Proanthocyanidins are the major anti-diabetic components of cinnamon water extract

Food and Chemical Toxicology 56 (2013) 398–405

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Proanthocyanidins are the major anti-diabetic components of cinnamon water extract Lihua Jiao a, Xin Zhang a, Lianqi Huang a, Hao Gong a, Biao Cheng a, Yue Sun b, Yixuan Li a, Qi Liu a, Ling Zheng b,⇑, Kun Huang a,c,⇑ a b c

Tongji School of Pharmacy, Huazhong University of Science and Technology, Wuhan 430030, PR China College of Life Sciences, Wuhan University, Wuhan 430072, PR China Centre for Biomedicine Research, Wuhan Institute of Biotechnology, Wuhan 430070, PR China

a r t i c l e

i n f o

Article history: Received 3 January 2013 Accepted 26 February 2013 Available online 7 March 2013 Keywords: Cinnamon water extract Proanthocyanidins Islet amyloid polypeptide Type 2 diabetes

a b s t r a c t Cinnamon consumption has been found to associate with the attenuation of diabetes mellitus. The misfolding of human islet amyloid polypeptide (hIAPP) is regarded as a causative factor of type 2 diabetes mellitus (T2DM). Here, we investigated whether cinnamon has any beneficial effect on the toxic aggregation of hIAPP. We found that cinnamon water extract (CWE) inhibited the amyloid formation of hIAPP in a dose-dependent manner, and identified proanthocyanidins as the major anti-amyloidogenic compounds of CWE. Proanthocyanidins affected the secondary structures of hIAPP and delayed the structural transition from unstructured coils to b-sheet-rich structures. Further studies showed that proanthocyanidins not only inhibited the formation of hIAPP oligomers, but also significantly attenuated the membrane damaging and cytotoxic effects caused by the hIAPP aggregation. Together, these results suggest a possible way by which cinnamon shows beneficial effects on T2DM, and indicate a potential pharmacological usage of proanthocyanidins as an anti-diabetic drug candidate. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Cinnamon has been used in several cultures for centuries as a spice and as a traditional herbal medicine (Gruenwald et al., 2010). A clinic trial in multi-ethnic type 2 diabetes patients in the United Kingdom has shown that a daily intake of 2 g cinnamon for 12 weeks can reduce the levels of HbA1c, systolic blood pressure and diastolic blood pressure (Akilen et al., 2010). Cinnamon consumption also reduces postprandial intestinal glucose absorption, inhibits gluconeogenesis and stimulates glucose metabolism, glycogen synthesis and insulin release in vitro; in diabetic animal models, cinnamon shows multiple beneficial effects including

Abbreviations: CD, circular dichroism; CWE, cinnamon water extract; EGCG, ()epigallocatechin 3-gallate; HFIP, hexafluoroisopropanol; hIAPP, human islet amyloid polypeptide; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PA, proanthocyanidins; PBS, phosphate buffered saline; PICUP, photo-induced crosslinking of unmodified proteins; POPG, 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphorac-(1-glycerol) sodium salt; RP-HPLC, reverse phase high performance liquid chromatography; Ru(bpy), Tris(2,20 -bipyridyl)dichlororuthenium(II); TEM, transmission electron microscopy; ThT, thioflavin-T; T2DM, type 2 diabetes mellitus. ⇑ Corresponding authors. Address: Tongji School of Pharmacy, Huazhong University of Science and Technology, Wuhan 430030, PR China (K. Huang). E-mail addresses: [email protected] (L. Zheng), kunhuang2008@hotmail. com (K. Huang). 0278-6915/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fct.2013.02.049

attenuation of diabetes-associated weight loss, reduction of fasting blood glucose and HbA1c, and upregulation of circulating insulin levels (Ranasinghe et al., 2012). Diabetes is a common metabolic disease and the number of adult patients is expected to reach 439 million by 2030 (Shaw et al., 2010). Type 2 diabetes mellitus (T2DM) accounts for over 90% of the diagnosed diabetics (Clark et al., 1987) and is characterized by insulin resistance, progressive loss of pancreatic b-cell function, decrease in b-cell mass and accumulation of human islet amyloid peptide (hIAPP) deposits. hIAPP is a 37 residue peptide hormone (Fig. 1A) co-secreted with insulin by pancreatic b-cells (Scherbaum, 1998) and plays a role in regulating glucose metabolism (Westermark et al., 2011). In the disease state, hIAPP aggregates and causes dysfunction of pancreatic b-cells (Hebda and Miranker, 2009; Ritzel and Butler, 2003). During aggregation, hIAPP monomers first form b-sheet-rich oligomers which further assemble into fibrillar amyloid (Lopes et al., 2007). And the oligomeric species are thought to be cytotoxic in disrupting the cellular membrane permeabilization and causing cell dysfunction and death (Glabe, 2006; Haataja et al., 2008). Previous structural studies on the nontoxic rat IAPP and hIAPP in membrane-mimicking detergent micelles have shown that the N-terminus that deeply buried within the micelles causes most of the membrane damage and the conformal changes in the C-terminus, and may thus be the modulator of the amyloid formation (Nanga et al., 2011,

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Fig. 1. Primary sequence of hIAPP and chemical structures of the major compounds from CWE. (A) hIAPP has an amidated C terminus and a disulfide bridge between Cys-2 and Cys-7; (B) chemical structures of cinnamaldehyde, cinnamic acid and coumarin; (C) chemical structures of the flavan-3-ol units in PA; (D) major constituents of cinnamon PA.

2009, 2008). Therefore, inhibiting the formation of toxic hIAPP amyloid provides a plausible therapeutic approach for the prevention and treatment of T2DM (Scrocchi et al., 2002). Although the beneficial effects of cinnamon consumption on diabetes have been known for a long time (Khan et al., 1990), the effect of cinnamon on the toxic aggregation of hIAPP has not been studied. We hypothesize that cinnamon may exert its beneficial function on diabetes through affecting the amyloidogenecity of hIAPP. To test this idea, a series of assays were applied to measure the effect of cinnamon water extract (CWE) on the amyloidogenecity of hIAPP and to identify the active anti-amyloid components of CWE.

2.2. Preparation of the cinnamon water extract (CWE) Cinnamon powder (5 g) was soaked in 50 mL water for 5 h at 40 °C. The extract was centrifuged at 7000g for 10 min, and the supernatant (cinnamon water extract (CWE)) was lyophilized and stored at 20 °C until use. 2.3. RP-HPLC analysis RP-HPLC was used to identify the components of CWE. Proanthocyanidins, cinnamic acid, cinnamaldehyde and coumarin were used as standards. RP-HPLC was performed on a Hitachi L-2000 HPLC system (Hitachi, Tokyo, Japan) with an Apollo C18 column (Grace, USA) thermostatted at 40 °C and detected with a UV detector set at 280 nm. The mobile phase consists of 60% acetonitrile, and the flow rate was 1 mL/min. 2.4. Amyloid formation and Thioflavin-T (ThT) fluorescence assay

2. Materials and methods 2.1. Materials Synthetic hIAPP (Fig. 1A) was obtained from Genscript (Piscataway, NJ, USA). Proanthocyanidins (PA), cinnamic acid, cinnamaldehyde and coumarin were purchased from Aladdin-reagent INC. (Shanghai, China). Hexafluoroisopropanol (HFIP), thioflavin-T (ThT), carboxyfluorescein, Tris(2,20 -bipyridyl)dichlororuthenium(II) (Ru(bpy)) and 2-oleoyl-1-palmitoyl-sn-glycerol-3-phospho-rac (1-glycerol) sodium salt (POPG) were obtained from Sigma–Aldrich (St. Louis, USA). Fresh blood was drawn from healthy volunteers using heparin as anticoagulant. INS-1 cells were obtained from the China Center for Type Culture Collection (CCTCC). All other chemicals were of the highest grade available.

For amyloid formation, hIAPP (13 lM) was incubated at 25 °C in 25 mM phosphate buffered saline (PBS; pH 7.4, 50 mM NaCl) containing 1% (v/v) HFIP in the presence or in the absence of CWE or CWE-derived compounds. hIAPP was first dissolved in HFIP and sonicated for 2 min to homogenize. Concentrated hIAPP in HFIP was diluted to the solution containing different concentrations of CWE or compounds to start the aggregation. 10 lL of the reaction mixture was removed at designated time for thioflavin-T (ThT) fluorescence assay on a Hitachi FL-2700 fluorometer (Hitachi, Tokyo, Japan). The assay solution contains 25 mM PBS (pH 7.4, 50 mM NaCl) and 26 lM ThT. The excitation and emission wavelengths were set at 450 nm and 482 nm, respectively. All experiments were repeated at least three times. The kinetic curves were calculated as we previously described (Zhang et al., 2011).

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2.5. Transmission electron microscopy (TEM)

2.11. Statistical analysis

Samples (5 lL) from ThT fluorescence assay (incubated for 12 h) were collected for TEM as previously described (Zhang et al., 2011). Briefly, samples were applied onto a 300-mesh Formvar-carbon-coated copper grid (Shanghai, China), stained with 1% freshly prepared uranyl formate, air-dried and observed under a transmission microscope (Hitachi, Tokyo, Japan) operating at an accelerating voltage of 150 kV.

All data were expressed as mean ± SD. Each treatment was repeated at least three times. Data were analyzed by the nonparametric Kruskal–Wallis test followed by the Mann–Whitney test. P < 0.05 was considered significant.

2.6. Far-UV circular dichroism (CD) and data analysis A JASCO-810 spectropolarimeter (JASCO, Tokyo, Japan) was used to detect the CD spectra of 15 lM hIAPP in the presence or in the absence of CWE (30 lg/mL) or PA (30 lM) under a constant flow of N2 at 25 °C as we previously described (Gong et al., 2013). hIAPP was first dissolved in HFIP and then diluted to a final concentration of 15 lM in 25 mM PBS (pH 7.4, 50 mM NaCl) containing 1% (v/v) HFIP. All spectra were recorded from 260 nm to 190 nm with a 2 nm bandwidth, 1 s response time, 25 nm/min scanning speed and a 1 mm pathlength. All samples were measured at least three times and the spectra were averaged to give the final result. The data were converted to mean residue ellipticity [h] and further analyzed by the software package CDPro using the CONTINLL algorithm as previously described (Cheng et al., 2011).

3. Results 3.1. Identification of the components in CWE Previous studies have revealed proanthocyanidins, cinnamic acid, cinnamaldehyde and coumarin as the major constituents in cinnamon water extract (Khuwijitjaru et al., 2012; Mateos-Martin et al., 2012; Shan et al., 2005). By comparing the chromatographic profiles of CWE with those of the four authentic standards (Fig. S1, Supplementary information), we confirmed the CWE peaks eluting at 2.09, 3.83, 4.57 and 5.77 min as proanthocyanidins, cinnamic acid, coumarin and cinnamaldehyde, respectively (Fig. 2). 3.2. Anti-amyloidogenic effects of CWE and its components

2.7. Photo-induced cross-linking of unmodified proteins (PICUP) assay PICUP assay was performed as we previously described (Cheng et al., 2012b). Briefly, hIAPP was first dissolved in HFIP and then sonicated for 2 min to homogenize. The final reaction solution contained 41 lM hIAPP, 125 lM Ru(bpy), 2.5 mM ammonium persulfate, 1% (v/v) HFIP, and various concentrations of CWE or PA. The mixture was irradiated for 5 s with a 150 W incandescent lamp installed in a house-made darkbox. After irradiation, loading buffer was immediately added into the solution, followed by denaturation at 97 °C for 10 min. The denatured samples were separated on a 20% tricine-urea gel and visualized by a fast silver staining kit (Beyotime, Jiangsu, China).

2.8. Dye leakage assay POPG vesicles were prepared for the dye leakage assay as described (Brender et al., 2008). Briefly, POPG was dissolved in chloroform at a concentration of 10 mg/mL. The solvent was removed under a stream of nitrogen to form a thin lipid film on the wall of the glass tube and then lyophilized overnight to remove the residual chloroform. The carboxyfluorescein containing POPG vesicles were made by mixing dry lipid films with 25 mM PBS (pH 7.4, 50 mM NaCl) containing 40 mM carboxyfluorescein. Nonencapsulated carboxyfluorescein was removed by a PD-10 column (Sangon Biotech., Shanghai, China). For flourescence measurements, the prepared POPG vesicles were diluted in 25 mM PBS (pH 7.4, 50 mM NaCl). The excitation and emission wavelengths were set at 493 nm and 518 nm, respectively. Each sample was measured for 150 s after the addition of hIAPP (0.5 lM) in the presence or in the absence of different amounts of CWE or PA and the fluorescence intensity at 150 s was taken as F150 s in the equation. The fluorescence intensity of POPG vesicles alone was used as the Fbaseline, and that of the POPG vesicles with the addition of 0.2% of Triton X-100 was taken as the Fdetergent. The following equation was used to calculate the percentage of dye leakage:

Percentage of dye leakage ¼ ðF 150 s  F baseline Þ=ðF detergent  F baseline Þ

2.9. Hemolytic assay Hemolytic assay was conducted as we previously reported (Li et al., 2012). Briefly, washed erythrocytes were resuspended in 200 ll isotonic PBS (pH 7.4, 1% hematocrit). 15 lM hIAPP together with CWE or PA of various concentrations was then added. The cell suspensions were incubated at 37 °C for 5 h followed by centrifugation at 1000g for 10 min and the absorbance of the supernatant was determined at 540 nm. The hemolytic rate was calculated in relation to the hemolysis of erythrocytes in 10 mM phosphate buffer, which was taken as 100%.

2.10. MTT cell toxicity assay For MTT-based cell toxicity assays, INS-1 cells were seeded into a 96-well plate at a density of 5  105 cells/mL as described (Cheng et al., 2012a). After culturing for 24 h, the medium was replaced by fresh medium containing 15 lM hIAPP and various amounts of CWE or PA. The cells were cultured for another 24 h, and then 20 lL MTT (5 mg/mL) was added to each well. The cells were further incubated for 4 h. The cell viability was measured at 570 nm on a Multiskan MK3-microplate photometer (Thermo Scientific, USA).

By RP-HPLC, we found that the major components of CWE include cinnamic acid, coumarin, cinnamaldehyde and proanthocyanidins (Fig. 1B–D). The inhibitive effects of CWE and its components on amyloid formation of hIAPP were tested by ThT fluorescence assay. The fluorescence intensity of 13 lM hIAPP reached maximum at 10 h with a short lag time of 4.1 ± 1.1 h (Fig. 3A and Table 1). The kinetic profile of hIAPP amyloid formation was dose-dependently changed by the addition of CWE or PA. No significant changes on the maximum fluorescence intensity and lag time were observed in the presence of 0.26 lg/mL CWE or 1.3 lM PA (Fig. 3A and Table 1). When CWE was added at final concentrations of 2.6 lg/mL and 26 lg/mL, the fluorescence intensities decreased significantly (P < 0.01, Table 1) and the lag times were prolonged to 5.2 ± 0.6 h (P = 0.180) and 6.9 ± 1.4 h (P < 0.05), respectively (Fig. 3A and Table 1). PA at 13 lM also reduced the maximum fluorescence intensity significantly (P < 0.01, Table 1), although the lag time was slightly prolonged. When the PA concentration was further increased to 26 lM, the ThT emission was strongly inhibited (Fig. 3A). In contrast, cinnamic acid, cinnamaldehyde and coumarin at near saturated concentrations showed little effect on the maximum fluorescence intensity and the lag time of hIAPP (Fig. 3B and Table 1). TEM was used to study the morphologies of the hIAPP aggregations in the absence or in the presence of CWE and the four identified compounds. Consistent with previous report (Meng et al., 2010), hIAPP formed long linear fibrils after 12 h of incubation at 25 °C (Fig. 3C). However, when hIAPP was co-incubated with 0.26 lg/mL CWE, short hIAPP fibrils were observed (Fig. 3D). When the concentration of CWE was increased to 26 lg/mL, no aggregates were observed (Fig. 3E). Similar results were obtained in the presence of PA (Fig. 3F and G). In contrast, for hIAPP samples co-incubated with near-saturated concentrations of cinnamic acid, cinnamaldehyde or coumarin, typical fibrils were still observed (Fig. 3H–J), which agreed with the results of ThT fluorescence assay. 3.3. CWE and PA delayed the secondary structural change of hIAPP Previous studies have shown that hIAPP undergoes a conformational transition into b-sheet-rich structure during the formation of amyloid (Higham et al., 2000; Kayed et al., 1999). The effects of CWE and PA on the secondary structure transition of hIAPP were tested by far-UV circular dichroism (CD). At the beginning of the incubation, the secondary structures of hIAPP were predominantly unstructured coils, which is consistent with previous report (Higham et al., 2000; Kayed et al., 1999). After 4 h incubation

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L. Jiao et al. / Food and Chemical Toxicology 56 (2013) 398–405 Table 1 Effects of CWE and its components on the amyloidogenic property of hIAPP. Fibril formationa

hIAPP 13 lM hIAPP + CWE (0.26 lg/mL)b hIAPP + CWE (2.6 lg/mL) hIAPP + CWE (26 lg/mL) hIAPP + PA (1:0.1)c hIAPP + PA (1:1) hIAPP + PA (1:2) hIAPP + cinnamic acid (1:90) hIAPP + cinnamaldehyde (1:50) hIAPP + coumarin (1:30)

Fig. 2. RP-HPLC identification of major components of cinnamon water extract.

T50 (h)

Maximum intensityd

Lag time (h)

6.1 ± 0.2 6.4 ± 0.6 6.8 ± 0.3 9.0 ± 2.0 6.5 ± 0.5 8.7 ± 0.6 N 7.5 ± 1.3 6.7 ± 0.6

1.7 ± 0.1 1.4 ± 0.2 1.2 ± 0.2⁄⁄ 0.2 ± 0.1⁄⁄ 1.0 ± 0.5 0.6 ± 0.2⁄⁄ <0.1⁄⁄ 1.5 ± 0.4 1.6 ± 0.1

4.1 ± 1.1 3.2 ± 1.3 5.2 ± 0.6 6.9 ± 1.4⁄ 4.3 ± 1.2 6.8 ± 1.4 N 4.1 ± 2.0 4.7 ± 0.8

6.5 ± 0.9

1.7 ± 0.2

5.3 ± 1.5

⁄

(Fig. 4A), the spectrum of hIAPP alone indicated a conversion to bsheet-rich structure (as revealed by the negative peak around 220 nm). In the presence of 30 lg/mL CWE, the conversion to bsheet-rich structure occurred after incubation for 6 h (Fig. 4B). In the presence of 30 lM PA, the structural transition was further delayed to 8 h (Fig. 4C). Further CDPro deconvolution analysis suggested that at 0 h, the b-sheet-rich structure of hIAPP alone was 39.5%, while in the presence of 30 lg/mL CWE or 30 lM PA, the percentages of bsheet-rich structure of hIAPP were decreased to 31.5% and 13.6%, respectively (Table 2). After 24 h incubation, for hIAPP incubated with 30 lg/mL CWE or 30 lM PA, the percentages of b-sheet-rich structures were 44.0% and 38.4%, respectively, while the number for hIAPP alone was 53.6% (Table 2).

Asterisk indicates a significant difference between hIAPP alone and hIAPP co-incubated with CWE or its components. P < 0.05, P < 0.01. N, The lag time and T50 were too long to be accurately calculated during the test. a All assays were repeated at least three times. b The amount of CWE was expressed as lyophilized CWE weight/volume. c The amounts of CWE-derived compounds were expressed as molar ratio of hIAPP to the compound, the concentration of hIAPP was 13 lM. d The maximum intensity was expressed as arbitrary unit.

3.4. Inhibitory effects of CWE and PA on the oligomerization of hIAPP The oligomers of hIAPP have been shown to be highly cytotoxic in disrupting the cellular membrane of pancreatic b-cells (Haataja et al., 2008). The effects of CWE and PA on the oligomerization of hIAPP were tested by photo-induced cross-linking of unmodified proteins (PICUP) assay. Without irradiation, hIAPP (41 lM) mi-

Fig. 3. Effects of CWE and its components on the amyloid formation of hIAPP. (A) Relative ThT fluorescence intensities of hIAPP alone and hIAPP co-incubated with CWE or PA; (B) relative ThT fluorescence intensities of hIAPP alone and hIAPP co-incubated with CWE-derived compounds; (C) TEM image of hIAPP alone (13 lM); (D) TEM image of hIAPP co-incubated with 0.26 lg/mL CWE; (E) TEM image of hIAPP co-incubated with 26 lg/mL CWE; (F) TEM image of hIAPP co-incubated with 1.3 lM PA; (G) TEM image of hIAPP co-incubated with 26 lM PA; (H) TEM image of hIAPP co-incubated with cinnamic acid; (I) TEM image of hIAPP co-incubated with cinnamaldehyde; (J) TEM image of hIAPP co-incubated with coumarin. The scale bars represent 500 nm.

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Fig. 5. Oligomerization status of hIAPP studied by the photo-induced cross-linking of unmodified proteins (PICUP) assay. The concentration of hIAPP in this study was 41 lM. Lane 1, hIAPP without irradiation; lane 2, hIAPP irradiated for 5 s; lane 3–5, hIAPP mixed with 0.82 lg/mL, 8.2 lg/mL and 82 lg/mL CWE and irradiated for 5 s; lane 6–8, hIAPP mixed with 4.1 lM, 41 lM and 82 lM PA and irradiated for 5 s.

of 10 ng/mL CWE (Fig. 6A). When the amount of CWE was further increased to 0.1 lg/mL and 1 lg/mL, the percentages of dye leakage were further decreased to 34.7 ± 2.9% (P < 0.01) and 31.3 ± 2.5% (P < 0.01, Fig. 6A), respectively. The addition of 50 nM PA decreased the dye leakage level to 50.1 ± 4.4% (P < 0.01, Fig. 6A). And when the amount of PA was further increased to 0.5 lM and 1 lM, the levels of dye leakage were decreased to 43.1 ± 3.9% (P < 0.01) and 42.7 ± 4.6% (P < 0.01, Fig. 6A), respectively. We have previously shown that amyloid fibrils can damage erythrocytes and cause hemolysis (Wang et al., 2011). We found that the fibrillation of hIAPP also caused erythrocytes hemolysis. The hemolytic rate was 94.3 ± 1.9% in the presence of 15 lM hIAPP incubated at 37 °C for 5 h (Fig. 6B). When 0.3 lg/mL CWE or 1.5 lM PA was added, the hIAPP-induced hemolysis rates were not changed significantly (Fig. 6B). When the amount of CWE was increased to 3 lg/mL and 30 lg/mL, the hemolytic rates were decreased to 65.6 ± 4.6% (P < 0.01) and 38.5 ± 3.4% (P < 0.01, Fig. 6B), respectively. And the presence of PA at 15 lM and 30 lM decreased the hemolytic rates to 66.2 ± 12.3% (P < 0.05) and 34.5 ± 5.5% (P < 0.01, Fig. 6B), respectively. Fig. 4. Far-UV CD spectra of hIAPP alone and hIAPP co-incubated with CWE or PA. (A) hIAPP (15 lM) alone; (B) hIAPP (15 lM) co-incubated with 30 lg/mL CWE; (C) hIAPP (15 lM) co-incubated with 30 lM PA.

grated mostly as monomers (lane 1, Fig. 5). After 5 s of irradiation, hIAPP monomers assembled into dimer, trimer, tetramer, pentamer and higher oligomers (lane 2, Fig. 5). Low concentrations of CWE (0.82 lg/mL and 8.2 lg/mL) and PA (4.1 lM) showed only mild effect on the oligomerization of hIAPP (lanes 3, 4 and 6, Fig. 5), whereas the oligomerization of hIAPP was almost completely inhibited with no signs of dimer or higher oligomers being observed in the presence of 82 lg/mL CWE (lane 5, Fig. 5). In contrast, PA effectively inhibited the oligomerization of hIAPP both at 41 lM and 82 lM (lanes 7 and 8, Fig. 5).

3.6. Protection effects of CWE and PA on hIAPP-induced cell toxicity The effects of CWE and PA on the cytotoxicity of hIAPP were evaluated on pancreatic INS-1 cells by MTT assay. INS-1 cells cultured with 15 lM hIAPP showed a cell viability of 40.8 ± 2.0% (Fig. 6C). The presence of low concentrations of CWE (0.3 lg/mL and 3 lg/mL) and PA (1.5 lM) showed little effect on the cell viability (Fig. 6C). When the concentration of CWE was increased to 30 lg/mL, the cell viability increased to 53.0 ± 4.7% (P < 0.05, Fig. 6C). And the presence of 15 lM PA and 30 lM PA increased the cell viabilities to 48.5 ± 4.3% (P < 0.05) and 62.6 ± 3.0% (P < 0.05, Fig. 6C), respectively. In control studies, CWE or PA alone did not change the cell viability significantly (Fig. 6D). 4. Discussion

3.5. Protection effects of CWE and PA on hIAPP-induced membrane damage It has been shown that hIAPP can cause dye leakage from vesicles prepared with POPG (Brender et al., 2008). In our study, we found approximately 95% of the POPG vesicles were damaged by 0.5 lM hIAPP (Fig. 6A). In contrast, the percentage of dye leakage was significantly reduced to 44.2 ± 5.4% (P < 0.01) in the presence

The aggregation of hIAPP into amyloid deposits has been regarded to be a causative factor of b-cell dysfunction (Hebda and Miranker, 2009; Ritzel and Butler, 2003). One of the proposed mechanisms is hIAPP-induced membrane disruption and fragmentation, and recent NMR studies have revealed that the interaction between hIAPP and cellular membrane involves multiple factors, such as calcium and phosphatidylethanolamine (Brender et al.,

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L. Jiao et al. / Food and Chemical Toxicology 56 (2013) 398–405 Table 2 Deconvolution results of CD spectra.a

a-Helix

Time (h) IAPP 0 2 4 6 8 24 a b c d

The The The The

5.5 7.2 6.3 5.5 5.0 3.6

b

IAPP + CWE 6.9 3.5 4.8 3.7 4.5 4.6

b-Sheet-rich structures c

IAPP + PA 6.9 10.2 4.6 5.2 4.3 5.2

d

Unstructured coils

IAPP

IAPP + CWE

IAPP + PA

IAPP

IAPP + CWE

IAPP + PA

39.5 44.7 51.7 55.4 57.4 53.6

31.5 29.0 35.4 43.9 43.0 44.0

13.6 24.4 16.3 37.8 36.5 38.4

54.9 48.1 42.1 39.1 37.7 42.7

61.7 67.5 59.8 52.4 52.6 51.5

79.4 65.4 79.1 57.0 59.1 56.4

data were analyzed using the software package CDPro. concentration of hIAPP was 15 lM. concentration of CWE was 30 lg/mL. concentration of PA was 30 lM.

Fig. 6. Protection effects of CWE and PA on hIAPP amyloid related membrane damage, cell toxicity and oligomerization. (A) Levels of dye leakage caused by hIAPP alone and hIAPP co-incubated with CWE or PA. (B) Levels of hemolysis caused by hIAPP alone and hIAPP co-incubated with CWE or PA. (C) INS-1 cell viabilities after treatments of hIAPP alone or hIAPP co-incubated with CWE or PA. (D) Control study of the effects of CWE and PA on the growth of INS-1 cells. P < 0.05, P < 0.01 versus the groups treated with hIAPP alone.

2007, 2011; Sciacca et al., 2012, 2013). Therefore, inhibition of amyloid formation by hIAPP may provide a novel way to prevent this disease. A number of chemicals have been found to be able to inhibit the hIAPP amyloid formation, such as polyphenols and zinc ions (Brender et al., 2010; Salamekh et al., 2011; Suzuki et al., 2012). Here, we investigated the anti-diabetes effect of cinnamon by studying its inhibitory activity on hIAPP amyloid formation and further identified its anti-amyloidogenic components. We first observed that CWE significantly reduced the amount of amyloid formed by hIAPP (Fig. 3A). Among the four major CWE components identified by RP-HPLC (Fig. 2), PA effectively inhibited the hIAPP amyloid formation (Fig. 3A), while cinnamic acid, coumarin and cinnamaldehyde showed little or none inhibitory effect on hIAPP amyloid formation (Fig. 3B). The predominant constituents of cinnamon PA are procyanidins which are characterized by A-type linkage of (epi)catechins (Fig. 1C and D) (Gu et al., 2003). Previous study has shown that (–)-epigallocatechin 3-gallate (EGCG), which has a similar polyphenolic structure to PA, is an effective inhibitor of hIAPP amyloid formation (Meng et al., 2010). We therefore speculate that PA may exert amyloid inhibitory effect partially through the various flavan-3-ol com-

ponents with trihydroxyphenyl rings or gallate esters that similar to EGCG. On the other hand, the polyphenolic and aromatic ring rich structure of PA may also play an important role in affecting the secondary structure transition of hIAPP, since many polyphenol inhibitors share similar dicyclic or tricyclic structures (Porat et al., 2006). The far-UV CD results indicated that PA immediately changed the secondary structure of hIAPP and delayed the transition from unstructured coils to b-sheet-rich structures (Fig. 4), which suggested that PA may directly bind to the hIAPP monomers and inhibit the formation of toxic oligomers. And the results of PICUP assay further confirmed PA as an efficient inhibitor of hIAPP oligomerization (Fig. 5). Consistent with the generally accepted theory that membrane damage is mostly caused by the toxic hIAPP oligomers (Glabe, 2006; Haataja et al., 2008), the data from dye leakage and hemolytic assays and cytotoxicity analysis showed that PA can effectively reduce the hIAPP-induced membrane damage, and protect INS-1 cells against hIAPP-induced toxicity (Fig. 6). Since all studies herein have been carried out in vitro, the bioavailability of cinnamon PA becomes an important issue. It has been shown that the physiological circulating concentration of hIAPP ranges from 1.6 to 20 pM in nondiabetic people (Cheng

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et al., 2011; Young, 2005). The concentration of cocoa procyanidin dimer in human plasma can reach 16 ± 5 nM at 30 min after cocoa consumption (0.375 g/kg body weight) (Holt et al., 2002). And the concentration of cranberry procyanidin dimer in rats plasma can even reach 0.9 lM at 1 h after cranberry consumption (1 g/kg body weight) (Rajbhandari et al., 2011). So under physiological conditions, the stoichiometry of procyanidin to hIAPP may actually be much higher than those used in the present study. Therefore, more significant anti-amyloidogenic effects are expected for people who consume cinnamon regularly. In summary, CWE shows strong anti-amyloidogenic effects with PA as the major active components. PA can effectively inhibit the formation of toxic hIAPP oligomers and significantly reduce cytotoxicity through alleviating hIAPP-induced membrane damage. Therefore, PA may serve as a potential anti-diabetic drug candidate. Conflict of Interest None. Acknowledgments This work was supported by the National Basic Research Program of China (2009BC918304 and 2012CB524901), the Natural Science Foundation of China (Nos. 81222043, 30970607, 81172971, 81100687 and 31271370), the Program for New Century Excellent Talents in University (NECT10-0623 and NECT110170). Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.fct.2013.02.049. References Akilen, R., Tsiami, A., Devendra, D., Robinson, N., 2010. Glycated haemoglobin and blood pressure-lowering effect of cinnamon in multi-ethnic type 2 diabetic patients in the UK: a randomized, placebo-controlled, double-blind clinical trial. Diabetic Medicine 27, 1159–1167. Brender, J.R., Durr, U.H., Heyl, D., Budarapu, M.B., Ramamoorthy, A., 2007. Membrane fragmentation by an amyloidogenic fragment of human islet amyloid polypeptide detected by solid-state NMR spectroscopy of membrane nanotubes. Biochimica et Biophysica Acta 1768, 2026–2029. Brender, J.R., Hartman, K., Nanga, R.P., Popovych, N., de la Salud Bea, R., Vivekanandan, S., Marsh, E.N., Ramamoorthy, A., 2010. Role of zinc in human islet amyloid polypeptide aggregation. Journal of the American Chemical Society 132, 8973–8983. Brender, J.R., Lee, E.L., Cavitt, M.A., Gafni, A., Steel, D.G., Ramamoorthy, A., 2008. Amyloid fiber formation and membrane disruption are separate processes localized in two distinct regions of IAPP, the type-2-diabetes-related peptide. Journal of the American Chemical Society 130, 6424–6429. Brender, J.R., Salamekh, S., Ramamoorthy, A., 2011. Membrane disruption and early events in the aggregation of the diabetes related peptide IAPP from a molecular perspective. Accounts of Chemical Research 45, 454–462. Cheng, B., Gong, H., Li, X., Sun, Y., Chen, H., Zhang, X., Wu, Q., Zheng, L., Huang, K., 2012a. Salvianolic acid B inhibits the amyloid formation of human islet amyloid polypeptide and protects pancreatic beta-cells against cytotoxicity. Proteins. Cheng, B., Gong, H., Li, X., Sun, Y., Zhang, X., Chen, H., Liu, X., Zheng, L., Huang, K., 2012b. Silibinin inhibits the toxic aggregation of human islet amyloid polypeptide. Biochemical and Biophysical Research Communications 419, 495–499. Cheng, B., Liu, X., Gong, H., Huang, L., Chen, H., Zhang, X., Li, C., Yang, M., Ma, B., Jiao, L., Zheng, L., Huang, K., 2011. Coffee components inhibit amyloid formation of human islet amyloid polypeptide in vitro: possible link between coffee consumption and diabetes mellitus. Journal of Agricultural and Food Chemistry 59, 13147–13155. Clark, A., Cooper, G.J., Lewis, C.E., Morris, J.F., Willis, A.C., Reid, K.B., Turner, R.C., 1987. Islet amyloid formed from diabetes-associated peptide may be pathogenic in type-2 diabetes. Lancet 2, 231–234. Glabe, C.G., 2006. Common mechanisms of amyloid oligomer pathogenesis in degenerative disease. Neurobiology of Aging 27, 570–575.

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