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The anti-diabetic effects and pharmacokinetic profiles of berberine in mice treated with Jiao-Tai-Wan and its compatibility
Phytomedicine 20 (2013) 780–786
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The anti-diabetic effects and pharmacokinetic profiles of berberine in mice treated with Jiao-Tai-Wan and its compatibility Guang Chen a , Fuer Lu b,∗ , Lijun Xu b , Hui Dong b , Ping Yi a , Fang Wang c , Zhaoyi Huang a , Xin Zou b a b c
Department of Integrative Traditional & Western Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science & Technology, Wuhan 430030, China Institute of Integrative Traditional & Western Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science & Technology, Wuhan 430030, China School of Medical and Life Sciences, Jianghan University, Wuhan 430056, China
a r t i c l e
i n f o
Keywords: Jiao-Tai-Wan Berberine Diabetes mellitus Pharmacokinetic differences Rhizoma Coptidis Cortex Cinnamomi cassiae
a b s t r a c t Jiao-Tai-Wan (JTW), a classical Chinese prescription, has been clinically employed to treat diabetes mellitus in recent years. To investigate the comparative evaluations on anti-diabetic effects and pharmacokinetics of the active ingredient berberine in mice treated with JTW in various combinations of its constituent herbs. In our study, the anti-diabetic study was carried out in diabetic mice induced by intraperitoneal injection of streptozotocin. The diabetic mice were randomly assigned to three therapy groups and orally administered with different prescription proportions of Rhizoma Coptidis and Cinnamomum cassia respectively. The level of plasma glucose, lipid profile and parameters related to oxidative stress were determined. The concentrations of berberine in non-diabetic mice plasma were determined using HPLC, and main pharmacokinetic parameters were investigated. The results indicated that the compatibility effects of ingredients present in Cinnamomum cassia could affect the anti-diabetic ability and pharmacokinetics of berberine in JTW. © 2013 Elsevier GmbH. All rights reserved.
Introduction Glycaemia and diabetes are rising globally, driven both by population growth and aging and by increasing age-specific prevalences (Danaei et al. 2011). The total number of adults (aged 20–79 years) globally estimated to have diabetes by the International Diabetes Federation (IDF) in 2011 is about 366 million (Whiting et al. 2011). For this reason, pharmacologic agents that control diabetes mellitus have received considerable attention. In China, herbal medicines have been widely used in the treatment of diabetes mellitus and its complications before the discovery of insulin. Jiao-Tai-Wan (JTW) is an important herbal formula in traditional Chinese medicine (TCM). JTW was first described by Han Yi (in the Chinese Ming Dynasty) in his treatise
Abbreviations: FBG, fasting blood glucose; OGTT, oral glucose tolerance test; BG, blood glucose; TC, total cholesterol; TG, triglyceride; LDL-c, low density lipoprotein; HDL-c, high density lipoprotein; Apo B, apolipoprotein B; STZ, streptozotocin; wt, weight; SD, standard deviation; TCM, traditional Chinese medicine; HPLC, high performance liquid chromatograph; AUC, area under curve; t1/2 , half-life; MRT, mean residence time; DM, diabetes mellitus; JTW, Jiao-Tai-Wan; CMC, Na carboxymethyl cellulose sodium salt. ∗ Corresponding author at: Institute of Integrative Traditional & Western Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science & Technology, Jiefang Road 1095#, Wuhan 430030, China. Tel.: +86 27 83663237; fax: +86 27 83663237. E-mail address: [email protected] (F. Lu). 0944-7113/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.phymed.2013.03.004
“Han Si Yi Tong”. This prescription, consisted of Rhizoma Coptidis and Cortex Cinnamomi cassiae, was mainly applied to treat insomnia in the past hundreds of years. However, a lot of clinical and laboratory researches have drawn people’s attention to its therapeutic effects on diabetes mellitus in the recent years (Jiang et al. 2001). The traditional remedy is a pill of Rhizoma Coptidis (Coptis chinensis Franch, Ranunculaceae) and Cortex Cinnamomi (Cinnamomum cassia, Lauraceae) with the ratio of 10:1 and both the herbs are officially listed in the Chinese pharmacopeia (Pharmacopoeia of PR China 2005). It is speculated that the ratio of Rhizoma Coptidis and Cinnamomum cassia could be changed in treating diabetes mellitus. Yet, data are still quite deficient to define precisely what ratio is superior or whether the mixture of two herbs is prior to single herb. Rhizoma Coptidis (Huang-Lian in Chinese), a key ingredient herb in JTW (Minister), has been widely used in TCM for the treatment of intestinal infection, fever, hypertension, tumor, etc. (Yu et al. 2006). Modern pharmacology has confirmed that isoquinoline alkaloids are the main active ingredients. Among them, berberine, the main isoquinoline alkaloid, has been used as a phytochemical marker for the quality control of Rhizoma Coptidis in Chinese pharmacopeia (Pharmacopoeia of PR China 2005). Pharmacological studies have indicated that berberine shares many beneficial activities with respect to antidiarrhea, antitumour and antimicrobial properties (Amin et al. 1969; Anis et al. 2001; Jantova et al. 2003; Taylor and Greenough 1989). Recently many researches have indicated that berberine possesses anti-diabetic effect (Leng et al. 2004;
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Lee et al. 2006; Tang et al. 2006), but the mechanism of its action is not quite clear. It is generally assumed that berberine has low bioavailability in human and rats (Sheng et al. 1993), resulting in poor antihyperglycaemic effect. Given the multi-ingredient character of herbal medicines, the likelihood of herb–drug interactions is theoretically higher than drug–drug interactions (Lu et al. 2007). In the recent years, multiple cases of herb–drug interactions have been reported (Hu et al. 2005; Izzo 2005). Herb–herb interaction has also been found in the decocting process of Rhizoma Coptidis and Cinnamomum cassia (Zhou et al. 1999; Xu et al. 2001). The aim of this study is to explore whether the Cinnamomum cassia in JTW can affect the pharmacokinetic behavior of berberine in Rhizoma Coptidis (herb–herb interaction). Due to the lack of standards of other conjugated metabolites, berberine was selected to compare the pharmacokinetic differences after oral administration of Rhizoma Coptidis, Jiao-Tai-Wan (Rhizoma Coptidis:Cinnamomum cassia = 1:0.25, JTW1) and Jiao-Tai-Wan (Rhizoma Coptidis:Cinnamomum cassia = 1:0.5, JTW2). We supposed that Cinnamomum cassia might influence the absorption of berberine and consequently affect the antihyperglycaemic effect in Rhizoma Coptidis, JTW1 and JTW2. To test this hypothesis, pharmacokinetic differences of berberine following oral administration of Rhizoma Coptidis, JTW1 and JTW2 were investigated in male Kunming mice. Materials and methods Materials Streptozotocin (STZ) was purchased from Sigma–Aldrich Inc. (USA). The detection kits of total cholesterol (TC), triglycerides (TG), high density lipoprotein-cholesterol (HDL-c), low density lipoprotein-cholesterol (LDL-c) and apolipoprotoein B (Apo B) were purchased from Wenchow Dong-Ou Bioengineering Company Ltd. (China). The detection kits of NO, GSH-px, SOD activities and MDA in plasma were purchased from Jiancheng Biotechnology Co., Nanjing, China. Berberine standard was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Ethylether and acetonitrile were of chromatographic grade, and other chemicals were of analytical grade.
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5 m) eluted with the mobile phases of 3 mM sodium dodecyl sulfate in 0.1% phosphoric acid (A) and acetonitrile (B) with isocratic eluting (A:B = 64:36). And the flow rate was 1.0 ml/min, the detection wavelength was 265 nm. Detection of berberine content in herbal extracts To calculate the dose administered to mice, content of berberine in Rhizoma Coptidis, JTW1 and JTW2 extracts were quantitatively determined. The aqueous extracts of Rhizoma Coptidis (1.87 g), JTW1 (2.5 g) and JTW2 (5 g) were extracted respectively by 100 ml of 1:100 mixture of hydrochloric acid and methanol at 60 ◦ C for 15 min, ultrasounded for 30 min and then diluted 100 times. The mixture was centrifuged for 10 min at 15,000 rpm. Ten microliters of the supernatant was injected into the HPLC system. The HPLC analysis of the berberine was a modified version of a previously published method (Qu et al. 2007). Berberine contents were determined to be 2.61% in Rhizoma Coptidis extract, 1.60% in JTW1 extract and 0.69% in JTW2 extract, respectively. Induction of experimental diabetic mice The animal studies were overseen and approved by the Animal Ethics Committee of Tongji Medical College, Huazhong University of Science & Technology before and during the experiment. Male kunming mice, weighing 30–40 g, provided by Hubei Province Experimental Animal Center (Grade SPF, Certificate No. SCXK (E2003-0005)) (Wuhan, China), were employed in the research. Animals were kept in an environmentally controlled breeding room (temperature: 20 ± 2 ◦ C, humidity: 60 ± 5%, 12-h dark/light cycle) with 4 mice per cage. All the mice had free access to food and water. The mice were adapted to diet for 2 weeks before beginning of the experiment. After a 16 h fasting, diabetes was induced by intraperitoneal injection of streptozotocin at 60 mg/kg in citrate buffer (0.1 mol/l sodium citrate and 0.1 mol/l citric acid, pH 4.5) for 3 consecutive days. The blood glucose levels were detected using the glucose-oxidase method on the third day after STZ injection. Animals with blood glucose above 16.7 mmol/l were considered as being diabetic and were taken for the experimenta l tests. Animals grouping and treatment
Preparation of herbal extracts Rhizoma Coptidis and Cinnamomum cassia were mixed in the ratio of 1:0.25 (JTW1) or 1:0.5 (JTW2), and the weight of Cinnamomum cassia was 45 g. The mixture was decocted by refluxing with water (1:10, w/v) for two times, 2 h for the first time and 1 h for the second time, respectively. Combined decoctions were filtered, and the solution obtained was concentrated to 450 g Cinnamomum cassia (45 g) and Rhizoma Coptidis (270 g) were extracted in the same way as above and gave two extracts of 225 g and 505 g. The extracts were stored at 4 ◦ C before use. All composed decoction pieces in JTW were purchased from Hubei Province Traditional Chinese Medicine Company (Wuhan, China). HPLC fingerprint of the extracts HPLC fingerprint was performed with the extracts of JTW1, JTW2, Rhizoma Coptidis and Cinnamomum cassia to identify the main chemical constituents in the samples. Berberine, coptisine, jateorhizine, palmatine, cinnamaldehyde and cinnamic acid were taken as standard substances. The extracts was dissolved in water at concentration of 5, 5, 5, 2 g/ml (w/v), and then diluted with methanol–water (50:50) to 2.5, 2.5, 2.5, 1 g/ml (w/v). HPLC analysis was performed on an outstand C18 column (4.6 mm × 250 mm,
Animals were randomly divided into 5 groups (n = 6 in each group): normal control, diabetic control, and three therapy groups. The mice in therapy groups were treated separately with Rhizoma Coptidis, JTW1, JTW2 extracts for 4 weeks. The therapy diabetic mice were gavaged by different aqueous extracts such as Rhizoma Coptidis extract (Rhizoma, contained berberine at the dosage of 100 mg/kg/d), JTW1 (contained berberine at the dosage of 100 mg/kg/d), JTW2 (contained berberine at the dosage of 100 mg/kg/d) respectively and correspondingly, and one time at 8:30–9:30 am per day. The diabetic control mice were gavaged by 0.5% carboxymethyl cellulose sodium salt (CMC-Na). The above extracts were prepared with 0.5% CMC-Na. Raw power solution or distilled water was fed by gavage administration once a day during the experiment period, respectively. The body weight and water consumption of all the mice were recorded every day. The blood glucose levels of the mice were monitored before and at 0, 1, 2, 3 and 4 weeks after STZ injection. The blood glucose levels were measured by the glucose-oxidase method. Biochemical analysis At the end of the experiment (4 weeks), the blood was taken from the retro-orbital plexus of the eye under mild ether
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Fig. 1. HPLC fingerprint chromatograms of the extracts of JTW1(A), JTW2(B), Rhizoma Coptidis(C) and Cinnamomum cassia(D). In the chromatograms: (1) cinnamic acid; (2) cinnamaldehyde; (3) jateorhizine; (4) coptisine; (5) palmatine and (6) berberine.
anesthesia and was prepared for measurement of insulin, lipid profile and parameters related to oxidative stress. The insulin level in plasma was estimated using commercially available kits. TC and TG were detected by enzyme end-point method, HDL-c and LDL-c by PTA-Mg2+ method and Apo B by immunoturbidimetric method. All the above targets were determined with semi-automatic biochemistry analysator (Shandong Rainbow Company, China). Serum insulin level was estimated by radioimmunoassay kit (Beijing Institute of Atomic Energy, Beijing, China). NO, GSH-px, SOD and MDA levels in plasma were estimated using commercially available kits. Pharmacokinetic study of berberine in mice After 2 weeks of adaptive feeding, male Kunming mice were randomly and averagely divided into 3 groups. The mice were fasted for 12 h, but were given access to water prior to the oral administration of 3.83 g/kg Rhizoma Coptidis extract (100 mg berberine/kg), 6.25 g/kg JTW1 (100 mg berberine/kg) and 14.49 g/kg JTW2 (100 mg berberine/kg). The dose of berberine to mice was referred to a former report (Shen and Xie 1993). All above extracts were prepared with 0.5% CMC-Na. Blood samples (1.2 ml) were collected from the eyes under aether anesthesia at the time points of 0.167, 0.25, 0.333, 0.5, 1, 1.5, 2, 2.5, 3, 5, 8 and 12 h after administration, then were put into 1.5 ml EP tube and centrifugated for 10 min at 3000 rpm in 4 ◦ C to separate serum. The serum was stored at −70 ◦ C after separation until assayed as described below. Serum samples (400 l) were alkalified with approximately 200 l of 0.1 mol/l NaOH added to each of them. Then, each portion
was shaken with 6 ml of ethylether for 10 min and centrifuged at 2500 rpm for 1 min, and the organic layer was transferred into an empty tube. This procedure was repeated for three times and the organic layer collected was dried at 35 ◦ C under a nitrogen stream. The residue was dissolved in 100 l mobile phase. After centrifuged at 15,000 rpm for 10 min, 50 l supernatant was analyzed with Waters High Performance Liquid Chromatography (HPLC) system consisting of a Waters 600 controller, Waters 600 pump, Waters 2487 dual absorbance and 717 plus autosampler (Waters Assoc., Milford, MA, USA). The chromatograph condition is as follows, chromatographic column: XTerra® RP18 (4.6 mm × 250 mm, 5 m); mobile phase: 0.1 mol/l KH2 PO4 , 0.05 mol/l NaOH (adjusted to a pH of 10 with triethylamine) – acetonitrile (30:70, v/v); flow rate: 0.8 ml/min; the detection wavelength: 345 nm; Column temperature: 40 ◦ C. The LLOQs (lower limit of quantification) of the assay was 6.4 g/l for berberine. Good linearity was obtained from 10 to 640 g/l for berberine.
Statistical analysis Data were expressed as the mean ± standard deviation (SD). Statistical analysis was performed using the SPSS Version 13.0 (SPSS Inc., Chicago, IL). Differences in numerical variables among groups were analyzed with one-way analysis of variance (ANOVA) LSD-t and SNK-q. A P value of less than 0.05 was considered statistically significant. The obtained berberine concentration was analyzed with DAS (Drug and Statistics) 2.0 pharmacokinetic program (Chinese Pharmacology Society) to obtain the relative pharmacokinetic parameters.
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Table 1 Effects of oral administration with the aqueous extracts of Rhizoma, JTW1 and JTW2 on fasting blood glucose in diabetic mice. Group
Week 0
Normal Diabetic Rhizoma JTW1 JTW2
4.9 19.3 19.1 19.4 19.2
± ± ± ± ±
Week 1 1.2 2.5** 2.3 2.5 2.2
5.1 19.2 18.7 18.3 18.5
± ± ± ± ±
Week 2 1.5 2.9** 2.4 2.2 2.3
5.4 20.5 17.5 16.6 15.8
± ± ± ± ±
Week 3 1.6 3.1** 2.1# 2.5# 2.1##
5.3 21.9 16.4 15.2 13.6
± ± ± ± ±
Week 4 1.5 3.3** 2.6## 2.2## 1.9##
5.3 20.8 15.7 13.5 11.8
± ± ± ± ±
1.7 3.5** 2.6## 2.3## 2.1##
Values are given as means ± SD of 6 mice. *p < 0.05, compared with normal control group. ** p < 0.01, compared with normal control group. # p < 0.05, compared with diabetic control group. ## p < 0.01, compared with diabetic control group.
Table 2 Effects of oral administration with the aqueous extracts of Rhizoma, JTW1 and JTW2 on insulin level and serum lipid profile in diabetic mice. Group
Insulin (mU/l)
Normal Diabetic Rhizoma JTW1 JTW2
44.37 26.16 29.92 34.26 38.45
± ± ± ± ±
7.56 2.54** 5.07 6.92## 6.63##
TG (mmol/l) 0.52 0.94 0.85 0.75 0.68
± ± ± ± ±
TC (mmol/l)
0.14 0.21** 0.16 0.19 0.18#
1.22 2.09 1.75 1.51 1.33
± ± ± ± ±
HDL-c (mmol/l)
0.36 0.53** 0.42 0.39# 0.32##
1.57 0.79 0.86 0.95 1.03
± ± ± ± ±
0.39 0.17** 0.22 0.20 0.24#
LDL-c (mmol/l) 0.65 1.41 1.13 1.08 0.96
± ± ± ± ±
0.12 0.32** 0.25 0.22# 0.16##
ApoB (g/l) 0.67 1.46 1.12 0.91 0.73
± ± ± ± ±
0.13 0.25** 0.22# 0.18## 0.16##
Values are given as means ± SD of 6 mice. *p < 0.05, compared with normal control group. ** p < 0.01, compared with normal control group. # p < 0.05, compared with diabetic control group. ## p < 0.01, compared with diabetic control group.
Results
Influence of extracts on serum insulin level and lipid metabolic parameters in diabetic mice
HPLC fingerprints of extracts The HPLC fingerprint chromatograms of JTW1, JTW2, Rhizoma Coptidis and Cinnamomum cassia are shown in Fig. 1. By comparing both the retention times and the UV spectra of the reference standards, six compounds (cinnamic acid, cinnamaldehyde, jateorhizine, coptisine, palmatine, berberine) in the extracts were well identified.
The levels of TC, TG, LDL-c and Apo B in mice treated with Rhizoma, JTW1 and JTW2 were lower than those in the diabetic control mice, while the levels of insulin and HDL-c were higher (Table 2, p < 0.05 or p < 0.01). Among them, the antihyperlipidemic effect was the most remarkable in the JTW2 group (Table 2).
Influence of extracts on blood glucose in diabetic mice
Influence of extracts on NO, GSH-px, SOD, MDA and CAT activities in diabetic mice
The levels of plasma glucose in diabetic mice injected by STZ (60 mg/kg) were increased significantly (19.3 ± 2.5 mM vs. 4.9 ± 1.2 mM, p < 0.01), compared to those in normal mice. Administration of Rhizoma, JTW1 and JTW2 for 4 weeks significantly reduced the blood glucose of diabetic mice (Table 1, p < 0.05 or p < 0.01). In addition, JTW1 and JTW2 groups showed a statistically significant difference of serum glucose level compared with the Rhizoma group (Table 1). Among them, the antihyperglycaemic effect was the most remarkable in the JTW2 group.
It was shown that the activities of NO, GSH-px, SOD and CAT were significantly decreased in diabetic group compared to normal group (Table 3, p < 0.01). Rhizoma, JTW1 and JTW2 could increase the activities of NO, GSH-px, SOD and CAT (Table 3, p < 0.05 or p < 0.01). A significant increase in NO, GSH-px, SOD and CAT activities in plasma were observed in the JTW1 and JTW2 group compared with those of diabetic group (Table 3, p < 0.01). The MDA level in plasma was raised by 65.52% in STZ-diabetic mice compared to that in normal control mice (p < 0.01). The administration of Rhizoma, JTW1 and JTW2 reduced the increased MDA level (Table 3,
Table 3 Effects of oral administration with the aqueous extracts of Rhizoma, JTW1 and JTW2 on plasma NO, GSH-px, SOD, MDA and CAT activity in diabetic mice. Group
NO (mol/l)
Normal Diabetic Rhizoma JTW1 JTW2
24.52 15.37 18.44 20.58 21.96
± ± ± ± ±
3.76 2.24** 2.86# 2.78## 3.51##
Values are given as means ± SD of 6 mice. *p < 0.05, compared with normal control group. ** p < 0.01, compared with normal control group. # p < 0.05, compared with diabetic control group. ## p < 0.01, compared with diabetic control group.
GSH-px (U/ml) 114.88 73.75 80.88 85.49 98.85
± ± ± ± ±
10.15 7.28** 7.89 8.55# 8.83##
SOD (U/ml) 186.95 139.86 155.74 165.92 176.55
± ± ± ± ±
16.63 12.67** 14.59# 13.85## 14.68##
MDA (nmol/l) 5.83 9.65 8.58 8.02 6.17
± ± ± ± ±
0.62 0.79** 0.67# 0.65## 0.59##
CAT (U/ml) 27.63 14.34 17.56 20.16 23.67
± ± ± ± ±
3.85 2.52** 2.59# 2.75## 3.06##
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Table 4 Pharmacokinetic differences of oral administration of the aqueous extract of Rhizoma Coptidis (100 mg/kg berberine), JTW1 (100 mg/kg berberine) and JTW2 (100 mg/kg berberine) to mice (n = 5). Parameters
Rhizoma Coptidis
JTW1
JTW2
AUC0–t (g h/l) AUC0–∞ (g h/l) AUMC0–t AUMC0–∞ MRT0–t (h) MRT0–∞ (h) VRT0–t (h2 ) VRT0–∞ (h2 ) t1/2 ˛ (h) t1/2 ˇ (h) Tmax (h) Cmax (g/l) CLz/F (l/h/kg) Vz/F (l/kg)
742.92 789.91 3193.19 3968.84 4.29 5.02 8.89 17.86 0.662 7.09 1.0 202.79 126.59 565.97
1309.20 1507.69 5393.52 6959.12 4.12 4.62 7.61 20.09 3.75 3.75 2.0 241.27 66.33 368.54
1562.25 1670.67 6533.02 8267.06 4.18 4.95 7.96 17.04 0.59 3.08 2.0 290.73 59.86 242.77
p < 0.05 or p < 0.01) and JTW2 group showed no significant difference compared with normal control (p > 0.05). Pharmacokinetic comparison of berberine in mice after oral administration of Rhizoma Coptidis and JTW1 Serum concentration of berberine was determined after oral administration of Rhizoma Coptidis extract to mice and the serum profile demonstrated bimodal phenomenon (Fig. 3). The first peak occurred at about 0.25 h and the second at 1 h. Compared with Rhizoma Coptidis extract, bimodal phenomenon of berberine in mice serum also existed, but the concentration of the second peak and the values of AUC(0–∞) /dose were significantly increased after oral administration of JTW1, and two peak times were delayed (Table 4 and Fig. 2). These results indicated that existence of some ingredients in JTW1 might affect the pharmacokinetic behavior of berberine. Pharmacokinetic comparison of berberine in mice after oral administration of JTW1 and JTW2 The results of the above researches indicated that Cinnamomum cassia increased the absorption of berberine. We thought that this effect was resulted from the interaction between Rhizoma Coptidis and Cinnamomum cassia. In order to determine whether Cinnamomum cassia could contribute to the increase of berberine absorption, we compared the pharmacokinetics of berberine with 400
Serum concentration (µg/L)
350 300 250 200 150 100 50 0
0
2
4
6
8
10
12
14
Time(h) Fig. 2. Mean serum concentration–time curves of berberine after oral administration of the Rhizoma Coptidis aqueous extract (100 mg/kg berberine) (䊉), JTW1 (100 mg/kg berberine) ( ) and JTW2 (100 mg/kg berberine) ( ) to mice (n = 5).
JTW1 and JTW2 in mice. The serum concentrations of berberine were determined and pharmacokinetic parameters were estimated (Table 4). Results were also compared with those acquired from mice given Rhizoma Coptidis extract, which contains the same dose of berberine (Fig. 2). As shown in Fig. 2 and Table 4, the serum profile still demonstrated bimodal phenomenon after intake of JTW2, which confirmed that the ability of rapid absorption of berberine resulting in the appearance of the first peak. Similar to the mice given JTW1, higher serum concentration and increased AUC(0–∞) of berberine were found in mice given JTW2 compared to normal control. These results further suggested that ingredients in Cinnamomum cassia affected the pharmacokinetic behavior of berberine.
Discussion With multiple applications and remarkable curative effect, Rhizoma Coptidis and Cinnamomum cassia are commonly used in clinical practice of TCM. Rhizoma Coptidis, the representative medicine for heat-clearing and detoxicating, has been used to treat diabetes for thousands of years in China. In the past few years, the research work in this field is much more popular. However, the clinical applications of Rhizoma Coptidis were limited due to its mild antihyperglycaemic effect. Recently, Anderson reported that, Cinnamomum cassia, a drug for dispelling internal cold, has favorable curative effect on diabetes mellitus. We assumed Cinnamomum cassia could strengthen the therapeutic function of Rhizoma Coptidis on diabetes mellitus. Therefore, this study compared the effects of Rhizoma Coptidis and JTW composed of different proportions of Rhizoma Coptidis and Cinnamomum cassia on serum glucose level and lipid profile in diabetic mice. In this study, fasting blood glucose in the diabetic mice was significantly higher than those in the normal mice, indicating that the diabetic mice met the characteristics of diabetes mellitus. The results of this study showed that all of Rhizoma Coptidis, Cinnamomum cassia and JTW could decrease fasting blood glucose level, and improve insulin level. Moreover, the effect of JTW was superior to that of single Rhizoma Coptidis or Cinnamomum cassia. Among treatment groups, the antidiabetic effect in JTW2 group was the best. These results indicated that the compatibility of Cinnamomum cassia and Rhizoma Coptidis could strengthen Rhizoma Coptidis’s antihyperglycaemic effect. Lipids play a vital role in the pathogenesis of diabetes mellitus. The most common lipid abnormalities in diabetes are the increase in TC, TG, LDL-c, Apo B and fall of HDL-c accompanied with the rise in blood sugar (Sharma et al. 2003). In this study, we have noticed elevated levels of serum lipids such as cholesterol and triglycerides in diabetic mice. Meanwhile, it is encouraging that the treatment of Rhizoma Coptidis and JTW lowered the elevated levels of TC, TG, LDL-c and Apo B in diabetic mice. There was increase in HDL-c also, which indicated that Rhizoma Coptidis and JTW might be beneficial to diabetic individuals with coronary heart disease for HDL-c acts as a protective factor against coronary heart disease. In parallel with the above antihyperglycaemic effects, the antihyperlipidemic effect in JTW2 was the best among treatment groups. Oxidative stress in diabetes is coupled to a decrease in the antioxidant status, which can increase the deleterious effects of free radicals. We observed that activities of SOD, GSH-px and CAT in the treated diabetic groups were increased significantly, compared with diabetic group. This suggested that Rhizoma Coptidis and JTW had exerted the antioxidant effect by activation of SOD, GSH-px and CAT activity, at least in part, Rhizoma Coptidis and JTW decrease lipid peroxidation products and MDA in plasm. Our data indicated that Rhizoma Coptidis and JTW could increase NO production of diabetic mice in serum. Rhizoma Coptidis and JTW might
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Fig. 3. Typical chromatograms for the determination of berberine in serum samples: (A) chromatogram of a blank serum sample, (B) chromatogram of a serum sample spiked with berberine, and (C) chromatogram of the serum sample of a mouse taken 1 h after the oral administration of the Rhizoma Coptidis aqueous extract.
prevent the oxidative damage and increase a protective effect on postponing the cause of diabetic complications. Massive clinical and experimental studies have already proved that berberine was the active component of Rhizoma Coptidis to decrease blood glucose (Chen and Xie 1986; Ni 1988; Leng et al. 2004; Lee et al. 2006; Tang et al. 2006), while that of Cinnamomum cassia was indefinite. Therefore, this experiment only studied the pharmacokinetics of berberine to explore the causes underlying the difference of antihyperglycaemic and antihyperlipidemic effects in the treated groups. So far, the facts that berberine shows poor absorption through the gut wall and has low bioavailability have been regarded as key factors leading to unsatisfied clinical curative effect on diabetes mellitus. Some researches reported that the bioavailability of berberine is usually lower than 5% and one of the main factors leading to poor absorption is due to the external secretion of entero-epithelium P-glucoprotein. Hence, using P-glucoprotein catastaltica could improve the absorption of berberine (Pan et al. 2003). Our data showed that berberine demonstrated bimodal phenomenon in the serum profile, the weight was 1/C2 , indicating that the endosomatic metabolic process of berberine was confirmed to the two-compartment model. There was berberine
absorption in mice body after oral administration with Rhizoma Coptidis extract or JTW, and the bimodal model was observed. The Cmax and AUC(0–∞) of the JTW were both higher than those of Rhizoma Coptidis extract. It was reported that, AUC(0–∞) and Cmax /AUC were used as indexes for absorption extent and absorption rate respectively (Endrenyi et al. 1991). The AUC(0–∞) among the three groups were significantly different, while Cmax /AUC was similar among the groups, indicating that Cinnamomum cassia promoted the absorption extent of berberine rather than affect its absorption rate. The Cmax , AUC(0–t) , AUC(0–∞) were significantly different among the 3 groups, reaching their maximums in JTW2 group. Our data demonstrated that with the rise of Cinnamomum cassia proportion, the bioavailability of berberine increased significantly in mice body. It might because some components in the Cinnamomum cassia enhanced the berberine absorption when used together with Rhizoma Coptidis, which resulted in increased blood berberine’s concentration in mice body relative to single use of Rhizoma Coptidis, and enhanced the therapeutic effects of berberine. Cinnamomum cassia showed no significant influence on other behaviors in the pharmacokinetics of berberine in mice. To conclude, Cinnamomum cassia facilitates the antidiabetic effects
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of Rhizoma Coptidis in two ways, firstly, Cinnamomum cassia in itself could improve insulin resistance and promote glucose absorption and utilization as well as modulate lipid metabolism (Khan et al. 2003; Kim et al. 2006); secondly, according to this study, Cinnamomum cassia enhances berberine’s absorption after oral administration. Rhizoma Coptidis and Cinnamomum cassia is the classic medicine couple in TCM. It had been reported that precipitation phenomena occurred in decocting process of TCM containing this medicine couple (Zhou et al. 1999; Xu et al. 2001). In this study, we also found that Cinnamomum cassia could decrease the content of berberine in decocting process (data not shown). Combining the experimental data with literature reported, we could conclude that the mechanism of precipitation phenomena might be the complexation between the main active ingredients, phenolic acids and alkaloid, result the decreased contents of these active ingredients significantly. From this point of view, the combination of Rhizoma Coptidis and Cinnamomum cassia was rather unsuitable. On the other hand, however, under the guidance of the theory of TCM, Cinnamomum cassia could enhance the efficacy (“restoring normal coordination between heart and kidney”) and decrease the adverse effect (“wounding stomach with bitter and cold factors”) of Rhizoma Coptidis, which were confirmed by the clinical application for hundreds of years and by modern pharmacology (Cui and Zhao 2003). In addition, this study also indicated that Cinnamomum cassia could enhance the berberine’s absorption in mice. The huge number of active ingredients in TCM makes it suitable for multi-target actions. Different from Western medicine, TCM focuses on the overall functional state of the patient, which is becoming a trend in modern medicine for treating complicated diseases (Jiang 2005). Therefore, considering this aspect, the multiherb remedy might be reasonable prescription in spite of the existence of chemical interaction between Rhizoma Coptidis and Cinnamomum cassia. Moreover, our experimental data indicated that the better antidiabetic effects of JTW1 and JTW2 were related to the increased berberine bioavailablity. This result further confirmed that the multiherb remedy might be reasonable. Conclusions Our findings suggested that Cinnamomum cassia improved the antidiabetic effect of Rhizoma Coptidis in diabetic mice and berberine absorption in mice after oral administration with JTW extract, though the complexation between phenolic acids in Cinnamomum cassia and alkaloids in Rhizoma Coptidis might decrease the exposure of berberine. Berberine demonstrated bimodal phenomenon in the serum profile. Some ingredients in Cinnamomum cassia had pharmacokinetic interaction with berberine. Although we still cannot ascertain what the ingredient is or whether the material generated by interaction between two herbs influence the absorption of berberine, the mechanism for the increased berberine absorption is worthy of consideration. It should be worthwhile to find out if there is some type of p-glycoprotein suppressant at work as well. All these are still ambiguous and further profound researches are required. Acknowledgement This work was supported by the National Natural Science Foundation of China (No. 30801492). References Amin, A.H., Subbaiah, T.V., Abbasi, K.M., 1969. Berberine sulfate: antimicrobial activity, bioassay, and mode of action. Canadian Journal of Microbiology 15, 1067–1076.
Anis, K.V., Rajeshkumar, N.V., Kuttan, R., 2001. Inhibition of chemical carcinogenesis by berberine in rats and mice. Journal of Pharmacy and Pharmacology 53, 763–768. Chen, Q.M., Xie, M.Z., 1986. Studies on the hypoglycemic effect of Coptis chinensis and berberine. Yao Xue Xue Bao 21, 401–406. Committee of the Pharmacopoeia of PR China, 2005. Pharmacopoeia of PR China Part I. Chemical Industry Press, Beijing213–214. Cui, S.R., Zhao, L.P., 2003. Applied in conjunction with Rhizoma Coptidis. He Bei Zhong Yi 25, 758–759. Danaei, G., Finucane, M.M., Lu, Y., Singh, G.M., Cowan, M.J., Paciorek, C.J., Lin, J.K., Farzadfar, F., Khang, Y.H., Stevens, G.A., Rao, M., Ali, M.K., Riley, L.M., Robinson, C.A., Ezzati, M., 2011. National, regional, and global trends in fasting plasma glucose and diabetes prevalence since 1980: systematic analysis of health examination surveys and epidemiological studies with 370 country-years and 2.7 million participants. Lancet 378, 31–40. Endrenyi, L., Fritsch, S., Yan, W., 1991. Cmax /AUC is a clearer measure than Cmax for absorption rates in investigations of bioequivalence. International Journal of Clinical Pharmacology, Therapy and Toxicology 29, 394–399. Hu, Z., Yang, X., Ho, P.C., Chan, S.Y., Heng, P.W., Chan, E., Duan, W., Koh, H.L., Zhou, S., 2005. Herb–drug interactions: a literature review. Drugs 65, 1239–1282. Izzo, A.A., 2005. Herb–drug interactions: an overview of the clinical evidence. Fundamental and Clinical Pharmacology 19, 1–16. Jantova, S., Cipak, L., Cernakova, M., Kost’alova, D., 2003. Effect of berberine on proliferation, cell cycle and apoptosis in HeLa and L1210 cells. Journal of Pharmacy and Pharmacology 55, 1143–1149. Khan, A., Safdar, M., Ali, Khan, M.M., Khattak, K.N., Anderson, R.A., 2003. Cinnamon improves glucose and lipids of people with type 2 diabetes. Diabetes Care 26, 3215–3218. Kim, S.H., Hyun, S.H., Choung, S.Y., 2006. Anti-diabetic effect of cinnamon extract on blood glucose in db/db mice. Journal of Ethnopharmacology 104, 119–123. Jiang, Z.S., Zhang, S.L., Yan, H., Zhang, X.Y., He, Y., 2001. Effect of Jiaotai Decoction on the level of blood lipid and the sensitivity of insulin in rats with type 2 diabetes. Zhongguo Zhong Yi Yao Xin Xi Za Zhi 8, 36–37. Jiang, W.Y., 2005. Therapeutic wisdom in traditional Chinese medicine: a perspective from modern science. Trends in Pharmacological Sciences 26, 558–563. Leng, S.H., Lu, F.E., Xu, L.J., 2004. Therapeutic of berberine in impaired glucose tolerance rats and its influence on insulin secretion. Acta Pharmacologica Sinica 25, 496–502. Lee, Y.S., Kim, W.S., Kim, K.H., Yoon, M.J., Cho, H.J., Shen, Y., Ye, J.M., Lee, C.H., Oh, W.K., Kim, C.T., Hohnen-Behrens, C., Gosby, Alison, Kraegen, E.W., James, D.E., Kim, J.B., 2006. Berberine, a natural plant product, activates AMP-Activated Protein Kinase with beneficial metabolic effects in diabetic and insulin-resistant states. Diabetes 55, 2256–2264. Lu, T., Song, J., Huang, F., Deng, Y., Xie, L., Wang, G., Liu, X., 2007. Comparative pharmacokinetics of baicalin after oral administration of pure baicalin, Radix scutellariae extract and Huang-Lian-Jie-Du-Tang to rats. Journal of Ethnopharmacology 110, 412–418. Ni, Y.X., 1988. Therapeutic effect of berberine on 60 patients with type II diabetes mellitus and experimental research. Zhong Xi Yi Jie He Za Zhi 8, 711–713. Pan, G.Y., Huang, Z.J., Wang, G.J., Fawcett, J.P., Liu, X.D., Zhao, X.C., Sun, J.G., Xie, Y.Y., 2003. The antihyperglycaemic activity of berberine arises from a decrease of glucose absorption. Planta Medica 69, 632–636. Qu, H., Ma, Y., Yu, K., Cheng, Y., 2007. Simultaneous determination of eight active components in Chinese medicine ‘YIQING’ capsule using high-performance liquid chromatography. Journal of Pharmaceutical and Biomedical Analysis 43, 66–72. Sharma, S.B., Nasir, A., Prabhu, K.M., Murthy, P.S., Dev, G., 2003. Hypoglycaemic and hypolipidemic effect of ethanolic extract of seeds of Eugenia jambolana in alloxan-induced diabetic rabbits. Journal of Ethnopharmacology 85, 201–206. Shen, Z.F., Xie, M.Z., 1993. Determinationofberberineinbiological specimen by high performance TLC and fluoro-densitometric method. Yao Xue Xue Bao 28, 532–536. Sheng, M.P., Sun, Q., Wang, H., 1993. Studies on the intravenous pharmacokinetics and oral absorption of berberine HCl in beagle dogs. Zhongguo Yao Li Xue Tong Bao 9, 64–67. Tang, L.Q., Wei, W., Chen, L.M., Liu, S., 2006. Effects of berberine on diabetes induced by alloxan and a high-fat/high-cholesterol diet in rats. Journal of Ethnopharmacology 108, 109–115. Taylor, C.E., Greenough III, W.B., 1989. Control of diarrheal diseases. Annual Review of Public Health 10, 221–244. Whiting, D.R., Guariguata, L., Weil, C., Shaw, J., 2011. IDF diabetes atlas: global estimates of the prevalence of diabetes for 2011 and 2030. Diabetes Research and Clinical Practice 94, 311–321. Xu, Y.C., Wei, L.X., Wang, Y.L., 2001. Determination of cinnamic acid in Cinnamomum cassia presl after compatibility in Coptis chinensis Franch. Zhongguo Zhong Yao Za Zhi 26, 47–49. Yu, Y.Y., Wang, B.C., Peng, L., Wang, J.B., Zeng, C., 2006. Advances in pharmacological studies of Coptis chinesis. Chongqing Da Xue Xue Bao (Zi Ran Ke Xue Ban) 29, 107–111. Zhou, H.L., Wei, L.X., Yang, D.H., 1999. Determination of alkaloids in Coptis Cinnamormum compatibility by capillary electrophoresis. Zhongguo Zhong Yao Za Zhi 24 (308–310), 320.
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Phytomedicine journal homepage: www.elsevier.de/phymed
The anti-diabetic effects and pharmacokinetic profiles of berberine in mice treated with Jiao-Tai-Wan and its compatibility Guang Chen a , Fuer Lu b,∗ , Lijun Xu b , Hui Dong b , Ping Yi a , Fang Wang c , Zhaoyi Huang a , Xin Zou b a b c
Department of Integrative Traditional & Western Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science & Technology, Wuhan 430030, China Institute of Integrative Traditional & Western Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science & Technology, Wuhan 430030, China School of Medical and Life Sciences, Jianghan University, Wuhan 430056, China
a r t i c l e
i n f o
Keywords: Jiao-Tai-Wan Berberine Diabetes mellitus Pharmacokinetic differences Rhizoma Coptidis Cortex Cinnamomi cassiae
a b s t r a c t Jiao-Tai-Wan (JTW), a classical Chinese prescription, has been clinically employed to treat diabetes mellitus in recent years. To investigate the comparative evaluations on anti-diabetic effects and pharmacokinetics of the active ingredient berberine in mice treated with JTW in various combinations of its constituent herbs. In our study, the anti-diabetic study was carried out in diabetic mice induced by intraperitoneal injection of streptozotocin. The diabetic mice were randomly assigned to three therapy groups and orally administered with different prescription proportions of Rhizoma Coptidis and Cinnamomum cassia respectively. The level of plasma glucose, lipid profile and parameters related to oxidative stress were determined. The concentrations of berberine in non-diabetic mice plasma were determined using HPLC, and main pharmacokinetic parameters were investigated. The results indicated that the compatibility effects of ingredients present in Cinnamomum cassia could affect the anti-diabetic ability and pharmacokinetics of berberine in JTW. © 2013 Elsevier GmbH. All rights reserved.
Introduction Glycaemia and diabetes are rising globally, driven both by population growth and aging and by increasing age-specific prevalences (Danaei et al. 2011). The total number of adults (aged 20–79 years) globally estimated to have diabetes by the International Diabetes Federation (IDF) in 2011 is about 366 million (Whiting et al. 2011). For this reason, pharmacologic agents that control diabetes mellitus have received considerable attention. In China, herbal medicines have been widely used in the treatment of diabetes mellitus and its complications before the discovery of insulin. Jiao-Tai-Wan (JTW) is an important herbal formula in traditional Chinese medicine (TCM). JTW was first described by Han Yi (in the Chinese Ming Dynasty) in his treatise
Abbreviations: FBG, fasting blood glucose; OGTT, oral glucose tolerance test; BG, blood glucose; TC, total cholesterol; TG, triglyceride; LDL-c, low density lipoprotein; HDL-c, high density lipoprotein; Apo B, apolipoprotein B; STZ, streptozotocin; wt, weight; SD, standard deviation; TCM, traditional Chinese medicine; HPLC, high performance liquid chromatograph; AUC, area under curve; t1/2 , half-life; MRT, mean residence time; DM, diabetes mellitus; JTW, Jiao-Tai-Wan; CMC, Na carboxymethyl cellulose sodium salt. ∗ Corresponding author at: Institute of Integrative Traditional & Western Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science & Technology, Jiefang Road 1095#, Wuhan 430030, China. Tel.: +86 27 83663237; fax: +86 27 83663237. E-mail address: [email protected] (F. Lu). 0944-7113/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.phymed.2013.03.004
“Han Si Yi Tong”. This prescription, consisted of Rhizoma Coptidis and Cortex Cinnamomi cassiae, was mainly applied to treat insomnia in the past hundreds of years. However, a lot of clinical and laboratory researches have drawn people’s attention to its therapeutic effects on diabetes mellitus in the recent years (Jiang et al. 2001). The traditional remedy is a pill of Rhizoma Coptidis (Coptis chinensis Franch, Ranunculaceae) and Cortex Cinnamomi (Cinnamomum cassia, Lauraceae) with the ratio of 10:1 and both the herbs are officially listed in the Chinese pharmacopeia (Pharmacopoeia of PR China 2005). It is speculated that the ratio of Rhizoma Coptidis and Cinnamomum cassia could be changed in treating diabetes mellitus. Yet, data are still quite deficient to define precisely what ratio is superior or whether the mixture of two herbs is prior to single herb. Rhizoma Coptidis (Huang-Lian in Chinese), a key ingredient herb in JTW (Minister), has been widely used in TCM for the treatment of intestinal infection, fever, hypertension, tumor, etc. (Yu et al. 2006). Modern pharmacology has confirmed that isoquinoline alkaloids are the main active ingredients. Among them, berberine, the main isoquinoline alkaloid, has been used as a phytochemical marker for the quality control of Rhizoma Coptidis in Chinese pharmacopeia (Pharmacopoeia of PR China 2005). Pharmacological studies have indicated that berberine shares many beneficial activities with respect to antidiarrhea, antitumour and antimicrobial properties (Amin et al. 1969; Anis et al. 2001; Jantova et al. 2003; Taylor and Greenough 1989). Recently many researches have indicated that berberine possesses anti-diabetic effect (Leng et al. 2004;
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Lee et al. 2006; Tang et al. 2006), but the mechanism of its action is not quite clear. It is generally assumed that berberine has low bioavailability in human and rats (Sheng et al. 1993), resulting in poor antihyperglycaemic effect. Given the multi-ingredient character of herbal medicines, the likelihood of herb–drug interactions is theoretically higher than drug–drug interactions (Lu et al. 2007). In the recent years, multiple cases of herb–drug interactions have been reported (Hu et al. 2005; Izzo 2005). Herb–herb interaction has also been found in the decocting process of Rhizoma Coptidis and Cinnamomum cassia (Zhou et al. 1999; Xu et al. 2001). The aim of this study is to explore whether the Cinnamomum cassia in JTW can affect the pharmacokinetic behavior of berberine in Rhizoma Coptidis (herb–herb interaction). Due to the lack of standards of other conjugated metabolites, berberine was selected to compare the pharmacokinetic differences after oral administration of Rhizoma Coptidis, Jiao-Tai-Wan (Rhizoma Coptidis:Cinnamomum cassia = 1:0.25, JTW1) and Jiao-Tai-Wan (Rhizoma Coptidis:Cinnamomum cassia = 1:0.5, JTW2). We supposed that Cinnamomum cassia might influence the absorption of berberine and consequently affect the antihyperglycaemic effect in Rhizoma Coptidis, JTW1 and JTW2. To test this hypothesis, pharmacokinetic differences of berberine following oral administration of Rhizoma Coptidis, JTW1 and JTW2 were investigated in male Kunming mice. Materials and methods Materials Streptozotocin (STZ) was purchased from Sigma–Aldrich Inc. (USA). The detection kits of total cholesterol (TC), triglycerides (TG), high density lipoprotein-cholesterol (HDL-c), low density lipoprotein-cholesterol (LDL-c) and apolipoprotoein B (Apo B) were purchased from Wenchow Dong-Ou Bioengineering Company Ltd. (China). The detection kits of NO, GSH-px, SOD activities and MDA in plasma were purchased from Jiancheng Biotechnology Co., Nanjing, China. Berberine standard was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Ethylether and acetonitrile were of chromatographic grade, and other chemicals were of analytical grade.
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5 m) eluted with the mobile phases of 3 mM sodium dodecyl sulfate in 0.1% phosphoric acid (A) and acetonitrile (B) with isocratic eluting (A:B = 64:36). And the flow rate was 1.0 ml/min, the detection wavelength was 265 nm. Detection of berberine content in herbal extracts To calculate the dose administered to mice, content of berberine in Rhizoma Coptidis, JTW1 and JTW2 extracts were quantitatively determined. The aqueous extracts of Rhizoma Coptidis (1.87 g), JTW1 (2.5 g) and JTW2 (5 g) were extracted respectively by 100 ml of 1:100 mixture of hydrochloric acid and methanol at 60 ◦ C for 15 min, ultrasounded for 30 min and then diluted 100 times. The mixture was centrifuged for 10 min at 15,000 rpm. Ten microliters of the supernatant was injected into the HPLC system. The HPLC analysis of the berberine was a modified version of a previously published method (Qu et al. 2007). Berberine contents were determined to be 2.61% in Rhizoma Coptidis extract, 1.60% in JTW1 extract and 0.69% in JTW2 extract, respectively. Induction of experimental diabetic mice The animal studies were overseen and approved by the Animal Ethics Committee of Tongji Medical College, Huazhong University of Science & Technology before and during the experiment. Male kunming mice, weighing 30–40 g, provided by Hubei Province Experimental Animal Center (Grade SPF, Certificate No. SCXK (E2003-0005)) (Wuhan, China), were employed in the research. Animals were kept in an environmentally controlled breeding room (temperature: 20 ± 2 ◦ C, humidity: 60 ± 5%, 12-h dark/light cycle) with 4 mice per cage. All the mice had free access to food and water. The mice were adapted to diet for 2 weeks before beginning of the experiment. After a 16 h fasting, diabetes was induced by intraperitoneal injection of streptozotocin at 60 mg/kg in citrate buffer (0.1 mol/l sodium citrate and 0.1 mol/l citric acid, pH 4.5) for 3 consecutive days. The blood glucose levels were detected using the glucose-oxidase method on the third day after STZ injection. Animals with blood glucose above 16.7 mmol/l were considered as being diabetic and were taken for the experimenta l tests. Animals grouping and treatment
Preparation of herbal extracts Rhizoma Coptidis and Cinnamomum cassia were mixed in the ratio of 1:0.25 (JTW1) or 1:0.5 (JTW2), and the weight of Cinnamomum cassia was 45 g. The mixture was decocted by refluxing with water (1:10, w/v) for two times, 2 h for the first time and 1 h for the second time, respectively. Combined decoctions were filtered, and the solution obtained was concentrated to 450 g Cinnamomum cassia (45 g) and Rhizoma Coptidis (270 g) were extracted in the same way as above and gave two extracts of 225 g and 505 g. The extracts were stored at 4 ◦ C before use. All composed decoction pieces in JTW were purchased from Hubei Province Traditional Chinese Medicine Company (Wuhan, China). HPLC fingerprint of the extracts HPLC fingerprint was performed with the extracts of JTW1, JTW2, Rhizoma Coptidis and Cinnamomum cassia to identify the main chemical constituents in the samples. Berberine, coptisine, jateorhizine, palmatine, cinnamaldehyde and cinnamic acid were taken as standard substances. The extracts was dissolved in water at concentration of 5, 5, 5, 2 g/ml (w/v), and then diluted with methanol–water (50:50) to 2.5, 2.5, 2.5, 1 g/ml (w/v). HPLC analysis was performed on an outstand C18 column (4.6 mm × 250 mm,
Animals were randomly divided into 5 groups (n = 6 in each group): normal control, diabetic control, and three therapy groups. The mice in therapy groups were treated separately with Rhizoma Coptidis, JTW1, JTW2 extracts for 4 weeks. The therapy diabetic mice were gavaged by different aqueous extracts such as Rhizoma Coptidis extract (Rhizoma, contained berberine at the dosage of 100 mg/kg/d), JTW1 (contained berberine at the dosage of 100 mg/kg/d), JTW2 (contained berberine at the dosage of 100 mg/kg/d) respectively and correspondingly, and one time at 8:30–9:30 am per day. The diabetic control mice were gavaged by 0.5% carboxymethyl cellulose sodium salt (CMC-Na). The above extracts were prepared with 0.5% CMC-Na. Raw power solution or distilled water was fed by gavage administration once a day during the experiment period, respectively. The body weight and water consumption of all the mice were recorded every day. The blood glucose levels of the mice were monitored before and at 0, 1, 2, 3 and 4 weeks after STZ injection. The blood glucose levels were measured by the glucose-oxidase method. Biochemical analysis At the end of the experiment (4 weeks), the blood was taken from the retro-orbital plexus of the eye under mild ether
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Fig. 1. HPLC fingerprint chromatograms of the extracts of JTW1(A), JTW2(B), Rhizoma Coptidis(C) and Cinnamomum cassia(D). In the chromatograms: (1) cinnamic acid; (2) cinnamaldehyde; (3) jateorhizine; (4) coptisine; (5) palmatine and (6) berberine.
anesthesia and was prepared for measurement of insulin, lipid profile and parameters related to oxidative stress. The insulin level in plasma was estimated using commercially available kits. TC and TG were detected by enzyme end-point method, HDL-c and LDL-c by PTA-Mg2+ method and Apo B by immunoturbidimetric method. All the above targets were determined with semi-automatic biochemistry analysator (Shandong Rainbow Company, China). Serum insulin level was estimated by radioimmunoassay kit (Beijing Institute of Atomic Energy, Beijing, China). NO, GSH-px, SOD and MDA levels in plasma were estimated using commercially available kits. Pharmacokinetic study of berberine in mice After 2 weeks of adaptive feeding, male Kunming mice were randomly and averagely divided into 3 groups. The mice were fasted for 12 h, but were given access to water prior to the oral administration of 3.83 g/kg Rhizoma Coptidis extract (100 mg berberine/kg), 6.25 g/kg JTW1 (100 mg berberine/kg) and 14.49 g/kg JTW2 (100 mg berberine/kg). The dose of berberine to mice was referred to a former report (Shen and Xie 1993). All above extracts were prepared with 0.5% CMC-Na. Blood samples (1.2 ml) were collected from the eyes under aether anesthesia at the time points of 0.167, 0.25, 0.333, 0.5, 1, 1.5, 2, 2.5, 3, 5, 8 and 12 h after administration, then were put into 1.5 ml EP tube and centrifugated for 10 min at 3000 rpm in 4 ◦ C to separate serum. The serum was stored at −70 ◦ C after separation until assayed as described below. Serum samples (400 l) were alkalified with approximately 200 l of 0.1 mol/l NaOH added to each of them. Then, each portion
was shaken with 6 ml of ethylether for 10 min and centrifuged at 2500 rpm for 1 min, and the organic layer was transferred into an empty tube. This procedure was repeated for three times and the organic layer collected was dried at 35 ◦ C under a nitrogen stream. The residue was dissolved in 100 l mobile phase. After centrifuged at 15,000 rpm for 10 min, 50 l supernatant was analyzed with Waters High Performance Liquid Chromatography (HPLC) system consisting of a Waters 600 controller, Waters 600 pump, Waters 2487 dual absorbance and 717 plus autosampler (Waters Assoc., Milford, MA, USA). The chromatograph condition is as follows, chromatographic column: XTerra® RP18 (4.6 mm × 250 mm, 5 m); mobile phase: 0.1 mol/l KH2 PO4 , 0.05 mol/l NaOH (adjusted to a pH of 10 with triethylamine) – acetonitrile (30:70, v/v); flow rate: 0.8 ml/min; the detection wavelength: 345 nm; Column temperature: 40 ◦ C. The LLOQs (lower limit of quantification) of the assay was 6.4 g/l for berberine. Good linearity was obtained from 10 to 640 g/l for berberine.
Statistical analysis Data were expressed as the mean ± standard deviation (SD). Statistical analysis was performed using the SPSS Version 13.0 (SPSS Inc., Chicago, IL). Differences in numerical variables among groups were analyzed with one-way analysis of variance (ANOVA) LSD-t and SNK-q. A P value of less than 0.05 was considered statistically significant. The obtained berberine concentration was analyzed with DAS (Drug and Statistics) 2.0 pharmacokinetic program (Chinese Pharmacology Society) to obtain the relative pharmacokinetic parameters.
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Table 1 Effects of oral administration with the aqueous extracts of Rhizoma, JTW1 and JTW2 on fasting blood glucose in diabetic mice. Group
Week 0
Normal Diabetic Rhizoma JTW1 JTW2
4.9 19.3 19.1 19.4 19.2
± ± ± ± ±
Week 1 1.2 2.5** 2.3 2.5 2.2
5.1 19.2 18.7 18.3 18.5
± ± ± ± ±
Week 2 1.5 2.9** 2.4 2.2 2.3
5.4 20.5 17.5 16.6 15.8
± ± ± ± ±
Week 3 1.6 3.1** 2.1# 2.5# 2.1##
5.3 21.9 16.4 15.2 13.6
± ± ± ± ±
Week 4 1.5 3.3** 2.6## 2.2## 1.9##
5.3 20.8 15.7 13.5 11.8
± ± ± ± ±
1.7 3.5** 2.6## 2.3## 2.1##
Values are given as means ± SD of 6 mice. *p < 0.05, compared with normal control group. ** p < 0.01, compared with normal control group. # p < 0.05, compared with diabetic control group. ## p < 0.01, compared with diabetic control group.
Table 2 Effects of oral administration with the aqueous extracts of Rhizoma, JTW1 and JTW2 on insulin level and serum lipid profile in diabetic mice. Group
Insulin (mU/l)
Normal Diabetic Rhizoma JTW1 JTW2
44.37 26.16 29.92 34.26 38.45
± ± ± ± ±
7.56 2.54** 5.07 6.92## 6.63##
TG (mmol/l) 0.52 0.94 0.85 0.75 0.68
± ± ± ± ±
TC (mmol/l)
0.14 0.21** 0.16 0.19 0.18#
1.22 2.09 1.75 1.51 1.33
± ± ± ± ±
HDL-c (mmol/l)
0.36 0.53** 0.42 0.39# 0.32##
1.57 0.79 0.86 0.95 1.03
± ± ± ± ±
0.39 0.17** 0.22 0.20 0.24#
LDL-c (mmol/l) 0.65 1.41 1.13 1.08 0.96
± ± ± ± ±
0.12 0.32** 0.25 0.22# 0.16##
ApoB (g/l) 0.67 1.46 1.12 0.91 0.73
± ± ± ± ±
0.13 0.25** 0.22# 0.18## 0.16##
Values are given as means ± SD of 6 mice. *p < 0.05, compared with normal control group. ** p < 0.01, compared with normal control group. # p < 0.05, compared with diabetic control group. ## p < 0.01, compared with diabetic control group.
Results
Influence of extracts on serum insulin level and lipid metabolic parameters in diabetic mice
HPLC fingerprints of extracts The HPLC fingerprint chromatograms of JTW1, JTW2, Rhizoma Coptidis and Cinnamomum cassia are shown in Fig. 1. By comparing both the retention times and the UV spectra of the reference standards, six compounds (cinnamic acid, cinnamaldehyde, jateorhizine, coptisine, palmatine, berberine) in the extracts were well identified.
The levels of TC, TG, LDL-c and Apo B in mice treated with Rhizoma, JTW1 and JTW2 were lower than those in the diabetic control mice, while the levels of insulin and HDL-c were higher (Table 2, p < 0.05 or p < 0.01). Among them, the antihyperlipidemic effect was the most remarkable in the JTW2 group (Table 2).
Influence of extracts on blood glucose in diabetic mice
Influence of extracts on NO, GSH-px, SOD, MDA and CAT activities in diabetic mice
The levels of plasma glucose in diabetic mice injected by STZ (60 mg/kg) were increased significantly (19.3 ± 2.5 mM vs. 4.9 ± 1.2 mM, p < 0.01), compared to those in normal mice. Administration of Rhizoma, JTW1 and JTW2 for 4 weeks significantly reduced the blood glucose of diabetic mice (Table 1, p < 0.05 or p < 0.01). In addition, JTW1 and JTW2 groups showed a statistically significant difference of serum glucose level compared with the Rhizoma group (Table 1). Among them, the antihyperglycaemic effect was the most remarkable in the JTW2 group.
It was shown that the activities of NO, GSH-px, SOD and CAT were significantly decreased in diabetic group compared to normal group (Table 3, p < 0.01). Rhizoma, JTW1 and JTW2 could increase the activities of NO, GSH-px, SOD and CAT (Table 3, p < 0.05 or p < 0.01). A significant increase in NO, GSH-px, SOD and CAT activities in plasma were observed in the JTW1 and JTW2 group compared with those of diabetic group (Table 3, p < 0.01). The MDA level in plasma was raised by 65.52% in STZ-diabetic mice compared to that in normal control mice (p < 0.01). The administration of Rhizoma, JTW1 and JTW2 reduced the increased MDA level (Table 3,
Table 3 Effects of oral administration with the aqueous extracts of Rhizoma, JTW1 and JTW2 on plasma NO, GSH-px, SOD, MDA and CAT activity in diabetic mice. Group
NO (mol/l)
Normal Diabetic Rhizoma JTW1 JTW2
24.52 15.37 18.44 20.58 21.96
± ± ± ± ±
3.76 2.24** 2.86# 2.78## 3.51##
Values are given as means ± SD of 6 mice. *p < 0.05, compared with normal control group. ** p < 0.01, compared with normal control group. # p < 0.05, compared with diabetic control group. ## p < 0.01, compared with diabetic control group.
GSH-px (U/ml) 114.88 73.75 80.88 85.49 98.85
± ± ± ± ±
10.15 7.28** 7.89 8.55# 8.83##
SOD (U/ml) 186.95 139.86 155.74 165.92 176.55
± ± ± ± ±
16.63 12.67** 14.59# 13.85## 14.68##
MDA (nmol/l) 5.83 9.65 8.58 8.02 6.17
± ± ± ± ±
0.62 0.79** 0.67# 0.65## 0.59##
CAT (U/ml) 27.63 14.34 17.56 20.16 23.67
± ± ± ± ±
3.85 2.52** 2.59# 2.75## 3.06##
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Table 4 Pharmacokinetic differences of oral administration of the aqueous extract of Rhizoma Coptidis (100 mg/kg berberine), JTW1 (100 mg/kg berberine) and JTW2 (100 mg/kg berberine) to mice (n = 5). Parameters
Rhizoma Coptidis
JTW1
JTW2
AUC0–t (g h/l) AUC0–∞ (g h/l) AUMC0–t AUMC0–∞ MRT0–t (h) MRT0–∞ (h) VRT0–t (h2 ) VRT0–∞ (h2 ) t1/2 ˛ (h) t1/2 ˇ (h) Tmax (h) Cmax (g/l) CLz/F (l/h/kg) Vz/F (l/kg)
742.92 789.91 3193.19 3968.84 4.29 5.02 8.89 17.86 0.662 7.09 1.0 202.79 126.59 565.97
1309.20 1507.69 5393.52 6959.12 4.12 4.62 7.61 20.09 3.75 3.75 2.0 241.27 66.33 368.54
1562.25 1670.67 6533.02 8267.06 4.18 4.95 7.96 17.04 0.59 3.08 2.0 290.73 59.86 242.77
p < 0.05 or p < 0.01) and JTW2 group showed no significant difference compared with normal control (p > 0.05). Pharmacokinetic comparison of berberine in mice after oral administration of Rhizoma Coptidis and JTW1 Serum concentration of berberine was determined after oral administration of Rhizoma Coptidis extract to mice and the serum profile demonstrated bimodal phenomenon (Fig. 3). The first peak occurred at about 0.25 h and the second at 1 h. Compared with Rhizoma Coptidis extract, bimodal phenomenon of berberine in mice serum also existed, but the concentration of the second peak and the values of AUC(0–∞) /dose were significantly increased after oral administration of JTW1, and two peak times were delayed (Table 4 and Fig. 2). These results indicated that existence of some ingredients in JTW1 might affect the pharmacokinetic behavior of berberine. Pharmacokinetic comparison of berberine in mice after oral administration of JTW1 and JTW2 The results of the above researches indicated that Cinnamomum cassia increased the absorption of berberine. We thought that this effect was resulted from the interaction between Rhizoma Coptidis and Cinnamomum cassia. In order to determine whether Cinnamomum cassia could contribute to the increase of berberine absorption, we compared the pharmacokinetics of berberine with 400
Serum concentration (µg/L)
350 300 250 200 150 100 50 0
0
2
4
6
8
10
12
14
Time(h) Fig. 2. Mean serum concentration–time curves of berberine after oral administration of the Rhizoma Coptidis aqueous extract (100 mg/kg berberine) (䊉), JTW1 (100 mg/kg berberine) ( ) and JTW2 (100 mg/kg berberine) ( ) to mice (n = 5).
JTW1 and JTW2 in mice. The serum concentrations of berberine were determined and pharmacokinetic parameters were estimated (Table 4). Results were also compared with those acquired from mice given Rhizoma Coptidis extract, which contains the same dose of berberine (Fig. 2). As shown in Fig. 2 and Table 4, the serum profile still demonstrated bimodal phenomenon after intake of JTW2, which confirmed that the ability of rapid absorption of berberine resulting in the appearance of the first peak. Similar to the mice given JTW1, higher serum concentration and increased AUC(0–∞) of berberine were found in mice given JTW2 compared to normal control. These results further suggested that ingredients in Cinnamomum cassia affected the pharmacokinetic behavior of berberine.
Discussion With multiple applications and remarkable curative effect, Rhizoma Coptidis and Cinnamomum cassia are commonly used in clinical practice of TCM. Rhizoma Coptidis, the representative medicine for heat-clearing and detoxicating, has been used to treat diabetes for thousands of years in China. In the past few years, the research work in this field is much more popular. However, the clinical applications of Rhizoma Coptidis were limited due to its mild antihyperglycaemic effect. Recently, Anderson reported that, Cinnamomum cassia, a drug for dispelling internal cold, has favorable curative effect on diabetes mellitus. We assumed Cinnamomum cassia could strengthen the therapeutic function of Rhizoma Coptidis on diabetes mellitus. Therefore, this study compared the effects of Rhizoma Coptidis and JTW composed of different proportions of Rhizoma Coptidis and Cinnamomum cassia on serum glucose level and lipid profile in diabetic mice. In this study, fasting blood glucose in the diabetic mice was significantly higher than those in the normal mice, indicating that the diabetic mice met the characteristics of diabetes mellitus. The results of this study showed that all of Rhizoma Coptidis, Cinnamomum cassia and JTW could decrease fasting blood glucose level, and improve insulin level. Moreover, the effect of JTW was superior to that of single Rhizoma Coptidis or Cinnamomum cassia. Among treatment groups, the antidiabetic effect in JTW2 group was the best. These results indicated that the compatibility of Cinnamomum cassia and Rhizoma Coptidis could strengthen Rhizoma Coptidis’s antihyperglycaemic effect. Lipids play a vital role in the pathogenesis of diabetes mellitus. The most common lipid abnormalities in diabetes are the increase in TC, TG, LDL-c, Apo B and fall of HDL-c accompanied with the rise in blood sugar (Sharma et al. 2003). In this study, we have noticed elevated levels of serum lipids such as cholesterol and triglycerides in diabetic mice. Meanwhile, it is encouraging that the treatment of Rhizoma Coptidis and JTW lowered the elevated levels of TC, TG, LDL-c and Apo B in diabetic mice. There was increase in HDL-c also, which indicated that Rhizoma Coptidis and JTW might be beneficial to diabetic individuals with coronary heart disease for HDL-c acts as a protective factor against coronary heart disease. In parallel with the above antihyperglycaemic effects, the antihyperlipidemic effect in JTW2 was the best among treatment groups. Oxidative stress in diabetes is coupled to a decrease in the antioxidant status, which can increase the deleterious effects of free radicals. We observed that activities of SOD, GSH-px and CAT in the treated diabetic groups were increased significantly, compared with diabetic group. This suggested that Rhizoma Coptidis and JTW had exerted the antioxidant effect by activation of SOD, GSH-px and CAT activity, at least in part, Rhizoma Coptidis and JTW decrease lipid peroxidation products and MDA in plasm. Our data indicated that Rhizoma Coptidis and JTW could increase NO production of diabetic mice in serum. Rhizoma Coptidis and JTW might
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Fig. 3. Typical chromatograms for the determination of berberine in serum samples: (A) chromatogram of a blank serum sample, (B) chromatogram of a serum sample spiked with berberine, and (C) chromatogram of the serum sample of a mouse taken 1 h after the oral administration of the Rhizoma Coptidis aqueous extract.
prevent the oxidative damage and increase a protective effect on postponing the cause of diabetic complications. Massive clinical and experimental studies have already proved that berberine was the active component of Rhizoma Coptidis to decrease blood glucose (Chen and Xie 1986; Ni 1988; Leng et al. 2004; Lee et al. 2006; Tang et al. 2006), while that of Cinnamomum cassia was indefinite. Therefore, this experiment only studied the pharmacokinetics of berberine to explore the causes underlying the difference of antihyperglycaemic and antihyperlipidemic effects in the treated groups. So far, the facts that berberine shows poor absorption through the gut wall and has low bioavailability have been regarded as key factors leading to unsatisfied clinical curative effect on diabetes mellitus. Some researches reported that the bioavailability of berberine is usually lower than 5% and one of the main factors leading to poor absorption is due to the external secretion of entero-epithelium P-glucoprotein. Hence, using P-glucoprotein catastaltica could improve the absorption of berberine (Pan et al. 2003). Our data showed that berberine demonstrated bimodal phenomenon in the serum profile, the weight was 1/C2 , indicating that the endosomatic metabolic process of berberine was confirmed to the two-compartment model. There was berberine
absorption in mice body after oral administration with Rhizoma Coptidis extract or JTW, and the bimodal model was observed. The Cmax and AUC(0–∞) of the JTW were both higher than those of Rhizoma Coptidis extract. It was reported that, AUC(0–∞) and Cmax /AUC were used as indexes for absorption extent and absorption rate respectively (Endrenyi et al. 1991). The AUC(0–∞) among the three groups were significantly different, while Cmax /AUC was similar among the groups, indicating that Cinnamomum cassia promoted the absorption extent of berberine rather than affect its absorption rate. The Cmax , AUC(0–t) , AUC(0–∞) were significantly different among the 3 groups, reaching their maximums in JTW2 group. Our data demonstrated that with the rise of Cinnamomum cassia proportion, the bioavailability of berberine increased significantly in mice body. It might because some components in the Cinnamomum cassia enhanced the berberine absorption when used together with Rhizoma Coptidis, which resulted in increased blood berberine’s concentration in mice body relative to single use of Rhizoma Coptidis, and enhanced the therapeutic effects of berberine. Cinnamomum cassia showed no significant influence on other behaviors in the pharmacokinetics of berberine in mice. To conclude, Cinnamomum cassia facilitates the antidiabetic effects
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of Rhizoma Coptidis in two ways, firstly, Cinnamomum cassia in itself could improve insulin resistance and promote glucose absorption and utilization as well as modulate lipid metabolism (Khan et al. 2003; Kim et al. 2006); secondly, according to this study, Cinnamomum cassia enhances berberine’s absorption after oral administration. Rhizoma Coptidis and Cinnamomum cassia is the classic medicine couple in TCM. It had been reported that precipitation phenomena occurred in decocting process of TCM containing this medicine couple (Zhou et al. 1999; Xu et al. 2001). In this study, we also found that Cinnamomum cassia could decrease the content of berberine in decocting process (data not shown). Combining the experimental data with literature reported, we could conclude that the mechanism of precipitation phenomena might be the complexation between the main active ingredients, phenolic acids and alkaloid, result the decreased contents of these active ingredients significantly. From this point of view, the combination of Rhizoma Coptidis and Cinnamomum cassia was rather unsuitable. On the other hand, however, under the guidance of the theory of TCM, Cinnamomum cassia could enhance the efficacy (“restoring normal coordination between heart and kidney”) and decrease the adverse effect (“wounding stomach with bitter and cold factors”) of Rhizoma Coptidis, which were confirmed by the clinical application for hundreds of years and by modern pharmacology (Cui and Zhao 2003). In addition, this study also indicated that Cinnamomum cassia could enhance the berberine’s absorption in mice. The huge number of active ingredients in TCM makes it suitable for multi-target actions. Different from Western medicine, TCM focuses on the overall functional state of the patient, which is becoming a trend in modern medicine for treating complicated diseases (Jiang 2005). Therefore, considering this aspect, the multiherb remedy might be reasonable prescription in spite of the existence of chemical interaction between Rhizoma Coptidis and Cinnamomum cassia. Moreover, our experimental data indicated that the better antidiabetic effects of JTW1 and JTW2 were related to the increased berberine bioavailablity. This result further confirmed that the multiherb remedy might be reasonable. Conclusions Our findings suggested that Cinnamomum cassia improved the antidiabetic effect of Rhizoma Coptidis in diabetic mice and berberine absorption in mice after oral administration with JTW extract, though the complexation between phenolic acids in Cinnamomum cassia and alkaloids in Rhizoma Coptidis might decrease the exposure of berberine. Berberine demonstrated bimodal phenomenon in the serum profile. Some ingredients in Cinnamomum cassia had pharmacokinetic interaction with berberine. Although we still cannot ascertain what the ingredient is or whether the material generated by interaction between two herbs influence the absorption of berberine, the mechanism for the increased berberine absorption is worthy of consideration. It should be worthwhile to find out if there is some type of p-glycoprotein suppressant at work as well. All these are still ambiguous and further profound researches are required. Acknowledgement This work was supported by the National Natural Science Foundation of China (No. 30801492). References Amin, A.H., Subbaiah, T.V., Abbasi, K.M., 1969. Berberine sulfate: antimicrobial activity, bioassay, and mode of action. Canadian Journal of Microbiology 15, 1067–1076.
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