Jiao Tai Wan (JTW), a Chinese herbal formula containing Rhizoma Coptidis and Cortex Cinnamomi, has been used for diabetic treatment for many years. The aim of this study was to determine the main components in JTW and to investigate the effects of JTW on hepatic lipid accumulation in diabetic rats and humans. JTW extract was prepared and the main components were assayed by HPLC. An animal model of diabetes mellitus was established and JTW was administered intragastrically. In the clinical study, diabetic patients with poor glycemic control were treated with JTW. Blood glucose and lipid parameters, liver histology, hepatic triglyceride content and lipogenic gene expression were examined. Our data demonstrated that JTW significantly improved hyperglycemia, hyperlipidemia and hepatic lipid accumulation in diabetic rats. This was accompanied by the down-regulation of acetyl coenzyme A carboxylase (ACC) and fatty acid synthase (FAS) protein expressions, and the up-regulation of AMP-activated protein kinase (AMPK) and phosphorylated-ACC (pACC) protein expressions in the liver tissues. Diabetic patients also exhibited decreases in their hepatic triglyceride content. The results suggest that JTW attenuates hepatic lipid accumulation in diabetic rats and humans. These beneficial effects are possibly associated with the inhibition of lipogenic gene expression in the liver.
Obesity, the abnormal or excessive accumulation of fat within the body, has become a global epidemic secondary to high caloric food intake and sedentary life style [
Excessive lipid accumulation in the liver of patients who drink little or no alcohol is called nonalcoholic fatty liver disease (NAFLD). NAFLD refers to a wide spectrum of liver diseases ranging from simple hepatic steatosis to nonalcoholic steatohepatitis to cirrhosis. NAFLD is highly prevalent in subjects with T2DM; approximately 70% of type 2 diabetic patients are estimated to have NAFLD [
Jiao Tai Wan (JTW) is a classical traditional Chinese prescription first mentioned in the book Han-Shi-Yi-Tong in the Ming Dynasty that consists of Rhizoma Coptidis (
Male Wistar rats (8 weeks,
Seven rats were selected randomly as the control group and were injected with saline into the tail vein. The remaining rats were injected with streptozotocin (STZ; Sigma Chemical Co. MO, USA; 30 mg/kg) into the tail vein. An oral glucose tolerance test (OGTT) was performed two weeks later. Impaired glucose tolerance (IGT) was diagnosed when the plasma glucose levels of rats at two time points were higher than the upper limit (4.62 mmol/L before glucose-loading, 7.56 mmol/L at 1 h after glucose-loading, and 6.29 mmol/L at 2 h after glucose-loading, resp., which is the 95% range of confidence calculated according to plasma glucose levels in the rats of control group) or when the plasma glucose levels at one time point exceeded the upper limit by more than 20% (5.54 mmol/L, 9.07 mmol/L, and 7.55 mmol/L, resp.). Thereafter, twenty-one rats with IGT were randomly divided into 3 groups. Rats receiving saline served as diabetic controls. Rats receiving JTW (3 g/kg/day) were taken as the JTW group, and rats receiving metformin hydrochloride (183 mg/kg/day) were taken as the metformin (Met) group. Oral gavage was performed once a day between 8:00 and 10:00 a.m. The doses were adjusted to the body weight, which was recorded once a week. Meanwhile, the high-fat chow (containing 72.7% standard laboratory rat chow, 20% lard, 5% egg yolk powder, 2% cholesterol, and 0.3% bile salts) was provided to the animals with IGT while the rats in the control group continued their standard diet. At the end of the 8-week period, overnight-fasted rats were weighed and then anaesthetized by pentobarbital sodium. Blood samples were obtained from the abdominal aorta. The liver was quickly excised and divided into two parts. One sample was immediately stored in liquid nitrogen, while the other was fixed with 4% paraformaldehyde.
Rhizoma Coptidis and Cortex Cinnamomi used for animal experiments were obtained from the Traditional Chinese Medicine Company in Hubei Province (Wuhan, China). The voucher specimen was deposited at the herbarium of the Pharmacy Faculty at the Hubei University of Chinese Medicine and authenticated by Department of Pharmacognosy, Hubei University of Chinese Medicine (Wuhan, China). The rat doses of Rhizoma Coptidis and Cortex Cinnamomi were obtained by the conversion of the human doses (Chinese Pharmacopoeia, 2010) to rat equivalent doses based on body surface areas. The JTW preparation process was as follows: ninety grams Rhizoma Coptidis and forty-five grams Cortex Cinnamomi were mixed together. The mixture was decocted twice by refluxing it with water (1 : 10 w/v), 2 hours for the first time and 1 hour for the second time. The volatile oil was collected by the volatile oil extractor during the decocting procedure. The volatile oil and the solution obtained were concentrated to 450 grams. This extract represented the solution for rat administration and was stored at 4°C until use.
In the clinical study, we used another form of JTW for the convenience of oral administration. JTW, containing Rhizoma Coptidisand Cortex Cinnamomi concentrated granules, was purchased from China Resources Sanjiu Medical and Pharmaceutical Co., Ltd. Rhizoma Coptidis concentrated granules largely contain berberine (>104.0 mg/g, HPLC), coptisine (>26.0 mg/g, HPLC), palmatine (>26.0 mg/g, HPLC), and epiberberine (>17.0 mg/g, HPLC). Cortex Cinnamomi is mainly composed of cinnamaldehyde (>1.0 mg/g, HPLC) and cinnamic acid (>0.5 mg/g, HPLC). Each pack of JTW contains two grams of Rhizoma Coptidis concentrated granules (equal to six grams of Rhizoma Coptidis) and one gram of Cortex Cinnamomi concentrated granules (equal to three grams ofCortex Cinnamomi). Patients mixed the granules together by boiling water and drank two packs each day.
Berberine, cinnamaldehyde, and cinnamic acid, the main components in JTW extracts and concentrated granules, were all measured using the high-performance liquid chromatography (HPLC) method. HPLC (Waters, USA) equipped with Waters 600 Pump, and Waters 2487 Dual
The rats were fasted overnight and given a single dose of 50% glucose solution one week before being sacrificed. The glucose solution was administered by gavage at a dose of 2.0 g/kg body weight. Blood samples for glucose measurement were obtained from the tail vein at 0 hour (before glucose-loading), 1 hour, and 2 hours (after glucose-loading) by glucose-oxidase method using a glucose monitor (LifeScan Inc., J&J Company, Milpitas, CA, USA).
The plasma levels of total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), nonesterified fatty acid (NEFA), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were determined using commercial reagents (Jiancheng Bioengineering Institute, Nanjing, China). The fasting insulin concentration (FINS) was measured by radioimmunoassay kit (Northern Institute of Biotechnology, Beijing, China). The homeostasis model assessment index (HOMA-IR) was calculated using the formula of fasting glucose (mmol/L) × fasting insulin (
Serial 4
Protein lysates extracted from liver homogenates were subjected to 10% SDS-PAGE gel (100 v, 2 h–2.5 h) and then transferred to nitrocellulose membranes. The membranes were blocked with 5% (w/v) nonfat milk in tris-buffered saline with Tween 20 (TBST) for 2 h at room temperature. The blocking solution was then removed, and the membranes were washed by TBST, followed by overnight incubation at 4°C with the primary antibodies (rabbit anti-rat AMPK, pAMPK, ACC, pACC, and FAS) (Abcam, Hong Kong, China) and
Type 2 diabetic patients with poor glycemic control were enrolled in this study based on the following inclusion criteria: (1) aged 25–70 years; (2) use of stabilizing oral hypoglycemic drugs or lifestyle modification to control their blood glucose for at least 8 weeks; (3) fasting plasma glucose (FPG) 7.0–13.0 mmol or 2-hour postprandial blood glucose (2hPPG) 11.1–16.6 mmol/L and hemoglobin A1c (HbA1c) >6.5%; (4) no known acute or chronic disease based on history, physical examination, and standard laboratory tests; (5) alcohol consumption <20 g/day; and (6) no evidence of hepatitis A, B, or C as well as no evidence of autoimmune hepatitis, clinical signs or symptoms of inborn errors of metabolism, and no history of the use of toxins or drugs known to induce hepatitis. Exclusion criteria included thyroid disease, the use of antihypertensive agents that could possibly influence glucose metabolism (
A total of forty patients with T2DM were enrolled. None of patients withdrew or dropped out of the study during the 12 weeks. The patients were randomly divided into two groups: JTW group (treated with one pack of JTW two times a day for 12 weeks) and the control group. Their previous diabetes management was maintained during the 12-week experimental period except for the addition of JTW in patients of the JTW-treated group. The following indicators before and after treatment were measured: body mass index (BMI), waist circumference, waist-to-hip ratio (WHR), blood pressure (BP), FPG, 2hPPG, HbA1c, plasma TC, TG, LDL-C, HDL-C, ALT, and AST.
All of the patients received proton magnetic resonance spectroscopy (1H-MRS) scans of their livers before and after treatment, as described previously [
Normally distributed data are presented as the mean ± SD, whereas nonnormally distributed data are presented as the median with the 25th and 75th percentiles in parenthesis. Statistical significance was determined by one-way analysis of variance (ANOVA) followed by Dunnett’s T3 test for data with equal variances not assumed. For data with equal variances assumed, ANOVA followed by LSD test was used. Nonparametric tests were used to compare median values between the groups. Calculations were performed using SPSS 14.0 software. Statistical significance was defined as
The contents of berberine, cinnamaldehyde, and cinnamic acid in the JTW extract used in the animal experiments were 23.03 mg/g, 3.12 mg/g, and 0.27 mg/g, respectively. In clinical trials, the contents of berberine, cinnamaldehyde, and cinnamic acid in the JTW concentrated granules were 79.51 mg/g, 13.39 mg/g, and 1.06 mg/g, respectively. The chromatograms of berberine, cinnamaldehyde, and cinnamic acid in JTW were presented in Figure
The chromatograms of berberine, cinnamaldehyde, and cinnamic acid in JTW. (a) The chromatogram of berberine in JTW (peak A is berberine), (b) the chromatogram of cinnamaldehyde in JTW (peak B is cinnamaldehyde), and (c) the chromatogram of cinnamic acid in JTW (peak C is cinnamic acid).
As shown in Table
The effect of JTW on body weight, OGTT, insulin levels, and HOMA-IR in diabetic rats.
Group | Body weight | FPG | 1hPPG | 2hPPG | FINS | HOMA-IR |
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(g) | (mmol/L) | (mmol/L) | (mmol/L) | ( | ||
Control |
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Diabetic |
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JTW |
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Values are expressed as mean ± SD.
As shown in Table
The effect of JTW on plasma lipid profiles in diabetic rats.
Group | TC | TG | LDL-C | HDL-C | NEFA |
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(mmol/L) | (mmol/L) | (mmol/L) | (mmol/L) | (mmol/L) | |
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Diabetic |
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JTW |
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Met |
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Values are expressed as mean ± SD.
As shown in Table
The effect of JTW on plasma liver enzyme activity and HTC in diabetic rats.
Group | ALT | AST | HTC |
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(U/L) | (U/L) | ( | |
Control |
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JTW |
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Met |
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Values are expressed as mean ± SD.
The effect of JTW on liver pathology in diabetic rats. Representative H&E stained images in liver sections from the control group (a), diabetic group (b), JTW group (c), and metformin group (d). Original magnification ×400.
As shown in Figure
The effect of JTW on hepatic lipogenic gene expression in diabetic rats. pAMPK and AMPK protein expressions ((a), (c), and (e)), pACC and ACC protein expressions ((b), (d), and (f)), and FAS protein expression ((g), (h)). Values are expressed as mean ± SD. #
No significant differences were noted between the baseline markers of diabetic patients in the JTW group and the control group. Patients with poor glycemic control were treated with JTW for 12 weeks. These patients exhibited decreasing trend in their BMI, waist circumference, WHR, FPG, 2hPPG, FINS, HbA1c, TC, TG, and LDL-C at the end of the treatment period (
General characteristics and laboratory data of diabetic patients at baseline and 12 weeks after the therapy.
Group | Control | JTW | ||
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Subjects (M/F) | 9/11 | 9/11 | ||
Age (years) |
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BMI (kg/m2) |
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WC (cm) (F) |
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WC (cm) (M) |
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WHR (F) |
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WHR (M) |
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SBP (mmHg) |
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DBP (mmHg) |
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FPG (mmol/L) | 7.64 (6.20, 9.37) | 7.67 (6.41, 9.25) | 7.89 (6.10, 9.22) | 6.78 (4.01, 8.69) |
2hPPG (mmol/L) |
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FINS ( |
7.59 (4.33, 10.42) | 7.61 (4.57, 9.99) | 7.52 (4.11, 12.92) | 6.04 (4.20, 9.61) |
HbA1c (%) |
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TC (mmol/L) |
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TG (mmol/L) | 1.32 (1.05, 2.38) | 1.31 (1.01, 2.23) | 1.29 (1.02, 2.75) | 1.18 (0.94, 2.13) |
HDL-C (mmol/L) | 1.24 (0.98, 1.58) | 1.25 (1.03, 1.60) | 1.27 (1.03, 1.90) | 1.26 (0.96, 1.87) |
LDL-C (mmol/L) |
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ALT (U/L) | 26 (18, 35) | 25 (17, 35) | 21 (18, 40) | 24 (17, 35) |
AST (U/L) | 22 (15, 31) | 21 (16, 32) | 19 (11, 35) | 21 (10, 30) |
HTC (%) | 9.63 (5.87, 11.18) | 10.79 (6.75, 19.99) |
9.81 (5.76, 15.09) | 6.54 (4.82, 13.08) |
Normally distributed data are presented as the mean ± SD; nonnormally distributed data are presented as the median with the 25th and 75th percentiles in parenthesis.
No serious adverse events were observed when JTW was used. Plasma creatinine concentrations and electrocardiography (ECG) were also monitored in the clinical study. No significant changes in plasma creatinine were observed during the 12 weeks of JTW treatment. No severe hypoglycemia was observed.
Chinese medicine is an excellent alternative and complementary medicine in treating NAFLD. In traditional Chinese medicine, NAFLD falls into the category of symptoms caused by phlegm/dampness retention, qi/blood stasis, and spleen/kidney deficiency. Increasing evidence indicates that herbs that remove accumulated pathogenic phlegm and dampness are useful in treating NAFLD [
In the animal study, the diabetic rats exhibited the characteristics of hyperglycemia, hyperinsulinemia, and hyperlipidemia. Plasma liver enzymatic activity and HTC also significantly increased. Liver histology demonstrated severe fatty degeneration and inflammation infiltration. The oral administration of JTW not only attenuated the biochemical disturbances in peripheral blood but also alleviated TG elevation and the morphological changes in liver tissues. We suggest that JTW could be an effective herbal prescription to treat nonalcoholic steatohepatitis of diabetic rats. These results were further confirmed by the clinical study, with the exception of the effects of JTW on liver histology and enzymatic activity. Invasive liver biopsy was not used in the clinical study to obtain histology data for ethical considerations. As an alternative, we applied noninvasive 1H-MRS scans of liver as an assessment tool. Previous studies have demonstrated that, although this method gives no information of liver histology, HTC obtained by spectroscopy closely coincides with biopsy or autopsy-derived triglyceride concentrations [
With respect to the constituents of Rhizoma Coptidis and Cortex Cinnamomi, many substances have been identified. Among the constituents effective in treating diabetes, berberine (which is contained in Rhizoma Coptidis), cinnamaldehyde, and cinnamic acid (which are contained in Cortex Cinnamomi) have attracted significant attention [
However, one limitation of our study must be considered. The effect of JTW on the fatty liver was evaluated using different formulations in the animal and clinical studies. In the animal experiments, the traditional Chinese herbal decoction was used, whereas concentrated herbal granules were chosen in the clinical trial. These different preparations account for the differences in the content of berberine, cinnamaldehyde, and cinnamic acid in JTW detected by HPLC. However, the daily dosage of JTW intake in diabetic rats was nearly identical to that in diabetic patients. Herbal granules, extracted from herbs with modern pharmaceutical technology, are convenient for those who cannot make an herbal decoction every day. We believe that the application of JTW granules assures quality control in the clinical trials. Nonetheless, we cannot confirm that there are any other differences in the constituents of JTW without the process of boiling. However, we have provided preliminary data that suggest that JTW is promising in the treatment of NAFLD.
In summary, our study demonstrates that oral treatment with JTW attenuates hepatic lipid accumulation in diabetic rats and humans. These beneficial effects are possibly associated with the inhibition of lipogenic gene expression in the liver.
Acetyl coenzyme A carboxylase
Alanine aminotransferase
AMP-activated protein kinase
Body mass index
Diastolic blood pressure
Fatty acid synthase
Free fatty acid
Fasting insulin level
Fasting plasma glucose
Hemoglobin A1c
High-density lipoprotein cholesterol
Proton magnetic resonance spectroscopy
Homeostasis model assessment index
High-performance liquid chromatography
2-hour postprandial blood glucose
Hepatic triglyceride content
Impaired glucose tolerance
Jiao Tai Wan
Low-density lipoprotein cholesterol
Metformin
Nonalcoholic fatty liver disease
Nonesterified fatty acid
Oral glucose tolerance test
Phosphorylated-ACC
Phosphorylated-AMPK
Systolic blood pressure
Streptozotocin
Total cholesterol
Type 2 diabetes mellitus
Triglycerides
Waist circumference
Waist-to-hip ratio.
The authors declare no conflict of interests.
Zhaoyi Huang and Xiaohu Xu contributed equally to this work.
This work was supported by Grants from National Natural Science Foundation of China (nos. 81273683, 30801492).