Islet Protection and Amelioration of Type 2 Diabetes Mellitus by Treatment with Quercetin from the Flowers Ofedgeworthia Gardneri

Journal name: Drug Design, Development and Therapy Article Designation: Original Research Year: 2018 Volume: 12 Drug Design, Development and Therapy Dovepress Running head verso: Zhuang et al Running head recto: Pharmacological activities of quercetin from the flowers of Edgeworthia gardneri open access to scientific and medical research DOI: 153898 Open Access Full Text Article ORIGINAL RESEARCH Islet protection and amelioration of type 2 diabetes mellitus by treatment with quercetin from the flowers of Edgeworthia gardneri Manjiao Zhuang1 Background and purpose: The traditional Chinese medicine – the flower of Edgeworthia Honghong Qiu2 gardneri – is reported as an effective therapeutic for type 2 diabetes mellitus (T2DM). Ping Li2 Nevertheless, most constituents of the flowers of E. gardneri have not yet been stud- Lihua Hu2 ied. This study was conducted to investigate the effect of quercetin extracted from the Yayu Wang3 flowers of E. gardneri on islet protection and amelioration in T2DM and explore its Lei Rao2 mechanism. Method: Quercetin was extracted from the flowers of E. gardneri and verified by high-per- 1 Department of Pharmacy, Sichuan formance liquid chromatography. Quercetin or crude extract’s effect on insulin secretion was University, Chengdu, People’s Republic of China; 2Collaborative Innovation investigated. ERK1/2 and phospho-ERK1/2 were detected by Western blot analysis, and fluo-3 Center of Sichuan for Elderly Care AM was used to detect intracellular Ca2+. The anti-apoptosis effect of quercetin or crude extract and Health, Chengdu Medical College, on MIN-6 cells was investigated by thiazolyl blue tetrazolium bromide (MTT) assay and flow Chengdu, China; 3Department of Cell Biology, Institute of Biological cytometry analysis. Activation of caspases and expression of Bcl-2 and BAX were tested by Medicine, Jinan University, Guangzhou, Western blot analysis. In addition, the mitochondrial membrane potential was determined by People’s Republic of China JC-1 probe. Moreover, in vivo activity was also tested in db/db mice. Results: A quercetin level of .10 μmol/L could induce insulin secretion. Intracellular Ca2+ and ERK1/2 were involved in the signaling pathway of quercetin-induced insulin secretion. We also observed that quercetin could inhibit palmitic acid-induced cell apoptosis via suppressing the activation of caspase-3, -9, -12; increasing the ratio of Bcl-2/BAX and reversing the impaired mitochondrial membrane potential. Crude extract’s effect on insulin secretion was similar to that of pure extracted quercetin, while it possessed higher anti-apoptosis activity. Additionally, intraperitoneal glucose tolerance, plasma insulin level, hepatic triglyceride, hepatic glycogen and the pathological histology of both pancreatic islet and liver in db/db mice were significantly improved by the administration of the extracted quercetin. Conclusion: Our study indicated that quercetin extracted from the flowers of E. gardneri exerted excellent properties in islet protection and amelioration. Keywords: quercetin, Edgeworthia gardneri, insulin secretion, anti-apoptosis, type 2 diabetes mellitus Introduction Correspondence: Lei Rao Collaborative Innovation Center of Type 2 diabetes mellitus (T2DM), a common chronic disease, has spread rapidly Sichuan for Elderly Care and Health, as a grave threat to people’s health in the past few decades.1 It is characterized by Chengdu Medical College, Number 783 -cell dysfunction and chronic insulin resistance.2,3 The decline in -cell function is Xindu Road, Chengdu 610500, Sichuan, β β People’s Republic of China characterized as impaired insulin secretion and increased β-cell decrease.4 Therefore, Tel +86 28 6273 9582 improving insulin secretion as well as inhibiting -cell apoptosis are considered as Fax +86 28 6273 9583 β Email [email protected] two main strategies to treat T2DM. submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2018:12 955–966 955 Dovepress © 2018 Zhuang et al. This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms.php and incorporate the Creative Commons Attribution – Non Commercial (unported, v3.0) License (http://creativecommons.org/licenses/by-nc/3.0/). By accessing the work you http://dx.doi.org/10.2147/DDDT.S153898 hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms (https://www.dovepress.com/terms.php). Zhuang et al Dovepress The traditional Chinese medicine – the flower of antibody were purchased from Cell Signaling Technol- Edgeworthia gardneri – is a class of herbal teas found ogy (Danvers, MA, USA). Annexin V-FITC/PI Apoptosis in Tibet that is believed to provide a range of benefits. It Detection Kit was purchased from Merck. MitoProbe JC-1 contains various active components including coumarins, Assay Kit was a product from Thermo Fisher Scientific flavones, edgeworin and daphnoretin.5 Previous studies have (Waltham, MA, USA). Triglyceride Quantification Assay demonstrated that the extracts of the flowers of E. gardneri Kit (Colorimetric/Fluorometric) and Glycogen Colori- display broad pharmacological activities for the treatment metric/fluorometric Assay Kit were obtained from Abcam of diabetes, inflammation and cardiovascular disease.6,7 For (Cambridge, UK). example, Zhao et al demonstrated that coumarin from the flowers ofE. gardneri exhibits α-glucosidase and α-amylase Extraction and isolation inhibitory activities.8 Ma et al showed that phenolics from the Dried powder of the flowers of E. gardneri (500 g) was flowers ofE. gardneri possess α-glucosidase inhibition and extracted by using methanol at room temperature for 3 days. antihyperglycemic activity.9 Nevertheless, most constituents The resultant extracts were combined and concentrated of the flowers of E. gardneri have not yet been studied. under reduced pressure, and the residue was partitioned Quercetin is a compound of flavones in many Chinese into water and extracted with petroleum ether, ethyl acetate traditional herbal medicines with good biologic value. It is and n-butano in succession. Each fraction was evaporated known to exhibit a broad variety of biological effects that in vacuo to yield residue extracts. The ethyl acetate frac- have potential applications in medical treatment, includ- tion was used to isolate quercetin (3 mg) by using col- ing anticancer, antioxidant, anti-inflammatory and, in umn chromatography over Sephadex LH-20 according to particular, protective effect in diabetes.10–12 Quercetin was Ma et al’s study.9 also found to be abundant in the flowers of E. gardneri, while its antidiabetic activity has yet to be investigated High-performance liquid chromatography systematically.9 (HPLC) analysis Therefore, in the present study, the isolation and biologi- HPLC analysis of quercetin extracted from the flowers of cal activity of quercetin from the flowers ofE. gardneri were E. gardneri was implemented by a Waters Alliance 2695- reported. This is the original report on islet protection and 2487 HPLC system with an Agilent C18 column (Waters, amelioration of T2DM by treatment with quercetin from the Milford, MA, USA) in gradient elution. The effluent was flowers of E. gardneri both in vitro and in vivo. detected at 280 nm.13 Materials and methods MIN-6 cell culture Materials The insulin-secreting cell line MIN-6 was cultured in RPMI- The flowers of E. gardneri were purchased from 1640 in accordance with previous studies. MIN-6 cells were ZangXiTang Co., Ltd (Tibet, People’s Republic of China). cultured and treated with palmitic acid for 24 h to establish Quercetin standard, rutin standard and isoquercetin stan- oxidative damage model.14 dard were purchased from Solarbio (Beijing, People’s Republic of China). MIN-6 cell line was purchased from Measurement of insulin secretion Cell Bank of Chinese Academy Sciences (Beijing, People’s For insulin secretion studies, 1×104 MIN-6 cells were plated Republic of China). Both RPMI-1640 medium and fetal in a 96-well microplate and cultured for 48 h. After that, bovine serum were purchased from Gibco (Gaithersburg, the medium was removed from each well and 1 mL of fresh MD, USA). Glimepiride, AZD8330 (inhibitor of ERK1/2), medium was added. Increasing concentrations of quercetin nifedipine (inhibitor of Ca2+ channel) and fluo-3 AM were from E. gardneri were added to the medium, and after 1 h of obtained from Sigma-Aldrich (St Louis, MO, USA). incubation, the medium was collected. The insulin concentra- Thiazolyl blue tetrazolium bromide (MTT) was purchased tion in the medium was measured by a commercial Mouse Insu- Merck (Darmstadt, German). Insulin concentrations in lin Elisa Kit in accordance with the manufacturer’s instructions. cell supernatant were determined using the Mouse Insulin Glimepiride was taken as a positive control. Additionally, to Elisa Kit obtained from Crystal Chem (Downers Grove, IL, test the role of Ca2+ channel and ERK1/2 in quercetin-induced USA). Rabbit anti-ERK1/2 antibody, rabbit anti-phospho- insulin secretion, relevant inhibitors (AZD8330 or Nifedipine) ERK1/2 antibody (Thr202/Tyr204) and apoptosis-related were added in the medium together with quercetin.15 956 submit your manuscript | www.dovepress.com Drug Design, Development and Therapy 2018:12 Dovepress Dovepress Pharmacological

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