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Mulberry leaf has attracted much attention due to its excellent curative effect on diabetes and its complications, whether the combination of its effective components have protective and synergistic effect on diabetic nephropathy (DN) in vivo remain unclear.The aim of this study was to investigate the protective and synergistic effect of the combination (MAFA step by step method consisted of network pharmacological prediction, animal in vivo validation and metabolic mechanism research was used to construct the multi-component-target-pathway network of mulberry leaf against DN. Firstly, the potential components and mechanism of mulberry leaf against DN was explored by network pharmacology analysis. Secondly, DN animal model was established to validate the anti-DN activity of these potential compounds. Thirdly, the metabolomics of serum and urine samples from animal experiments was analyzed to explore the anti-DN mechanism of these potential compounds.The results of network pharmacology demonstrated that a total of 7 compounds detected in MA and MF exhibited anti-DN activity, their mechanism were strongly in connection with metabolic pathways, arachidonic acid metabolism, sphingolipid signaling pathway, etc. The results of animal experiment indicated that MAFMAF

Danshen (Salviae Miltiorrhizae Radix et Rhizoma, SMR) has been used as a traditional Chinese medicine in clinic for treatment of coronary heart diseases. Previous works have shown that the chronic renal failure (CRF) is closely related to changes of intestinal bacteria. The aim is to explore the interaction between active components of SMR and intestinal bacteria from normal and CRF rats. The changes of intestinal bacteria were evaluated among normal rats, CRF model rats and SMR-treated rats via 16S rRNA gene sequencing technology. UPLC-QTOF/MS was applied for the analysis and identification of metabolites. RESULTS: Results showed that the following intestinal bacteria varied significantly in CRF rats, including Mucispirillum, Kurthia, Clostridium, Blautia, Butyrivibrio, Shuttleworthia, Peptococcus, Ruminococcus, Bradyrhizobium, Methylobacterium, Azospirillum, Thalassospira, Methylophilus, Pseudomonas, peptostreptococcaceae and bacteroidales. The ethanol extract of SMR (DS) significantly regulated Shuttleworthia, peptostreptococcaceae and Pseudomonas, while the water extract (DSS) significantly affected Peptococcus, peptostreptococcaceae and Ruminococcus. Methylation, demethylation, dehydrogenation, hydrogenation and hydroxylation were the major metabolic transformation of tanshinones in vitro by intestinal bacteria. Glucuronidation, methylation and hydrogenation were the main metabolic transformation of salvianolic acids. These results showed that the bioactive components of SMR, including tanshinones and salvianolic acids, might exert the medical effect via regulation intestinal bacteria.

Diabetic nephropathy (DN) is a complication of diabetes that is caused by uncontrolled high blood sugar. It has been reported that Salvia miltiorrhiza (SM) possesses the ability to prevent kidney damage, although the mechanisms remain unclear. The study was to investigate whether and how SM improved DN injury via regulation of metabolome and the molecular mechanisms. In this study, SD rats were fed a high glucose / high fat diet accompanied by 0.5% glucose water. Three weeks later, the rats were given one intraperitoneal injection of 30 mg/kg STZ each day for three days for DN model. The biochemical indicators and metabolomics of plasma, urine and renal tissue were analyzed. Then the western blotting analysis of renal tissue and glomerular mesangial cells were investigated. The results showed that Salvia miltiorrhiza extracts improved the renal injury and regulation of abnormal glycolipid metabolism. The metabolites in serum, urine and renal tissues have been changed significantly. The involved metabolic pathways mainly include phospholipid, arachidonic acid, and pyrimidine metabolisms. Meanwhile, SM inhibited the relative expression levels of wnt4, β-catenin and TGF-β in renal tissue and high-glucose induced glomerular mesangial cells.

Ethnopharmacological relevance The flower of Abelmoschus manihot (Linn.) Medicus ( A. manihot), as a traditional Chinese Herbal medicine, was used widely in China with efficacy of inducing diuresis for treating strangurtia, and subdhing swelling and detoxicating. It has been reported that Huangkui capsule, prepared by the extract of the flower of A. manihot , can reduce the content of urinary protein, serum creatinine and serum urea nitrogen in nephropathy rats and processes renoprotective activity, while the action mechanism need to illuminate deeply. Aims of the study In this study, we investigated the protection effect of Huangkui capsule on tubulointerstitial fibrosis in chronic renal failure (CRF) rats and its mechanism against high glucose-induced epithelial to mesenchymal transition (EMT) in renal tubular epithelial cells (HK-2) of its bioactive components. Materials and methods The animals were divided into normal group, CRF model group and Huangkui capsule-treated group. Hematoxylin eosin (HE) staining and Masson staining were applied to observe pathological changes in renal tissue of different groups. Biochemical indicators including serum urea nitrogen (BUN), urine protein (UP) and serum creatinine (Scr) were measured according to the manufacturer's instructions of kits. HK-2 cell damaged model was established to access the protection effect and action mechanism of five main flavonoids from Huangkui capsule. The experimental cells were divided into eight groups: control group, model group, positive drug group and five main flavonoids treated groups. The dichlorodihydrofluorescein diacetate (DCFH-DA) assay was used to determine the reactive oxygen species (ROS) in different groups. Western blot was applied to analyze the expression of pathogenesis-related proteins in different groups. Results The results stated that Huangkui capsule significantly inhibited the elevation of Scr, BUN, UP, the expression of α-smooth muscle actin (α-SMA), phosphorylation-extracellular signal-regulated kinase (p-ERK1/2), NADPH Oxidase 1, NADPH Oxidase 2 and NADPH Oxidase 4 in adenine-induced CRF rats. The main bioactive components of quercetin (QT), hyperoside (HY), isoquercitrin (IQT), gossypetin-8- O - β -D-glucuronide (GG) and quercetin-3′- O -glucoside (QG) at the dosage of 100 µM, like NADPH oxidase inhibitor diphenyleneiodonium, exhibited a significant effect on inhibiting the expression of α-SMA, p-ERK1/2, NADPH Oxidase 1, NADPH Oxidase 2 and NADPH Oxidase 4 in high glucose-induced HK-2 cells, especially GG. Conclusions These results demonstrated that Huangkui capsule and the flavonoids components prevent tubulointerstitial fibrosis in CRF rat involvement in the action mechanism of inhibiting NADPH oxidase/ROS/ERK pathway.

Rehmannia glutinosa Libosch (RG), is officially listed in the Chinese Pharmacopoeia and is widely used in China. The leaves of RG (LR) is an important vegetative organ of the plant. At present, the total glycosides of RG (TLR) were extracted from RG, and developed a national second class of new drugs to the Dihuangye total glycoside capsule (DTG). Additionally, DTG has the effect of nourishing yin and tonifying kidney, promoting blood circulation and blood cooling, and applicable to chronic glomerulonephritis mild to Qi and Yin Deficiency. Moreover, diabetic nephropathy (DN) rats model was induced by intraperitoneal injection of a small dose of streptozotocin (45 mg/kg) and high-fat diet and plus 5% glucose drinking water. Over 15 days, after oral administration TLR and DTG in DN rats, samples from serum, urine and kidney were collected for biochemical indicators measurements, pathological analysis, western blotting and metabolomics. Therefore, the analytical results of biochemical indicators, histopathological observations and western blotting showed that TLR and DTG exhibited a significant effect in renal protection. And 27 endogenous metabolites (12 in serum and 15 in urine) could be tentatively identified in the process of DN in rats using metabolomics method. Those endogenous metabolites were chiefly involved in sphingolipid metabolism; pentose, glucuronate interconversion; terpenoid backbone biosynthesis; purine metabolism and retinol metabolism. After drug intervention, these endogenous metabolites turned back to normal level some extent (P < 0.05). Furthermore, TLR and DTG prevent high glucose-induced glomerular mesangial cells (GMCs) by inhibiting TGF-β1 and Wnt/β-catenin signaling pathway, providing a powerful supports to develop a new therapeutic agent for DN. This study paved the way for further exploration of the pathogenesis of DN, early diagnosis and the evaluation of curative effect.

Ethnopharmacological relevance Chronic renal failure (CRF) is defined as a progressive and irreversible loss of renal function and associated with inflammation and oxidative stress. Salvia miltiorrhiza (SM) is an important Chinese herb used in traditional Chinese medicine for treating cardiovascular diseases. The previous studies showed the SM exhibited significant protective effects on CRF. In this present study, the metabolic profiling changes and action mechanism of SM on CRF were explored. Aims of the study The aims of this study were to illustrate the metabolic profiling changes of adenine induced CRF and analyze the protective effects and action mechanisms of SM ethanol extract (SMEE) and water extract (SMWE). Materials and methods The animals were divided into normal group, CRF model group, Huangkui capsule-treated group, SMEE-treated group and SMWE-treated group. The UPLC−QTOFMS coupled with multivariate statistical methods were used to explore the changes of metabolic profile in plasma, urine and renal tissue from CRF rats simultaneously after treatment with SMEE and SMWE. Hematoxylin eosin (HE) staining and Masson staining were applied to observe pathological changes in renal tissue. Biochemical indicators including serum urea nitrogen (BUN), urine protein (UP) and serum creatinine (Scr) were measured according to the manufacturer's instructions of kits. Furthermore, HK-2 cell damaged model induced by ISF was established to access the protective effects and action mechanism. The dichlorodihydrofluorescein diacetate (DCFH-DA) assay was used to determine the reactive oxygen species (ROS) and Western blot was applied to analyze the expression of pathogenesis-related proteins in different groups. Results The results showed that the ethanol extract (SMEE) and water extract (SMWE) of SM significantly inhibited the elevation of serum creatinine (Scr), blood urea nitrogen (BUN), urine protein (UP) and indoxyl sulfate (ISF) in adenine-induced CRF rats, especially SMEE exhibited more significant effects. Moreover, SM extracts obviously improved the symptoms of glomerular and tubular atrophy, focal calcium deposits, interstitial fibrosis, interstitial inflammation, and renal tissues. By metabolomics analysis, fifty-nine metabolites (thirteen in plasma, twenty-seven in urine and nineteen in kidney tissue) were up-regulated or down-regulated and contributed to CRF progress. After treatment of SM extracts, the altered metabolites were restored back to normal level. These potential biomarkers underpinning the metabolic pathways are including phenylalanine metabolism, pyrimidine metabolism, purine metabolism and tryptophan metabolism. Furthermore, SM extracts prevent epithelial-mesenchymal transition (EMT) of human renal tubular epithelial (HK-2) cell by inhibiting NADPH oxidase/ROS/ERK and TGF-β/Smad signaling pathways. Conclusions SMEE and SMWE can significantly alleviate adenine-induced CRF via regulation of the metabolic profiling and modulation of NADPH oxidase/ROS/ERK and TGF-β/Smad signaling pathways, which provided important supports for the development of protective agent of SM for CRF.

Salvia miltiorrhiza, one of the major traditional Chinese medicines, is commonly used and the main active ingredients—tanshinones—possess the ability to improve renal function. In this paper, the UPLC-TQ/MS method of simultaneously determining four tanshinones—tanshinone IIA, dihydrotanshinone I, tanshinone I, and cryptotanshinone—was established and applied to assess the pharmacokinetics in normal and chronic renal failure (CRF) rat plasma. The pharmacokinetics of tanshinones in rats were studied after separately intragastric administration of Salvia miltiorrhiza ethanol extract (SMEE) (0.65 g/kg), SMEE (0.65 g/kg) combined with Salvia miltiorrhiza water extract (SMWE) (1.55 g/kg). The results showed Cmax and AUC0–t of tanshinone IIA, tanshinone I, cryptotanshinone reduced by 50%~80% and CLz/F increased by 2~4 times (p < 0.05) in model group after administrated with SMEE. Nevertheless, after intragastric administration of a combination of SMWE and SMEE, the Cmax and AUC0–t of four tanshinones were upregulated and CLz/F was downregulated, which undulated similarity from the model group to the normal group with compatibility of SMEE and SMWE. These results hinted that SMWE could improve the bioavailability of tanshinones in CRF rats, which provides scientific information for further exploration the mechanism of the combination of SMWE and SMEE and offers a reference for clinical administration of Salvia miltiorrhiza.

Alpinia oxyphylla (Zingiberaceae) is a well-known medicinal plant. Its fruit ("Yi-Zhi-Ren" in Chinese) is used as an anti-diuretic and traditionally used for the treatment of enuresis and reduce urination. Chronic kidney disease (CKD) is a disease with the characteristic of the slowly loss of kidney function and has a prevalence of up to 7-10% in adults. Recent advances in its etiology and pathogenesis are providing more speculative hypotheses focused on integral systems. Using a UPLC-QTOF-MS/MS-based metabolomic platform, we explored the changes of metabolic profiling in plasma/urine simultaneously between chronic kidney disease (CKD) induced from adenine excess and the protective effects of A. oxyphylla extract (AOE). The total twenty-one metabolites (twelve in urine and nine in plasma), up-regulated or down-regulated, were identified and contributed to CKD progress. Among these biomarkers, agmatine, CAMP, 7-methylguanine, hippuric acid, indoxyl sulfate, asparagines, kynurenic acid and p-cresol sulfate were restored back to the control-like level after the treatment of AOE (p

Background Galangin (3,5,7-trihydroxyflavone) is present in high concentrations in herbal medicine such as Alpinia officinarum Hance. Galangin shows multifaceted in vitro and in vivo biological activities. The number and position of hydroxyl groups in this molecule play an important role in these biological activities. However, these hydroxyl groups undergo glucuronidation and sulfation in in vitro assay system. However, the systemic exposure to galangin after dosing in animals and/or humans remains largely unknown. Thus it is not clear whether the galangin exists in the body at concentrations high enough for the biological effects. Furthermore, the metabolite identification and the corresponding plasma pharmacokinetics need to be characterized. Results Two LC-MS/MS methods were developed and validated and successfully applied to analyze the parent drug molecules and aglycones liberated from plasma samples via β-glucuronidase hydrolysis. Our major findings were as follows: (1) The routes of administration showed significant influences on the systemic exposure of galangin and its metabolites. (2) Galangin was preferentially glucuronidated after p.o. dosing but sulfated after i.v. medication. (3) Kaempferol conjugates were detected demonstrating that oxidation reaction occurred; however, both glucuronidation and sulfation were more efficient. (4) Oral bioavailability of free parent galangin was very low. Conclusions Systemic exposure to galangin and its metabolites was different in rat plasma between oral and intravenous administration. Further research is needed to characterize the structures of galangin conjugates and to evaluate the biological activities of these metabolites.

Rehmannia glutinosa Libosch (RG), is officially listed in the Chinese Pharmacopoeia and is widely used in China. In this paper, a sensitive and rapid ultra-performance liquid chromatography-mass spectrometry method including multiple-reaction monitoring mode was developed and applied to study the pharmacokinetic effect of acteoside from total glycoside extracted from the leaves of Rehmannia (TLR) and Dihuangye total glycoside capsule (DTG) in normal and diabetic nephropathy rats. The diabetic nephropathy rat model was induced by intraperitoneal injection of a small dose of streptozotocin and high-fat diet and plus 5% glucose drinking water. Samples of plasma of rats were obtained at different times after rats were administered TLR (7.2 g/kg) and DTG (360 mg/kg). After deproteinization by acetonitrile, the concentrations of acteoside in rats at different time points were detected by UPLC-TQ-MS method and pharmacokinetics parameters were calculated using DAS 3.2.8 software. A good linearity of acteoside was shown in the range of 8.51-3404.8 ng/m L (r2 = 0.9987). The mean extraction recovery of analyte was in the range of 63.55-79.49%, and the intra- and inter-day RSD values were