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8from 1-methyl-4-phenylpyridinium-induced neuronal apoptosis in rat forebrain culture. Neuroscience 144, 46-55.
WHO, 2004. Radix Eleutherococci. In:WHO Monographs on selected medicinal plants,vol.2. WHO, Geneva, pp. 83-96.
Zhang, A., Sun, H., Wang, Z., Sun, W., Wang, P., Wang, X., 2010. Metabolomics: towards understanding traditional Chinese medicine. Planta Med 76, 2026-2035.
Zhang, L., Liu, S.M., 2006. Recent developments of traditional Chinese medicine in treatment of Parkinson disease. Chinese Journal of Clinical Rehabilitation, 152-154.
Zhang, Z. F., Liu, Y., Luo, P., Zhang, H., 2009. Separation and purification of two flavone glucuronides from Erigeron multiradiatus (Lindl.) Benth with macroporous resins. J Biomed Biotechnol 2009, 875629.
Zheng, S., Yu, M., Lu, X., Huo, T., Ge, L., Yang, J., Wu, C., Li, F., 2010. Urinary metabonomic study on biochemical changes in chronic unpredictable mild stress model of depression. Clin Chim Acta 411, 204-209.
The clinical chemistry and metabonomic profiles of the efficacy of a Chinese traditional prescription Tianqi Jiangtang Capsule on fat emulsion and alloxan-
induced type 2 diabetes mullitus rat model
Shuxiang Zhanga'b, Hui Suna, Yimin Niu a, Guozheng Jiaoa, Kun Yang a, Xijun Wanga' *
aDepartmentofPharmacognosy,bInstitute of Traditional Chinese Medicine, HeilongjiangUniversityofChineseMedicine,No.24HepingRoad,Harbin,China 150040
Abstract
In this research, clinical chemistrycombined with ultra-performance liquid chromatography/ electrospray ionization high definition mass spectrometry (UPLC/ESI-HDMS) were used to analysis urinary samples from an improved rat model of type 2 diabetes mellitus (T2DM) induced by daily ig constant dosage of fat emulsion for 10 days and twice ip low dosage of alloxan. The indexes of clinical chemistry and the metabonomic profiles of PCA illustrated the panorama of T2DM disorders, and proved the success of this kind of T2DM rat model as well as the efficacy of Tianqi Jiangtang Capsule to redress metabolite disorders.
Keywords: Metabonomics; rat model for type 2 diabetes mellitus; Tianqi Jiangtang Capsule;
1.Introduction
Type 2 diabetes mellitus (T2DM), characterized as a kind of multiple, complex molecular disorders, is one of the most prevalent metabolic diseases [1]. Lots of animal models have been developed for better understanding T2DM, including genetically spontaneous models and experimentally nonspontaneous ones [2], of which model evaluation methods mostly focus on clinical chemistry and histopathology.
Metabonomics as a systemic biological approach for studying profiles of small-molecule metabolites in vivo can provide us information on disease processes at several stages[3]. It tries to fully interpret the influences of diseases or drugs on organisms through metabolite variations [4-5]. The mtabonomic data can be obtained by mass spectrometry coupled with gas chromatography (GC-MS) or liquid chromatography (LC-MS) [6], and nuclear magnetic resonances (NMR) [7], and finally give out the information of small-molecule metabolites in vivo that will reflect the internal overall changes in state of disease.
Nowadays, people pay more and more attention to herbal drugs in the role of diabetes prevention and therapy. Tianqi Jiangtang(TQJT) Capsule, licensed as a Grade V New Drugs in China, is a traditional Chinese materia medica preparation. The bulk drug is aqueous extraction of
10 traditional Chinese herbals, including Panax Ginseng, Radix Astragali, Herba Dendrobii, Rhizoma Coptidis, Galla Chinensis, Fructus Corni, Frucus Ligustri lucidi, Cortex Lycii, Radix Trichosanthis, Herba Ecliptae Prostratae, which is mainly used for treating type 2 diabetes mellitus.
In this research we proposed to assess an improved T2DM rat model and tested the efficacy of Tianqi Jiangtang Capsule on this model by metabonomic method combined with clinical chemistry.
2. Materials and methods
2.1 Chemicals and reagents
Lard 20 g, alloxan 1 g, cholesterol 5 g, sodium glutamate 1 g, sugar 5 g, fructose 5 g, Tween 80 20 ml , propylene glycol 30 ml were prepared to a constant volume of 100 ml fat emulsion by adding distilled water and stored at 4 °C. Tianqi Jiangtang Capsule was provided by Heilongjiang Baoquan Pharmaceutical Company, China. ACCU-CHEK Active blood sugar detector and test papers were purchased from Roche Diagnostics (Shanghai) Ltd., China. Alloxan, acetonitrile (HPLC grade), and leucine enkephaline were purchased from Sigma-Aldrich, UK. Formic acid (analytical grade) was purchased from Beijing Reagent Company, China. The deionized water was purified by Milli-Q system (Millipore, Bedford, MA, USA). The assay kits of cholesterol, triglyceride (TG) were purchased from Zhongsheng Beikong Biologic Technology Ltd., China. Insulin, the assay kit of free fatty acid (FFA) and malondialdehyde (MDA) were purchased from Nanjing Jiancheng Bioengineering Institute, China.
2.2 Animal treatment and sample collection
Male Wistar rats weighted 200~230 g were provided by Good Laboratory Practices Center, Heilongjiang University of Chinese Medicine. All rats were reared in animal room in a 12h dark to light circle at the 25±2 °C and 60±5% relative humidity for 1 week before experiment.
60 Wistar rats were randomly divided into normal control group (CG) of 20, model group (MG) of 40. All rats were fed on standard laboratory chow and drank ad libitum. The details of modeling method can be referred in Table 1 [14] . At day 15 (72 h after the last injection of alloxan), we collected sight blood from rat tail wound. Blood sugar was detected in 10 CG rats and all MG rats using a blood sugar detector and test papers. In MG rats, the judgement of the accomplished T2DM rat model was that fasting serum glucose value (FSG value)>16.7 mmolL-1. Only 29 in 40 MG rats reached that standard. We randomly divided these 29 model rats with FSG value into three groups (group differences<1.1 mmolL-1). One group remains model group 1 (MG1, 9 rats) for the following indicators assay; the other two groups, each had 10 rats, separately as therapy group and model group 2 (MG2) for pharmacodynamic experiment of TQJT.
2 ml orbital venous blood was collected from 10 CG and 9 MG1 rats, centrifuged at 4000 r/min for 10 minutes, and then the serum indicators involving TC, TG, FAA, MDA were determined. In £ITT definition, blood sugar in CG and MG1 rats were detected six times after ip insulin (0.05 U/kg) with blood sugar detector and test papers mentioned above.
In the pharmacodynamic experiment of TQJT, another normal control group was still needed to be set up, in which 10 rats were from the CG not disturbed last time. Therapy group and model group 2 (MG2) were established as defined above. Details of pharmacodynamic experiment method can be seen in Table 2. The concentration of aqueous solution of the bulk drug in the capsule was 1.7280 g/kg/day (four times clinical equvalent efficacy dose). Clinical chemical parameters, including FSG, TC, TG, FAA, MDA, £ITT, were detected after the 20 days of oral administration of TQJT.
Urine samples were collected from rat individual urine collection cages from 18:00 p.m. to 6:00 a.m. every day and stored at -20 °C. Body mass, food, and water consumption were monitored daily.
2.3 UPLC-ESI-MS conditions
UPLC-MS data were acquired using a Waters ACQUITY UPLC system (Waters Corporation, Milford, MA, USA) coupled with a Waters Synapt High Definition MS™ (HDMS) System. The LC-MS system was run in binary gradient mode. Solvent A was 0.1% formic acid in water and solvent B was 0.1% formic acid in acetonitrile; the flow rate was 0.4 ml/min. A ACQUITY UPLCTM BEH C18 column (50 mmx2.1 mm i.d., 1.7 ^m, Waters Corp, Milford, USA) was used
and column temperature was maintained at 4 °C. The column was eluted with a linear gradient (Table 3). The mass scanning range was m/z100-1000 carried out under negative ion mode (tried both positive and negative mode but in negative mode the chemical compounds easy to lose protons are well identified). Mass measurement was externally calibrated before initiating the experiment; leucine enkephalin was employed as lock mass.
2.4 Data processing
Data of clinical chemistry were analyzed by Student's i-test, which were expressed as mean±SD. Metabonomic data and multivariate analysis were processed by MarkerLynx Application Manager (Waters Corporation, Milford, MA, USA) for the peak detection, PCA.
3. Results and discussion
3.1 Clinical chemistry
At 15 th day, 29 among 40 MG rats had FSG value>16.7 mmolL-1 with a modeling success ratio of 72.5%. All model rats exhibited a notable diabetic phenotype, because rats body weights and urine volumes were obviously changed. The contents of FSG, TG, TC, MDA, and FFA in serum were markedly increased; KITT value was notably decreased compared with CG rats after 15 days modeling accomplishment (Fig. 1a). All these changes were obviously intervened by administration of TQJT for 20 days. TG rats gained weights and reduced urine volumes when compared with MG and CG rats. The contents of FSG, TG, TC, and FFA were significantly decreased; KITT was notably increased compared with MG rats. The content of MDA had a tendency to drop down but was not decreased significantly (Fig. 1b).
T2DM rat models always include genetically spontaneous models and experimentally nonspontaneous ones. The genetically spontaneous models usually have less relevant natural pathologic history in human T2DM process, and the experimental cycles always require 4-30 weeks [8-11]. Compared with genetically spontaneous models, experimentally nonspontaneous ones are relatively lower cost, easier operations, and shorter modeling cycles, which are consequently more popular for use, including STZ/alloxan rat models, high-fat-fed models, partial pancreatectomy models, and etc [12]. Now in fat-fed and alloxan-treated rat model we further take fat emulsion administration instead of fat food feeding, which can ensure us a better control of daily fat intake and a shorter modeling time [13, 14].
3.2 Analysis of metabolic profiling
Principal components analysis (PCA) was adopted to analyze the data of urine samples in different days of CG, MG1, MG2, TG rats, which have been well distinguished from PCA score plot and further proved a modeling success through the obvious clustering tracks of MG rats with fat emulsion in day 1 to 10 and with alloxan in day 11 to 12 until model accomplishment in day 15; presented a pullback trend after admininstration of Tianqi Jiangtang Capsule as well (Fig. 2).
4. Conclusion
In this research, we successfully applied metabonomic analysis combined with clinical biochemistry to evaluate a fat emulsion and alloxan-induced T2DM rat model based on the platform of UPLC/ESI-HDMS. Through metabolic profiles of urine samples from CG and MG rats, the biomarkers can be contributed to diabetes diagnosis and treatment, and their metabolic pathways elucidated a panoramic view of metabolic disorders in T2DM and its complications. By comparing with other T2DM rat models, fat emulsion and alloxan-induced one was demonstrated to be a cheap, effective, and simple animal model of T2DM. Further on account of metabonomic results, we discovered that this kind of rat model is especially fit for the study of T2DM complications for its severe protein disorder. In conclusion, it is recommended that this model and evaluation method can be applied for T2DM early diagnosis, prevention and treatment in the future.
References
[1] Stumvoll M, Goldstein BJ, van Haeften TW. (2005) Type 2 diabetes: principles of pathogenesis and therapy. Lancet. 365(9467):1333-1346.
[2] Srinivasan K, Ramarao P. (2007) Animal models in type 2 diabetes research: an overview. Indian J Med Res. 125(3):451-472.
[3] Nicholson J K, Oflynn M P, Sadler P J. (1984) Proton-nuclear-magnetic-resonance studies of serum, plasma and urine from fasting normal and diabetic subjects. Biochem. J 217: 365 - 375.
[4] Yuan K L, Kong H W, Guan Y F, et al. (2007) A GC-based metabonomics investigation of type 2 diabetes by organic acids metabolic profile. J Chromatogr B 850 (1-2): 236-240.
[5] Li X, Xu Z, Lu X, Yang X, Yin P, Kong H, Yu Y, Xu G. (2009) Comprehensive two-dimensional gas chromatography/time-of-flight mass spectrometry for metabonomics: Biomarker discovery for diabetes mellitus. Anal Chim Acta. 633(2):257-262.
[6] Wang C, Kong H W, Guan Y F, et al. (2005) Plasma phospholipid metabolic profiling and biomarkers of type 2 diabetes mellitus based on high-performance liquid chromatography/electrospray mass spectrometry and multivariate statistical analysis. Anal Chem. 77 (13): 4108-4116.
[7] Zhang S, Nagana Gowda GA, Asiago V, Shanaiah N, Barbas C, Raftery D. (2008) Correlative and quantitative (1)H NMR-based metabolomics reveals specific metabolic pathway disturbances in diabetic rats. Anal Biochem. 383(1):76-84.
[8] Rebecca S, Joan F, et al. (2004) The BBZDR/Wor rat model for investigating the complications of type 2 diabetes mellitus. ILAR J 45(3), 292-302.
[9] Park S H, Marso S P, Zhou Z M, et al. (2001) Neointimal hyperplasia after arterial injury is increased in a rat model of non-insulin-dependent Diabetes Mellitus. Circulation 104, 815-819.
[10] Haluzik M, Colombo C, Gavrilova O, Chua S, Wolf N, Chen M, Stannard B, Dietz KR, Le Roith D, Reitman ML. (2004) Genetic background (C57BL/6J versus FVB/N) strongly influences the severity of diabetes and insulin resistance in ob/ob mice. Endocrinology 145: 3258-3264.
[11] Lombardo YB, Drago S, Chicco A, Fainstein-Day P, Gutman R, Gagliardino JJ, Gomez Dumm CL. (1996) Long-term administration of a sucrose-rich diet to normal rats: relationship between metabolic and hormonal profiles and morphological changes in the endocrine pancreas. Metabolism.45: 1527-1532.
[12] Islam MS, Loots du T. (2009) Experimental rodent models of type 2 diabetes: a review. Methods Find Exp Clin Pharmacol. 31(4):249-261.
[13] Tang LQ, Wei W, Chen LM, Liu S. (2006) Effects of berberine on diabetes induced by alloxan and a high-fat/high-cholesterol diet in rats. Journal of Ethnopharmacology 108(1):109-115.
[14] Ai J, Wang N, Yang B F, et al.(2005) Development of Wistar rat model of insulin resistance. World J Gastroenterology 11(24), 3675-3679.
Fig. 1a
Fig. 1b
Fig. 1 The measurement of serum biochemical indexes. a) after modeling accomplishment; b) after administration of Tianqi Jiangrang Capsule for 20 days. The data were expressed by Mean±SD.
***significant difference model group compared with control group p<0.001; **p<0.01; *p<0.05; #signigicant difference therapy group compared with model group p<0.05.
Scores Comp[l] vs. Comp[S] colored by Sample Group
«11
Fig. 2 PCA score plot of rat urinary metabolites in each group rats. KB: control group; MX: model group 1; GY: therapy group; MXHF: model group 2. Numbers represent days of each experimental stage.
EmergingChinmedomicstotraditional Chinese medicine: Present and future
Ai-hua Zhang*, Hui Sun, Guangli Yan, Ping Wang, Ying Han, Xi-jun Wang*.
(National TCM Key Lab of Serum Pharmacochemistry, Key Lab of Chinmedomics, Heilongjiang University of Chinese Medicine, and Key Pharmacometabolomics Platform of Chinese Medicines, Heping Road 24, Harbin 150040, China. Tel. & Fax +86-451-82110818, email: aihuaz@yeah.net and xijunwangls@126.com)
Abstract
For World Health Organization proposed 'Health for All', we shouldpromote the usage of traditional medicine.With the development of society and people's health attention, TCM is getting more and more popular in the whole world for improving health condition of human beings and preventing or healing diseases. However, TCM is also facing severe challenges or problemes. The largest obstacle suffers from insufficient modern scientific research, that not only lowering the position of TCM also restricting the development of TCM in the abroad. Therefore, it is indispensable to strengthen TCM research, which undoubtedly demands significant amount of analytical power and efforts. Through system studies, we have establishedand advanced the concept of Chinmedomics that is suitable for effective evaluationandbasic research of prescription and practice characteristics of TCM, may beneficially provide an opportunity to scientifically express the meaning of the syndrome and medical formulae of TCM. Chinmedomics apporoaches as pillars of the potential bridge between chinese and western medicine, may beneficially influence and provide an opportunity to explain the true meaning of evidence-based chinese medicine. TCM can
