Liuwei Dihuang Pills Research Articles

Bioinformatics and network pharmacology discover the molecular mechanism of Liuwei Dihuang pills in treating cerebral palsy.

A collection of chronic central motor, postural, and activity restriction symptoms are referred to as cerebral palsy (CP). Previous research suggests that a number of perinatal variables, including hypoxia, may be linked to CP. And the pathophysiological process that causes brain injury in growing fetuses is mostly caused by amniotic fluid infection and intra-amniotic inflammation. Still, there is still much to learn about the molecular mechanism of CP. The goal of this study was to identify the molecular mechanism of Liuwei Dihuang pill (LWDHP) in the treatment of CP using network pharmacology and bioinformatics. The Chinese medicine database provided the LWDHP components and targets, the CP illness gene data set was gathered from a disease, and the expression profile of children with CP was chosen from anther database. Using the Kyoto Encyclopedia of Genes and Genomes and gene ontology databases, a network of interactions between proteins was created, and functional enrichment analysis was carried out. Analysis of traditional Chinese medicine found that the key active ingredients of LWDHP are quercetin, Stigmasterol and kaempferol. Through enrichment analysis, it was found that the hub genes for LWDHP treatment of CP are CXCL8, MMP9, EGF, PTGS2, SPP1, BCL2L1, MMP1, and AR. K EGG analysis found that LWDHP treatment of CP mainly regulates PI3K-Akt signaling pathway, IL-17 signaling pathway, Jak-STAT signaling pathway, NF-kappa B signaling pathway, etc. To summarize, LWDHP regulates immunological and inflammatory variables through a variety of components, targets, and signaling pathways, which plays a significant role in the development and management of CP.

Liuwei Dihuang Pills alleviates postmenopausal osteoporosis and fatigue in rats by inhibiting the epigenetic regulatory molecule BRD4 pathway

To explore the role of epigenetic signal molecule bromodomain protein 4 (BRD4) in mediating the therapeutic effect of Liuwei Dihuang (LWDH) Pills on postmenopausal osteoporosis (PMOP) and fatigue. Thirty rat models of PMOP induced by bilateral ovariectomy were randomized equally into two groups for treatment with normal saline (model group) or LWDH Pills (385.7 mg/kg), with another 15 sham-operated rats as the sham operation group. After 12 weeks of treatment, femoral samples were taken to determine the bone density and BRD4 protein expression. The weight-bearing exhaustive swimming time of the rat models was recorded, and serum levels of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) were measured using enzyme-linked immunosorbent assay. In cultured primary osteoblasts the changes in the expressions of BRD4, MAPK and NF-κB proteins were detected by immunofluorescence staining following treatment with LWDH Pills. The rat models of PMOP showed significantly up-regulated expression of BRD4 protein in the femoral tissue (P < 0.05), which was obviously lowered by treatment with LWDH Pills. The rats treated with LWDH Pills also showed significant improvement of fatigue. Immunofluorescent staining of the osteoblasts showed that treatment with LWDH Pills significantly decreased the protein expressions of BRD4, MAPK and NF-κB. Analysis of the GSE56116 dataset revealed that that patients with kidney-yin deficiency had significantly higher BRD4 expression than those in the kidney-yang-deficiency group and non-kidney-deficiency group (P < 0.05). The upregulation of BRD4 expression involved multiple signaling pathways including neural ligand receptor response, cytoskeleton rearrangement, cytokine interaction, and granulocyte colony-stimulating factor chemotaxis pathways. LWDH can alleviate PMOP and fatigue by decreasing BRD4 signaling pathway, suggesting that potential of BRD4 as a promising therapeutic target for PMOP.

Multicomponent Analysis of Liuwei Dihuang Pills by a Single Marker Quantification Method and Chemometric Discrimination of Fingerprints.

An effective and comprehensive quality evaluation method for Liuwei Dihuang pills (LDP) was established by the simultaneous determination of 8 active components in LDP by the quantitative analysis of multicomponents by single marker (QAMS) method and high-performance liquid chromatography (HPLC) fingerprint combined with chemometrics. These 8 active components were determined by QAMS and the external standard method (ESM), and the quantitative results of the two methods were compared to validate the accuracy and feasibility of the QAMS method. 8 active components showed good linear relationships within their ranges, whose average recoveries were 99.7∼102.3%. No significant difference was found (P > 0.05) in the quantitative results determined by QAMS and ESM. Furthermore, the fingerprint of LDP was also established, with 11 common peaks identified, and the similarity of the fingerprints of 21 batches of LDP was greater than 0.95. The 21 batches of LDP were basically divided into 3 groups by hierarchical cluster analysis (HCA) and principal component analysis (PCA), and 3 differential markers were screened out by orthogonal partial least squares discriminant analysis (OPLS-DA). The established QAMS method is accurate, economical, fast, and convenient and can simultaneously determine the content of 8 active components in LDP. HPLC fingerprint combined with chemometric analysis more comprehensively evaluated the quality consistency of different batches of LDP and analyzed the markers that cause quality differences between batches. It can provide a scientific basis and reference of quality consistency evaluation for the manufacturers and drug regulatory departments of the preparation.

Study on the Mechanism of Liuwei Dihuang Pill in Treating Hypothyroidism Based on Network Pharmacology

Objective: To study the mechanism of Liuwei Dihuang Pill (LWDP) in the treatment of hypothyroidism from the perspective of network pharmacology. Methods: The active ingredients of Liuwei Dihuang pill were screened by TCMSP platform database and the target prediction was carried out. GeneCards database was used to screen genes associated with hypothyroidism.The target of Liuwei Dihuang pill in treating hypothyroidism was obtained by Venny platform. The protein-protein interaction (PPI) network, gene ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of these targets were performed to reveal the possible mechanism of Liuwei Dihuang pill in treating hypothyroidism. Results: A total of 28 active components and 189 targets were identified from Liuwei Dihuang Pills, and 4502 targets were identified from GeneCard database. The PPI network contains 124 nodes and 323 edges. 662 biological processes (BP), 72 cell components (CC) and 127 molecular functions (MF) were obtained by GO enrichment. A total of 149 signaling pathways were obtained by KEGG enrichment analysis, and the top 20 signaling pathways were visualized and analyzed.The signal pathways related to hypothyroidism include AGE-RAGE signal pathway, lipid and atherosclerosis signal pathway, HIF-1 signal pathway, PI3K-Akt signal pathway and MAPK signal pathway. Conclusion: The main components of Liuwei Dihuang pill in treating hypothyroidism are quercetin, kaempferol, β-sitosterol, diosgenin, kadsurenone, hederagenin, sitosterol and stigmasterol. The main targets of Liuwei Dihuang pill are MAPK1, TP53, TNF, AKT1 and EGFR, and they play a therapeutic role through AGE-RAGE, lipid and atherosclerosis, HIF-1, PI 3K-Akt and MAPK signal pathways.

Liuwei Dihuang Pills in the Treatment of Senile Dementia: A Review

Objective To elucidate the synergistic mechanism of Liuwei Dihuang Pills (LWDHP), with multi-components, multi-targets and multi-channels in the treatment of senile dementia. Methods We searched Chinese and English databases to obtain the blood components of LWDHP, its clinical and preclinical mechanism research progress in the treatment of senile dementia. Based on the network pharmacology of the key pathophysiological process of Alzheimer’s disease (AD), we screened the anti-AD target of LWDHP and its blood components. Results The therapeutic effect of LWDHP on senile dementia is accurate, with few adverse reactions. Its mechanism includes regulation of cholinergic system, autophagy, cerebral microvascular function, neuron apoptosis, insulin signal transduction pathway, etc. In this review, 35 blood components of LWDHP based on HPLC were collected, and their drug toxicity and safety were evaluated. The network pharmacology analysis based on the pathophysiological process of AD shows that the key components of LWDHP in the treatment of AD are Alisol A monoacetate, Linoleic acid, Alisol F, Tumulosic acid, Methyl eugenol, Palmitic acid and Pachymic acid. Conclusion This article summarizes 35 blood components of LWDHP, summarizes the synergistic mechanism of LWDHP in the treatment of senile dementia, uses network pharmacology to provide a series of potential lead compounds for the development of senile dementia’s drugs, and provides a theoretical basis for further expanding the clinical application of LWDHP.

The potential mechanism of Liu-Wei-Di-Huang Pills in treatment of type 2 diabetic mellitus: from gut microbiota to short-chain fatty acids metabolism.

Type 2 diabetes mellitus (T2DM) has already become a global pandemic. Recently, reports showed its pathogenesis was closely related to a disorder of gut microbiota. In China, the Liu-Wei-Di-Huang Pills (LWDH) have treated T2DM for thousands of years. However, its therapeutic mechanism associated with gut microbiota is worthy of further study. This study aims to investigate the effects of LWDH on T2DM by regulating gut microbiota and short-chain fatty acids (SCFAs) in Goto-Kakizaki (GK) rats. T2DM models were successfully established based on GK rats and administrated with LWDH. The changes in fasting blood glucose (FBG), oral glucose tolerance test (OGTT), and serum insulin (INS) were determined, and the immunohistochemical (IHC) method was used to test INS expression in pancreas. The 16S-ribosomal DNA (16S rDNA) sequencing analysis assessed gut microbiota structural changes; a gas chromatography-mass spectrometer (GC-MS)-based metabolomics method was adopted to detect SCFA levels. The pathological morphology of jejunum was detected by hematoxylin-eosin (H&E) staining, and the expression of GPR43, GPR41, GLP-1, and GLP-1R was evaluated by qRT-PCR and ELISA, respectively. We observed that GK rats treated with LWDH: (a) has altered the microbial structure and promoted the abundance of bacteria in Firmicutes, including Lactobacillus, Allobaculum, and Ruminococcus_2, (b) increased SCFAs levels involving acetic acid, propionic acid, and butyric acid and (c) alleviated T2DM and jejunum injuries potentially based on SCFAs-GPR43/41-GLP-1 pathway. LWDH could improve T2DM by regulating gut microbiota and SCFAs, and the therapeutic mechanism might be related to the SCFAs-GPR43/41-GLP-1 pathway.

Study on the Mechanism of Liuwei Dihuang Pills in Treating Parkinson's Disease Based on Network Pharmacology.

Background Parkinson's disease (PD) is a common neurodegenerative disease in middle-aged and elderly people. Liuwei Dihuang (LWDH) pills have a good effect on PD, but its mechanism remains unclear. Network pharmacology is the result of integrating basic theories and research methods of medicine, biology, computer science, bioinformatics, and other disciplines, which can systematically and comprehensively reflect the mechanism of drug intervention in disease networks. Methods The main components and targets of herbs in LWDH pills were obtained through Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP). Its active components were screened based on absorption, distribution, metabolism, and excretion (ADME); the PD-related targets were obtained from the Genecards, OMIM, TTD, and DRUGBANK databases. We used R to take the intersection of LWDH- and PD-related targets and Cytoscape software to construct the drug-component-target network. Moreover, STRING and Cytoscape software was used to analyze protein–protein interactions (PPI), construct a PPI network, and explore potential protein functional modules in the network. The Metascape platform was used to perform KEGG pathway and GO function enrichment analyses. Finally, molecular docking was performed to verify whether the compound and target have good binding activity. Results After screening and deduplication, 210 effective active ingredients, 204 drug targets, 4333 disease targets, and 162 drug-disease targets were obtained. We consequently constructed a drug-component-targets network and a PPI-drug-disease-targets network. The results showed that the hub components of LWDH pills were quercetin, stigmasterol, kaempferol, and beta-sitosterol; the hub targets were AKT1, VEGFA, and IL6. GO and KEGG enrichment analyses showed that these targets are involved in neuronal death, G protein-coupled amine receptor activity, reactive oxygen species metabolic processes, membrane rafts, MAPK signaling pathways, cellular senescence, and other biological processes. Molecular docking showed that the hub components were in good agreement with the hub targets. Conclusion LWDH pills have implications for the treatment of PD since they contain several active components, target multiple ligands, and activate various pathways. The hub components possibly include quercetin, stigmasterol, kaempferol, and beta-sitosterol and act through pairing with hub targets, such as AKT1, VEGFA, and IL6, to regulate neuronal death, G protein-coupled amine receptor activity, reactive oxygen species metabolic process, membrane raft, MAPK signaling pathway, and cellular senescence for the treatment of PD.

Exploring the Molecular Mechanism of Liuwei Dihuang Pills for Treating Diabetic Nephropathy by Combined Network Pharmacology and Molecular Docking.

Background Diabetic nephropathy (DN) is a common and serious complication of diabetes, but without a satisfactory treatment strategy till now. Liuwei Dihuang pills (LDP), an effective Chinese medicinal formula, has been used to treat DN for more than 1000 years. However, its underlying mechanism of action is still vague. Methods Active compounds and corresponding targets of LDP were predicted from the TCMSP database. DN disease targets were extracted from the OMIM, GeneCards, TTD, DisGeNET, and DrugBank databases. Subsequently, the “herbal-compound-target” network and protein-protein interaction (PPI) network were constructed and analyzed via the STRING web platform and Cytoscape software. GO functional and KEGG pathway enrichment analyses were carried out on the Metascape web platform. Molecular docking utilized AutoDock Vina and PyMOL software. Results 41 active components and 186 corresponding targets of LDP were screened out. 131 common targets of LDP and DN were acquired. Quercetin, kaempferol, beta-sitosterol, diosgenin, and stigmasterol could be defined as five crucial compounds. JUN, MAPK8, AKT1, EGF, TP53, VEGFA, MMP9, MAPK1, and TNF might be the nine key targets. The enrichment analysis showed that common targets were mainly associated with inflammation reaction, oxidative stress, immune regulation, and cell apoptosis. AGE-RAGE and IL-17 were the suggested two significant signal pathways. Molecular docking revealed that the nine key targets could closely bind to their corresponding active compounds. Conclusion The present study fully reveals the multicompound's and multitarget's characteristics of LDP in DN treatment. Furthermore, this study provides valuable evidence for further scientific research of the pharmacological mechanisms and broader clinical application.

A randomized controlled clinical trial study protocol of Liuwei Dihuang pills in the adjuvant treatment of diabetic kidney disease.

A large number of patients with diabetic kidney disease (DKD) approach traditional Chinese medicine (TCM) owing to discontent with standard treatments. Based on TCM theory and clinical experience, the syndrome of kidney yin deficiency is a common type of DKD. Liuwei Dihuang pills (LDPs) is a common prescription of a Chinese herbal formula for patients presenting this syndrome of DKD. However, well-established data supporting the efficacy and safety of LDP in DKD treatment are lacking. We have designed a double-blind, placebo-controlled, randomized trial. After a 2-week run-in period, 124 eligible participants with DKD will be assigned to either the experimental or the control group in a 1:1 ratio. Patients in the experimental group will receive LDP, while patients in the control group will receive a matched placebo. As the basic treatment in the 2 groups, metformin hydrochloride sustained-release tablets, for blood glucose control, and irbesartan tablets, for blood pressure regulation, will be provided. All participants will undergo 4 weeks of treatment and 12 weeks of follow-up. The primary outcome is the change in 24 hours urinary protein levels, measured from the baseline to the end of the treatment phase (week 24). The secondary outcomes to be assessed include the change in serum creatinine and estimated glomerular filtration rate, urinary albumin excretion rate, improvement of TCM syndromes and symptoms, fasting blood glucose and postprandial 2-hour blood glucose, blood lipids (total cholesterol, triglyceride, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol, from baseline to weeks 12 and 24. The results of this study will provide high-quality evidence of the effects of LDP in DKD treatment, which will provide an alternative treatment strategy in patients with DKD.

Effects of Compound Active Peptides on Protecting Liver and Intestinal Epithelial Cells from Damages and Preventing Hyperglycemia.

Active peptides have good effectiveness in controlling or preventing many diseases. Compound active peptides (CAP) obtained from animal, plant, and sea food proteins were used in this study to explore their effects on antioxidation, anti-inflammation, and antihyperglycemia in vitro and in vivo. The results demonstrated that 10 μg/mL CAP could increase cell viability (P < 0.05) and decrease reactive oxygen species (ROS) levels and cell apoptosis (P < 0.05) when WRL68 cells were induced by H2O2 for 6 h. Moreover, incubation with 20 μg/mL CAP for 6 h significantly increased cell viability and Bcl-2 expression level (P < 0.05) and decreased expression levels of IL-6, IL-8, TNF-α, Bax, and Caspase 3 and the ratio of Bax/Bcl-2 (P < 0.05) when swine jejunal epithelial cells (IPEC-J2) were induced by deoxynivalenol (DON). In addition, adding CAP individually or combined with Liuweidihuang pills (LDP, Chinese medicine) and low-dose glibenclamide could lower blood glucose levels in alloxan-induced hyperglycemic model mice. These results suggested that CAP was probably a beneficial ingredient for alleviating H2O2-induced oxidative stress and DON-induced cell inflammation and apoptosis and preventing hyperglycemia.

ALWPs Improve Cognitive Function and Regulate Aβ Plaque and Tau Hyperphosphorylation in a Mouse Model of Alzheimer's Disease.

Recently, we reported that ALWPs, which we developed by combining Liuwei Dihuang pills (LWPs) with antler, regulate the LPS-induced neuroinflammatory response and rescue LPS-induced short- and long-term memory impairment in wild-type (WT) mice. In the present study, we examined the effects of ALWPs on Alzheimer’s disease (AD) pathology and cognitive function in WT mice as well as 5x FAD mice (a mouse model of AD). We found that administration of ALWPs significantly reduced amyloid plaque levels in 5x FAD mice and significantly decreased amyloid β (Aβ) levels in amyloid precursor protein (APP)-overexpressing H4 cells. In addition, ALWPs administration significantly suppressed tau hyperphosphorylation in 5x FAD mice. Oral administration of ALWPs significantly improved long-term memory in scopolamine (SCO)-injected WT mice and 5x FAD mice by altering dendritic spine density. Importantly, ALWPs promoted spinogenesis in primary hippocampal neurons and WT mice and modulated the dendritic spine number in an extracellular signal-regulated kinase (ERK)-dependent manner. Taken together, our results suggest that ALWPs are a candidate therapeutic drug for AD that can modulate amyloid plaque load, tau phosphorylation, and synaptic/cognitive function.

Bioinformatics Analysis Reveals the Altered Gene Expression of Patients with Postmenopausal Osteoporosis Using Liuweidihuang Pills Treatment.

Postmenopausal osteoporosis (PMOP), as well as its associated increased risk for fragility fracture, is one of the most disabling consequences of aging in women. This present study aimed to identify candidate genes that involve pathogenesis of PMOP and the therapeutic mechanism of Liuweidihuang (LWDH) pills on PMOP. We integrated microarray datasets of PMOP derived from the Gene Expression Omnibus (GEO) to screen differentially expressed genes (DEGs) between PMOP and normal controls as well as patients with PMOP and patients after treatment of LWDH pills. GO and KEGG enrichment analysis for DEGs were performed. The shared DEGs, associated with both the pathogenesis of PMOP and the therapeutic mechanism of LWDH, were further analyzed by protein-protein interaction (PPI) network. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to verify the DEGs obtained by our integrated analysis. Compared with normal controls, 1732 DEGs in PMOP were obtained with p<0.05. According to the qRT-PCR results, expression of ATF2, FBXW7, RDX, and RBBP4 was consistent with that in our integrated analysis, generally. GO and KEGG enrichment analysis showed that those DEGs were significantly enriched in regulation of transcription, DNA-dependent, cytoplasm, protein binding, and MAPK signaling pathway. A total of 58 shared DEGs in PMOP versus normal control and in patients with PMOP versus patients after LWDH treatment were identified, which had opposite expression trend in these two comparisons. In the PPI network, CSNK2A1, ATF2, and FBXW7 were three hub proteins. Three genes including ATF2, FBXW7, and RDX were speculated to be therapeutic targets of LWDH for PMOP based on BATMAN-TCM database. We speculated that three genes of ATF2, FBXW7, and RDX may play crucial roles in both pathogenesis of PMOP and therapeutic mechanism of LWDH on PMOP. Our results may provide clues for the molecular pathogenesis of PMOP and offer new possibilities for treatment of PMOP.

An Aqueous Extract of Herbal Medicine ALWPs Enhances Cognitive Performance and Inhibits LPS-Induced Neuroinflammation via FAK/NF-κB Signaling Pathways.

Recent studies have shown that Liuwei Dihuang pills (LWPs) can positively affect learning, memory and neurogenesis. However, the underlying molecular mechanisms are not understood. In the present study, we developed ALWPs, a mixture of Antler and LWPs, and investigated whether ALWPs can affect neuroinflammatory responses. We found that ALWPs (500 mg/ml) inhibited lipopolysaccharide (LPS)-induced proinflammatory cytokine IL-1β mRNA levels in BV2 microglial cells but not primary astrocytes. ALWPs significantly reduced LPS-induced cell-surface levels of TLR4 to alter neuroinflammation. An examination of the molecular mechanisms by which ALWPs regulate the LPS-induced proinflammatory response revealed that ALWPs significantly downregulated LPS-induced levels of FAK phosphorylation, suggesting that ALWPs modulate FAK signaling to alter LPS-induced IL-1β levels. In addition, treatment with ALWPs followed by LPS resulted in decreased levels of the transcription factor NF-κB in the nucleus compared with LPS alone. Moreover, ALWPs significantly suppressed LPS-induced BV2 microglial cell migration. To examine whether ALWPs modulate learning and memory in vivo, wild-type C57BL/6J mice were orally administered ALWPs (200 mg/kg) or PBS daily for 3 days, intraperitoneally injected (i.p.) with LPS (250 μg/kg) or PBS, and assessed in Y maze and NOR tests. We observed that oral administration of ALWPs to LPS-injected wild-type C57BL/6J mice significantly rescued short- and long-term memory. More importantly, oral administration of ALWPs to LPS-injected wild-type C57BL/6J mice significantly reduced microglial activation in the hippocampus and cortex. Taken together, our results suggest that ALWPs can suppress neuroinflammation-associated cognitive deficits and that ALWPs have potential as a drug for neuroinflammation/neurodegeneration-related diseases, including Alzheimer’s disease (AD).

Holistic analysis of Liuwei Dihuang pills using ultrasonic cell grinder extraction and ultra-performance liquid chromatography

An efficient holistic analysis strategy was developed and validated for quality control of the traditional Chinese medicine (TCM) preparation, Liuwei Dihuang pills (LWDHPs). In this strategy, eight bioactive components, i. e. allantoin, morroniside, loganin, peoniflorin, acteoside, paeonol, alisol B 23-acetate and pachymic acid from the six medicinal herbs composing LWDHPs were selected as the evaluation markers, and then they were simultaneously extracted by an ultrasonic cell grinder extraction (UCGE) method and separated in a single run by an ultra-performance liquid chromatographic method coupled with photodiode array detection (UPLC-PDA). Response surface methodology (RSM), a multivariate experimental design technique, was used to optimize the UCGE parameters based on Box-Behnken design (BBD) model. The optimum extraction conditions were obtained at a ratio of solvent to solid 45 mL/g, extraction time of 40 min and extraction temperature of 20℃. Under such conditions, the higher yields of the eight analytes were acquired compared with the traditional extraction methods, indicating that UCGE was a fast, easy and efficient method for extracting bioactive constituents from LWDHPs. Chromatographic separation was achieved on an HSS T3 column (50 mm×2.1 mm, 1.8 μm) using a linear gradient elution of acetonitrile and water. Because of the different UV characteristics of these components, five detection wavelengths were used for quantitative analysis. The proposed method was validated through linearity, limits of detection, limits of quantification, precision, stability, repeatability, and accuracy. The validated method was applied to analyze LWDHPs, which provided a reference for the quality evaluation of LWDHPs.

Liuwei Dihuang Pills Enhance the Effect of Western Medicine in Treating Diabetic Nephropathy: A Meta-Analysis of Randomized Controlled Trials.

Objectives. To assess the effectiveness and adverse effects of adding Liuwei Dihuang Pills (LDP) to Western medicine for treating diabetic nephropathy. Methods. Studies were retrieved from seven electronic databases, including PubMed, Embase, The Cochrane Library, CBM, CNKI, Chinese Scientific Journal Database (VIP), and Wanfang Data until November 2015. Study selection, data extraction, quality assessment, and data analyses were conducted according to Cochrane standards. Meta-analysis was performed on the overall therapeutic efficacy of hyperglycemia and renal functions, and the study also analyzed adverse events. Results. A total of 1,275 patients from 18 studies were included. The methodological quality of these included trials was generally low. We found that adding LDP can lower patients' FBG (MD: −0.36 [−0.46, −0.25], P < 0.00001), PBG (MD: −1.10 [−1.35, −0.85], P < 0.00001), and HbA1c (MD: −0.14 [−0.49, 0.21], P = 0.43). There were also improvements in lowering patients' BUN (MD: −0.67 [−0.89, −0.45], P < 0.00001), SCr (MD: −0.96 [−1.53, −0.39], P < 0.00001), 24 h UTP (SMD: −1.26 [−2.38, −0.15], P < 0.00001), UAER (MD: −26.18 [−27.51, −24.85], P < 0.00001), and UmAlb (SMD: −1.72 [−2.67, −0.77], P < 0.00001). Conclusion. There is encouraging evidence that adding LDP to Western medicine might improve treatment outcomes of diabetic nephropathy, including hyperglycemia and renal functions. However, the evidence remains weak. More rigorous high-quality trials are warranted to substantiate or refute the results.

Enhanced Estrogenic Activity of Soybean Isoflavones by Coadministration of Liuwei Dihuang Pills in Ovariectomized Rats.

Soybean isoflavones are beneficial for treating hormone-related diseases. Simultaneous consumption of soybean isoflavones and Liuwei Dihuang pills (LWPs) is effective for treating perimenopausal period syndrome. However, why the combination of isoflavones and LWPs is more effective than ingestion of each component alone remains unknown. Here, we show that enhanced estrogenic activities would appear when the ovariectomized rats were fed with a soybean diet in combination of LWPs treatment. Our further studies explored enhancements of Lactobacillus (19-fold) and Bifidobacterium (12-fold) contents in the intestine of rat and 1.84-fold higher intestinal β-glucosidase activity in LWPs treatment group compared with the control group. As a result, steady-state concentrations of genistein (1.20-fold), daidzein (1.36-fold), and equol (1.43-fold) in serum were significantly elevated in the combination group compared with the soybean alone group. The results present the first evidence of the mechanism of enhanced estrogenic activity of dietary soybean isoflavones in combination with LWPs. Our study indicates that alterations of gut bacteria after LWPs treatment play a key role in the enhanced estrogenic effect of dietary soybean, suggesting a direct relationship between dietary soybean, LWPs, and gut flora.

Effects of Liuwei-Dihuang Pills combined with Ginkgo Biloba Leaves Extract Tablets on complications of type 2 diabetes mellitus

Objective To investigate the effects of Liuwei-Dihuang Pills combined with Ginkgo Biloba Leaves Extract Tablets on diabetic complications in patients with type 2 diabetes mellitus (T2DM). Methods Seventy-eight patients with T2DM were randomized into a TCM treatment group and a conventional treatment group by random number table method, 39 in each group. The patients in the conventional treatment group were regularly using antihyperglycemic drugs or insulin. On the basis of conventional treatment group, the patients in the TCM treatment group were additionally treated with Liuwei-Dihuang Pills and Ginkgo Biloba Leaves Extract Tablets. Intima-media thickness (IMT) of internal carotid artery, vibration perception threshold (VPT) and diabetic retinopathy in both groups were detected before the treatment, 12 and 36 months after the treatment. Results The VPT in the left foot 12 months after the treatment in the TCM treatment group were significantly lower than that in the conventional treatment group (13.98 ± 4.38 V vs. 18.70 ± 2.43 V; t=2.764, P=0.008). IMT of internal carotid artery (left: 0.81 ± 0.16 mm vs. 0.70 ± 0.10 mm, t=3.120, P=0.003; right: 0.81 ± 0.17 mm vs. 0.73 ± 0.12 mm, t=2.286, P=0.026), VPT (left foot: 14.82 ± 6.45 V vs. 20.63 ± 9.75 V, t=2.714, P=0.009; right foot: 16.73 ± 7.10 V vs. 20.73 ± 10.35 V, t=2.001, P=0.048) and incidence of diabetic retinopathy (35.9% vs. 53.8%; χ2=5.804, P=0.016) 36 months after the treatment in the TCM treatment group were significantly lower than those in the conventional treatment group. Conclusions Liuwei-Dihuang Pills combined with Ginkgo Biloba Leaves Extract Tablets can effectively reduce the progression of diabetic complications in patients with T2DM. Key words: Liu Wei Di Huang Wan; Folium Ginkgo; Diabetes mellitus, type 2; Diabetes complications

Application of metabonomic strategy to discover an unreported active ingredient in LiuWeiDiHuang pills suppressing beta-glucuronidase.

Identification of the bioactive ingredient from traditional Chinese medicine (TCM) remains a challenging task by traditional approach that focuses on chemical isolation coupled with biological activity screening. Here, we present a metabonomics-based approach for bioactive ingredient discovery in LiuWeiDiHuang pills (LWPs). First, a non-targeted high-performance liquid chromatography ultraviolet (HPLC-UV) profiling of rat urine was used to discriminate urinary profiling intervened by LWPs. Orthogonal partial least-squares discriminant analysis (OPLS-DA) revealed that eight chromatographic peaks made a significant contribution to the classification of the LWPs group and the control group. Five of these chromatographic peaks were successfully isolated and identified as hippurate, genistein (GT), daidzein (DZ), and glucuronide conjugate of GT and that of DZ by mass spectroscopy (MS). Subsequently, we found that LWPs significantly decreased the activity of intestinal β-glucuronidase by 18% and exerted a dose-dependent inhibitory effect on rat liver lysosomal fraction, suggesting that LWPs were a β-glucuronidase inhibitor. In the end, by inhibiting β-glucuronidase-guided isolation, D-glucaro-1,4-lactone, a previously unreported ingredient of LWPs, was identified by MS, MS/MS, and nuclear magnetic resonance spectroscopy. Our findings indicated that metabonomics might increase research productivity toward the drug targets and/or bioactive compounds from TCM.

Serum metabonomic study on liuwei dihuang pills in the treatment of hyperthyroidism

Aim: To explore the underlying mechanism of hyperthyroidism induced by subcutaneous injection of L-levothyroxine sodium and and intervention effects of Liuwei Dihuang pills on sprague dawley rats via serum metabolomics. Methods: After rats were injected with L-levothyroxine sodium at a dosage of 35 μg·(100 g)-1 every day for 1 week,and then Liuwei dihuang pills(LDPs) were treated at a dose of 4.7 g·kg-1 for 3 consecutive weeks.The serum samples for rats were obtained after the treatment of LDPS and the metabolic profiles of serum were obtained with ultra performance liquid chromatography coupled with time-of-flight mass spectrometry(UPLC-QTOF-MS). Results: The levels of oxaloacetic acid,Dihydroxyacetone phosphate(DHAP),ethyl glucuronide,triglyceride,lysophospholipids and cholesterol were significantly increased in the model group as compared to the normal rats.Moreover,these levels could be regulated to be close to normal after the treatment of LDPS. Conclusions: There is severe disturbance in the tricarboxylic acid cycle as well as sugar and lipid metabolism for hyperthyroidism rats.LDPS can exhibite obvious effects on the treatment of hyperthyroidism.Serum metabonomics analysis reveals the changes of metabolites traits during the treatment of LDPS,thus providing a supplementary evaluation method of LDPS in the treatment of hyperthyroidism.

A simple HPLC method for simultaneous analysis of 7 bioactive constituents for Liuwei Dihuang Pill and its application in quality consistency evaluation

A combinative method using HPLC fingerprinting and quantitative analysis was developed and validated for manufacturer-to-manufacturer quality consistency evaluation of Liuwei Dihuang pills (LDP). Seven bioactive constituents of LDP, including gallic acid, 5-hydroxymethyl furfural, morroniside, paeoniflorin, sweroside, loganin and paeonal, were selected as markers to evaluate the similarities of 20 samples from 16 manufacturers. The similarity values for the 20 LDP samples were all greater than 0.92, indicating that there is a general quality consistency for different manufacturers. Additionally, the 7 selected bioactive compounds were in accordance with the compatibility principle of “the monarch drug, the ministerial drug, the adjunctive drug and the messenger drug” of Traditional Chinese Medicine (TCM) prescription and the multi components quality evaluation requirements of TCM as well. The detection and quantification limits for these 7 bioactive components were all less than 0.11 μg mL−1 and 0.38 μg mL−1, respectively. The mean recovery for all the investigated constituents was acceptable with an accuracy of 98–102%. The intra- and inter-day precisions of this method were less than 4.73%, and stability and repeatability were less than 3.23%. This method is simple and reliable, and also shows that the combination of the chromatographic fingerprint and quantitative analysis offers an efficient solution to quality consistency evaluation of herbal preparations.