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Overdose taking curcumin initiating the unbalanced state of bodies Peiyu Qiu, Shuli Man, Jing Li, Jing Liu, Liming Zhang, Peng Yu, and Wenyuan Gao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b00053 • Publication Date (Web): 15 Mar 2016 Downloaded from http://pubs.acs.org on March 15, 2016
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Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
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Journal of Agricultural and Food Chemistry
Curcumin is a widely used spice and coloring agent in food, while its safety evaluation has been few investigated. Overdose or long-term taking curcumin could initiate the unbalanced state of bodies through oxidative stress, inflammation and metabolic disorders, which induces liver injury. 254x190mm (96 x 96 DPI)
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Overdose taking curcumin initiating the unbalanced state of bodies
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Peiyu Qiu,†, ‡, & Shuli Man,*, †, ‡ Jing Li,†, & Jing Liu,† Liming Zhang,† Peng Yu,† Wenyuan Gao,∗, #
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†
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Tianjin University of Science & Technology, Tianjin, 300457, China
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‡
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University of Science & Technology, Tianjin, 300457, China
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#
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Pharmaceutical Science and Technology, Tianjin University, Tianjin, 300072, China
∗
Key Laboratory of Industrial Microbiology, Ministry of Education, College of Biotechnology,
Tianjin Key Laboratory of Industry Microbiology, College of Biotechnology, Tianjin
Tianjin Key Laboratory for Modern Drug Delivery and High Efficiency, School of
Corresponding authors: Shuli Man and Wenyuan Gao. Tel./fax, 86-022-87401895.
E-mail addresses:
[email protected] (Gao WY),
[email protected] (Man SL). &
Both authors are regarded as co-first authors and contributed equally to this work.
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Abstract
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Curcumin is the major active component of turmeric and widely used as spice and
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coloring agent in food. However, its safety evaluation has been few investigated.
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To evaluate the 90-day subchronic toxicity of curcumin in rats, its general
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observation, clinical biochemistry, pathology and metabolomics were evaluated.
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The results showed that curcumin induced liver injury through the generation of
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the over-expression of reactive oxygen species (ROS) and pro-inflammatory
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cytokines IL6, and the decrease of the levels of antioxidant enzyme SOD and
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detoxified enzyme GST. Meanwhile, as the self-protection of rats, curcumin
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treatment activated the transcription of Nrf2 and elevated the expression of HO-1
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to reduce tissues damage. What’s more, curcumin significantly increased key
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mRNA levels of HK2, PKM2, LDHA, CES, Cpt1, Cpt2, FASN, and ATP5b, and
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decreased levels of GLUT2 and ACC1 to enhance glycolysis and inhibit lipid
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metabolism and TCA cycle. Therefore, overdose or long-term taking curcumin
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could initiate the unbalanced state of bodies through oxidative stress, inflammation
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and metabolic disorders, which induces liver injury. It’s necessary for intermittent
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administration of curcumin in our daily life.
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Keywords: curcumin; subchronic toxicity; oxidative stress; self-protection;
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metabolomics
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Introduction Curcuma longa is a famous and important food spice1 with a rich source of
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polyphenolic
compounds,
namely
curcuminoids,
such
as
curcumin,
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demethoxycurcumin, bis-demethoxycurcumin and so forth. Among these
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compounds, curcumin as a major active constituent possesses a wide range of
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pharmacological activities like hepatoprotective2, anti-inflammatory3, antioxidant4,
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pro-oxidant5, anticarcinogenic6 and so on. It’s estimated that adult Indians
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everyday consume 80-200 mg of curcumin which is equivalent up to 5 g of
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turmeric power7.
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Previous research reported that the administration of turmeric for 90 days
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showed a significant reduction in body weight gain and hepatotoxicity both in mice
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and rats8. Meanwhile, the main compounds of turmeric including polysaccharide9
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and essential oil10 were both safe in rats. However, using in silico methods
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predicted that 64 compounds isolated from turmeric were hepatotoxic, such as
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curcumin and its derivatives
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based on the toxicity of curcumin in animals. For curcumin has been widely used
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as preservative, flavoring and coloring agents in beverages and foods including
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curry, mustard, and margarine12, it’s critical to clear whether it’s safe and edible
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used as a functional food ingredient.
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Materials and methods
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. Up to now, there have been few detailed reports
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Drugs
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Curcuminoids (90% of curcumin) were obtained from Zhongda Co. (China)
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and deposited at College of Biotechnology at Tianjin University of Science &
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Technology, Tianjin, China.
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Experimental animals
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Eighteen male Sprague-Dawley rats weightting 170 ± 10 g were purchased
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from the Laboratory Animal Center of academy of Military Medical Sciences
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(Beijing, China quality certification number: SCXK (Jun) 2012-0004). This animal
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study was approved by the Institutional Animal Care and Use Committee of China.
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After one week of acclimatization, rats were randomly divided into three groups.
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The curcumin groups (Curcumin & Curcumin-L) were fed orally 100 and 25 mg of
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curcumin in 10 mL of 0.9% sodium chloride per kg of body weight five days per
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week for 90 days, respectively. Normal control rats (Normal) were administered
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appropriate vehicles. During the experiment, the body weight was measured every
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week. Blood samples (0.5 mL) were collected into heparinized tubes by the
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puncture of the retro-orbital sinus on the 90th and 120th days. Then three rats from
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each group were sacrificed. Portions of each tissue were fixed in 10% formalin (pH
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7.4) or snap frozen in liquid nitrogen.
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Biochemical analyses
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Serum levels of alkaline phosphatase (ALP), aspartate transaminase (AST),
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alanine transaminase (ALT), gamma glutamyl transpeptidase (γ-GGT), catalase
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(CAT), blood urea nitrogen (BUN), creatinine (Cr), high-density lipoprotein
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cholesterol (HDL-C),
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cholesterol (T-CHO), triglyceride (TG) and glucose (GLU) were measured by the
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detection kits according to the manufacturer’s instructions obtained from Nanjing
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Jiancheng Institute of Biotechnology (Nanjing, China).
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Histopathological examination
low-density lipoprotein cholesterol (LDL-C),
total
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Portions of the liver, kidney, heart, spleen and lung tissues were fixed in 10%
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formalin. After the proper dehydration, the tissues were embedded in paraffin wax.
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Five-µm-thick sections were prepared and stained with hematoxylin and eosin.
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Histopathology examination was performed by a pathologist who was unaware of
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whether tissues were treated.
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Oxidative stress
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Livers fixed for snap frozen were rapidly washed and homogenized in ten
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volumes (v/w) of ice-cold saline solution. The homogenate was centrifuged at 3000
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rpm for 10 min. Its supernatant was measured for analyzing the levels of
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malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT) and
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reduced glutathione (GSH) based on the commercially available kits (Nanjing
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Jiancheng Bioengineering Institute, Nanjing, China).
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Reactive oxygen species (ROS) release assay
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The liver tissue samples were prepared following the ROS assay kit (Applygen
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Technologies Inc., Beijing, China) according to the manufacturer's instruction. The
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oxidation product dichlorofluorescein (DCF) fluorescence was imaged by C6
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Software.
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Detection of nuclear 8-hydroxy-2-deoxyguanosine (8-OHdG) level
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Each urine was centrifuged at 3000 rpm for 20 min. The supernatant was
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measured on behalf of 8-OHdG concentration, using an enzyme-linked
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immunosorbent assay (ELISA) kit following the manufacturer’s instruction
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(Huiying Co., Shanghai, China).
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Measurements of COX-2, TNF-α and IL-6 levels in plasma
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Plasma samples were analyzed for COX-2 (Huole Co., Shanghai, China),
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TNF-α and IL-6 (Suer Co., Shanghai, China) with rat ELISA kits according to the
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manufacturer's instructions. Each value was measured within the linear portion of
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the standard curve.
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Western blot assay
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Total proteins from livers were isolated using the tissue protein extraction kit
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(Bio-Rad, California, USA), and quantified by the Bradford Assay Kit (BioRad,
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Hercules, CA). The protein samples (20-100 µg) were separated on 12%
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SDS-PAGE. Proteins were transferred to PVDF membranes (Millipore Co., Boston,
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USA) and probed with GST-α, GAPDH, HO-1 (Bioworld Technology, Minnesota,
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USA), GST-µ, GST-π (Boster Co., Wuhan, China), and Nrf2 (Santa Cruz
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Biotechnoligy, Shanghai, China) primary antibodies and followed by appropriate
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secondary antibody. The relative optical densities of the bands were quantified
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using Odyssey infrared imaging system (LI-COR Biotechnology, Nebraska, USA).
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Reverse transcription PCR
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Total RNA was isolated from rats liver tissues in each group using TRIzol
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(Sangon Biotech Co., Shanghai, China) according to the manufacturer's instruction.
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Polymerase chain reaction products were electrophorized on 3.0% agarose gel and
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visualized after ethidium bromide staining. The intensity of bands was quantified
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by ImageJ software.
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GC/MS sample collection and detection
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Referring to previous methods
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, liver tissues separated into aqueous and
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lipophilic phases were performed on an Agilent 7890A gas chromatography
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(Agilent Technologies Co., Ltd., California, USA) coupled to an Agilent 5975C
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mass selective detector (Agilent Technologies Co., Ltd., California, USA)
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performed in the electron ionization (EI) mode.
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Statistical Analysis
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Statistical analysis was evaluated by using SPSS 17.0 for Windows. The
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reconstruction of the metabolic network was mainly based on the information from
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the metabolic databases KEGG.
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Results
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General observations and organ examination
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As previous reported, higher dose administration of curcumin for 90 days
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showed a significant reduction in body weight gain and hepatotoxicity in rats.8
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Especially at the 12th week, Curcumin-treatment significantly reduced the body
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weight gain (p
