Article pubs.acs.org/JAFC
Dicaffeoylquinic Acid-Enriched Fraction of Cichorium glandulosum Seeds Attenuates Experimental Type 1 Diabetes via Multipathway Protection Jing Tong,† Bingxin Ma,† Lanlan Ge,† Qigui Mo,† Gao Zhou,† Jingsheng He,† and Youwei Wang*,†,‡ †
Institute of TCM and Natural Products, School of Pharmaceutical Sciences, Wuhan University, Wuhan 430071, P. R. China Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education, Wuhan University, Wuhan 430072, P.R. China
‡
ABSTRACT: Chicory has a major geographical presence in Europe and Asia. Cichorium glandulosum Boiss. et Huet, a genus Cichorium, is used for medicinal and food purposes in Asia. In this study, a dicaffeoylquinic acid-enriched fraction of C. glandulosum seeds n-BuOH fraction (CGSB) could ameliorate type 1 diabetes mellitus (T1DM) in streptozotocin (STZ)induced diabetic mice with continuous administration for 2 weeks. CGSB treatment showed significantly higher plasma insulin levels but lower free fatty acids in adipose tissue and liver. Moreover, CGSB improved pancreatic islet mass. In vitro, different fractions of C. glandulosum seed (CGS) induced the differentiation of 3T3-L1 preadipocytes. The mRNA level for peroxisome proliferator-activated receptor alpha increased in high glucose treatment group in HepG2 cells, while CGSB significantly downregulated the mRNA expression. The main compound of CGSB, 3,5-dicaffeoylquinic acid, was isolated and identified, which exhibited α-glucosidase inhibitory activity. These findings demonstrated that CGSB attenuated experimental T1DM via multipathway protection. KEYWORDS: Cichorium glandulosum seed, 3,5-dicaffeoylquinic acid, diabetes, multipathway protection
■
with major distribution areas in Europe and Asia.8 Chicory (C. intybus L.) was grown by the ancient Egyptians as a medical plant, coffee substitute, vegetable crop, and animal forage. Chicory is cultivated for numerous applications and can be divided into four main varieties or cultigroups: “root” chicory as coffee substitute or for inulin extraction, “witloof” chicory as etiolated buds, “leaf” chicory as fresh or cooked vegetables, and “forage” chicory.9 Aerial parts of chicory are often consumed as salads or to prepare meals, whereas the root is used as coffee substitutes or food ingredients such as chewing gum.10 The extracts of chicory are regarded as safe for both food and nonfood utilization by the Food and Drug Administration (FDA).11 In recent years, numerous medical functions of chicory have been researched. The traditional use and pharmacological activities were reviewed by Street et al.9 C. glandulosum is widely distributed plant in Xinjiang (Xinjiang Uyghur Autonomous Region, China). The aerial and roots of C. intybus and C. glandulosum are named “juju” or chicory in Chinese, which are all recorded in the Chinese Pharmacopoeia of the People’s Republic of China.12 The two species may share similar chemical composition and bioactivities.13 C. intybus L. is extensively used as a folk remedy for treating diabetes mellitus in Bulgaria, India, Italy, Pakistan, and Serbia (reviewed by Street),9 and the antidiabetic effects was investigated by Pushparaj et al.,14 but for the hypoglycemic activity of C. glandulosum was not reported. The aerial parts and
INTRODUCTION Type 1 diabetes mellitus (T1DM) in humans is considered to have a T-cell mediated autoimmune etiology in which the insulin-secreting pancreatic β-cells are selectively destroyed by β-cell antigens.1 Although the exact causes of T1DM development are not completely understood, it appears to result from a complex interaction between genetic and environmental factors.2 The World Health Organization estimated the global prevalence of diabetes at 381.3 million people in 2013, which rises by approximately 3−5% per year.3 T1DM accounts for 10% of all diabetes patients. Exogenous insulin injections, hormone substitution, and oral hypoglycemic agents are the main treatment options; however, these methods are not considered cures for diabetes but methods to deal with the symptoms associated with diabetes.4 T1DM treatment has certainly improved but too slowly. Human islet transplantation has been a successful form of treatment, but it is significantly limited by a lack of suitable donor material. Although new therapeutic strategies have been developed, including cell therapy5 and enteroviruses-based vaccine,6 these strategies still need more research. Therefore, better treatments or methods are necessary to remove the heavy burden on the health care in the community. Ludvigsson advised that combination therapy is useful for T1DM therapy, such as immune suppression combined with antigen treatment, β-cell protection, and antiinflammatory treatment, to stimulate β-cell regeneration.7 Cichorium glandulosum Boiss. et Huet, belonging to the Asteraceae family, is a biennial or perennial plant. The genus Cichorium consists of six species, namely, Cichorium intybus L., Cichorium endivia L., C. glandulosum Boiss, Cichorium spinosum L. Cichorium bottae Defl, and Cichorium calvum Schultz-Bip, © 2015 American Chemical Society
Received: Revised: Accepted: Published: 10791
September 17, 2015 November 19, 2015 November 20, 2015 November 20, 2015 DOI: 10.1021/acs.jafc.5b04552 J. Agric. Food Chem. 2015, 63, 10791−10802
Article
Journal of Agricultural and Food Chemistry
and partitioned with petroleum ether, chloroform, ethyl acetate, and nBuOH (four times per solution). These four fractions of CGS were prepared for use (CGSP, CGSC, CGSE, and CGSB, respectively). On the basis of the pre-experiment and bioactivity in vitro, CGSB was selected and submitted to evaluate the antidiabetes activity. The fraction showed inhibition of α-glucosidase. The pure molecule was subjected to nuclear magnetic resonance (NMR) and mass spectroscopy for structure elucidation. Experimental Animals. The study received clearance from the Institutional Animal Ethical Committee of the Committee for the Purpose of Control and Supervision of Experiments on Animals, Wuhan University, Wuhan, China. As previously described,18 fourweek-old male KM mice (n = 80) were purchased from the Laboratory Animal Center (LAC) of Wuhan University. The mice had free access to purified water and a standard rodent diet ad libitum (purchased from the LAC) housed in a room with a 12 h light/dark cycle. After 1 week of acclimatization, mice were designed to initiate the study, which lasted for 24 days. First, mice were divided randomly into two groups, namely the control group (n = 20) and multiple-low-dose of STZ-induced group (n = 60). The control group was further randomly subdivided into two groups. One was the control group (group CTRL), and one was the CGSB control group (200 mg/kg body weight, group CGSB 200). The multiple-low-dose of STZ-induced group (n = 60), which was treated with STZ at 40 mg/kg body weight intraperitoneally for five consecutive days.18 Blood glucose levels were measured on day 5 after the final dose of STZ. The blood samples were obtained from the tail vein of fasting mice, and glucose was measured by a blood glucose meter (Accu-CHEK, Roche, GER). Mice were considered diabetic when fasting blood glucose levels were >200 mg/dL. STZ-treated mice were further randomly subdivided into four groups (n = 15 per group), receiving CGSB (100 mg/kg body weight, STZ+CGSB 100 group; 200 mg/kg body weight, STZ+CGSB 200 group) or Rosi (4 mg/kg body weight, group STZ+Rosi group) or vehicle group (STZ group) by oral gavages once per day for another 2 weeks. The CTRL group was only given the vehicle. At the end of the study, animals were not fed (from 17 p.m. to 9 a.m.). On the following day, oral glucose tolerance test (OGTT) was conducted. The mice were then sacrificed immediately, and blood and tissue (livers, pancreas, and epididymal adipose tissue) were collected for further analyses. Body Weight and Body Weight Loss Analyses. Throughout the study, body weight was recorded daily. Body weight loss is defined as the difference between day 1 (the first day of treatment) and the weight before sacrifice. OGTT. The blood glucose level of mice was measured after an overnight fast (16 h, from 17 p.m. to 9 a.m.). This time was designated as Time 0. Mice were orally administered 2 g/kg of glucose solution. Blood glucose levels were assessed at 30, 60, 120, and 180 min following the intragastric administration.19 Blood Biochemical Analysis. Blood samples were collected from retro-orbital sinuses of fasting mice. Plasma TG, TC, ALT, and AST were determined using commercial assay kits (purchased from Nanjing Jiancheng Technology Co. Ltd.) (Nanjing, China). Analysis of FFA in Liver and Adipose Tissue. Liver and epididymal adipose tissue samples were collected. FFA was determined using commercial assay kits (purchased from Nanjing Jiancheng Technology Co. Ltd.) (Nanjing, China). Plasma Insulin Determination. Nonfasting blood samples were collected on day before fast in heparinized tubes. Plasma was separated and assayed for insulin concentration. The inulin level of plasma was determined using a commercial Mouse Insulin ELISA kit (purchased from Huawei Biotech Co., Ltd.) (Shanghai, China). Histopathological Examination. A portion of the collected pancreas, liver tissues, and epididymal adipose tissue were fixed with formalin (10%, v/v) solution and embedded in paraffin. Sections (6 μm) were cut and stained with hematoxylin and eosin. Identification of Phenolic Compounds in CGSB. Liquid Chromatography−Tandem Mass Spectrometry. Analytical HPLC was conducted using Shimadu LC-20A with dual wavelength detector and a 20 μL injector. The analytical column was a 5 μm × 4.6 mm ×
seeds of C. glandulosum are widely used by the Uyghur residents in Xinjiang as a functional food or a traditional treatment for chronic diseases such as jaundice, liver enlargement, and urinary tract infection.13 Seeds of C. intybus L. (Compositae) are commonly known as chicory and mixed with coffee seeds to prepare coffee powder.15 Multiple research papers have been published describing several health properties of C. glandulosum, including hepatoprotective effect, antihepatic fibrosis, lipid modulatory activity, antiultraviolat B, and diabetes (Table 1). Table 1. Activities of Cichorium glandulosum Boiss. et Huet plant part root aerial part whole plant seeds
therapeutic effect
active ingredient
refs
hepatoprotective effect hepatic fibrosis hepatoprotective effect lipid modulatory activities diabetes antiultraviolet b
phenolic acids extract of root sesquiterpene quercetin/derivatives total flavonoids butanol fraction
42 43−45 46 47 17 30
This study focused on the antidiabetic activity of C. glandulosum seed (CGS) in streptozotocin (STZ)-induced diabetic mice. STZ is currently the most frequently used drug to induce experimental type 1 diabetes in rodent.16 This study provided evidence for the first time that supplementation of C. glandulosum seeds n-BuOH fraction (CGSB) can ameliorate hyperglycemia via multi pathways.
■
MATERIALS AND METHODS
Plant Material. CGSs were purchased from the experimental base of Xinjiang Uygur Medical College, Hetian (Xinjiang Uygur Autonomous Region, China). The seeds were identified by Prof. Youwei Wang of the School of Pharmaceutical Sciences at the Wuhan University. A voucher specimen number (no. 703) was deposited in the herbarium of the Institute of Traditional Chinese Medicine and Natural Products at the School of Pharmaceutical Sciences, Wuhan University, Wuhan, China. Reagents. 3-(4,5-Dimethyl thiazol-2-yl)-5-diphenyl tetrazolium bromide (MTT), 4-nitrophenyl α-D-glucopyranoside (pNPG), 3isobutyl-1-methylxanthine (IBMX), dexamethasone, insulin, Oil Red O, and α-glucosidase were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Rosiglitazone (Rosi) was obtained from GlaxoSmithKline. Acarbose was obtained from Bayer. All other reagents were obtained from the Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Cell culture reagents were obtained from Thermo Fisher Scientific Inc. (USA). The primers used in this study were synthesized by Invitrogen Biotechnology (USA). Diagnostic kits for alanine aminotransaminase (ALT), aspartate aminotransferase (AST), triacylglycerols (TGs), total cholesterol (TC), and free fatty acids (FFAs) were purchased from Nanjing Jiancheng Technology Co. Ltd. (Nanjing, China). An ELISA kit for insulin was purchased from Cusabio Biotech Co. Ltd. (Wuhan, China). A kit for glucose consumption was purchased from Shanghai Rongsheng Biotech Co., Ltd. (Shanghai, China). High-performance liquid chromatography (HPLC)-grade methanol was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA) and filtered through a 0.45 μm membrane before use. Deionized water was obtained from a Milli-Q reagent water system (Miilpore Co., Milford, MA). Preparation of the Plant Extracts. The extracts were prepared as previously described.17 In brief, the dried seeds were crushed to a coarse powder (1.0 kg), which were exhaustively extracted with 70% ethanol at 80 °C for 2 h by reflux extraction on three occasions (solvent/seed ratio of 10:1, v/w). The crude alcoholic extracts were concentrated under reduced pressure in a rotary evaporator at 45 °C to obtain a viscous mass (145 g), which was suspended in H2O (1 L) 10792
DOI: 10.1021/acs.jafc.5b04552 J. Agric. Food Chem. 2015, 63, 10791−10802
Article
Journal of Agricultural and Food Chemistry
Figure 1. CGSB alleviates T1DM in STZ-induced diabetic mice. Weight loss (A), oral glucose tolerance test (B), liver index (C), and kidney (D) were measured. Data are expressed as mean ± SD (n = 10 in control groups, or n = 14 in STZ-induced groups). Statistically significant differences are indicated by pounds and asterisks and determined via ANOVA followed by LSD test. (###p < 0.001, #p < 0.05 compared with the control group, ***p < 0.001, **p < 0.01, *p < 0.05 compared with the model group). CGSB 200, supplemented with 200 mg/kg body weight; STZ+Rosi, STZ supplemented with 4 mg/kg body weight; STZ+CGSB 200, STZ supplemented with 200 mg/kg body weight; STZ+CGSB 100, STZ supplemented with 100 mg/kg body weight. 250 mm C18 (Agilent, USA). The HPLC mobile phase consisted of (A) methanol and (B) water containing 0.1% formic acid. A gradient HPLC method was used. This method consisted of a 10 min increase of A from 0 to 30%, a 10−20 min increase of A from 30 to 42%, 20− 30 min of sustaining A at 42%, a 30−35 min increase of A from 42 to 55%, and a 35−60 min increase of A from 55 to 100% with a flow rate of 1 mL/min and ultraviolet detection at 330 nm. Mass analyses were performed using an ESI interface in the positive ion mode. The data were set as follows. Positive mode: desolvation temperature, 250 °C; source temperature, 120 °C; capillary voltage, 1.2 kV; cone voltage, 30 V; desolvation gas (N2) flow rate, 900 L/h; cone gas (He) flow rate, 40 L/h; and scan range, m/z 100−1000 amu. The MS/MS2 spectra were acquired using a collision energy of 40 V. Isolation and Purification of Dicaffeoylquinic Acid. The n-BuOH fraction (1 g) prepared from the previously described steps was further isolated on a preparative HPLC system. Each chromatographic run was conducted at a flow rate of 12 mL/min with a binary mobile phase consisting of methanol containing 0.1% formic acid (A) and water with 0.25% formic acid (B) using a step gradient profile. The gradient started with 20% A, varied to 80% A at 60 min, and then decreased to 20% A in 0.1 min. After re-equilibration at 20% A for 12 min, the next sample was injected. The temperature of the column oven was 25 °C, and 500 μL was injected into the system every time. The peaks that adsorbed at 330 nm were recorded. Compounds 11 (82.4 mg) was obtained at a retention time of 39.5 min. NMR Measurements. 1H NMR (400 MHz) and 13C NMR (100 MHz) measurements were performed using Bruker DPX-400 spectrometer (Bruker BioSpin GmbH, Rheinstetten, Germany). Compound 11 was identified by using distortionless enhancement by polarization transfer, double quantum filtered correlated spectroscopy (DQF-COSY), heteronuclear multiple-quantum coherence (HMQC), and heteronuclear multiple-bond connectivity (HMBC).
NMR spectra were recorded from samples in CDCl3. Chemical shifts (δ) were reported in parts per million (ppm) relative to tetramethylsilane. Inhibition Assay for α-Glucosidase Activity in Vitro. Using a previously described method with minor modification,20 the different fractions were diluted to range of concentrations of dimethyl sulfoxide (DMSO) and then diluted in 100 times with sodium phosphate buffer (pH 6.9). Acarbose was diluted of ddH2O (15.6−500 μg/mL). CGS fractions including CGS ethanol extracts (CGSEE), CGSP, CGSC, CGSE and CGSB (62.5−2000 μg/mL) and acarbose (100 μL) were added in a 96-well plate. Subsequently, 1 U/mL of α-glucosidase (10 μL) mixed with 2 mM pNPG (30 μL) was added. The remaining 40 μL comprised sodium phosphate buffer (pH 6.9). The control well contained enzyme and substrate, with DMSO replacing CGS fractions. In the positive control well, acarbose replaced the CGS fractions. Mixtures without enzyme, CGS fractions, and acarbose served as blanks. The reactions were incubated at 37 °C for 10 min, after which the reaction was stopped by adding 0.5 M Na2CO3 solution (100 μL). Absorbance of the resulting p-nitrophenol (pNP) was determined at 405 nm using a spectrophometer (F 50, Infinite, CH). The absorbance reading was compared with that of the control, which included buffer solution instead of the enzyme solution. The assay was performed in triplicate. The results are reported as inhibition (%) ± SD. The percentage of enzyme inhibition was calculated as follows: inhibition (%) = (1 − ΔAbssample/ΔAbsenzyme) × 100. Cell Culture. 3T3-L1 preadipocytes and human hepatoma cell line (HepG2) cells (China Center for Type Culture Collection, CCTCC, Wuhan, China) were cultured in DMEM-high-glucose and DMEMlow-glucose with 10% fetal bovine serum, respectively. Cell Viability. 3T3-L1 and HepG2 were harvested at the exponential growth phase, seeded into 96-well plates at 2 × 104 cells/well in 100 μL, and incubated for 24 h. The samples were then 10793
DOI: 10.1021/acs.jafc.5b04552 J. Agric. Food Chem. 2015, 63, 10791−10802
Article
Journal of Agricultural and Food Chemistry Table 2. Effects of CGSB on Blood Variable Valuesa after 14 Days Treatment groupsa CTRL Insulin (mUI/mL) ALT (U/L) AST (U/L) TC (mmol/L) TG (mmol/L)
58.72 42.86 98.86 3.10 1.80
± ± ± ± ±
7.33 4.99 6.15 0.23 0.71
CGSB 200 63.26 45.75 108.75 3.03 1.30
± ± ± ± ±
4.01 2.25 4.42 0.34 0.11##c
STZ 25.24 106.20 191.80 3.49 2.32
± ± ± ± ±
STZ+Rosi ###d
3.45 8.36##c 10.20##c 0.19 0.24#b
30.97 107.78 177.89 3.63 2.44
± ± ± ± ±
3.09 10.10 9.30 0.25 0.21
STZ+CGSB 200 40.44 96.28 160.14 3.33 1.16
± ± ± ± ±
e
3.82* 18.60 17.23**f 0.20 0.10***g
STZ+CGSB 100 33.10 99.85 198.61 3.76 2.10
± ± ± ± ±
6.44 11.10 16.82 0.25 0.25
a All values are mean ± SD (n = 10). Statistically significant differences are indicated by pounds and asterisks and determined via ANOVA followed by LSD test. CGSB 200: CGSB 200 mg/kg body weight, CGSB 100: CGSB 100 mg/kg body weight; Rosi: rosiglitazone (4 mg/kg body weight). ALT, alanine aminotransferase; AST, aspartate transaminase; TC, total cholesterol; TG, triacylglycerols. b#p < 0.05. c##p < 0.01. d###p < 0.001 compared with the control (CRTL) group. e*p < 0.05. f**p < 0.01. g***p < 0.001 compared with model (STZ) group.
followed: PPAR-α (NM_001001928.2) (forward) 5′CCTCGGTGACTTATCCTGTGGT-3′ and (reverse) 5′-GACATCCCGACAGAAAGGCAC-3′, and β-actin (NM_001101) (forward) 5′-CACCCAGCACAATGAAGATCAAGAT-3′ and (reverse) 5′-CCAGTTTTTAAATCCTGAGTCAAGC-3′. Target gene mRNA levels were normalized to those of β-actin using the 2−ΔΔCT method. The fold change was calculated in comparison with the control group, and the fold change of >1.5 was considered statistically significant. Statistical Analysis. All data were expressed as the mean ± SD. Statistical significance was determined by one-way ANOVA, followed by least significant difference (LSD) post hoc testing, performed using SPSS 20.0. p < 0.05 was considered statistically significant.
treated with CGS extract fractions, Rosi, and 3,5-dicaffeoylquinic acid (DCQA) at selected concentrations for 48 h. The solution were prepared the medium containing 0.1% DMSO (v/v). The control culture was treated in basal medium (DMEM with 0.1% DMSO). Cell viability was determined by MTT assay.21 The media and test samples were replaced with MTT (5 mg/mL) and incubated in the dark for 2− 4 h. Formazan crystals were formed and solubilized using DMSO. Absorbance was measured using a microplate reader at 492 nm. Cell viability (%) was calculated as Absample/Abcontrol × 100%. 3T3-L1 Preadipocyte Differentiation. The differentiation assay followed method of Zebisch et al.,22 3T3-L1 preadipocytes were seeded in six-well plates at a density of 2 × 104 cells/mL. Two days after reaching confluence (day 0), the 3T3-L1 cells were differentiated in DMEM containing 10 μg/mL insulin, 1 μM dexamethasone, 0.5 mM IBMX, and 10% FBS (differentiation induction medium, DIM) for 2 days. After 2 days (day 2), the cells were maintained in DMEM containing 10 μg/mL insulin and 10% FBS (differentiation maintenance medium, DMM). The DMM was changed to basic medium (DMEM containing 10% FBS) every 2 days until the cells were harvested (day 10). To test effects in adipogenesis, the test samples were added to the DIM (day 0), DMM and basic medium until the cells were harvested. Lipid droplets were stained with Oil Red O, and lipid accumulation was determined as previously described.23 Cells treated with Rosi served as positive control. The concentrations of samples used in this assay were determined to be noncytotoxic to the 3T3-L1, as established in the MTT viability assay. Oil Red O Staining. The differentiation cells were washed with PBS twice, fixed with formalin for 30 min, stained with Oil Red O (0.5% w/v Oil Red O in 60% 2-propanol) for 10 min. Then cells washed with 40% 2-propanol. The degree of lipid accumulation were visualized under microscopy and dissolved in 2-propanol. The absorbance 2-propanol was measured at 490 nm. Glucose Consumption Determination. 3T3-L1 adipocyte culture supernatant samples were collected. Glucose consumption was determined using commercial kit (Shanghai Rongsheng Biotech Co., Ltd.). HepG2 Cell Treatment.24 HepG2 cells were seeded in six-well plates at the density of 1 × 106 cells/well in 2 mL for 24 h. HepG2 cells were grown to confluence and made quiescent in serum-free DMEM-low-glucose for 12 h. The cells were then maintained at low glucose (5.5 mM) or high glucose (30 mM) in the presence or absence of CGSB or 3,5-DCQA for 48 h. Six groups were present: the low glucose group as control group, the CGSB (100 μg/mL) with low glucose group, the 3,5-DCQA (10 μM) with low glucose group, the high glucose (30 mM)-induced group as the model group, the CGSB (100 μg/mL) with high glucose group, and the 3,5-DCQA (10 μM) with high glucose group. RNA Isolation and Quantitative RT-PCR. Total RNA from each well treated as above was isolated using Trizol reagent (Invitrogen), following the manufacturer’s protocol. Complementary cDNA were synthesized using RevertAid First Strand cDNA Synthesis Kit (Thermo). Quantitative reverse transcription PCR was conducted using FastStart Universal SYBR Green Master (Roche) to determine the gene expression level. The primer sequences used were as
■
RESULTS
CGSB Improves Weight Loss and Glucose Tolerance in T1DM Mice. Body Weight. The loss of body weights (for STZ treatment group) or weight gain (for control group) is shown in Figure 1A. No difference was observed in the body weight of the CGSB 200-treated and control mice. The average weight of the STZ-treated mice decreased compared with before STZ ip, which was significantly lower (p < 0.001) than the average weight of the control group mice. The body weight of treatment groups were significantly improved compared with that of STZ only group mice, with statistically significant differences after two week of treatment. The consumptions of food and water of all STZ treatment groups were almost the same during the experimental period (data not shown). Hence, these differences were not due to the food consumption but to the effects of CGSB and Rosi in T1DM mice. Moreover, CGSB did not affect the body weight of control group mice, which suggested that CGSB improved weight loss in T1DM mice only. OGTT. The OGTT was performed after 2 weeks of administration. Treatment with CGSB 200 lowered blood glucose significantly (p < 0.05 or p < 0.01) during 180 min of the glucose tolerance test (Figure 1B). However, Rosi, the positive control, could not significantly lower blood glucose. Liver Index and Kidney Index. When mice were sacrificed, livers and kidneys were collected and weighed to calculate viscera index. In Figure 1C,D, STZ treatment could significantly increase the liver index (p < 0.05) and kidney index (p < 0.001) compared with the control group. STZ with CGSB 200 could significantly reduce liver index (p < 0.001) compared with STZ treatment only. Both Rosi with STZ and CGSB 200 with STZ decreased kidney index significantly (p < 0.01, p < 0.05, respectively). Analysis the Blood Variables. The plasma concentration of insulin and the serum concentrations of TC, TG, ALT, and AST were assayed (Table 2.). To determine if the improved blood glucose results from increased insulin secretion, we 10794
DOI: 10.1021/acs.jafc.5b04552 J. Agric. Food Chem. 2015, 63, 10791−10802
Article
Journal of Agricultural and Food Chemistry
Figure 2. CGSB treatment improves pancreatic islet mass in STZ-induced diabetic mice. Pancreatic sections were stained with hematoxylin and eosin. Original magnification, ×100 (A); ×400 (B).
Figure 3. Effect of CGSB on FFA in epididymal adipose tissues (A), liver (B), and epididymal adipose tissues morphology (C). Statistically significant differences are indicated by pounds and asterisks and determined via ANOVA followed by LSD test. (###p < 0.001 compared with the control group, ***p < 0.001, *p < 0.05 compared with the model group). Original magnification, ×100. 10795
DOI: 10.1021/acs.jafc.5b04552 J. Agric. Food Chem. 2015, 63, 10791−10802
Article
Journal of Agricultural and Food Chemistry
Figure 4. HPLC chromatograms of n-BuOH fraction from Cichorium glandulosum Boiss. et Huet.
Table 3. Putative Identification of Compounds Using Mass Spectrometry in CGSB peak
TR (min)
MS+ (m/z)
major fragments (positive ion mode) (m/z)
tentative identification
1 2 3 4 5 6 7 8 9 10 11 12 13 14
15.618 16.730 18.077 18.724 19.106 19.972 23.813 24.813 25.636 26.816 27.488 33.152 37.509 40.754
405.9 354.8 179.0 516.5 445.7 505.9 556.2 556.2 181.6 516.5 516.5 516.5 449 403.1
405.9, 354.8; 224.9 354.8, 191, 135 179.0, 150 516.5, 353, 335 445.7, 354.8, 192.4 505.9, 354.8, 301.4, 162.8 556.2, 498.8, 336.8 556.2, 498.8, 336.8, 225 181.6, 177, 163.7 516.5, 353, 299, 317 516.5, 353, 191 516.5, 353 449, 327, 284 403, 257
unknown 5-O-caffeoylquinic acid esculetin 1,3-dicaffeoylquinic acid unknown quercitrin-3-glucoside unknown unknown unknown 3,4-dicaffeoylquinic acid 3,5-dicaffeoylquinic acid 4,5-dicaffeoylquinic acid kaempferal-7-O-glucoside pinocembrin-O-rhamnoside
Islet size, number, and structure were improved compared with the STZ treatment group. Histological specimens were prepared from epididymal adipose tissues and observed microscopically (Figure 3C). Sizes of the adipocytes in the control group and CGSB 200 control group were nearly the same. Meanwhile, the adipocyte cells were reduced in the STZ treatment group but improved in the STZ+CGSB 200 and STZ+CGSB 100 treatment groups. But in the Rosi treatment group, the size of adipocytes was atrophy obviously. In T1DM mice, the body weight decreased because of the reduction in adipose tissues. In this experiment, the administration of CGSB may prevent the reduction in adipocytes and normalize them in T1DM mice, but exert no effects on normal mice. Analysis FFA Released from Adipose and Liver Tissues. The FFA contents of epididymal adipose tissue were compared among groups (Figure 3A). Significant difference was observed between the STZ group and the control group, and the FFA contents of the STZ group was significantly higher than that of the control group. However, the FFA contents of adipose tissues of the treatment groups, namely, Rosi, CGSB 200, and CGSB 100 groups, were significantly lower compared with the STZ group (p < 0.001). A similar tendency was observed in liver (Figure 3B). The FFA contents of the liver were compared among groups. A significant difference between the STZ group and the control group was observed. The FFA content of the STZ group was significantly higher than those of the control group (p < 0.05). However, the FFA levels of the liver tissues of the treatment groups (i.e., Rosi, CGSB 200, and CGSB 100 groups) were significantly lower than those of the STZ group (p < 0.001).
measured and compared plasma insulin levels. Mice administrated with multiple low dose of STZ had significantly lower plasma insulin levels (25.24 ± 3.45 mIU/mL vs 58.72 ± 7.33 mIU/mL, p < 0.001) compared with the mice in the control group. Mice treated with CGSB (200 mg/kg body weight) exhibited significantly improved secretion of insulin (40.44 ± 3.82 vs 25.24 ± 3.45 mIU/mL, p < 0.05). Consistent with this result, CGSB treatment improved pancreatic islet mass (Figure 2). Meanwhile, we evaluated biochemical parameters (ALT, AST, TG, and TC) in serum (Table 2). Mice administrated with STZ had significantly higher ALT levels (106.2 ± 8.36 vs 42.86 ± 4.99 U/L, p < 0.01) compared with control group. Mice administrated with STZ displayed increased the serum AST levels (191.8 ± 10.2 U/L vs 98.86 ± 6.15 U/L, p < 0.01) compared with control. Mice treated with CGSB (200 mg/kg body weight) had significantly decreased AST (160.14 ± 17.23 vs 98.86 ± 6.15 U/L, p < 0.01). For TG in serum, mice administered with STZ showed increased TG levels (2.325 ± 0.243 mmol/L vs 1.796 ± 0.714 mmol/L, p < 0.05) compared with the control. Mice treated with CGSB (200 mg/kg body weight) demonstrated significantly decreased TG (1.157 ± 0.097 mmol/L, p < 0.001). Notably, TG in the CGSB control group significantly decreased (1.303 ± 0.111 mmol/L, p < 0.001) as well. Histopathologic Effects of CGSB on Pancreatic Islet and Size of Adipocytes in STZ-Induced Mice. Following STZ treatment, the destruction of pancreatic islets was observed (Figure 2). STZ-induced diabetic mice treated with CGSB displayed improved pancreatic islets and preserved islet architecture. Histological findings indicated that CGSB protected against STZ-induced destruction of pancreatic tissue. 10796
DOI: 10.1021/acs.jafc.5b04552 J. Agric. Food Chem. 2015, 63, 10791−10802
Article
Journal of Agricultural and Food Chemistry
the presence of two trisubstituted aromatic ring protons at δ 6.98 (2H, s, H-2′,2″), 6.70 (2H, d, J = 8.0 Hz, H-5′,5″), δ 6.88 (2H, d, J = 8.0 Hz, H-6′, 6″), and four protons in the trans configuration for double bonds at δ 7.50 (2H, d, J = 16.0 Hz, H-7′,7″), 6.19 (1H, d, J = 16.0 Hz, H-8″), and 6.23 (1H, d, J = 16.0 Hz, H-8′). The presence of a quinic acid moiety, four methylene protons δ 2.50 (1H, d, J = 10.8 Hz, H-2a), 2.36 (2H, m, H-6), 1.98 (1H, dd, J = 9.3, 13.5 Hz, H-2b), and three oxygenated methane protons [5.32 (1H, td, J = 8.0, 2.0 Hz, H3), 4.21 (1H, d, J = 3.3 Hz, H-5), 3.71 (1H, dd, J = 3.1, 8.1 Hz, H-4)] were present. Therefore, compound 11 was suggested to be dicaffeoylquinic acid with two caffeic acids and a quinic acid. The spectroscopic data were the same as previously published data.27 Combined with the analysis of HMBC, HMQC, and DQF-COSY data, compound 11 was identified as 3,5-DCQA. On the basis of spectral data results and comparisons of retention times with authentic standards using HPLC analysis, isolated compound 4 was identified as 1,3-DCQA. 3T3-L1 Preadipocyte Differentiation. First, the cytotoxicity of the CGS fractions were evaluated in Figure 5A. Cell viability was reduced at high concentrations (>50 μg/mL, except for CGSC and CGSE), so three dosages (25, 12.5 6.25 μg/mL) were chosen for the following study. The 3T3-L1 preadipocytes were incubated with CGS fractions and Rosi during adipogenesis, and Oil-red O staining was performed at 10 days after induction of differentiation. The absorbance of Oil Red O-stained lipid droplets was measured (Figure 5B). Induced differentiation was confirmed by TG contents in the cytoplasm. Rosi treatment induced significant differentiation compared with induced control group (p < 0.001). The differentiation rate of CGS fractions increased in the following order: CGSP (p < 0.001) > CGSEE (p < 0.001) > CGSC (p < 0.001), CGSB (p < 0.01), or CGSE (p < 0.05) at 25 μg/mL. The lipid accumulation in different groups was visualized in Figure 5C (only high concentration for each group). Glucose consumption was increased significantly in Rosi group (p < 0.01) and CGSB groups (p < 0.01, p < 0.05) at concentration of 50 and 25 μg/mL (Table 6). Quantification of PPAR-α at Gene Expression Level. To study the effect and corresponding mechanism of CGSB and 3,5-DCQA in HepG2 cells exposed to high glucose, PPARα expression involved in fatty acid metabolism in liver was determined. The results of qRT-PCR analysis showed that the expression of PPAR-α was up-regulated in high glucoseexposure HepG2 cells (Figure 6A). High glucose exposure significantly increased the mRNA expression of PPAR-α by 2.5fold compared with the control group (p < 0.001). CGSB (100 μg/mL) and 3,5-DCQA (10 μM) decreased gene expression compared with the control group, but no significant difference was noted. In the high glucose treated groups, CGSB (100 μg/ mL) decreased gene expression compared with the model group by 0.6-fold for PPAR-α (p < 0.05). Meanwhile, 10 μM 3,5-DCQA did not show significant effects in gene expression. Histopathologic Effects of CGSB on the Liver. STZinduced diabetes caused damage to mice liver, and steatosis was observed (Figure 6B). Treatment of STZ-induced diabetic mice with 200 and 100 mg/kg body weight CGSB for 14 days caused a reduction in fat accumulation (Figure 6B).
α-Glucosidase Inhibitory Activities. The different fractions of CGS were dissolved in H2O successively to obtain different solvent-soluble fractions. The α-glucosidase inhibitory activities of the individual fractions were then measured, and the results are shown in Table 5. CGSB showed the highest αglucosidase inhibition (IC50 = 231.26 ± 1.64 μg/mL). The main compound, 3,5-DCQA, showed the highest α-glucosidase inhibition (IC50 = 92.43 ± 4.21 μg/mL). The positive control acarbose, which is a well-known α-glucosidase inhibitor, showed α-glucosidase inhibition IC50 values of 154.69 ± 24.09 μg/mL. CGSE showed moderate α-glucosidase inhibitory activity potential, with an IC50 value of 328.34 ± 19.63 μg/mL. Putative Characterization and Identification of CGSB Compounds. The chemical compositions in the CGSB were analyzed by HPLC coupled with HPLC-ESI/MS. The chromatogram is shown in Figure 4. The results of HPLCESI/MS and tentative identification are shown in Table 3. On the basis of previous reports, these compounds exhibited quasimolecular ions [M + H]+. 5-O-Caffeoylquinic acid (2), esculetin (3), 1,3-dicaffeoylquinic acid (4), quercitin-3-glucoside (6), 3,4-dicaffeoylquinic acid (10), 3.5-dicaffeoylquinic acid (11), 4,5-dicaffeoylquinic acid (12), kaempferal-7-O-glucoside (13), and pinocembrin-O-rhamnoside (15).25,26 Putative Characterization and Identification of Compound. Compound 11 was isolated as yellow amorphous solid, with the molecular formula of C25H24O12, was deduced from the molecular ion peak at m/z 516.9 [M + H] + in its ESI-MS and supported by the 1H NMR and 13C NMR spectral data (Table 4). The 1H NMR spectrum of compound 11 showed Table 4. 1H-NMR and 13C-NMR Data of Compound 11 (in CD3OD, dppm) 1
position
H NMR
13
C NMR
Quinic Acid 1 2 3 4 5 6 7
2.50 5.32 3.71 4.21 2.36
(1H, (1H, (1H, (1H, (2H,
d, 10.8); 1.98 (1H, dd, 9.3, 13.5) td, 8.0, 2.0) dd, 3.1, 8.1) d, 3.3) m)
79.90 35.67 70.18 71.63 68.28 34.41 175.7
3-Caffeoyl 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′
6.98 (1H, s)
6.70 6.88 7.50 6.23
(1H, (1H, (1H, (1H,
d, d, d, d,
8.0) 8.0) 16.0) 16.0)
126.45 113.86 145.34 148.18 115.19 121.82 146.13 113.99 167.44
5-Caffeoyl 1″ 2″ 3″ 4″ 5″ 6″ 7″ 8″ 9″
6.98 (1H, s)
6.70 6.88 7.50 6.19
(1H, (1H, (1H, (1H,
d, d, d, d,
8.0) 8.0) 16.0) 16.0)
126.42 113.78 145.34 148.17 113.99 121.77 145.96 113.95 166.79
■
DISCUSSION T1DM is a chronic incurable metabolic disease characterized by T cell-mediated autoimmune process. The immune system attacks the pancreatic β-cells, eventually leading to insulin 10797
DOI: 10.1021/acs.jafc.5b04552 J. Agric. Food Chem. 2015, 63, 10791−10802
Article
Journal of Agricultural and Food Chemistry
Figure 5. Effect of CGS fractions on 3T3-L1 preadipocyte differentiation. (A) MTT assay. (B) Absorbance values of Oil Red O staining in differentiation adipose cells [HC, MC, and LC represent high, medium, and low doses; CGS fractions (25, 12.5, and 6.25 μg/mL); Rosi (25, 5, and 1 μM); 3,5-DCQA (20, 10, and 5 μM); control induced as induced control]. Values are shown as the mean ± SD (n = 3). Statistically significant differences are indicated by pounds and asterisks and determined via ANOVA followed by LSD test. (##p < 0.01 compared with the control group, ***p < 0.001, **p < 0.01, *p < 0.05 compared with the induced control group). (C) Lipid droplets were stained by Oil Red O. Differentiation in high concentration was performed.
deficiency.1 In the present study, we found that the CGSB could partly alleviate hyperglycemia symptoms and some impairments caused by STZ-induced T1DM in mice. CGSB treatment significantly ameliorated glucose tolerance and body weight loss, which were associated with increased circulating
insulin levels, preserved pancreatic islet mass, improved FFA utilization, and inhibited α-glucosidase activity. These findings provided evidence that CGSB may be a natural agent with potential hypoglycemic constituents to alleviate T1DM. 10798
DOI: 10.1021/acs.jafc.5b04552 J. Agric. Food Chem. 2015, 63, 10791−10802
Article
Journal of Agricultural and Food Chemistry Table 5. α-Glucosidase Inhibitory Activities of CGS Fractions samples CGSP CGSC CGSE CGSB CGSB 3,5-dicaffeoylquinic acid acarbosee
development of delayed but progressive hyperglycemia only in thymus competent mice.28 STZ can induce inflammation of the islets by immune cells and destroy β-cells progressive hyperglycemia within a few days when STZ is administered in multiple low doses. This models is similar to human T1DM and requires the participation of macrophages and T cells.29 In the present study, treatment with CGSB enhanced the exogenous insulin in circulating levels compared with treatment STZ only. Furthermore, CGSB treatment could improve fasting blood glucose levels and oral glucose tolerance. Meanwhile, islet size and structure improved in the CGSB treatment group compared with those in the STZ treatment group (Figure 2), thereby reflecting a direct response of β-cells to circulating glucose. Oxidative stress may play a potential role in the initiation chronic inflammation, which usually causes many diseases including diabetes. CGSB is considered to have antioxidant activities.17,30 These findings suggest that CGSB could partly restore β-cells function via an antioxidant mechanism. Controlling postprandial hyperglycemia is an effective way to treat diabetes. One approach is to suppress the absorption of glucose in the intestine. The intestine is the first key organ involved in glucose homeostasis, where dietary carbohydrates are digested and glucose is released through the retardation of carbohydrate-hydrolyzing enzymes into circulation.31 α-Glucosidase is the key enzyme in the digestive process, and it is located at the brush-border surface membrane of intestinal cells. Hence, α-glucosidase inhibitors can reduce the rate of glucose absorption, resulting in postprandial hyperglycemia glucose levels and suppressed diabetes.32 Some studies showed that added acarbose in patients with T1DM could modestly decrease HbA1c concentrations and decrease glucose levels. αGlucosidase inhibitory might be a useful adjunctive therapies to insulin treatment in patients with T1DM.33 In our previous study, flavonoids from CGS exhibited α-glucosidase inhibitory activity.17 The α-glucosidase inhibitory activities of CGS fractions were evaluated. The inhibitory activity of CGSB was close to acarbose. To elucidate whether the main compound of CGSB play inhibitory activity, 3,5-DCQA was isolated and identified. 3,5-DCQA exhibited similar inhibition activity with
IC50 (μg/mL)a 3153.5 1153.0 328.3 231.3 2035.8 92.4 154.7
± ± ± ± ± ± ±
293.8**c 37.1***d 19.6*b 1.6*b 256.6**c 4.2 24.1
All values are mean ± SD (n = 3). Statistically significant differences are indicated by asterisks and determined via ANOVA followed by LSD test. b*p < 0.05. c**p < 0.01. d***p < 0.001 compared with positive control. eUsed as positive control in each assay. a
Table 6. Glucose Consumption of CGSB in 3T3-L1 Adipocyte groups CTRL Rosi (1 μM) CGSB (6.25 μg/mL) CGSB (12.5 μg/mL) CGSB (25 μg/mL) CGSB (50 μg/mL)
glucose consumption (mmol/L)a 10.75 12.50 11.64 11.71 12.02 12.30
± ± ± ± ± ±
0.52 0.23**c 0.22 0.35 0.05*b 0.67**c
All values are mean ± SD (n = 3). Statistically significant differences are indicated by asterisks and determined via ANOVA followed by LSD test. b*p < 0.05. c**p < 0.01 compared with CTRL group. a
Given that no safe methods exist for sampling or visualizing the human endocrine pancreas, rodent models of diabetes are extensively used models. The common rodent models of T1DM classify spontaneous diabetes such as Bio-Breeding diabetic-resistant rat and nonobese diabetic mice and inducible diabetic rodent such as the STZ-induced diabetic rodents.16 STZ is the most frequently used drug to induce diabetes. STZ is a nitrosourea analogue or a donor of nitric oxide, which is known to selectively damage pancreatic islet cells. In this study, multiple doses (40 mg/kg per day for five consecutive days) were given because multiple low doses of STZ cause the
Figure 6. CGSB reduces hepatic steatosis in STZ-induced diabetic mice (B) and effect of CGSB on mRNA expression of PPAR-α in HepG2 cells under hyperglycemic condition (A). Statistically significant differences are indicated by pounds and asterisks and determined via ANOVA followed by LSD test. (###p < 0.001 compared with the low glucose group; *p < 0.05 compared with the high glucose group). Fat accumulation was significant in STZ administration. Original magnification, ×100. 10799
DOI: 10.1021/acs.jafc.5b04552 J. Agric. Food Chem. 2015, 63, 10791−10802
Article
Journal of Agricultural and Food Chemistry
discrepancy between these results, which was explained by Yan et al., results from the fact that different animal models were employed.41 In conclusion, we provided evidence that CGSB could partly alleviate T1DM in STZ-induced diabetic mice. This antidiabetic effect was likely because CGSB repaired damaged β-cells and enhanced exogenous insulin in STZ-diabetes mice. CGSB induced the differentiation of adipocyte to decrease the release of FFA from adipose tissue. Furthermore, the α-glucosidase inhibitory activity was evaluated. The main compound 3,5DCQA showed α-glucosidase inhibiting activity but moderate adipocyte differentiation. CGSB accommodated the mRNA level of PPAR-α to intervene with glucolipid metabolism. Thus, CGSB provided multipathway protection from STZ-induced type 1 diabetes. However, further research on combination pharmacology is necessary.
acarbose without significant difference. The interaction between 3,5-DCQA and α-glucosidase might be a spontaneous and hydrophobic force.34 The secretion of insulin is response to the fluctuations of glucose and FFA in circulation. Once pancreatic β-cells are destroyed, the deficiency of insulin secretion is initiated. Typical signs of T1DM are high glucose levels in blood and urine, accompanied by inefficient glucose tolerance. The adipose tissue weight was markedly reduced in mice with diabetes. In STZ-induced diabetes, high blood glucose content is accompanied by elevated of lipid levels, and alterations in the lipid profile are also common.35 In the STZ alone group, an increased TG was observed. After supplementation of CGSB, the serum level of TG decreased. In addition, hepatic FFA extraction and FFA in adipose tissue were significantly lower. The most frequent pathologic condition associated with T1DM is massive mobilization of adipocytes. Thus, adipose supplies glycerol and FFA to the other tissues via blood. Glycerol and FFA in the circulation were uptaken by hepatocytes. Glycerol is used for gluconeogenesis to produce glucose by the liver. FFA is used for TG synthesis and secretion.36 The results showed that FFA contents in liver and adipose tissue were significantly high in the STZ treatment group. Meanwhile, FFA levels were remarkably reduced in the CGSB treatment groups. The size of adipocytes was restored simultaneously, and the capability of lipogenesis improved. To determine whether lipid metabolism is the second action of the improvement in β-cellular function, an adipocyte differentiation assay was designed in 3T3-L1 cells. The results showed that CGS fractions could promote preadipocyte differentiation in varying degrees. Rosi, as positive control, could significantly induce preadipocyte differentiation because of lipid accumulation. CGSH strongly induced the differentiation of 3T3-L1 preadipocytes. The CGSP fraction contained oleic acid (based on the result of GC/MS, result not showed). Oleic acid could induce 3T3-L1 cells differentiation. Our study showed the same tendency as with Cheguru et al.37 Other fractions and the main compound 3,5DCQA showed moderate induction at high concentration. Thus the induction of preadipocyte differentiation might not be an effective mechanism. Further experiments were designed to explain the influence of CGSB on FFA metabolism. PPAR-α, a ligand-activated transcription factor, is a key gene involved in the β-oxidation of fatty acids, and it is highly expressed in the liver.38 In T1DM, given the occurrence of insulin deficiency, impaired glucose utilization renders tissues more dependent on lipid utilization, which leads to the activation of mitochondrial β-oxidation of fatty acids in tissues, especially in the liver. In the following experiment, HepG2 cells were used to study the effects of CGSB and 3,5-DCQA on mRNA expression with high glucose levels. High glucose up-regulated PPAR-α expression (the same results were showed in animal study, data not shown). Moreover, FFA contents increased in adipose tissue and liver. Considering that fatty acids are natural ligands for PPAR-α,39 FFA induced the expression of PPAR-α. Activated PPAR-α induces hepatic steatosis in STZ-induced diabetic mice, which could be proved in the biopsy (Figure 6B). CGSB, instead of Rosi, could improve hepatic steatosis effectively. Interestingly, some results showed different trends from our research.24 The effects of PPAR-α on glucose metabolism are ambiguous in other reports.40 PPAR-α activation involved liver FFA uptake, fatty acid β-oxidation, and accelerated lipid mobilization. The
■
AUTHOR INFORMATION
Corresponding Author
*Phone: +86-27-68759010. Fax: +86 27 68759010. E-mail:
[email protected]. Funding
This work was supported by the National Natural Science Funds of China (81503356), the Project of the National Twelve-Five Year Research Program of China (2012BAI29B03), the Commonweal Specialized Research Fund of China Agriculture (201103016), and the Nanjing 321 plan for Bringing in technological leading talents (2013A12011). Notes
The authors declare no competing financial interest.
■
REFERENCES
(1) Morgan, N. G.; Leete, P.; Foulis, A. K.; Richardson, S. J. Islet inflammation in human type 1 diabetes mellitus. IUBMB Life 2014, 66, 723−734. (2) Xie, Z.; Chang, C.; Zhou, Z. Molecular mechanisms in autoimmune type 1 diabetes: a critical review. Clin. Rev. Allergy Immunol. 2014, 47, 174−192. (3) Patterson, C. C.; Dahlquist, G. G.; Gyurus, E.; Green, A.; Soltesz, G. Incidence trends for childhood type 1 diabetes in Europe during 1989−2003 and predicted new cases 2005−20: a multicentre prospective registration study. Lancet 2009, 373, 2027−2033. (4) Lin, H. P.; Chan, T. M.; Fu, R. H.; Chuu, C. P.; Chiu, S. C.; Tseng, Y. H.; Liu, S. P.; Lai, K. C.; Shih, M. C.; Lin, Z. S.; Chen, H. S.; Yeh, D. C.; Lin, S. Z. Applicability of adipose-derived stem cells in type 1 diabetes mellitus. Cell transplant. 2015, 24, 521−532. (5) Muir, K. R.; Lima, M. J.; Docherty, H. M.; Docherty, K. Cell therapy for type 1 diabetes. QJM 2014, 107, 253−259. (6) Drescher, K. M.; von Herrath, M.; Tracy, S. Enteroviruses, hygiene and type 1 diabetes: toward a preventive vaccine. Rev. Med. Virol. 2015, 25, 19−32. (7) Ludvigsson, J. Combination therapy for preservation of beta cell function in Type 1 diabetes: new attitudes and strategies are needed! Immunol. Lett. 2014, 159, 30−35. (8) Bais, H. P.; Ravishankar, G. A. Cichorium intybus L-cultivation, processing, utility, value addition and biotechnology, with an emphasis on current status and future prospects. J. Sci. Food Agric. 2001, 81, 467−484. (9) Street, R. A.; Sidana, J.; Prinsloo, G. Cichorium intybus: Traditional Uses, Phytochemistry, Pharmacology, and Toxicology. Evid-based Compl. Alt. 2013, 2013, 1−13. (10) Rasmussen, M. K.; Klausen, C. L.; Ekstrand, B. Regulation of cytochrome P450 mRNA expression in primary porcine hepatocytes
10800
DOI: 10.1021/acs.jafc.5b04552 J. Agric. Food Chem. 2015, 63, 10791−10802
Article
Journal of Agricultural and Food Chemistry by selected secondary plant metabolites from chicory (Cichorium intybus L.). Food Chem. 2014, 146, 255−263. (11) Schmidt, B. M.; Ilic, N.; Poulev, A.; Raskin, I. Toxicological evaluation of a chicory root extract. Food Chem. Toxicol. 2007, 45, 1131−1139. (12) China Pharmacopoeia Committee. Pharmacopoeia of the People’s Republic of China, the first division of 2015 ed.; China Medical Science and Technology Press: Beijing, 2015; p310. (13) Wu, H.; Su, Z.; Yang, Y.; Ba, H.; Aisa, H. A. Isolation of three sesquiterpene lactones from the roots of Cichorium glandulosum Boiss. et Huet. by high-speed counter-current chromatography. J. Chromatogr. A 2007, 1176, 217−222. (14) Pushparaj, P. N.; Low, H. K.; Manikandan, J.; Tan, B. K.; Tan, C. H. Anti-diabetic effects of Cichorium intybus in streptozotocininduced diabetic rats. J. Ethnopharmacol. 2007, 111, 430−4. (15) Wealth of India, A Dictionary of Indian Raw Materials and Industrial Products; Council of Scientific and Industrial Research: New Delhi, 1950, Vol. 2, p 161. (16) Novikova, L.; Smirnova, I. V.; Rawal, S.; Dotson, A. L.; Benedict, S. H.; Stehno-Bittel, L. Variations in rodent models of type 1 diabetes: islet morphology. J. Diabetes Res. 2013, 2013, 1−13. (17) Yao, X.; Zhu, L.; Chen, Y.; Tian, J.; Wang, Y. In vivo and in vitro antioxidant activity and alpha-glucosidase, alpha-amylase inhibitory effects of flavonoids from Cichorium glandulosum seeds. Food Chem. 2013, 139, 59−66. (18) Amirshahrokhi, K.; Dehpour, A. R.; Hadjati, J.; Sotoudeh, M.; Ghazi-Khansari, M. Methadone ameliorates multiple-low-dose streptozotocin-induced type 1 diabetes in mice. Toxicol. Appl. Pharmacol. 2008, 232, 119−124. (19) Park, J. S.; Rhee, S. D.; Kang, N. S.; Jung, W. H.; Kim, H. Y.; Kim, J. H.; Kang, S. K.; Cheon, H. G.; Ahn, J. H.; Kim, K. Y. Antidiabetic and anti-adipogenic effects of a novel selective 11betahydroxysteroid dehydrogenase type 1 inhibitor, 2-(3-benzoyl)-4hydroxy-1,1-dioxo-2H-1,2-benzothiazine-2-yl-1-phenylethanone (KR66344). Biochem. Pharmacol. 2011, 81, 1028−1035. (20) Matsui, T.; Tanaka, T.; Tamura, S.; Toshima, A.; Tamaya, K.; Miyata, Y.; Tanaka, K.; Matsumoto, K. alpha-glucosidase inhibitory profile of catechins and theaflavins. J. Agric. Food Chem. 2007, 55, 99− 105. (21) Borenfreund, E.; Babich, H.; Martin-Alguacil, N. Comparisons of two in vitro cytotoxicity assays-The neutral red (NR) and tetrazolium MTT tests. Toxicol. In Vitro 1988, 2, 1−6. (22) Zebisch, K.; Voigt, V.; Wabitsch, M.; Brandsch, M. Protocol for effective differentiation of 3T3-L1 cells to adipocytes. Anal. Biochem. 2012, 425, 88−90. (23) Ramirez-Zacarias, J. L.; Castro-Munozledo, F.; Kuri-Harcuch, W. Quantitation of adipose conversion and triglycerides by staining intracytoplasmic lipids with Oil red O. Histochemistry 1992, 97, 493− 497. (24) Wang, W.; Wang, C.; Ding, X. Q.; Pan, Y.; Gu, T. T.; Wang, M. X.; Liu, Y. L.; Wang, F. M.; Wang, S. J.; Kong, L. D. Quercetin and allopurinol reduce liver thioredoxin-interacting protein to alleviate inflammation and lipid accumulation in diabetic rats. Br. J. Pharmacol. 2013, 169, 1352−1371. (25) Mascherpa, D.; Carazzone, C.; Marrubini, G.; Gazzani, G.; Papetti, A. Identification of phenolic constituents in Cichorium endivia var. crispum and var. latifolium salads by high-performance liquid chromatography with dio.de array detection and electrospray ioniziation tandem mass spectrometry. J. Agric. Food Chem. 2012, 60, 12142−12150. (26) Carazzone, C.; Mascherpa, D.; Gazzani, G.; Papetti, A. Identification of phenolic constituents in red chicory salads (Cichorium intybus) by high-performance liquid chromatography with diode array detection and electrospray ionisation tandem mass spectrometry. Food Chem. 2013, 138, 1062−1071. (27) Kim, J. Y.; Cho, J. Y.; Ma, Y. K.; Park, K. Y.; Lee, S. H.; Ham, K. S.; Lee, H. J.; Park, K. H.; Moon, J. H. Dicaffeoylquinic acid derivatives and flavonoid glucosides from glasswort (Salicornia herbacea L.) and their antioxidative activity. Food Chem. 2011, 125, 55−62.
(28) Paik, S. G.; Fleischer, N.; Shin, S. I. Insulin-dependent diabetes mellitus induced by subdiabetogenic doses of streptozotocin: obligatory role of cell-mediated autoimmune processes. Proc. Natl. Acad. Sci. U. S. A. 1980, 77, 6129−6133. (29) Papaccio, G.; Pisanti, F. A.; Latronico, M. V.; Ammendola, E.; Galdieri, M. Multiple low-dose and single high-dose treatments with streptozotocin do not generate nitric oxide. J. Cell. Biochem. 2000, 77, 82−91. (30) Huang, B.; Chen, Y.; Ma, B.; Zhou, G.; Tong, J.; He, J.; Wang, Y. Protective effect of Cichorium glandulosum seeds from ultraviolet Binduced damage in rat liver mitochondria. Food Funct. 2014, 5, 869− 875. (31) Knauf, C.; Cani, P. D.; Kim, D. H.; Iglesias, M. A.; Chabo, C.; Waget, A.; Colom, A.; Rastrelli, S.; Delzenne, N. M.; Drucker, D. J.; Seeley, R. J.; Burcelin, R. Role of central nervous system glucagon-like Peptide-1 receptors in enteric glucose sensing. Diabetes 2008, 57, 2603−2612. (32) Ceriello, A. Postprandial hyperglycemia and diabetes complications: is it time to treat? Diabetes 2005, 54, 1−7. (33) Lebovitz, H. E. Adjunct therapy for type 1 diabetes mellitus. Nat. Rev. Endocrinol. 2010, 6, 326−334. (34) Xu, D.; Wang, Q.; Zhang, W.; Hu, B.; Zhou, L.; Zeng, X.; Sun, Y. Inhibitory activities of caffeoylquinic acid derivatives from Ilex kudingcha C.J. Tseng on alpha-glucosidase from Saccharomyces cerevisiae. J. Agric. Food Chem. 2015, 63, 3694−3703. (35) Kim, H. K.; Kim, M. J.; Shin, D. H. Improvement of lipid profile by amaranth (Amaranthus esculantus) supplementation in streptozotocin-induced diabetic rats. Ann. Nutr. Metab. 2006, 50, 277−281. (36) Prentki, M.; Madiraju, S. R. Glycerolipid/free fatty acid cycle and islet beta-cell function in health, obesity and diabetes. Mol. Cell. Endocrinol. 2012, 353, 88−100. (37) Cheguru, P.; Chapalamadugu, K. C.; Doumit, M. E.; Murdoch, G. K.; Hill, R. A. Adipocyte differentiation-specific gene transcriptional response to C18 unsaturated fatty acids plus insulin. Pfluegers Arch. 2012, 463, 429−447. (38) Issemann, I.; Green, S. Activation of a member of the steroid hormone receptor superfamily by peroxisome proliferators. Nature 1990, 347, 645−550. (39) Berger, J.; Moller, D. E. The mechanisms of action of PPARs. Annu. Rev. Med. 2002, 53, 409−435. (40) Wang, C.; Xu, C. X.; Krager, S. L.; Bottum, K. M.; Liao, D. F.; Tischkau, S. A. Aryl hydrocarbon receptor deficiency enhances insulin sensitivity and reduces PPAR-alpha pathway activity in mice. Environ. Health Persp. 2011, 119, 1739−1744. (41) Yan, F.; Wang, Q.; Xu, C.; Cao, M.; Zhou, X.; Wang, T.; Yu, C.; Jing, F.; Chen, W.; Gao, L.; Zhao, J. Peroxisome proliferator-activated receptor alpha activation induces hepatic steatosis, suggesting an adverse effect. PLoS One 2014, 9, e99245. (42) Upur, H.; Amat, N.; Blazekovic, B.; Talip, A. Protective effect of Cichorium glandulosum root extract on carbon tetrachloride-induced and galactosamine-induced hepatotoxicity in mice. Food Chem. Toxicol. 2009, 47, 2022−2030. (43) Qin, D.; Nie, Y.; Wen, Z. Protection of rats from thioacetamideinduced hepatic fibrosis by the extracts of a traditional Uighur medicine Cichorium glandulosum. Iran. J. Basic Med. Sci. 2014, 17, 879−885. (44) Qin, D.; Wen, Z.; Nie, Y.; Yao, G. Effect of Cichorium Glandulosum Extracts on CCl4-Induced Hepatic Fibrosis. Iran Red Crescent Med. J. 2013, 15, e10908. (45) Qin, D. M.; Hu, L. P.; Nie, Y. R.; Chen, W. Effects of Cichorium glandulosum Boiss. et Huet. on expression of fibronectin, Smad3, IGFBP-rPl, and TGFbeta1 in a liver fibrosis rat model. Chin. J. Hepatol. 2013, 21, 776−777. (46) Yang, W. J.; Luo, Y. Q.; Aisa, H. A.; Xin, X. L.; Totahon, Z.; Mao, Y.; Hu, M. Y.; Xu, L.; Zhang, R. P. Hepatoprotective activities of a sesquiterpene-rich fraction from the aerial part of Cichorium glandulosum. Chin. Med. 2012, 7 (21), 21. (47) Ding, L.; Liu, J. L.; Hassan, W.; Wang, L. L.; Yan, F. R.; Shang, J. Lipid modulatory activities of Cichorium glandulosum Boiss et Huet are 10801
DOI: 10.1021/acs.jafc.5b04552 J. Agric. Food Chem. 2015, 63, 10791−10802
Article
Journal of Agricultural and Food Chemistry mediated by multiple components within hepatocytes. Sci. Rep. 2014, 4, 4715.
10802
DOI: 10.1021/acs.jafc.5b04552 J. Agric. Food Chem. 2015, 63, 10791−10802
