Investigation of the Lipid-Lowering Mechanisms and Active Ingredients of Danhe Granule on Hyperlipidemia Based on Systems Pharmacology

Objective Investigate the active ingredients and underlying hypolipidemic mechanisms of Danhe granule (DHG). Methods The lipid-lowering effect of DHG was evaluated in hyperlipidemic hamsters induced by a high-fat diet. The ingredients absorbed into the blood after oral administration of DHG in hamsters were identified by ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF/MS). A systems pharmacology approach incorporating target prediction and network construction, gene ontology (GO) enrichment and pathway analysis was performed to predict the active compounds and map the compounds-targets-disease network. Real-time polymerase chain reaction (RT-PCR) and Western blot were utilized to analyze the mRNA and protein expression levels of predicted targets. Results DHG remarkably lowered the levels of serum total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-c), and arteriosclerosis index (AI), at the same time, elevated the levels of serum high-density lipoprotein cholesterol (HDL-c) and HDL-c/TC ratio in hyperlipidemic hamsters. Sixteen ingredients absorbed into blood after oral administration of DHG were identified as the possible components interacted with targets. Moreover, 65 potential targets were predicted after targets intersection and compounds–targets–disease network mapping. Then, compounds–targets–pathways network mapping revealed that six active compounds (emodin, naringenin, etc.) compounds could interact with 10 targets such as sterol regulatory element binding protein (SREBP) 1c, SREBP-2 and peroxisome proliferation-activated receptor (PPAR) α, regulate three lipid metabolism-related pathways including SREBP control of lipid synthesis pathway, PPAR signaling pathway and nuclear receptors in lipid metabolism and toxicity pathway, and further affect lipid metabolic processes including fatty acid biosynthesis, low-density lipoprotein receptor (LDLR)-mediated cholesterol uptake, bile acid biosynthesis, and cholesterol efflux. Experimental results indicated that DHG significantly increased SREBP-2, LDLR, PPARα, liver X receptor alpha (LXRα), cholesterol 7α-hydroxylase (CYP7A1), and ATP binding cassette subfamily A member 1 (ABCA1) mRNA and protein expressions while decreased SREBP-1c and fatty acid synthase (FAS) mRNA, and protein expressions. Conclusion DHG possessed a good hypolipidemic effect that may be through affecting the mRNA and protein expressions of SREBP-1c, FAS, SREBP-2, LDLR, PPARα, LXRα, CYP7A1, and ABCA1, involving in fatty acid synthesis, LDLR-mediated cholesterol uptake, bile acid biosynthesis, and cholesterol efflux. This study further provided experimental evidence about its practical application for treating hyperlipidemia and its complications.

Batch numbers of herbs in ten batches of Danhe granules 2. Instrumentation and chromatographic conditions Chromatographic analysis was performed on an Agilent 1260 Infinity highperformance liquid chromatograph system (Agilent Technologies, Waldbronn, Germany). The chromatographic data were processed with Agilent Chem Station. Samples separation was based on a ZORBAX Eclipse XDB-C18 column (4.6×250 mm, 5 μm) with column temperature at 30 ℃. The mobile phase was composed of solvent A (water-0.1% phosphoric acid) and solvent B (acetonitrile), and the flow rate was kept at 1.0 mL/min. The injection volume was 10 μL and the UV detection wavelength was set at 286 nm. The gradient elution was as follows: 0-55 min, 5-30% B; 55-60 min, 30-60% B; 60-70 min, 60-95% B; 70-75 min, maintained at 95% B.

Preparation of standard solutions and DHG samples
Stock solutions of six reference standards were prepared at a concentration of 1.0 mg/mL in methanol, respectively. Serial mixed standard working solutions with different concentrations were prepared through blends and dilutions of the stock solutions with methanol. The stock solutions were filtered through 0.45μm nylon membranes before injection. The DHG sample was weighed 0.20 g and extracted with methanol 50 mL at room temperature by ultrasonic extraction for 30 min, then filtered through 0.45μm nylon membranes before injection.

Precision, stability and repeatability validation
Precision validation: The sample solution (batch No: XS190318-01) was injected for 6 times continuously. Salvianolic acid B was used as the correction peak. The results showed that the RSD of the relative retention time of the common peaks was less than 1.20% and the relative peak area RSD was less than 1.52%. Repeatability validation: The sample (batch No: XS190318-01) was used to made six independently sample solutions. Salvianolic acid B was used as the correction peak. After analyzing the six sample solutions, results showed that the RSD of the relative retention time of the common peaks was less than 0.93% and the relative peak area RSD was less than 1.90%. Stability validation: The sample solution (batch No: XS190318-01) was analyzed at 0, 2, 4, 6, 12 and 24 h. Salvianolic acid B was taken as the correction peak. The results showed that the RSD of the relative retention time of the common peaks was less than 2.83% and the relative peak area RSD was less than 2.61%. The above results indicated that the method was with good accuracy, reliable and reproducible for the similarity and quantification analysis of DHG samples.

Establishment of chromatographic fingerprint
Based on the Similarity Evaluation System for Chromatographic Fingerprint of TCM (2012 edition), the similarities of the 11 batches of DHG samples (batch XS190312-01 as S1, batch XS190312-02 as S2, batch XS190318-01 as S3, batch XS190326-01 as S4, batch XS190409-01 as S5, batch XS190409-02 as S6, batch XS190412-01 as S7, batch XS190415-01 as S8, batch XS190420-01 as S9 and batch XS190420-02 as S10 and batch D1905007 as S11) was evaluated. After peak-picking, templatematching process, the peaks in the spectra were matched automatically (Shown in Figure S1). The reference template was set finally for spectra peak difference and entire similarity evaluation. The similarities of repeatability were greater than 0.94. The results showed that the preparation process of DHG was reasonable and feasible.
Comparability results of reproducibility of DHG samples 6. Quantitative analysis Under the above chromatographic conditions, based on the comparison of the standard references, six main HPLC chromatographic peaks in DHG sample were identified. In addition, the quantitative analysis of the six compounds in the DHG sample (batch D1905007) which was used in the pharmacological study was carried out. The results are as follows: the contents of gallic acid, polydatin, isoquercetin, hesperidin, salvianolic acid B and emodin-8-O-β-D-glucoside were 7.09 mg/g, 4.37 mg/g, 5.39 mg/g, 9.62 mg/g, 13.09 mg/g, and 2.15 mg/g, respectively. The chromatograms of mixed standards and DHG sample are listed in Supplementary Figure S1.    The detailed process of identification of serum ingredients Ten compounds including polydatin, hyperin, hesperidin, salvianolic acid b, nuciferine, quercetin, emodin 8-O-β-D-glucoside, nobiletin, emodin and tanshinone Ⅱ-A were identified by comparing with reference standards. In addition, other six compounds were identified based on consulting with literatures and fragment ions analysis. Compound 2 and compound 7 are isomers, exhibiting the [M+H] + ion at m/z 282.1495 (C18H19NO2). The differences between them are that compound 7 would produce obvious fragment ion at m/z 265 and compound 7 exhibited a longer retention time on a chromatographic column than compound 2. Then, compound 2 and compound 7 were identified as n-nornuciferine and o-nornuciferine, respectively, by consulting with literatures (Deng et al., 2016;Luo et al., 2005). Compound 4, the isomer of hyperin  (Pan et al., 2015). The information of the proposed fragment behaviors of the 16 compounds was also summarized in the following figures.