Acta Pharmacologica Sinica (2016) 37: 1083–1090 © 2016 CPS and SIMM All rights reserved 1671-4083/16 www.nature.com/aps Original Article Trimer procyanidin oligomers contribute to the protective effects of cinnamon extracts on pancreatic β-cells in vitro Peng SUN1, 2, #, Ting WANG1, #, Lu CHEN2, #, Bang-wei YU1, Qi JIA2, Kai-xian CHEN1, 2, Hui-min FAN3, Yi-ming LI2, *, He-yao WANG1, * 1Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China; 2School of Pharmacy, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China; 3Department of Cardiovascular and Thoracic Surgery, Shanghai Heart Failure Research Center, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200123, China Aim: Cinnamon extracts rich in procyanidin oligomers have shown to improve pancreatic β-cell function in diabetic db/db mice. The aim of this study was to identify the active compounds in extracts from two species of cinnamon responsible for the pancreatic β-cell protection in vitro. Methods: Cinnamon extracts were prepared from Cinnamomum tamala (CT-E) and Cinnamomum cassia (CC-E). Six compounds procyanidin B2 (cpd1), (–)-epicatechin (cpd2), cinnamtannin B1 (cpd3), procyanidin C1 (cpd4), parameritannin A1 (cpd5) and cinnamtannin D1 (cpd6) were isolated from the extracts. INS-1 pancreatic β-cells were exposed to palmitic acid (PA) or H2O2 to induce lipotoxicity and oxidative stress. Cell viability and apoptosis as well as ROS levels were assessed. Glucose-stimulated insulin secretion was examined in PA-treated β-cells and murine islets. Results: CT-E, CC-E as well as the compounds, except cpd5, did not cause cytotoxicity in the β-cells up to the maximum dosage using in this experiment. CT-E and CC-E (12.5–50 μg/mL) dose-dependently increased cell viability in both PA- and H2O2-treated β-cells, and decreased ROS accumulation in H2O2-treated β-cells. CT-E caused more prominent β-cell protection than CC-E. Furthermore, CT-E (25 and 50 μg/mL) dose-dependently increased glucose-stimulated insulin secretion in PA-treated β-cells and murine islets, but CC-E had little effect. Among the 6 compounds, trimer procyanidins cpd3, cpd4 and cpd6 (12.5–50 μmol/L) dose-dependently increased the cell viability and decreased ROS accumulation in H2O2-treated β-cells. The trimer procyanidins also increased glucose-stimulated insulin secretion in PA-treated β-cells. Conclusion: Trimer procyanidins in the cinnamon extracts contribute to the pancreatic β-cell protection, thus to the anti-diabetic activity. Keywords: cinnamon; procyanidin oligomers; cinnamtannin B1; procyanidin C1; cinnamtannin D1; diabetes; pancreatic β-cells; cultured murine islets; lipotoxicity; ROS; insulin secretion Acta Pharmacologica Sinica (2016) 37: 1083–1090; doi: 10.1038/aps.2016.29; published online 30 May 2016 Introduction mon extract enhanced the insulin sensitivity in normal rats[4]. As a common spice, cinnamon may be a potential treatment The blood glucose level in streptozotocin-induced diabetic rats for type 2 diabetes. Several clinical studies found that cin- or type 2 diabetic db/db mice was also reduced after cinnamon namon supplements decreased the fasting serum glucose, tri- administration[5–8]. Although these studies suggest a beneficial glyceride, total cholesterol and HbA1c levels in type 2 diabetic effect of cinnamon in treating diabetes, the biological effects patients[1–3]. Furthermore, cinnamon extracts improved the of cinnamon in the treatment of type 2 diabetes remain con- glucose profiles in animal models. Qin et al found that cinna- troversial. For example, some studies reported that cinnamon supplementation did not have any significant effects on type 2 diabetic patients[9, 10]. # These authors contributed equally to this work. Cinnamon is produced from the bark of multiple species of *To whom correspondence should be addressed. E-mail [email protected] (He-yao WANG); Cinnamomum, which contains a variety of compounds, such [11] [email protected] (Yi-ming LI) as cinnamaldehyde, eugenol, and polyphenols . Recently, Received 2016-01-17 Accepted 2016-03-28 active water-soluble components were isolated and found to Acta Pharmacologica Sinica www.nature.com/aps 1084 Sun P et al mediate beneficial effects on glucose metabolism[12–14]. These (Carlsbad, CA, USA). The INS-1 rat insulinoma cell line was components were identified as polyphenols, also named pro- cultured in Roswell Park Memorial Institute (RPMI)-1640 cyanidins, which usually occur as oligomers of epicatechin medium. The MIN6 mouse insulinoma cell line was cultured and the flavonoid catechin[13]. Our previous studies also in Dulbecco’s modified Eagle medium (DMEM) containing confirmed that procyanidin oligomer-rich cinnamon extracts 25 mmol/L glucose[18]. A concentration of 10% fetal bovine improved the glucose profiles in diabetic animals[7, 8]. In a serum (FBS) and 50 μmol/L of β-mercaptoethanol were added further study on the hypoglycemic effect of cinnamon extracts to the medium to maintain cell growth. Murine islets were on db/db mice, we found that extracts isolated from two spe- isolated from 8-week old male C57BL6 mice, described in our cies of the genus Cinnamomum, Cinnamomum cassia (C cassia) previous study[16]. All animal experiments were permitted by and Cinnamomum tamala (C tamala), both exerted different anti- the IACUC of Shanghai Institute of Materia Medica. All cells diabetic pharmacological effects, which improved not only the were kept at 37 °C and 5% CO2 in humidified air. insulin sensitivity but also the pancreatic β-cell function in db/ db mice[15]. Therefore, we hypothesized that β-cell protection Cell viability assay, Hoechst 33342 staining and reactive oxygen is an important mechanism for the anti-diabetic effect of cin- species (ROS) determination namon. Furthermore, our other study confirmed that a one Cell viability was determined using the 3-(4,5-dimethylthi- trimer procyanidin oligomer isolated from cinnamon extract, azol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT, Sigma- cinnamtannin D-1, protected pancreatic β-cells from lipotoxic- Aldrich, St Louis, MO, USA) assay as previously described[16]. ity[16]. To detect the cytotoxicity of each compound, different doses Therefore, to explore the mechanisms involved in the anti- of CC-E, CT-E and the six procyanidins were added into the diabetic effect of cinnamon, further investigations of β-cell medium and incubated for 48 h separately. To determine protection using cinnamon extracts or purified procyanidin the effect of the compounds on cell viability in palmitic acid oligomers are important. The objective of this study was to (PA) or H2O2 treated pancreatic β-cells, INS-1 and MIN6 cells verify, in vitro, the protective effects of cinnamon extracts from were treated with 0.4 mmol/L PA in the presence of different C cassia and C tamala on lipotoxic pancreatic β-cells, as well as doses of compounds for 48 h. For the H2O2 model, the cells the effects of different procyanidin oligomers isolated from were pre-incubated with compounds for 4 h and subsequently these two species of Cinnamomum. exposed to 0.5 mmol/L H2O2 with compounds for another 2 h. Then, cell viability was measured using the MTT assay. Materials and methods The value was calculated as the inhibition ratio (% of control Plants and sample preparation group). C cassia and C tamala were collected in 2014 from the Guangxi Hoechst 33342 staining was performed to observe the effect and Yunnan provinces, respectively. The samples were botan- of CC-E and CT-E on PA-induced apoptosis in INS-1 cells. ically authenticated by Professor Guan-yun GU at the School The cells were incubated with 0.4 mmol/L PA in the presence of Pharmacy, Fudan University. The voucher specimens, of different doses of extracts for 48 h. Then, Hoechst 33342 numbers RG012 and RG013, were deposited at the Herbarium staining was performed as previously described[16]. The apop- of the Department of TCM Chemistry, School of Pharmacy totic ratio in each group was calculated as the apoptotic cell of Shanghai University of Traditional Chinese Medicine number divided by the total cell number. (Shanghai, China). The extraction methods for the two cinna- For ROS determination, the cells were treated with different mon samples, C cassia and C tamala, and the analysis of these doses of CC-E, CT-E or procyanidins for 4 h. Then, the ROS two cinnamon extracts and six compounds isolated from the level was measured as previously described[16]. The fluores- extracts were described in our previous study[15, 17]. The bioac- cence of the intracellular ROS was observed using fluorescence tivity of CC-E (bark extract from C cassia), CT-E (bark extract microscopy (DP70, Olympus, Tokyo, Japan), and the fluores- from C tamala) and six compounds was investigated in this cence absorption of ROS was also measured using a micro- study. The high performance liquid chromatography (HPLC) plate reader (FlexStation III, Molecular device, Sunnyvale, CA, chromatographic profiles of CC-E and CT-E are provided in USA). the supplementary data, which was described in our previous study[15, 17]. The HPLC fingerprints and structures of the com- Glucose stimulated insulin secretion (GSIS) pounds, procyanidin B2 (cpd1), (–)-epicatechin (cpd2), cin- The cells/islets were seeded into 24-well plates (100 000 cells namtannin B1 (cpd3), procyanidin C1 (cpd4), parameritannin or 20 islets per well). Then, the cells/islets were incubated A1 (cpd5) and cinnamtannin D1 (cpd6), are shown in Figure with 0.4 mmol/L PA in the presence/absence of different 1. These compounds were analyzed by HPLC using a solvent doses of CC-E or CT-E for 48 h. The GSIS assay was per- system with 0.1% acetic acid and acetonitrile, and the purity of formed as previously described[16]. the compounds was greater than 95% (Supplementary Table 3 and 4).