|Year : 2019 | Volume
| Issue : 2 | Page : 224-228
Effect of ethyl acetate and aqueous fractions of Ziziphus jujuba extract on biochemical and hematological parameters in rat
Reza Mohebbati1, Yasamin Kamkar-Del1, Mohammad Naser Shafei2
1 Department of Physiology, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
2 Neurogenic Inflammation Research Center, Mashhad University of Medical Sciences, Mashhad, Iran
|Date of Web Publication||30-Oct-2019|
Dr. Mohammad Naser Shafei
Neurogenic Inflammation Research Center, Mashhad University of Medical Sciences, Mashhad.
Source of Support: None, Conflict of Interest: None
Introduction: Despite the numerous therapeutic effects of the Ziziphus jujuba (ZJ), its effect on biochemical and hematological parameters are unknown. Therefore, in this study, the effect of ethyl acetate (EA) and aqueous (AQ) fractions of the ZJ fruit extract on biochemical and hematological parameters were explored. Material and Methods: Thirty-six male Wistar rats randomly were divided into six groups as follows: (1) Control, (2) ZJ extract (200mg/kg), (3, 4) EA fraction of ZJ (EA150 and EA300mg/kg), and (5, 6) AQ fraction of ZJ (AQ150 and AQ300mg/kg). Saline, ZJ extract, and its fractions were gavage once a day for 4 weeks. At the day of the experiment (28th day), one blood sample was collected in citrated tube for the counting of the complete blood cell count calculation. The serum was used for biochemical parameters (cholesterol, triglyceride, and glucose concentrations) evaluation. Results: The results showed that the blood glucose, cholesterol, and triglyceride levels in all treated groups, especially in AQ groups, significantly decreased compared to the control (P < 0.01 to P < 0.001) group. Furthermore, the number of red blood cell and white blood cell in treating rats with ZJ extract and its AQ fraction significantly increased (P < 0.05), whereas the number of platelets significantly decreased (P < 0.05) compared to the control. Conclusion: In conclusion, both AQ and EA fractions of ZJ have beneficial effects on biochemical and hematological parameters. Since the effect of AQ fraction is higher than EA, it is suggested that these effects of ZJ mostly mediated by compounds of ZJ that soluble in the polar solvent.
Keywords: Biochemistry, ethyl acetate, fraction, hematology, ziziphus jujuba
|How to cite this article:|
Mohebbati R, Kamkar-Del Y, Shafei MN. Effect of ethyl acetate and aqueous fractions of Ziziphus jujuba extract on biochemical and hematological parameters in rat. J Rep Pharma Sci 2019;8:224-8
|How to cite this URL:|
Mohebbati R, Kamkar-Del Y, Shafei MN. Effect of ethyl acetate and aqueous fractions of Ziziphus jujuba extract on biochemical and hematological parameters in rat. J Rep Pharma Sci [serial online] 2019 [cited 2019 Nov 15];8:224-8. Available from: http://www.jrpsjournal.com/text.asp?2019/8/2/224/269948
| Introduction|| |
Medicinal plants have a wide historical background and are still used in the treatment and improvement of various diseases in traditional and complementary medicine. The World Health Organization (WHO) has estimated that 90% of people in developing countries use medicinal plants for primary health-care needs. The WHO also announced 21,000 plants are used for medicinal propose in the world. One of the well-known medicinal plants is Ziziphus jujuba (ZJ) that belongs to the Rhamnaceae family. This plant is native to Asia and Southern Europe. The local name of this plant varies from country to country. In Iran, it is commonly known as “Annab,” and in China named a red date. Fruit, seed, and leaves of this plant were used as a remedy, flavoring, and edible food. ZJ fruit has several pharmacological properties, including antifungal, antiulcer, anti-inflammatory, anti-oxidant, and cardio tonic effects. The important biological components of ZJ are polysaccharides, phenols, flavonoids, mucilage, iron, ascorbic acid, riboflavin, pectin, and different chemical components such as jujubosides A, B, and C, zizyberenalic acid, and betulinic acid.,,, In a previous study, the effect of ZJ seed on the biochemical parameters has been investigated, but effects of the ZJ fruit and its fractions on biochemical and hematological parameters are not yet investigating. The aim of this study was to explore the effect of the ZJ fruit extract and its two fractions (aqueous [AQ] and ethyl acetate [ EA ]) on these parameters.
| Materials and Methods|| |
Extract and fraction preparation
After the collection of ZJ fruits (Birjand, South Khorasan, Iran), it is identified by a botanist in the herbarium of Ferdowsi University of Mashhad. Then, 100g of the ZJ fruit was powdered and then soaked in 500mL ethanol (70%) and shacked for 72h in the standard temperature (37°C). Next, the mixture was passed through the filter papers with different filter sizes. The solvent (ethanol) was evaporated by an oven at 40°C., At the stage of fraction preparation, 25g of ZJ extract was dissolved in 100mL of distilled water. After that, the solution was poured into the decanter; the soluble ingredients were isolated three times by adding 25ml of EA. The remaining solution was AQ fraction. AQ and EA fractions were placed in the oven to remove the solvent (40°C).
Thirty-six Wistar male rats were kept in standard laboratory condition in the animal room (12:12 light-dark cycle, temperature 20°C–22°C, and the humidity 25%–35%). Animals were randomly divided into six groups as follows (n = 6):
1. Control; received saline
2. ZJ extract; received extract (200mg/kg; ip)
3, 4. Ethyl acetate (EA) fraction of ZJ (150 and 300mg/kg)
5, 6. Aqueous (AQ) fraction of ZJ (150 and 300mg/kg)
Saline, ZJ extract, and its fractions were gavage once a day for 4 weeks. All procedures were approved by the Animal Research Ethics Committee of Mashhad University of Medical Sciences (Code number: IR. MUMS.fm. REC.1396.589).
After treatment (4 weeks), the rats were deeply anesthetized with urethane (1.4g/kg, IP). Then the chest cavities of animals were opened, and the fresh blood samples were collected from the heart directly. One blood sample was collected in the citrated tube for the counting of the complete blood count calculation. Another blood sample was collected and its serums were separated by centrifuging at 3000rpm in 10min for analyzing the serum biochemical parameters (cholesterol, triglyceride, and glucose concentrations). These parameters were measured by related kits purchased from Pars Azmoon Co, Iran.
Data were explained as the mean ± standard error of the mean. After normalization survey, the data analysis was performed using one-way ANOVA followed by least significant difference post hoc comparison test. The statistical significance value was P < 0.05.
| Results|| |
The results showed that the glucose level same as the previous study was 183.3mg/dL in normal rat and treated with total extract and both fractions significantly decreased compared to the control (P < 0.01 to P < 0.001). The glucose level in lower doses of both fractions was significant than the ZJ extract group [P < 0.01, [Figure 1]]. The cholesterol level in all treated rats except ZJ extract group significantly decreased in comparison with the control (P < 0.05 to P < 0.01). This parameter in rats treated with two doses of EA and a higher dose of AQ fractions significantly reduced compared to the ZJ extract group (P < 0.05) [Figure 2]. The level of triglyceride in rats treated with higher doses of both fractions (AQ300 and EA300mg/kg) significantly decreased compared to the control, and two doses of both fractions are also significantly lower than the ZJ extract group (P < 0.05 to P < 0.01) [Figure 3].
|Figure 1: The comparative effects of glucose concentration in all treated groups. The data presented as mean ± standard error of the mean by one-way ANOVA test with post hoc least significant difference. **P < 0.01, ***P < 0.001 compared to the control, ##P < 0.01 compared to the Ziziphus jujuba extract group. A150/300: Aqueous fractions of the Ziziphus jujuba extract, EA150/300: Ethyl acetate fraction of the Ziziphus jujuba extract|
Click here to view
|Figure 2: The comparative effects of cholesterol concentration in all treated groups. The data presented as mean ± standard error of the mean by one-way ANOVA test with post hoc least significant difference. *P < 0.05, **P < 0.01 compared to the control, #P < 0.05 compared to the Ziziphus jujuba extract group. A150/300: Aqueous fractions of the Ziziphus jujuba extract, EA150/300: Ethyl acetate fraction of the Ziziphus jujuba extract|
Click here to view
|Figure 3: The comparative effects of triglyceride concentration in all treated groups. The data presented as mean ± standard error of the mean by one-way ANOVA test with post hoc least significant difference. *P < 0.05 compared to the control, #P < 0.05, ##P < 0.01 compared to the Ziziphus jujuba extract group. A150/300: Aqueous fractions of the Ziziphus jujuba extract, EA150/300: Ethyl acetate fraction of the Ziziphus jujuba extract|
Click here to view
As shown in [Table 1], the levels of white blood cell (WBC) and red blood cell (RBC) in treating rats with ZJ extract and its AQ fraction significantly increased compared to the control (P < 0.05) while the platelet level significantly decreased (P < 0.05) and this effect in higher doses of AQ extract was statistically significant in comparison with the ZJ extract group (P < 0.05).
|Table 1: The comparative effects of hematological parameters in all treated groups|
Click here to view
| Discussion|| |
The results indicate that total extract and two fractions of ZJ significantly decreased the glucose level. Serum levels of cholesterol and triglyceride decreased by both higher doses of fractions, but a dose of 200mg/kg of total extract has not significant effect on these parameters. Total extract and fractions of ZJ also have effect on hematologic parameters and increased RBC and WBS while decreasing platelet amount.
The ZJ fruit is a rich source of the various vitamins, flavonoids, polyphenolic compounds, pectin, alkaloids, glycosides, and terpenoids. In this study, we used polar (AQ) and semi-polar fractions (EA) of the ZJ extract to determine the effect of polar and semi-polar ZJ ingredients on the biochemical and hematological parameters in the normal rats. In a previous study, Davarinejad et al. have been shown that the highest components of ZJ extract are polyphenols and flavonoids. The polyphenols and flavonoids are polar compounds, and in this study, the effect of the AQ fraction (polar) of the ZJ extract on the biochemical and hematological parameters was better than the EA fraction that it is conceivable that due to more polarity of the AQ than EA solvent. The numerous studies have been shown that polyphenols and flavonoids isolated of plants have hypoglycemic and hypolipidemic as well as antioxidant effects.,, Therefore, a better effect of AQ fraction in this study on biochemical and hematological parameters attributed the components of this fraction.
In a study, Liu et al. have been shown that anthocyanin, one of the derivatives of flavonoids, also has a hypolipidemic effect in humans treated with anthocyanin-rich supplementation. The possible mechanisms of this effect are increasing the fecal excretion of total cholesterol as well as down-regulation of 3-hydroxy-3-methyl-glutaryl-cholesterol O-acyltransferase reductase (HMG-CoA reductase) gene expression in the liver specially. Furthermore, anthocyanin decreases the apo B/C-containing triglyceride-rich particles. In another study also has been indicated that polyphenols such as flavonoids inhibit both acyl CoA and HMG-CoA reductase, modulate adipokines, increasing of the GLUT-4 gene expression, reduction of the oxidative stress, and activate AMP-activated protein kinase. One of the other ingredients in the ZJ extract is pectin that modulates the HMG-CoA reductase activity and increases the hepatic pool of the cholesterol results in a reduction of blood lipid profile. Saponins, glycosidic compounds, have hypoglycemic and hypolipidemic effects., Jujubosides are also in the saponins group structurally that isolated from ZJ extract. Therefore, part of the hypoglycemic and hypolipidemic properties of the ZJ attributes to the presence of jujubosides.
About hematological parameters, some studies have been revealed that flavonoids and polyphenols bind to the RBC/WBC membrane and lead to increasing of membrane integrity via reduction of lipid peroxidation. Therefore, flavonoids by its antioxidant properties result in decrease of hemolysis and increase the RBC/WBC count., In addition, flavonoids have an inhibitory effect on the platelet function and aggregation via its anti-inflammatory effects and also flavonoids effect on the secretory process, mitogenesis, and cell–cell interaction., These are probably the reasons for reducing platelet count. Furthermore, several studies have been reported that ZJ extract induces the expression and production of the erythropoietin via activation of the hypoxia-inducible factor 1 and other signaling factors that result in the hematopoiesis process enhancement. Briefly, both fractions had better effects than whole extract on biochemical parameters, which is due to the use of relatively high doses of extract fractions.
| Conclusion|| |
According to the recent results, AQ and EA fractions of ZJ have beneficial effects on biochemical and hematological parameters. Since these fractions are polar, it is suggested that these effects of ZJ mostly mediated by compounds of ZJ that soluble in polar solvents.
We would like to thank the Research Council of Mashhad University of Medical Sciences for their financial support.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Rizvi SI, Mishra N Traditional indian medicines used for the management of diabetes mellitus. J Diabetes Res 2013;2013:712092.
Sumanth M, Bhargavi Y Evaluation of woundhealing effect of Ziziphus mauritiana L. leaf extract in rats. Int J Green Pharm 2014;8:263-6. [Full text]
Yue Y, Wu S, Zhang H, Zhang X, Niu Y, Cao X, et al
. Characterization and hepatoprotective effect of polysaccharides from Ziziphus jujuba mill. var. spinosa (Bunge) hu ex H. F. Chou sarcocarp. Food Chem Toxicol 2014;74:76-84.
Sarfaraz A, Ansari S, Porchezhian E Antifungal activity of alcoholic extracts of Ziziphus vulgaris and Acacia concinna. Hamdard Med 2002;45:14-5.
Ganachari M, Kumar S Anti-ulcer properties of Ziziphus jujuba Lam leaves extract in rats. J Nat Remed 2004;4:103-8.
Huang L, Ye W, Cai B, Li D, Liu J, Liu M, et al
. A preliminary study on the pharmacology of the compound prescription huangqin tang and its component drugs. Zhongguo Zhong Yao Za Zhi 1990;15:115-7, 128.
Na MK, An R, Lee SM, Hong ND, Yoo JK, Lee CB, et al
. Screening of crude drugs for antioxidative activity. Korean J Pharmacog 2001;32:108-15.
Mahajan RT, Chopda M Phyto-pharmacology of Ziziphus jujuba mill – A plant review. Pharmacog Rev 2009;3:320-9.
Mohebbati R, Bavarsad K, Rahimi M, Rakhshandeh H, Khajavi Rad A, Shafei MN, et al
. Protective effects of long-term administration of Ziziphus jujuba fruit extract on cardiovascular responses in L-NAME hypertensive rats. Avicenna J Phytomed 2018;8:143-51.
Peng WH, Hsieh MT, Lee YS, Lin YC, Liao J Anxiolytic effect of seed of Ziziphus jujuba in mouse models of anxiety. J Ethnopharmacol 2000;72:435-41.
Tripathi S Ziziphus jujuba: A phytopharmacological review. Int J Res Dev Pharm Life Sci 2014;3:959-66.
Kim HS Effects of the Zizyphus jujuba seed extract on the lipid components in hyperlipidemic rats. J Food Sci Nutr 2002;7:72-7.
Mohebbati R, Iranmanesh M, Beheshti F, Mousavi M, Rohani M, Mogharrabi M, et al
. The effect of some herbal extracts on nitric oxide production in endothelial cells 3T3 cell line. Iran J Pharm Sci 2016;12:1-10.
Dolati K, Rakhshandeh H, Shafei MN Effect of aqueous fraction of Rosa damascena on ileum contractile response of guinea pigs. Avicenna J Phytomed 2013;3:248-53.
Shafei MN, Nasimi A Effect of glutamate stimulation of the cuneiform nucleus on cardiovascular regulation in anesthetized rats: Role of the pontine Kolliker-Fuse nucleus. Brain Res 2011;1385:135-43.
Mahajan RT, Chopda MZ Phyto-pharmacology of Ziziphus jujuba mill – A plant review. Pharmacog Rev 2009;3:320-9.
Davarinejad G, Taghizadeh S, Asili J Effect of different solvents on total phenolic contents and antioxidant activity of Zizyphus jujube miller fruits. J Horticul Sci 2017;31:158-66.
Sharma B, Viswanath G, Salunke R, Roy P Effects of flavonoid-rich extract from seeds of Eugenia jambolana (L.) on carbohydrate and lipid metabolism in diabetic mice. Food Chem 2008;110:697-705.
Nagasako-Akazome Y, Kanda T, Ikeda M, Shimasaki H Serum cholesterol-lowering effect of apple polyphenols in healthy subjects. J Oleo Sci 2005;54:143-51.
Rice-Evans CA, Miller NJ, Bolwell PG, Bramley PM, Pridham JB The relative antioxidant activities of plant-derived polyphenolic flavonoids. Free Radic Res 1995;22:375-83.
Liu C, Sun J, Lu Y, Bo Y Effects of anthocyanin on serum lipids in dyslipidemia patients: A systematic review and meta-analysis. PLoS One 2016;11:e0162089.
Jurgoński A, Juśkiewicz J, Zduńczyk Z, Matusevicius P, Kołodziejczyk K Polyphenol-rich extract from blackcurrant pomace attenuates the intestinal tract and serum lipid changes induced by a high-fat diet in rabbits. Eur J Nutr 2014;53:1603-13.
Marounek M, Volek Z, Synytsya A, Copíková J Effect of pectin and amidated pectin on cholesterol homeostasis and cecal metabolism in rats fed a high-cholesterol diet. Physiol Res 2007;56:433-42.
Rodrigues HG, Diniz YS, Faine LA, Galhardi CM, Burneiko RC, Almeida JA, et al
. Antioxidant effect of saponin: Potential action of a soybean flavonoid on glucose tolerance and risk factors for atherosclerosis. Int J Food Sci Nutr 2005;56:79-85.
Li M, Qu W, Wang Y, Wan H, Tian C Hypoglycemic effect of saponin from Tribulus terrestris. Zhong Yao Cai 2002;25:420-2.
Otsuka H, Akiyama T, Kawai KI, Shibata S, Inoue O, Ogihara Y The structure of jujubosides A and B, the saponins isolated from the seeds of Zizyphus jujuba. Phytochem 1978;17:1349-52.
Chaudhuri S, Banerjee A, Basu K, Sengupta B, Sengupta PK Interaction of flavonoids with red blood cell membrane lipids and proteins: Antioxidant and antihemolytic effects. Int J Biol Macromol 2007;41:42-8.
Noroozi M, Angerson WJ, Lean ME Effects of flavonoids and vitamin C on oxidative DNA damage to human lymphocytes. Am J Clin Nutr 1998;67:1210-8.
Asgary S, Naderi G, Askari N Protective effect of flavonoids against red blood cell hemolysis by free radicals. Exp Clin Cardiol 2005;10:88-90.
Middleton E Jr., Kandaswami C Effects of flavonoids on immune and inflammatory cell functions. Biochem Pharmacol 1992;43:1167-79.
Chen J, Lam CT, Kong AY, Zhang WL, Zhan JY, Bi CW, et al
. The extract of Ziziphus jujuba fruit (jujube) induces expression of erythropoietin via hypoxia-inducible factor-1 in cultured hep3B cells. Planta Med 2014;80:1622-7.
[Figure 1], [Figure 2], [Figure 3]