Purslane (Portulaca oleracea) Seed Consumption And Aerobic Training Improves Biomarkers Associated with Atherosclerosis in Women with Type 2 Diabetes (T2D)

The aim of this study was to investigate the responses of atherosclerosis plaque biomarkers to purslane seed consumption and aerobic training in women with T2D. 196 women with T2D were assigned into; (1) placebo (PL), (2) aerobic training+placebo (AT + PL), 3) purslane seeds (PS), aerobic training+purslane seeds (AT + PS). The training program and purslane seeds consumption (2.5 g lunch and 5 g dinner) were carried out for 16 weeks. The components of purslane seed were identified and quantified by GC–MS. Blood samples were withdrawn via venipuncture to examine blood glucose, low-density lipoprotein (LDL), high-density lipoprotein (HDL), cholesterol, triglycerides (TG), creatinine, urea, uric acid, NF-κB, GLP1, GLP1R, TIMP-1, MMP2, MMP9, CRP, CST3, and CTSS expressions. Blood glucose, LDL, cholesterol, TG, creatinine, urea, and uric acid levels in the (P), (AT), and (AT + PS) groups were significantly decreased compared to the pre-experimental levels or the placebo group, while HDL, significantly increased. Furthermore, the protein and mRNA levels of NF-κB, TIMP-1, MMP2 &9, CRP, CST3, and CTSS in the (P), (AT), (AT + PS) significantly decreased compared to pre-experimental or the placebo group, while level of GLP1 and GLP1-R increased drastically. Findings suggest that purslane seed consumption alongside exercising could improve atherosclerosis plaque biomarkers through synergistically mechanisms in T2D.

. Schematic illustration of some of the key pathological pathways that are involved in both type 2 diabetes and atherosclerosis. Hyperglycemia, hyperlipidemia, hyperinsulinia, and hyperuricimia increase ROS generation through oxidative stress. ROS activates a number of stress sensitive kinase which mediates insulin resistance via activation of NF-κ B and CRP. On the other hand, activation of NF-κ B and CRP affect the structure of extracellular matrix and increase the activity of MMP and cathepsin S which are involved in atherosclerosis and diabetes. Exercise and purslane intake improve the regulation of biomarkers involved in the activation of these pathways. TG; triglycerides, LDL; Low-density lipoprotein, ROS; reactive oxygen species, NF-κ B; nuclear factor kappa-light-chain-enhancer of activated B cells, CRP; C-reactive protein, MMPs; Matrix metalloproteinase, TIMP-1; metallopeptidase inhibitor 1.
(Iran). Characteristics and demographics of the patients are presented in Table 1. The average age and BMI of these women were 52.08 ± 3.45 years and 29.5 ± 6.5 kg/m 2 respectively. Eligible participants lacked any form of complications arising from acute and chronic diabetes and did not exercise regularly. The participants did not have any history of other diseases such as chronic cardiovascular and inflammatory diseases, diabetic ulcers, and hepatitis. Patients consuming vitamins and supplements or those who smoked were excluded from the study. Patients were well informed about the study prior to the experiment and written consent was obtained from them. A double-blind study method was applied and the participants were randomly assigned into 4 groups of 8; (1) placebo (PL), (2) aerobic training + placebo (AT + PL), (3) purslane seeds (PS), and (4) aerobic training + purslane seeds (AT + PS). All procedures involving experiments were carried out in strict accordance of the United States Institute of Research guidelines and approved by the Medical Centre Board of Tehran Hospital with Medical Ethics Number 4382. 30. Plant sample preparation. Purslane seeds were purchased from a grocery shop in Tehran (Iran). The seeds were washed and air dried at room temperature for seven days. The dry seeds were powdered and packaged in capsules (5 g each). Portulaca oleracea was identified and verified by a botanist and deposited at Herbarium of Department of Biochemistry, (voucher specimen no. . The consumer groups (AT + PS, PS) consumed 2.5 g of purslane seeds with lunch and 5 g with dinner daily for 16 weeks. The placebo group similarly received placebo pills (the flavored maltodextrin).

Sample preparation and identification of compounds by Gas Chromatography/Mass
Spectroscopy (GC/MS/MS). Dried powdered (100 g) purslane seed was added with 100 ml of methanol and left at room temperature overnight. The eluate was filtered through Whatman filter paper and shade dried to remove the solvent. The extract was then weighed (0.563 g) and kept in − 20 °C for further analysis. The extract was used in Gas Chromatography. The detail of Chromatography is available in supplementary file.
Aerobic training Guideline. Patients in groups AT + PL and AT + PS performed their training under a trainer's supervision for 16 weeks in a gym. The trainer is blinded to the subject's treatment group. Jogging was considered for this study as a moderate-intensity aerobic activity. Progressive training was performed for a minimum of 60 min per session for three days a week at 50 to 70% of maximum heart rate (MHR) during the 16 weeks of experiment. Written informed consent was obtained from all patients before any study-related procedures were performed. Patients were familiarized with the protocol and a heart rate strap was applied on them to monitor their heart rate within the first two sessions. The training included a 15 min warm-up through walking with light static and dynamic stretching and cooling down with stretching in standing and lying position in the final 15 min. All movements were performed with medium tempo and separate movement for arms, legs and trunk. The main training time increased 40 to 45 min and heart rate has reached 70% MHR. At the end of 16 weeks participants were able to perform muscle movements with greater coordination.
Blood Sample Collection. Blood samples were collected at the certain time of the trainings through the elbow antecubital vein of all patients, 24 hours before and after the 16 weeks training and purslane consumption for measurement of blood glucose, LDL, HDL, cholesterol, TG, creatinine, urea, uric acid, NF-κ B, GLP1, GLP1R, TIMP-1, MMP2, MMP9, CRP, CST3, and CTSS mRNA and protein expressions.

Measurement of Serum parameters.
Blood samples were collected in serum separating tubes (SST) and allowed to clot at room temperature for 30 min. The blood clot was then centrifuge at 3000 g for 15 min. Aliquots of the serum samples were stored at − 20 °C for further use. Fasting serum glucose and total cholesterol levels were determined using the glucose oxidase method 34 with a digital spectrophotometer (Spectronic, US). LDL level was calculated using the Friedewald equation 35 , and to measure HDL level, diagnosis kits were used following the photometric method. Serum triglycerides was measured by using the Fossati and Prencipe method 36 . Based on manufacturer's instructions of Fortress Diagnostics Limited, creatinine, urea, and uric acid were examined by alkaline picrate method, urease-hypochlorite and uricase-peroxidase methods respectively.

RNA purification and mRNA Expression Analysis by Real Time PCR (qPCR). QIAamp RNA
Blood Mini Kit (Qiagen, Germany) was used to isolate total cellular RNA from fresh whole blood. The concentration and purification of isolated RNA were evaluated by 260/280 UV absorption ratios (Gene Quant 1300, UK). Specific amplification fragments of DNA/RNA, Two-step Real time qPCR (quantitative Polymerase Chain Reaction) technique was used to calculate gene expression during the PCR amplification process with application of TaqMan reagent. This method was able to detect small differences between samples compared to other methods 37 . All reagents including probes and primers were obtained from Applied Biosystems, USA. TaqMan probe (known as fluorogenic 5′ nuclease) was chosen to perform qPCR. This probe has a sensitivity of 100% and a specificity of 96.67% 17 and is capable of detecting as few as 50 copies of RNA/ml and as low as 5-10 molecules 18 . The same company designed specific primers for specific targets. The primers detail information are available in supplementary file. All experiments were conducted in 3 biological replicates in a Step One Plus real time PCR machine (Applied Biosystems, USA). The Real time PCR program includes reverse transcription, at 48 °C for 15 min, activation of ampli Taq gold DNA polymerase at 95 °C for 10 min, denaturation at 95 °C for 15 sec and annealing at 60 °C for 1 min. Denaturation and annealing steps were performed for 40 cycles. The fold changes of each target per average of ACTB were calculated and considered as mRNA expression levels of the target gene. Data was analyzed according to Comparative Ct (2 −ΔΔCt ) method, where amplification of the target and the reference genes were measured in the sample and reference.
Statistical Analysis. Levene's equality of variable assumption was applied and the results revealed no significant differences among the observations. All data were presented as mean ± SEM. Hormone levels of each subject were analyzed by descriptive statistics. Two-way analysis of variance with repeated measure (ANOVA) was used to compare changes between groups. A Bonferroni post-hoc test was used to check for significant differences between training alone vs. purslane seeds alone vs. aerobic + purslane seeds combined. For variables with normal distribution, Pearson correlation coefficient and for non-normal distributed variable, Spearman correlation coefficient were applied. SPSS 18.0 was used in this study and p < 0.05 was considered as statistically significant.

Results
Gas Chromatography/Mass Spectroscopy of Purslane Seed Extract. The components of Purslane seed were identified and quantified by GC-MS. A total of 21 known compounds are presented in Table 2. Among these compounds, 27% are unsaturated fatty acid (linoleic and palmitoleic acid); 11.18% are phytosterols (stiotestrol), and 7.9% are saturated fatty acids (palmitic acid). Additionally, smaller amounts of unknown substances were also detected ( Fig. 2).
Serum Biochemical Parameters Analysis. The effects of purslane seed consumption and aerobic on the serum biochemicals of diabetic women are presented in Table 3. Blood glucose, LDL, Cholesterol, and TG concentration in the (PS), (AT + PL), (AT + PS) groups were significantly decreased after 16 weeks as compared to pre-experimental levels or the (PL) (p < 0.05). No significant differences observed between (PS) and (AT + PL) (p > 0.05), whereas significant interactions were detected between (PS) and (AT + PL) compared to (AT + PS) (p < 0.05).
The changes were more pronounced in (AT + PS) group. Furthermore, Creatinine, Urea, and Uric Acid in the (PS), (AT + PL), (AT + PS) groups were significantly decreased after 16 weeks as compared to the pre experimental levels or the (PL) (p < 0.05). No significant differences observed between (PS) and (AT + PL) (p > 0.05), while significant distinction were detected in (PS) and (AT + PL) compared to (AT + PS) (p < 0.05). The reductions in levels were more pronounced in (AT + PS) group.
Atherosclerosis Biomarkers. The effects of purslane seed consumption and aerobic on the serum protein and mRNA biomarkers changes in diabetic women are presented in Table 4     Furthermore, GLP1 and GLP1-R had profoundly increased in the (PS), (AT + PL), (AT + PS) as compared to the pre experimental levels or the (PL) (p < 0.05). No significant difference observed between (PS) and (AT + PL) in the protein and mRNA concentration levels of GLP1-R (p > 0.05), while a significant difference observed between (PS) and (AT + PL) in the protein and mRNA concentration levels of GLP1 (p < 0.05). Moreover, significant distinction were detected in the protein and mRNA concentration levels of GLP1 and GLP1-R in (PS) and (AT + PL) compared to (AT + PS) (p < 0.05). The changes were more pronounced in (AT + PS) group.

Discussion
The control and treatment of diabetes and its complications predominantly depends on chemical or biochemical agents. However, total recovery from diabetes has never been reported 38,39 . The data from the current study showed that 16 weeks of aerobic training or purslane seed consumption ere effective in reduction of markers of inflammations such as NF-κ B, CRP, CST3, CTSS, MMP 2 & 9 and TIMP-1 in diabetic patients. In addition, glucose, TG, LDL, urea, uric acid and creatinine levels in all treated groups were significantly reduced. Furthermore, there was an increase in the levels of HDL, GLP-1 and GLP-1R. The improvement was more remarkable in women who received training and supplementation simultaneously. The changes in the level of biomarkers were further evaluated by the expression of mRNA from blood samples of the same subjects. Our results show that the mRNA levels of NF-κ B, CRP, CST3, CTSS, MMP 2, 9 and TIMP-1 increased after intervention. GC-MS analyses results indicated the presence of 21 compounds, of which 27% are poly unsaturated fatty acid (linoleic and palmetoleic acid) and 11.18% are phytosterols (stiotestrol). The observed effects of purslane seeds may be due to the presence of these compounds. The findings suggest that glucose levels had decreased in all groups. However the decrease was more evident in the group consuming purslane seed and exercising simultaneously. Purslane seed consumption has been shown previously to impact glucose levels 27 . High levels of glucose in diabetes appears to be an initiating factor for the eventual cascade of biomarkers involved in inflammation and coronary heart diseases. Improvements in lipid profile were observed in the subjects after 16 weeks of aerobic training or purslane seed consumption or both ( Table 3). The reduction of lipid profile in the aerobic group could be due to the decrease of free fatty acids, which negatively affect insulin resistance and excess lipid availability [40][41][42][43][44][45] . Hence, aerobic training is beneficial for patients with T2D 40,42,46 . The results obtained from our study also highlights the positive effects of purslane seed  consumption on TG, LDL, cholesterol and HDL levels, which is consistent with earlier reports in T2D of Yemeni and Iranian patients 5,31 .
Levels of blood urea, uric acid and creatinine (Table 3) were reduced in all treated groups. In T2D overweight patients, uric acid and urea serve as biomarkers for impaired physical performances and are associated with blood glucose levels 47 . Purslane consumption in T2D mice 48 and T2D women has also been shown to reduce levels of creatinine, uric acid and urea 49 . Therefore, the reduction of blood nitrogen content seen in this study, may be lined to reduction in glucose levels (Table 3) following purslane seed consumption or aerobic training. In a study reported by Sousa 50 , twelve weeks of resistance training improved uric acid levels in T2D in Brazilian patients [50][51][52][53] . However, six months of aerobic training did not produce remarkable effects on creatinine and urea in untrained aged healthy women 54 .
The current study highlighted the beneficial effects of purslane seed consumption in alleviation of diabetes parameters. The beneficial effect may due to the presence of unsaturated fatty acids and beta-sistosterol as identified via GC-MS analysis ( Table 2 and Fig. 2). Previous reports have suggested that, unsaturated fatty acids are responsible in reducing levels of LDL and cholesterol synthesis 55 , enhancing insulin function 56 and improving glucose tolerance and lipid profile 57,58 . Another active substance in purslane seed is beta-sitosterol which is a phytosterol. Studies have found that beta-sitosterol has cholesterol 59 and LDL 60 lowering effects, increases the expression of VEGF (vascular endothelial growth factor) and FLK-1 (Fetal Liver Kinase 1) of VEGF receptors while modulating inflammation and regulation the immune systems. The effectiveness of purslane seed consumption is in lowering cholesterol levels explained by the synergistic effect of both phytosterols and unsaturated fatty acids 61 .
Hyperglycemia, hyperlipidemia, hyperinsulinemia 17,18,53 and hyperuricemia 62-64 of diabetics are common symptoms which enhance production of pro inflammatory markers such as CRP, IL6, TNFα , NF-κ B 17,18,53,62,64 , reactive oxygen species (ROS) and reduce anti-inflammatory cytokine and adiponectin, which are involved in insulin resistance 16,65 and diabetes 66 . Reduction in the level of NF-κ B and CRP in the inflammatory cascade was observed in treated groups of diabetics' subjects. Our experiments showed that purslane seed consumption resulted in reduction of CRP and NF-κ B (Table 4 and Fig. 3). This may be the result of inhibitory effects of unsaturated fatty acids on NF-κ B reported in diabetic patients 67 . The inhibitory effect of unsaturated fatty acids such as omega 3 on CRP 68 in diabetic patients was reported even though the changes in CRP level were not significant. However, the significant reduction of CRP in the present study may indicate synergistic effect of various compounds found in purslane seed. Reduction in the level of CRP following resistance or strength training has been reported in patients with kidney disease 45,69 , older adults with T2D 21 and diabetic men 70 and in improving insulin sensitivity 21 . Various types of exercise may affect levels of NFκ B differently. For instance, acute exercise did not change the level of NFκ B in T2D patients 53 , but acute treadmill running raised the level of NFκ B activity in rat skeletal muscles 71 . Therefore, reduction in levels of NFκ B may be linked to CRP level reduction following aerobic exercise (Table 4) by impact on different synergistically pathways 52,53,72 . The expression of NFκ B and CRP are interconnected whereby CRP enhances NFκ B levels 9 , thus aerobic training and/or purslane seed consumption may directly or indirectly reduce the levels of NFκ B via reducing the CRP.
The reduction of these two biomarkers is associated with the levels of inflammation and ROS in addition to regulation of ECM 9,18 . Matrix metalloproteinase (MMPs) and cathepsin S (CTSS) are proteases that degrade at least one component ECM and contribute for tissue remodeling and inflammation. Higher level of CRP and IL-6 are associated with higher level of CTSS 17 and higher levels of NFκ B enhance MMPs level 16,73 . In pathological conditions such as diabetes, the balance between the protease enzymes (MMPs and CTSS) and their inhibitors (metallopeptidase inhibitor 1; TIMP-1 and CST3) are dysregulated 74 . The concentrations of MMP-2, 9 and TIMP-1 has been shown higher in T2D patients as compared to non-diabetic subjects 75,76 but in the current study, their levels were significantly reduced in all treated groups following different interventions (Table 4 and Fig. 4). Positive effects of exercise improve the balance between pro-and anti-inflammatory and oxidative stress markers which could improve nitric oxide bioavailability and alter MMPs and/or tissue inhibitors of MMPs (TIMP) activity. Chronic exercise training, that acts as a mechanical stressor for the arteries, could exert its effects by altering MMP/ TIMPs activity directly and/or indirectly via the anti-inflammatory and oxidative stress responses of exercise 77 . The finding were concurrent with those of Kim et al. 18 which states that the level of MMP-2 was reduced after low intensity exercise training in diabetic mice 78 . The suppressive effect of purslane seed on MMP and TIMP activity may be caused by the presence of the unsaturated fatty acids in the seeds. Similar outcomes were observed in patients with multiple sclerosis 79 and in pregnant rats 80 .
CTSS is another important regulator of inflammation 14 that plays a role in pathological conditions such as diabetes 17,81 . This regulator and its inhibitor CST3 are higher in diabetic patients as compared to healthy subjects 82 . Nonetheless the current interventions applied in this study reduced the protein and mRNA of both CTSS and CST3 (Table 4 and Fig. 5). This reduction following exercise may changes energy balance in adipose tissues 83 . Moreover, CTSS is positively correlated with weight loss 83,84 and TG level in obese subjects 85 . Reduction in CTSS and LDL was also observed in diet induced weight loss in non-obese men and women 86 . This further supports the hypothesis that CTSS contributes to cardiovascular risks in obesity 83 . The reduction of CTSS and CST3 was also observed in the group consuming purslane seed alone which could be due to the presence of unsaturated fatty acids and beta-sistosterol found in the seeds. However, the mechanism of for their reduction is unknown.
This study also evaluated the effect of intervention on biomarkers for insulin and beta cell activity. The levels of GLP-1 and GLP-1R were measured. GLP-1 is an insulinotropic in T2D which improves insulin secretion via it's receptor and by stimulation of glucose dependent insulin secretion 19 . It also has protective effect against degradation of ECM 20 and inhibits apoptosis of beta cells 87 . In addition GLP-1 mimetics are potential drugs to treat T2D 88 . The present findings, showed improving effects of interventions on GLP-1 and its receptor levels (Table 4 and Fig. 3). This is possibly linked to effects of purslane and aerobic training on beta cells mediated by the AMPK activated pathway 88,89 . Exercise and physical activity induce the AMPK pathway and inhibit energy consuming pathways such as fatty acid and cholesterol synthesis and stimulate the ATP catabolic pathway 90 . Since AMPK Scientific RepoRts | 6:37819 | DOI: 10.1038/srep37819 signaling pathway is among the crucial pathways involved in inflammation, following activation of this pathway, inhibition of NFκ B and its downstream signaling pathway occurs 91 .
Thus the reduction in the inflammatory state of diabetes may be an important factor leading to improved insulin sensitivity and better metabolic control 21 . Increased physical activity and weight loss are associated with lower CRP and reduction in concentration of other markers of inflammation 33,92 . Therefore, both intervention alone or in combination might act through AMPK pathway and inhibit the activation of downstream inflammation through inhibition of CRP and NFκ B. Among different insulin signaling pathways, available treatments for diabetes usually trigger the AMPK pathway which is insulin independent 93 and metformin activated that results in the inhibition of NF-kB activity in endothelial cells 94,95 . In this study, the level of NF-kB as a master inflammation key was reduced with purslane intake and/or aerobic training which could be due to activation of the AMPK pathway. Previous reports have indicated that aerobic exercise 52,53 and the uptake of unsaturated fatty acids activate the AMP and AMPK pathways 96 .

Conclusion
The data obtained from this study indicated that 16 weeks of aerobic training or/and purslane seed consumption ere effective in regulation of diabetic parameters and biomarkers associated with atherosclerosis in women with T2D. This may be due to the synergistic effect of aerobic training and unsaturated fatty acids found in purslane seed which activate the AMP and AMPK pathways. This may result in the regulation of biomarkers involved in cardiovascular complications of T2D. The parallel effects of purslane intake and aerobic training in their simultaneous role were more prominent which could be a strong therapeutic effective factor on diabetic patients with CVD. Thus, with the benefits of this combination on reducing health risk factors, diabetic patients are advised to exploit this arrangement of alternative to control and manage their disease. However, further investigations are warranted, and such research should focus on identifying the specific properties of purslane seed, proper dose, role of various active compounds, as well as the possibilities for synergistic interactions not only between the various components of this seed, but also between various alternatives in treating and reducing symptoms of these diseases.