Studying the Effects of Capparis Spinosa Hydroalcoholic Extract on Glucose Metabolism Pathways in Rat Liver Cells

Introduction

Diabetes mellitus is a common disease in the world, which is associated with increased blood glucose, and disturbance in carbohydrate and lipid metabolism. It is estimated that the number of patients with diabetes in the world will increase to 366 million in 2030 (Roberts et al., 2017). Deficiency or relative reduction of insulin in this disease is associated with acute and chronic metabolic complications (Abdelsalam et al., 2019; Teimouri et al., 2022). One of the most important consequences of diabetes is the emergence of oxidative stress conditions in patients (Paul et al., 2022). Past studies have shown that increased production of free radicals is involved in the pathogenesis and complications of diabetes and high blood pressure (Byrne et al., 2021; Bhatti et al., 2022; Liu et al., 2022). Also, it has been shown in various studies that oxidative stress in diabetes has an effective role in causing disease complications (Kang & Yang, 2020).

Various factors are known in the production and increase of free radicals in diabetic patients, and one of the most important factors is hyperglycemia, which increases the oxidation of glucose through several pathways and leads to the production of more free radicals. The levels of some prooxidants such as homocysteine and ferritin increase in diabetes. There is a definite relationship between homocysteine levels and lipid peroxidation in diabetic patients, and probably homocysteine is effective in causing vascular damage in diabetes through systems related to increasing the production of oxygen free radicals. Also, a direct relationship between poor glucose control and other complications of diabetes such as retinopathy and nephropathy has been shown, and in contrast to accurate blood glucose control, it leads to a delay in the occurrence of microvascular complications (Linn et al., 2023). In diabetes, the balance between free radicals and antioxidants is lost, and due to the increase of oxidants in the body, the process of developing complications of diabetes accelerates (Liguori et al., 2018; Ahmad & Ahsan, 2020; Babel & Dandekar, 2021).

Today, many chemical drugs are used to treat diabetes. Although chemical drugs have many beneficial effects, pharmacological and pharmacology knowledge believes that there is no chemical drug that is without complications and harm. Therefore, pharmaceutical science is looking for ways to achieve drugs that have minimal side effects (Adwan & Omar, 2021). Currently, the use of herbal and natural medicines instead of chemical medicines is an issue that has gained importance. Today, it is known that medicinal plants have countless compounds, these compounds create a very balanced situation next to each other and adjust each other's properties, and some compounds have exactly the task of neutralizing the effects of other compounds (Adwan & Omar, 2021).

Capparis spinosa belongs to the Capparidaceae family. The Capparis spinosa has aromatic compounds that grow wild in hot and dry areas such as West and Central Asia and the Mediterranean region (Ouhammou et al., 2022). The used parts of the plant are its buds or flower-producing pieces, which are usually placed in vinegar for about three months after picking, or those that are kept in salt water and then consumed. Its fruit, root, and skin are mostly used for therapeutic purposes; the effective ingredient of this plant is quercetin (Ouhammou et al., 2022). The results of examining the chemical composition of Capparis spinosa fruit essential oil showed that terpenoids and sulfur compounds are the main components of the essential oil. Thymol (1.24%) included the most terpenoid compound. Different types of isothiocyanates (2.29%) were observed in the essential oil, which mainly included methylsulfonyl heptyl isothiocyanate (5.12%), and isopropyl isothiocyanate (1.6%) (Sanchooli et al., 2012).

Considering that finding new drugs with fewer side effects, more effectiveness and less cost to reduce blood sugar and fat in diabetic people seems necessary and considering that so far there has been very limited research on the properties of Capparis spinosa (Sanchooli et al., 2012; Kumari, R., Kumar et al., 2019; Adwan & Omar, 2021; Ouhammou et al., 2022), as a result of this study, to determine the effect of Capparis spinosa hydroalcoholic extract on the key enzymes of glucose metabolism in the liver cells of diabetic rats.

Materials and Methods

The current study was of an experimental type in 60 male Wistar rats were randomly selected from among male rats with an approximate weight of 250-300 grams, which were in the same climatic conditions with a temperature of 20-22 C° and a relative humidity of 30-70%. and the dark or light cycle of 12 hours was kept, were selected, and randomly divided into 6 groups of 10.

First, the fruits of the Capparis spinosa plant were washed and dried in the shade, and then they were powdered using a Moulinex mill (made in France) and 10 grams of powder were added to the volume of 100 ml. The intended solvent in this study was 70% ethanol, which was added to the powder and the mixture was kept in laboratory conditions for 48 hours, to prevent alcohol evaporation, the opening of the Erlenmeyer flask was closed with parafilm. Then the mixture was passed through S&S laboratory filter paper with a diameter of 125 mm made in Germany. First, the extract was placed in a vacuum oven and the solution was turned into a dry powder using a freeze dryer (Zirbus Technology, Germany). To prepare oral solutions for oral administration (gavage) to the studied animals, extract powder with doses of 200 and 800 mg per kilogram of animal body weight was dissolved and prepared in 5 ml of solvent (distilled water). The extract solutions were kept in the refrigerator until use. This procedure was performed just before administration and daily.

To induce diabetes in animals from Streptozotocin; STZ was used in the amount of 50 mg/kg as an intraperitoneal injection. 5 days after the injection, the blood sugar of the rats was measured, and to confirm diabetes, a blood sample was taken from the tail vein, and sugar above 180 mg/dL was considered as diabetic. Rats were divided into three groups. Considering the metabolic changes and differences in the bodies of diabetic and healthy rats, we also examined three groups of healthy rats.

60 mice were randomly divided into 6 groups. Group 1, normal rats that received only distilled water. Group 2, normal rats that received a concentration of 200 mg/kg extract. Group 3, normal rats that received a concentration of 800 mg/kg extract. Group 4, diabetic rats that received only distilled water. Group 5, diabetic rats that received a concentration of 200 mg/kg extract. Group 6, diabetic rats that received a concentration of 800 mg/kg extract. All procedures of working with laboratory animals were done by the instructions of the ethics committee of the university. To measure blood sugar levels, diabetic and healthy rats were anesthetized, and blood was taken from the corner of the eye. After 42 days of administration of Capparis spinosa fruit extract by injection, blood was taken from the hearts of rats and the experiments were repeated.

The tested sample was serum, and the blood sugar of rats was measured, sugar above 180 mg/dL was considered as diabetic, and also the serum level of insulin hormone was measured using a special rat insulin measurement kit (Sweden, Mercodia) and the results were between The groups were compared after 6 weeks.

After the completion of the study (sixth week), the liver tissue of the animals was taken out and aliquoted in 4 separate microtubes and immediately transferred to the liquid nitrogen tank. For complementary DNA (cDNA) synthesis, 1 microgram of total ribonucleic acid (RNA) was incubated with 1 microgram of random hexamer for 5 minutes at 70°C. Then this mixture was transferred to the tube containing the cDNA master mix. The mixture was placed in a thermocycler for 60 minutes at 42°C to synthesize cDNA, then it was placed at 95°C for 5 minutes to inactivate the reverse transcription enzyme. At this stage, the necessary amount of synthesized cDNA was removed and mixed with a master containing Taq DNA polymerase and the rest of the required materials, and RT-PCR was performed in 40 cycles. The PCR program included a temperature of 96°C for 10 seconds to denature the cDNA, and a temperature of 70°C for 5 seconds was used for annealing and synthesis. The primers used included the specific forward and reverse primers of each gene whose sequence was designed. Cybergreen was used to monitor each stage, which is a fluorescent reporter molecule that shows the state of proliferation in each stage. The target gene concentration was stated relative to the β-actin housekeeping gene concentration. β-actin was utilized as an internal standard. Data were investigated by BIORAD CFX manager software version 2015.

The activity of glucokinase and hexokinase enzymes was measured manually. Briefly, first, one gram of liver tissue was separated, fragmented, and homogenized with an ultrasonic homogenizer (NexTgen Lab500, France). After centrifugation (Hettich, Germany, EBA200), the upper clear solution was used. It should be noted that all the above steps were repeated to measure each of the enzymes. Then according to valid protocols for each enzyme (Sakrani et al., 2022), by providing the relevant substrate and drawing a standard curve, the level of enzyme activity was determined and compared.

Statistical analysis was done by SPSS software version 23. Results are reported as "(third quartile - first quartile) median". The homogeneity of the variance of the groups was evaluated by Levene's test and the normality of the data distribution was evaluated by the non-parametric Kolmogorov-Smirnov test. Due to the lack of normal distribution (P<0.05), the Kruskal-Wallis non-parametric test and Mann-Whitney post hoc test were used to compare the mean of the groups. The level of significance in the tests was considered 0.05.

Results and Discussion

The findings of the study showed that Capparis spinosa fruit extract significantly decreased the mean serum glucose level in all groups receiving the extract compared to the groups receiving distilled water (Table 1) (P<0.001). Also, the findings of the Mann-Whitney test revealed that there was a statistically significant difference in all pairwise comparisons (P<0.001), only the mean of the serum glucose levels of normal rats that received different concentrations of 200 and 800 mg/kg extract. There was no statistically significant difference (P=0.796).

The mean serum level of insulin was increased in all groups receiving the extract compared to the groups receiving distilled water (P<0.001) (Table 1). The findings of paired group comparisons revealed that the median insulin serum level between the groups of normal rats did not have a statistically significant difference (P<0.05) and in the paired comparisons of diabetic rats, the median insulin serum level that different doses of 200 and 800 mg/kg extract had received, there was no statistically significant difference (P=0.853). Other paired group comparisons showed significant differences between the two groups of healthy and diabetic rats (P<0.001).

The mean activity of the glucokinase enzyme was increased in both diabetic and healthy rats receiving the extract compared to the group receiving distilled water (P<0.001) (Table 1). The results of pairwise comparisons of normal mice did not show a statistically significant difference (P<0.05) and in the group of diabetic mice, only the median difference of the groups receiving the extract with different concentrations of 200 and 800 mg/kg was not statistically significant (P = 0.853) and other group pair comparisons and also between two groups of healthy and diabetic mice were statistically significant (P<0.001).

Table 1. Comparison of mean serum levels of glucose, insulin, and glucokinase enzyme activity levels in different concentrations of Capparis spinosa extract.

* Kruskal-Wallis non-parametric test

According to the Mann-Whitney post hoc test, in each variable, groups with different English letters have a statistically significant difference in the mean (P<0.001).

The findings of the study also revealed that Capparis spinosa fruit extract significantly increased the median expression of the phosphofructokinase-1 gene in all groups receiving the extract compared to the groups receiving distilled water (P<0.001) (Table 2). The findings of paired group comparisons revealed that in the group of healthy and diabetic rats, there is no significant difference between the mean expression of the phosphofructokinase-1 gene in the two groups receiving extracts with different concentrations of 200 and 800 mg/kg (P<0.05) and other comparisons Group pair is significant (P<0.001).

The mean expression of the glucose-6-phosphatase gene in the studied groups had a statistically significant difference (Table 2) (P<0.001). Also, the results of group pairwise comparisons showed that the average expression of this gene between healthy mice did not differ significantly (P<0.05). Pairwise group comparisons in the group of diabetic rats also showed that the only difference in the mean of this gene in the group receiving distilled water and the group receiving the extract with a concentration of 200 mg/kg was not significant (P=0.998). Other pairwise group comparisons were statistically significant (P<0.001).

The mean activity of the hexokinase enzyme in the studied groups had a significant difference (P<0.001) (Table 2). The results of paired group comparisons showed that in healthy mice, only the mean of hexokinase enzyme activity between the group receiving distilled water and the group receiving the extract with a concentration of 800 mg/kg was not significant (P=0.739). Group pair comparisons showed that in diabetic rats, only the mean of hexokinase enzyme activity between the group receiving distilled water and the group receiving extract with concentrations of 200 and 800 mg/kg was not significant (P<0.05). Other pairwise comparisons between the two groups of diabetic and healthy rats were statistically significant (P<0.001).

Table 2. Comparison of the average expression level of phosphofructokinase-1, glucose-6-phosphatase genes, and hexokinase enzyme activity due to different concentrations of Capparis spinosa extract.

* Kruskal-Wallis non-parametric test

Abdelsalam, S. S., Korashy, H. M., Zeidan, A., & Agouni, A. (2019). The role of protein tyrosine phosphatase (PTP)-1B in cardiovascular disease and its interplay with Insulin resistance. Biomolecules, 9(7), 286. doi:10.3390/biom9070286

Adwan, G. M., & Omar, G. I. (2021). Evaluation of antimicrobial activity and genotoxic potential of Capparis spinosa (L.) plant extracts. Microbiology Research Journal International, 31(1), 48-57.

Ahmad, A., & Ahsan, H. (2020). Biomarkers of inflammation and oxidative stress in ophthalmic disorders. Journal of Immunoassay & Immunochemistry, 41(3), 257-271.

Aliyazıcıoglu, R., Sahin, H., Erturk, O., Ulusoy, E., & Kolayli, S. (2013). Properties of phenolic composition and biological activity of propolis from Turkey. International Journal of Food Properties, 16(2), 277-287.

Archana, B., Bharath, P., Deepika, G., Kiran, M. D., & Pavani, B. (2022). Pharmacological, pharmacognostic and phytochemical review of Capparis spinosa L. Future Journal of Pharmaceuticals and Health Sciences, 2(1), 9-21.

Assadi, S., Shafiee, S. M., Erfani, M., & Akmali, M. (2021). Antioxidative and antidiabetic effects of Capparis spinosa fruit extract on high-fat diet and low-dose streptozotocin-induced type 2 diabetic rats. Biomedicine & Pharmacotherapy, 138(21), 111391.

Babel, R. A., & Dandekar, M. P. (2021). A review on cellular and molecular mechanisms linked to the development of Diabetes complications. Current Diabetes Reviews, 17(4), 457-473.

Bhatti, J. S., Sehrawat, A., Mishra, J., Sidhu, I. S., Navik, U., Khullar, N., Kumar, S., Bhatti, G. K., & Reddy, P. H. (2022). Oxidative stress in the pathophysiology of type 2 Diabetes and related complications: Current therapeutics strategies and future perspectives. Free Radical Biology & Medicine, 184, 114-134.

Byrne, N. J., Rajasekaran, N. S., Abel, E. D., & Bugger, H. (2021). Therapeutic potential of targeting oxidative stress in Diabetic cardiomyopathy. Free Radical Biology & Medicine, 169, 317-342.

Kang, Q., & Yang, C. (2020). Oxidative stress and diabetic retinopathy: Molecular mechanisms, pathogenetic role and therapeutic implications. Redox Biology, 37, 101799.

Kazemian, M., Abad, M., Reza Haeri, M., Ebrahimi, M., & Heidari, R. (2015). Anti-Diabetic effect of Capparis spinosa L. root extract in Diabetic rats. Avicenna Journal of Phytomedicine, 5(4), 325.

Kuate, D., Kengne, A. P. N., Biapa, C. P. N., Azantsa, B. G. K., & Wan Muda, W. A. M. B. (2015). Tetrapleura tetraptera spice attenuates high-carbohydrate, high-fat diet-induced obese and type 2 Diabetic rats with metabolic syndrome features. Lipids in Health and Disease, 14(1), 1-13.

Kumari, R., Kumar, A., & Kumar, B. (2019). Ethnobotanical investigation of medicinal plants used by rural communities of district Chatra, Jharkhand, India. Journal of Biotechnology and Biochemistry, 5(6), 34-49.

Liguori, I., Russo, G., Curcio, F., Bulli, G., Aran, L., Della-Morte, D., Gargiulo, G., Testa, G., Cacciatore, F., Bonaduce, D., et al. (2018). Oxidative stress, aging, and diseases. Clinical Interventions in Aging, 13, 757-772.

Linn, W., Persson, M., Rathsman, B., Ludvigsson, J., Lind, M., Andersson Franko, M., & Nyström, T. (2023). Estimated glucose disposal rate is associated with retinopathy and kidney disease in young people with type 1 Diabetes: A nationwide observational study. Cardiovascular Diabetology, 22(1), 61. 

Liu, H., Sridhar, V. S., Boulet, J., Dharia, A., Khan, A., Lawler, P. R., & Cherney, D. Z. I. (2022). Cardiorenal protection with SGLT2 inhibitors in patients with Diabetes mellitus: From biomarkers to clinical outcomes in heart failure and Diabetic kidney disease. Metabolism: Clinical and Experimental, 126, 154918.

Mollica, A., Zengin, G., Locatelli, M., Stefanucci, A., Mocan, A., Macedonio, G., Carradori, S., Onaolapo, O., Onaolapo, A., Adegoke, J., et al. (2017). Anti-Diabetic and anti-hyperlipidemic properties of Capparis spinosa L.: In vivo and in vitro evaluation of its nutraceutical potential. Journal of Functional Foods, 35, 32-42.

Moutia, M., El Azhary, K., Elouaddari, A., Al Jahid, A., Jamal Eddine, J., Seghrouchni, F., Habti, N., & Badou, A. (2016). Capparis spinosa L. promotes anti-inflammatory response in vitro through the control of cytokine gene expression in human peripheral blood mononuclear cells. BMC Immunology, 17(1), 1-12.

Okur, M. E., Özbek, H., Polat, D. Ç., Yılmaz, S., & Arslan, R. (2018). Hypoglycemic activity of Capparis ovata desf. var. palaestina zoh. methanol extract. Brazilian Journal of Pharmaceutical Sciences, 54, e18031.

Ouhammou, M., Adnany, E. M. E., Mourjane, A., Hammou, H. A., Bouchdoug, M., Jaouad, A., & Mahrouz, M. (2022). Physico-chemical analysis and antioxidant activity of Moroccan caper leaves (Capparis spinosa L.). Euro-Mediterranean Journal for Environmental Integration, 7(3), 407-414.

Paul, A. K., Hossain, M. K., Mahboob, T., Nissapatorn, V., Wilairatana, P., Jahan, R., Jannat, K., Bondhon, T. A., Hasan, A., de Lourdes Pereira, M., et al. (2022). Does oxidative stress management help alleviation of COVID-19 symptoms in patients experiencing Diabetes? Nutrients, 14(2), 321.

Roberts, E., Jones, L., Blackman, A., Dewhurst, T., Matcham, F., Kan, C., Mustafa, O., Thomas, T., Siddiqi, N., Ismail, K., et al. (2017). The prevalence of diabetes mellitus and abnormal glucose metabolism in the inpatient psychiatric setting: A systematic review and meta-analysis. General Hospital Psychiatry, 45, 76–84.

Sakrani, I., Ameddah, S., Benrebaia, M., Bouaroudge, A., Erenler, R., Benkiniouar, R., & Cacan, E. (2022). Algerian Capparis spinosa n-buoh extract alleviates Diabetic neuropathy induced with streptozotocin in rats. Egyptian Journal of Chemistry, 65(132), 519-538.

Sanchooli, M., Bagheri, R., Jaberi, S., Mohammadi, S., & Khedangi, Z. (2012). Comaring the essential oil composision of root, leave and fruit of Capparis spinosa in field and habitat. Plant and Ecosystem, 8, 27-40.

Soliman, A. M. (2016). Potential impact of Paracentrotus lividus extract on Diabetic rat models induced by high fat diet/streptozotocin. The Journal of Basic & Applied Zoology, 77, 8-20.

Sun, Y., Yang, T., & Wang, C. (2023). Capparis spinosa L. as a potential source of nutrition and its health benefits in foods: A comprehensive review of its phytochemistry, bioactivities, safety, and application. Food Chemistry, 409, 135258.

Teimouri, M., Hosseini, H., ArabSadeghabadi, Z., Babaei-Khorzoughi, R., Gorgani-Firuzjaee, S., & Meshkani, R. (2022). The role of protein tyrosine phosphatase 1B (PTP1B) in the pathogenesis of type 2 diabetes mellitus and its complications. Journal of Physiology and Biochemistry, 78(2), 307-322.

Van Veen, M. M., Kooij, J. S., Boonstra, A. M., Gordijn, M. C., & Van Someren, E. J. (2010). Delayed circadian rhythm in adults with attention-deficit/hyperactivity disorder and chronic sleep-onset insomnia. Biological Psychiatry, 67(11), 1091-1096.

Vickers, N. J. (2017). Animal communication: When i’m calling you, will you answer too? Current Biology, 27(14), R713-R715.

Wojdyło, A., Nowicka, P., Grimalt, M., Legua, P., Almansa, M. S., Amorós, A., Carbonell-Barrachina, Á. A., & Hernández, F. (2019). Polyphenol compounds and biological activity of caper (Capparis spinosa L.) flowers buds. Plants, 8(12), 539.

Yang, T., Wang, Y. L., Zhang, Y. L., Liu, Y. T., Tao, Y. Y., Zhou, H., & Liu, C. H. (2022). The protective effect of Capparis spinosa fruit on triptolide-induced acute liver injury: A metabolomics-based systematic study. Journal of Functional Foods, 90, 104989.

Zhang, H., & Ma, Z. F. (2018). Phytochemical and pharmacological properties of Capparis spinosa as a medicinal plant. Nutrients, 10(2), 116.