bonferroni’s multiple comparisons test with the level of significance at P < 0.05. *, ***, ****, ns = P < 0.05, P < 0.001, P < 0.0001, and nonsignificant, respectively. STZ-induced diabetic rats. Insulin-dependent diabetes mellitus conditions was detected in experimental animals following the 23,24 (IDDM) is a disorder initiated by progressive damage to insulin- administration of antioxidant compounds. secreting β cells. STZ is normally used to induce diabetes in Although wheatgrass germinated under normal conditions (under gravity) showed significant antioxidant potential and pancreatic rats by DNA alkylation and β cell fragmentation. STZ antihyperglycemic effects in STZ-induced diabetic rats as a result selectively devastates the β cells, which secrete insulin, decreasing 14,25 of its high TP and flavonoid concentrations, our results under the β cell efficiency and resulting in diabetes mellitus. Many microgravity conditions show even more promise in the field. The living β cells remain after treatment with low-dose STZ, and their microgravity environment created by a three-dimensional (3D) regeneration is also possible. clinostat produced wheatgrass with a higher concentration of In our study, significantly more β cells were observed in the antioxidant compounds and a higher potential for antioxidant group treated with the ethanol extract of WGM conditions, and activity than WGG. We hypothesize that the presence of a high stimulation due to insulin secretion could be responsible for most concentration of some bioactive compounds, such as phenolic of the observed metabolic effects. Bioactive compounds in compounds, flavonoids, and vitamin C, in WGM conditions medicinal plants may have insulin-like properties and have been produces an insulinotropic effect due to the antidiabetic effects shown to enhance insulin sensitivity, improve glucose-dependent of these compounds that facilitates a decrease in blood glucose insulin secretion, and activate the regeneration of the pancreatic levels. The elevated plasma glucose levels in diabetic rats were islets of Langerhans in STZ-induced diabetic rats. Currently, the decreased due to the administration of wheatgrass germinated only option for achieving normal glucose levels in diabetic extract under microgravity conditions, and the administration of patients is the stimulation of insulin secretion by medicinal plants WGM elevated C-peptide and plasma insulin levels compared to 20,21 or renewal of β cells. Increased free radical generation and those in diabetic control rats. The possible mechanism of action of oxidative stress together with hyperglycemia play a key role in the WGM conditions is its regeneration and stimulation of β cells to pathogenesis of diabetes and its late complications. β Cells are release insulin, which mediates a decrease in glucose levels, as highly sensitive to destruction by oxidative stress because of their shown in our results. low level of free radical-scavenging enzymes, which protect the Wheatgrass ethyl alcohol extract was shown to decrease cell from the damaging effects of oxidative stress. Decreased mononuclear cell infiltration in the β cells of the pancreas oxidative stress due to a reduced hyperglycemic status in diabetic compared to that in the untreated group. This effect coincides Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2020) 6 W.J. Al-Awaida et al. with the protective effect of wheatgrass extract on the diabetes blood sugar levels in experimental animal models. The antiox- parameters investigated in this study. This effect was reported idant activity of these phenolic and flavonoid compounds depends using germinated T. aestivum. on their redox properties, and these compounds can act as The induction of diabetes by STZ led to a reduction in body hydrogen donors, reducing agents, and singlet oxygen quench- weight due to structural protein degradation as a result of ers. Polyphenolics containing hydroxyl groups are essential plant disturbances in carbohydrate metabolism and thus energy ingredients that can defend the body against oxidative stress. The metabolism. Due to damage to β cells caused by STZ, a present study concluded that the microgravity environment decrease in body weight and an increase in water and food intake created by a 3D clinostat produced germinated T. aestivum with a are commonly detected in diabetes, and these effects may be high concentration of antioxidant compounds and high level of caused by metabolic changes due to the absence or deficiency of potential antioxidant activity; the produced wheatgrass has insulin. The nonsignificant changes in the body weights of potential as a diabetic treatment without any side effects and a diabetic rats treated with extracts of WGG and WGM conditions as low manufacturing cost. This method can be used to explore the well as metformin-treated diabetic rats compared to the control therapeutic potential of WGM conditions for other diseases. group revealed the effect of a normal blood glucose level, which in turn prevents the loss of body weight. HbA1c is used as an indicator to assess the degree of METHODS hemoglobin glycation in diabetes mellitus. HbA1c was found to The 3D clinostat represent the average fasting blood glucose level of diabetic Earth’s gravitational field at its surface at a certain location is a fixed vector patients over the previous 120 days. Under diabetic conditions, pointing downwards (towards the Earth’s center) whose average extra glucose in the blood is covalently bound to hemoglobin to magnitude is 9.8 m/s. Many plants and organisms contain cells that form HbA1c. Hence, HbA1c levels are increased in untreated sense gravity through sedimentation due to gravity. A procedure in which cells in plants or organisms are placed in a randomizing machine in which diabetic rats. Treatment with WGM conditions significantly the gravity vector loses its effect on these cells would simulate the reduced HbA1c levels in comparison to those in the gravity and conditions of weightlessness in outer space as the direction of the gravity diabetic control groups. vector would be lost. The rotational speed of such a machine is determined The liver plays a central role in the metabolism, storage, by the time scale of the movement of the cells inside the plant. There are excretion, and detoxification of xenobiotics and their metabolites, many such machines, and the one used in this study was a 3D clinostat. We and AST and ALT are effective markers of liver function and STZ- aimed to optimize a 3D clinostat for a life science experiment. Since a 3D induced diabetic liver injury in rats. For that reason, elevated clinostat is prepared with two motors, we fixed the angular velocity of one activities of SGOT and SGPT in the plasma might be due mostly to (primary) motor and varied for the angular velocity of the other the escape of these enzymes from the liver cell cytosol into the (secondary) motor. In this setup, each motor ran continuously and bloodstream, which is a signal of the hepatotoxic effect of STZ. constantly in one direction through the experiment; this device was used Treatment of the diabetic rats with the extract of WGM conditions to rotate plants in three dimensions at a speed of 4 rotations/min. Wheatgrass seeds were germinated under these conditions for 7 days. reduced the activities of these enzymes in plasma compared to those in the gravity and diabetic control groups and consequently alleviated liver damage caused by STZ-induced diabetes, as Preparation of plant extracts presented in our results. To limit microbial growth, all equipment and containers involved in the However, insulin deficiency deactivates the lipoprotein lipase germination process were treated with boiling water. Prior to germination, enzyme. Under conditions of insulin deficiency with low wheat grains of the same size were submerged in 1.25% sodium hypochlorite lipoprotein lipase, TG, cholesterol, and LDL levels were elevated, at room temperature for 30 min to disinfect and kill any contaminating and the HDL level was decreased. This hypertriglyceridemia was microorganisms on the grain surface. Distilled water at ~10 °C was then run over the grains for 15 min to thoroughly rinse off any remaining sodium reduced rapidly after insulin treatment therapy. In this study, the hypochlorite. The sterilized grains were steeped in tap water for 24 h at administration of WGM conditions significantly increased serum 21 °C. For germination, wheat grains were placed into a special container with insulin levels, a marked decrease in TC, TG, and LDL levels in rats drain holes at the bottom and irrigated with Hoagland’s solution. treated with WGM conditions and metformin-treated rats was Germination was carried out in a plant growth room at 21 °C for 7 days. observed in comparison to the diabetic control group. Addition- After harvest, the whole plants were washed carefully with distilled water for ally, an increased HDL level was observed compared to diabetic 16 3 min to remove any remaining perlite particles or nutrient elements. control rats. This activity of WGM conditions reduces hypertrigly- WGG and WGM conditions were dried under shade for 7 days at room ceridemia under diabetic conditions and therefore prevents temperature ranging from 29 to 32 °C with a relative humidity of 70–90% diabetic-associated complications. and then ground using a blender, while nongerminated seeds were Elevated plasma urea and creatinine levels signal the develop- discarded. An appropriate amount of the powdered plant material was extracted with ethanol using a Soxhlet extraction apparatus. The solvent ment of diabetic nephropathy in diabetic rats. The urea and was completely removed under reduced pressure in a rotary vacuum creatinine levels in rats with diabetic nephropathy are higher than evaporator to yield the crude extract. All extracts were packed in an air- those in normal rats. The observed maintenance of urea and proof container before being stored at −20 °C until use. creatinine levels closer to those in control rats using extracts of WGM and gravity conditions, as well as metformin treatment, Determination of the TP, flavonoid, and vitamin C contents suggests that germinated wheatgrass extract plays an either direct or indirect primary role in protecting against diabetic nephropathy The TP content of WGG and WGM conditions was estimated using the Folin–Ciocalteu assay. A total of 475 µl of a 5% sodium carbonate or retarding its development. solution was mixed with 50 µl of plant extract. The reaction mixture was An ethyl alcohol extract was selected because of the expected incubated for 3–5 min, and 475 µl of 50% Folin–Ciocalteu reagent was presence of phenolic and flavonoid compounds with antihypergly- added. The reaction mixture was mixed and incubated at room cemic properties. The presence of phenolic and flavonoid temperature in darkness for 1 h. The absorbance of the reaction product compounds in our extract with antioxidant potential, as seen in was measured at 724 nm using a ultraviolet–visible spectrophotometer. our results, further confirms this prediction. Phenolic and flavonoid The TP content was calculated from a standard calibration curve based on compounds were shown to have antihyperglycemic effects. gallic acid, and the results are expressed as mmol of gallic acid equivalents −1 Phytochemical analysis of germinated wheatgrass showed the (GAEs) per 100 mg of plant dry weight (mmol GAE 100 mg DW). presence of tannins, flavonoids, saponins, and sterols. Their The total flavonoid (TF) content of WGG and WGM conditions was antidiabetic effects and ability to regenerate pancreatic β cells determined according to Karadeniz et al. Fifty microliters of plant extract have already been demonstrated. Sterols were shown to decrease and 600 µl of ddH O were mixed with 40 µl of 5% potassium nitrite. The npj Microgravity (2020) 6 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA W.J. Al-Awaida et al. mixture was incubated at room temperature for 6 min, and then 70 µl of a overnight fasting on the 0th (before the start of the experiment), 3rd, 7th, 4.26% aluminum chloride solution was added. After 5 min of incubation at 14th, 21st, and 30th day, and glucose levels were measured by using an 25 °C, we added 240 µl of sodium hydroxide (1 M) to the reaction mixture, Accu-Chek active glucometer. and the solution was mixed well. The absorbance of the reaction product was measured at 510 nm, and the TF content was calculated from a Oral glucose tolerance test standard curve based on rutin. The results are expressed as mmol of rutin −1 Briefly, after overnight fasting, the rats were intragastrically administered equivalents (REs) per 100 mg of plant dry weight (mmol RE 100 mg DW). glucose (2 g/kg). Blood samples were withdrawn from the tail vein after 0, Five milliliters of dilute extracts of T. aestivum germinated under gravity 30, 60, 90, and 120 min, and blood glucose levels were determined using and microgravity conditions was added into a test tube, following which an Accu-Chek active glucometer. 1 ml of glacial acetic acid was added, the solution was titrated with a 2,6- dichlorophenolindophenol solution (0.2 g/l) until a faint permanent pink color was observed, after which the titer (T) was recorded. The titration Assay of biochemical parameters was repeated with 5 ml of water as a blank (B 1) and 5 ml of an ascorbic After 30 days of treatment, the rats were fasted overnight and sacrificed. acid standard solution (0.1 g/l). The vitamin C content of the test sample Blood was collected using EDTA as the anticoagulant, and whole blood was calculated according to the following equation: was used to measure HbA1c, urea, and creatinine levels. The plasma was Vitamin C content (mg/dl) = (T − B1/ ST − B1) × 2 × dilution factor. separated by collecting blood in a heparin-coated tube and centrifuging it at 1000 × g for 15 min at 4 °C. The plasma was used to estimate insulin, and Hydrogen peroxide- and nitric oxide-scavenging activity assays plasma C-peptide assays were performed using a radioimmunoassay (RIA) kit for rats. TC, TG, HDL-C, and LDL levels and the activities of the liver The hydrogen peroxide-scavenging activity of ethanolic extracts of WGG enzymes AST and ALT were determined spectrophotometrically using and WGM conditions was evaluated according to the method of Ruch commercial kits. et al. The extract at different concentrations (3.4 ml, pH 7.4) was mixed with a hydrogen peroxide solution (43 mM, 0.6 ml, pH 7.4). After 10 min, the absorbance of the reaction mixture was measured at 230 nm. The reaction Histological analysis mixture without sample was used as a blank. Ascorbic acid was used as a Specimens of the pancreatic tissue of the different groups were reference compound. The percentage inhibition activity was evaluated as immediately fixed in 10% formalin and then underwent standard [(Abs. of the control− Abs. of the sample)/Abs. of the control] × 100%. treatment with an alcohol gradient and xylene. For histopathological Nitric oxide was produced from sodium nitroprusside and measured by examination, 6-µm-thick pancreatic specimens were stained with hema- the Griess reaction. Nitric oxide was generated spontaneously from toxylin and eosin. The β cells were counted under a ×40 objective lens. Cell sodium nitroprusside compound dissolved in aqueous solution at densities are expressed as cells per pancreatic islet. physiological pH and interacted with oxygen to generate nitrite ions, which were measured by the use of Greiss reagent. Different concentra- tions of ethanolic extracts of WGG and WGM conditions dissolved in Statistical analysis dimethyl sulfoxide were mixed with sodium nitroprusside (5 mM) in Statistical analysis was performed using GraphPad Prism 7. Values are phosphate-buffered distilled water and incubated at 25 °C for 150 min. A expressed as the mean ± SEM, and data were analyzed by using two-way control experiment without the test compound but with the equivalent ANOVA, followed by Tukey’s multiple comparison test, with the level of amount of alcohol was conducted similarly. At intervals, samples (0.5 ml) of significance set at P < 0.05. the incubation solution were diluted with 0.5 ml of Griess reagent (1% sulfanilamide, 0.1% naphthyl ethylenediamine dihydrochloride and 2% H PO ). 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Antihyperglycaemic activity of aqueous leaf extract of adaptation, distribution and reproduction in any medium or format, as long as you give Combretum micranthum (Combretaceae) in normal and alloxan-induced diabetic appropriate credit to the original author(s) and the source, provide a link to the Creative rats. J. Ethnopharmacol. 129,34–37 (2010). Commons license, and indicate if changes were made. The images or other third party 37. Tanaka, M. et al. Identification of five phytosterols from Aloe vera gel as anti- material in this article are included in the article’s Creative Commons license, unless diabetic compounds. Biol. Pharm. Bull. 29, 1418–1422 (2006). indicated otherwise in a credit line to the material. If material is not included in the 38. Gülçin, İ., Elmastaş, M. & Aboul-Enein, H. Y. Determination of antioxidant and article’s Creative Commons license and your intended use is not permitted by statutory radical scavenging activity of Basil (Ocimum basilicum L. Family Lamiaceae) regulation or exceeds the permitted use, you will need to obtain permission directly assayed by different methodologies. Phytother. Res. 21, 354–361 (2007). from the copyright holder. To view a copy of this license, visit http://creativecommons. 39. Jing, L. J., Mohamed, M., Rahmat, A. & Bakar, M. F. A. Phytochemicals, antioxidant org/licenses/by/4.0/. properties and anticancer investigations of the different parts of several gingers species (Boesenbergia rotunda, Boesenbergia pulchella var attenuata and Boe- © The Author(s) 2020 senbergia armeniaca). J. Med. Plants Res. 4, 027–032 (2010). npj Microgravity (2020) 6 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA

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