Research Approval
This work is based on a thesis for a degree in pharmacy at the School of Pharmacy, Ardabil University of Medical Sciences, Ardabil, Iran. All the processes were approved by the Ethics Committee of Ardabil University of Medical Sciences (approval ID: IR.ARUMS.REC.1397.174).

Chemicals
Aluminum trichloride, bovine serum albumin (BSA), Dichloro-dihydro-fluorescein diacetate (DCFH-DA), Folin–Ciocalteu, gallicacid, HEPES sodium salt, 3-(4,5-dimethylthiazol- 2-yr)-2,5-diphenyltetrazolium bromide (MTT), rutin, and the RPMI 1640 medium were purchased from Sigma-Aldrich Co.

Plant Materials and Preparation
C. spinosa L. leaf, bud, and roots were used in this study. They were collected from the Moghan district of Ardabil, Iran, in June 2018. After cleaning the collected parts of the plant, they were dried under the shade at room temperature and then powdered. The maceration method was adopted to obtain alcoholic extracts of different parts of the plant. Briefly, 25 g of the plant powder from each part was treated with 250 mL of methyl alcohol (99%) in a tightly closed container for two days. The container was shaken one or two times a day. Then, the mixture was filtered and the solvent was removed by using a rotary evaporator. Hydroalcoholic extracts were also prepared by treating 25 g of the plant powder from each part with 250 mL of ethanol (80%) as the solvent, and the same procedures were followed. The residues were dissolved and used for the following experiments.

Phenolic Content Determination
The content of total phenols in the extracts was measured with the reagent Folin- Ciocalteu. In this experiment, 100 μL of the plant extracts and 500 μL of the Folin–Ciocalteu reagent were added to 1.5 mL of sodiumcarbonate (20%). After shaking the mixture, it was diluted with distilled water up to 10 mL and left for 2h. Subsequently, the absorbance of the mixture was determined at 765 nm. A standard curve was plotted from the different concentrations of gallic acid so that the content of total phenols would be expressed as mg/g gallic acid equivalents (GAE) (12).

Flavonoid Content Determination
We followed a method from the State Pharmacopeia of USSR for the measurement of flavonoid content in the extracts by using rutin as the reference compound. Rutin, which was available in our lab, is a common flavonoid commonly used as a standard in the determination of total flavonoid content (13). Briefly, one drop of acetic acid was added to a mixture containing 1 mL of the extract in methanol (10 mg/mL) and 1 mL of aluminum trichloride in ethanol (20 mg/mL), and then made up to 25 mL by ethanol. After 40 mins, the absorbance of the mixture was determined at 415 nm. To prepare the blank samples, one drop of acetic acid was added to 1 mL of the extract and then ethanol was added to 25 mL. A standard solution containing rutin (0.5 mg/mL) in ethanol was used to plot the standard curve. The rutin equivalent (RE) was used to quantify the flavonoid content in the extracts according to the following formula; X = (A ⁎ m₀) (A₀ ⁎m) where X means the content of flavonoids (mg/mg extract inRE), A and A₀ denote the absorbance of extract solution and standard solution, respectively, and m and m₀ express the weight of the plant extract (mg) and rutin (mg) in the solution, respectively (14).

Animals
Adult male Wistar rats weighting ~200-250 g were used (n=16); all the animal experimental procedures were approved by the Ethics Committee of Ardabil University of Medical Sciences (IR.ARUMS.REC.1397.174).

Isolation of Pancreatic Islets of Langerhans
Before starting the experimental procedure, a suitable period of time was given to the animals to spend in the lab environment. A solution containing ketamine and xylazine at a ratio of 10:1 (the administered dose of 100 mg/kg ketamine and 10 mg/kg xylazine) was intraperitoneally injected and, after anesthetization, the animals were subjected to laparotomy. The pancreatic duct was cannulated for perfusion with a cold and fresh Krebs buffer which made the pancreas swollen and easy to isolate from the other parts. Then, the pancreas was chopped and cut off into small pieces by using a small scissor in the cold Krebs buffer. Collagenase was added to the homogenate to breakdown the tissue around the islets. After the separation of fat tissues, the homogenate was washed with Krebs buffer in triplicate. Next, BSA was added and washed twice in order to stop digestion. Finally, islets of Langerhans were picked up by using a sampler under the stereomicroscope, grouped based on similarity in size, and cultured in RPMI 1640. The cultured islets were incubated for 24 h at 37°C to overcome stressful conditions and restore their viability in the nourished and refreshed medium (15).

Pancreatic Islets' Treatment with the Plant Extract
After 24 h of pretreatment time, the islets were incubated with logarithmic concentrations of hydroalcoholic extracts of the bud, including 10,10²,10³,10⁴ µg/mL in the RPMI medium for 24 h.

Viability Assay in the Pancreatic Islets
The MTT assay is based on the reducing capacity of yellow tetrazole into purple formazan by viable cells having the normal mitochondrial respiration. After 24 h of treatment of pancreatic islets with hydroalcoholic extracts, we removed the medium and washed the islets of Langerhans with Krebs buffer twice. Then, 50 µL of the MTT solution (0.5 mg/mL) was added to each vial of the islets and incubated for 4 h at 37°C. The islets having viable cells converted the yellow tetrazole into purple formazan, which was dissolved in the solution by adding DMSO and shaking for 30 min. Finally, a microplate reader was employed to read the absorbance of the samples at 570 nm (16).

Statistical Analysis
The quantitative variables were expressed as mean and standard deviation, and the categorical variables were described as absolute frequencies and percentages (11,12). Normality of the data was determined by means of the Shapiro–Francia test (13). The comparison of the categorical variables was performed using Chi-square test or Fisher's exact test when more than 20% of the cells with the expected count of < 5 were observed. Furthermore, the quantitative variables were compared via the t-test or Mann-Whitney U test. All statistical analyses were performed in SPSS 16.0 (SPSS Inc., Chicago, IL. USA). P-value equal to or less than 0.05 was regarded as statistically significant (14,15).
The variables used to assess the anticipation were gender, disease duration, age, and severity of Parkinson's disease (according to the Unified Parkinson's Disease Rating Scale (UPDRS)).

ROS Assay in the Pancreatic Islets
The ROS level was measured using the DCFH-DA. ROS deacetylate and oxidize non-fluorescent DCFH-DA into the fluorescent DCF inside the cells. The islets of Langerhans were sonicated in 75 µL of the extraction buffer (containing 5 mM HEPES, 20 mMKCl, 1mM EDTA, 0.25 mM sucrose) and 10 µL of the DTT solution (50 mM), and then centrifuged at 5000 rpm for 5 min. In the next step, we extracted 25 µL of the supernatant and mixed it with 80 µL of the assay buffer (containing 0.1 mM FeCl₃, 20 mM MgCl2, 20 mM NaH2PO4, 130 mMKCl, 20 mMTris–HCl, 0.1 mM NADPH, and 1.7 mM ADP). Finally, 5 µL of DCFH-DA was added to the mixture and incubated for 15 min at 37^°C. During this time, DCFH-DA could enter the cells and be hydrolyzed by intracellular esterase to produce free DCFH. Measurement of the rate of DCF production through the oxidation of DCFH is an indicator of the amount of ROS in the cells. In this way, the absorbance of the mixture was read at an excitation of 485/20 and an emission of 528/20 every 5 min for 1 h with a fluorometer (Biotec, Tecan US, Inc.) (17).

Protein Assay in the Pancreatic Islets
The amount of total protein was assessed by the Bradford method in which BSA was used as the standard. Briefly, we added 100 μL of the Bradford reagent to 5 μL of each sample supernatant and standard solutions. After 20 min of incubation, we read the absorbance of the solutions by the spectrophotometer at 595 nm.

Statistical Analysis
The data were analyzed by SPSS and expressed as mean ± SEMwith a replication number, n=4. The P-value of differences among the means were analyzed by one-way ANOVA followed by Tukey's test.

 

Discussion

Diabetes is one of the most prevalent diseases in the current century and is expected to increase more quickly in developing countries (18). Apart from the changed lifestyle and diet, it has been proved that exposure to some environmental pollutants can increase the risk of diabetes in susceptible people. Heavy metals and pesticides have been identified as the major diabetogenic toxicants for which the probable relationship between environmental release and the risk of diabetes has been well documented by determining biochemical and molecular mechanisms (19-21). Therefore, presenting new therapeutic strategies from natural sources based on the recently known etiologies of the disease can be a promising approach to both the prevention and treatment of diabetes.
Diabetes has many etiologies, including the damage and destruction of pancreatic β-cells. There has been sufficient evidence on the occurrence of oxidative stress parameters in the biological samples of patients with diabetes (22). Oxidative stress caused by excessive free radicals and hyperglycemia is believed to damage cellular macromolecules in the islets of Langerhans and disrupt insulin gene expression and insulin release. Thus, antioxidants can improve the viability and function of islets by counteracting overproduced oxidants and maintaining cellular homeostasis (23). In fact, sustained hyperglycemia due to uncontrolled diabetes can gradually lead to glucose toxicity manifested by oxidative stress and inflammatory responses. It has been reported that under such conditions, some intracellular compensatory cascades are activated, including the JNK pathway that has been shown to be involved in the dysfunction of pancreatic β-cell and induction of insulin resistance in peripheral tissues, both of which can deteriorate hyperglycemia and lead to following events (24,25).
Plants are one of the main natural sources of antioxidants and are rich ineffective ingredients with a high capacity for scavenging free radicals and ROS. Evidence has suggested that antioxidant compounds from natural sources such as plants can positively affect a wide range of biological functions in the cells by virtue of their radical scavenging properties (13). Polyphenols are considered as the secondary metabolites of plants and act as potent antioxidants that can provide defense against overproduced oxidants and oxidative stress. Polyphenol-rich diets have been reported to protect against the development of many pathological conditions with chronic progress, such as diabetes (26).
C. spinosaL. is a known polyphenol-rich plant that exhibits good antioxidant activities (27). Glucose-lowering effects have also been reported for this plant in experimental and clinical studies. The hypoglycemic and hypolipidemic effects of the C. spinosa extract in laboratory animal models of diabetes caused by insulin deficiency and insulin resistance have been documented in different studies (28-33). The inhibitory effect of the C. spinosa extract on the activity of pancreatic alpha-amylase has also been confirmed. Drugs inhibiting the enzymatic activity of α-amylase and α-glucosidase are helpful in the treatment of diabetes by lowering postprandial glucose (34). This evidence suggests the protective role of ingredients in the C. spinosa extract on the metabolic profile of carbohydrates and lipids. Moreover, histological studies have demonstrated that the C. spinosa extract can decrease cell necrosis and tissue damage in diabetic laboratory animals. This effect can be mediated by the active ingredients of the plants, most commonly phenols and flavonols that have antioxidant and anti-inflammatory activities (35).
The present study is the first report on the protective effects of C. spinosa on the pancreatic islets whose integrity and function are essential for the maintenance of glucose metabolism since the pancreas is the only source of insulin production in the body. The C. spinose extract increased the viability of rat pancreatic islets of Langerhans in a dose-dependent manner. Moreover, the amount of ROS, which is the main indicator of oxidative stress, was decreased in the islets treated with the C. spinose extract. Oxidative stress plays a major role in depleting cellular ATP and initiating cell death procedures, especially in pancreatic β-cells that are highly sensitive to oxidants and reactive metabolites. In this way, the agents having antioxidant capacity have been proposed to be useful against β-cell damage and insulin synthesis and secretion disruption. Previous research has also reported anti-inflammatory activities for the C. spinose extract in different animal models of experimental setups (36). Of course, these effects are predictable since oxidative stress and inflammation develop together in the cells and most of their elements interact with and augment one another. Given that the pathogenesis of diabetes involves the activation of both oxidative and inflammatory pathways, agents having antioxidant and anti-inflammatory effects can be helpful in the treatment of the disease. According to previous evidence on the glucose-lowering activity of C. spinosa, as well as its protective effects on the isolated islets of Langerhans observed in this study, it is concluded that the active ingredients of this plant can be regarded as a promising adjuvant therapy for the prophylaxis and management of diabetes, and merit further research to uncover the exact mechanisms responsible for this action.

 

Conclusion

The present findings showed that the C. spinose bud extract from the Moghan region is a source of polyphenol and can enhance the viability and reduce the production of ROS in the islets of Langerhans. Accordingly, C. spinosa can be evaluated for use in the prophylaxis and treatment of diabetes.

Acknowledgements

The authors wish to thank the Department of Toxicology and Diseases, The Institute of Pharmaceutical Sciences (TIPS) at Tehran University of Medical Sciences, Tehran, Iran for technical support.

 

Conflicts of Interest

Authors declared no conflict of interests.