| BackgroundChronic renal failure(CRF) is a clinical syndrome which is characterized by progressive and irreversible destruction of renal tissue until renal failure on the basis of various chronic kidney disease(CKD). The morbidity of CKD is rising over the last20years and CKD has become a major disease which seriously threatens the public health after angiocardiopathy, cancer, diabetes. But modern medicine still lack satisfying therapy method and drug. In recent years, traditional Chinese medicine(TCM) has displayed special effect on improving clinical symptom, the prevention and control of complication, delaying the progression of disease, increasing the quality of life and decreasing mortality, and can greatly reduced the burden of social and family as well. So, explore and enhance the application of TCM on treat CRF has far-reaching significance.The aim of this trial is to do a reseach of second generation product of Niaoduqing granule and set the stage for drug discovery of compound Huanggan(CHG)-a compound preparation of Nanfang hospital, which was filtered from the prescription of Niaoduqing granule, choosing the crude drug extract of CHG tablet as research object. Part I Effect of Compound Huanggan Extract on Oxidative Stress in Chronic Renal Failure RatsObjective:To investigate the effect of compound huanggan(CHG) extract on CRF rats. Discuss the mechanism of CHG by measuring and analyzing the malondialdehyde (MDA), advanced oxidation protein products(AOPPs) and advanced glycation end products(AGEs) level in serum, and the superoxide dismutase(SOD) activity of kidney as well.Methods:Thirty-five male SD rats were randomly divided into seven groups (n=5):normal group; model group; Niaoduqing granule group; low dose group of water extract; high dose group of water extract; low dose group of ethanol extract; high-dose group of ethanol extract. The whole trail was last8weeks. CRF model was prepared with a gavage of adenine(200mg-kg"1) once a day during the first2weeks, then once every two days during the follow2weeks, and then the dose of adenine was reduced to100mg-kg-1and ig twice a week during the last4weeks. Rats were treated with a gavage of Niaoduqing granule(4.5g-kg-1), extract of CHG (water extract13.26g-kg-1,6.63g-kg-1; ethanol extract5.24g-kg-1,2.62g-kg-1) or vehicle (0.5%CMC-Na) since day one, respectively. After8weeks, volume of24h urine was measured; the level of serum creatinine(SCr), blood usea nitrogen(BUN), malondialdehyde(MDA), advanced oxidation protein products (AOPPs), superoxide dismutase (SOD) activity of renal and kidney index were detected and the pathological changes of the kidney tissue were observed by making HE staining sections.Results: 1Changes of bodyweight:Bodyweight of model group was significantly lighter than normal group(P0.05), and there were no significant difference between treatment groups and model group(P>0.05).2Changes of SCr, BUN,24h urine and kidney index:Compared with normal group, SCr, BUN,24h urine and kidney index of model group were significantly increased (P＜0.05). Compared with model group, the SCr of all treatment groups were significantly reduced (P＜0.05), the BUN of low dose group of water extract and ethanol extract groups(high and low dose) were significantly reduced (P＜0.05);24h urine have no significant difference between each treatment group and model group (P>0.05); the kidney index of water extract groups(high and low dose) and ethanol extract groups(high and low dose) were significantly reduced (P＜0.05). The SCr and kidney index of low dose group of water extract and ethanol extract groups(high and low dose) were significantly lower than Niaoduqing granule group(P＜0.05). The SCr of ethanol extract groups(high and low dose) were significantly lower than water extract groups (high and low dose)(P＜0.05) and the BUN were significantly lower than Niaoduqing granule group(P<0.05). The SCr of low dose group of water extract was significantly lower than high dose group of water extract(P<0.05).3Changes of MDA, AOPPs, AGEs and SOD activity of kidney:Compared with normal group, MDA, AOPPs, AGEs of model group were significantly increased(P＜0.05) and SOD activity of kidney was significantly decreased(P<0.05). MDA and AOPPs of low dose group of water extract and ethanol extract groups(high and low dose) were significantly lower than model group(P＜0.05). MDA of ethanol extract groups(high and low dose) was significantly lower than Niaoduqing granule group(P<0.05); low dose group of ethanol extract was significant lower than water extract groups(high and low dose) and high dose group of ethanol extract(P<0.05). MDA of low dose group of water extract and high dose group of ethanol extract were significantly lower than high dose group of water extract(P<0.05). AOPPs of low dose group of water extract was significantly lower than high dose group of water extract, ethanol extract groups(high and low dose) and Niaoduqing granule group(P<0.05). AGEs has no significant difference between each treatment group and model group (P>0.05). Kidney SOD activity of water extract groups(high and low dose) and ethanol extract groups(high and low dose) were significantly increased in comparison with model group and Niaoduqing granule group(P<0.05).4Pathological changes of kidney:Pathological picture of model group showed that nephron was atrophyed or reduced regionally; glomerulus were reduced obviously, and have atrophy, sclerosis or even necrosis more or less; kidney tubules were destroyed seriously, some tubules diminished or expanded, protein cast and granular cast can be observed in expanded tubules; there were a large number adenine metabolites crystals in tubules and tubulointerstitial; lymphocyte and giant cell infiltration could be observed in tubulointerstitial and some could see proliferation of fibrous tissue. The basic pathological changes of each treatment groups were the same with model group, but the destruction of tubules and crystals in tubulointerstitial were decreased in varying degrees.Conclusions:1Water and ethanol extract of CHG can obviously reduce SCr, BUN of CRF rats, improve renal function. Water extract low-dose and ethanol extract(high and low-dose) can obviously inhibit oxidative stress in CRF rats and reduce the AOPPs, which maybe one of the major mechanism for improving the renal function.2The effect of water and ethanol extract of CHG on SCr, kidney index and kidney SOD activity were better than Niaoduqing granule; the effect of water extract on AOPPs and ethanol extract on BUN and MDA were better than Niaoduqing granule. Part Ⅱ Study on the Effective Part of Ethanol Extract of CHGObjective:1To investigate the effective part of ethanol extract of CHG which have influence on the progress of CRF. Screen different polar parts of ethanol extract of CHG by a pharmacodynamics study. Observe the effects of different polar parts on SCr, BUN and kidney index in CRF mouse.2To analyze the origin of chemical composition in acetic ether part of ethanol extract and some composition may contain in it.Methods:1Pharmacodynamics study of different polar parts of ethanol extract of CHG1.1Separation of different polar parts:Mix ethanol extract with silica gel by half-and-half, then put into soxhlet extractor. Use20times acetic ether, n-butyl alcohol, absolute ethyl alcohol to extract in turn. Then get different polar parts by removing the extract solvent.1.2Pharmacodynamics study:Eighty male KM mice were randomly divided into ten groups (n=8):normal group, model group, Niaoduqing granule group, ethanol extract group, acetic ether part high dose and low dose group, n-butyl alcohol part high dose and low dose group, ethyl alcohol part high dose and low dose group. The trial was last3weeks. CRF model was made by ig adenine once every two days for the first week(200mg-kg-1), once every three days for the last two weeks(100mg-kg-1). Mice were treated with Niaoduqing granule(6.5g-kg-1), ethanol extract (3.78), acetic ether part (1.13,0.56), n-butyl alcohol part (0.95,0.47), ethyl alcohol part(3.21,1.60) since day one, respectively. After executed, blood samples were immediately collected to detect SCr, BUN and body weight and kidney index was measured.2Study on the chemical composition of acetic ether part of ethanol extractRHPLC and Acetonitrile and water gradient elution was used to separate chemical composition. Chromatography of acetic ether part of CHG and each crude drug were detected respectively. Then the chromatography peaks of acetic ether part of CHG were sorted in comparison with chromatography peaks of each crude drug. LC-MS/MS was used to analyze the chemicals of acetic ether part.Results:1Pharmacodynamics study of different polar parts of ethanol extract of CHG:Compared with normal group, SCr, BUN and kidney index of model group were significantly increased (P＜0.05); SCr and BUN of ethanol extract group, acetic ether part high dose and low dose group and ethanol part high dose group were significantly lower than model group(P<0.05). SCr and BUN of ethanol extract group were significantly lower than Niaoduqing granual group(P<0.05). SCr of low dose group of acetic ether part was significantly lower than Niaoduqing granual group(P<0.05). BUN of high dose group of ethanol part was significantly lower than low dose group of ethanol part(P<0.05). No significance were observed between ethanol extract group and acetic ether part high dose and low dose group and ethanol part high dose group in SCr and BUN (P>0.05). There were no significance between treatment group and model group in kidney index(P>0.05).2Study on the chemical composition of acetic ether part of ethanol extract:Chromatography peaks were well separated in this analyze method. Each chromatography peak of acetic ether part could find their origin. By comparing the molecular weight and product ion spectra of reference subtences and the peaks in acetic ether part with the same retention time as the standards, we can get the conclusion that acetic ether part contains Liquiritin, Aloeemodi, Emodin, Chrysophanol and Physcion.Conclusions:1Acetic ether part and ethanol part maybe the effect part of ethanol extract of CHG.2The main composition of acetic ether part could find their origin. Aacetic ether part contain Liquiritin, Aloe-emodi, Emodin, Chrysophanol, Physcion. |
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