A four-component combination derived from Huang-Qin Decoction significantly enhances anticancer activity of irinotecan.

Huang-Qin Decoction (HQD) is a classic prescription for diarrhea in Chinese medicine treatment. Recent studies have demonstrated that HQD and its modified formulation PHY906 could ameliorate irinotecan (CPT-11) induced gastrointestinal (GI) toxicity and enhance its anticancer therapeutic efficacy. Nevertheless, which constituents in HQD are effective is still unclear so far. The study aims to screen out the key bioactive components combination from HQD that could enhance the anticancer effect of CPT-11. First, the potential bioactive constituents were obtained through system pharmacology strategy. Then the bioactivity of each constituent was investigated synthetically from the aspects of NCM460 cell migration, TNF-α release of THP-1-derived macrophage and MTT assay in HCT116 cell. The contribution of each constituent in HQD was evaluated using the bioactive index Ei, which taken the content and bioactivity into comprehensive consideration. And then, the most contributing constituents were selected out to form a key-component combination. At last, the bioefficacy of the key-component combination was validated in vitro and in vivo. As a result, a key-component combination (HB4) consisting of four compounds baicalin, baicalein, glycyrrhizic acid and wogonin was screened out. In vitro assessment indicated that HB4 could enhance the effect of CPT-11 on inhibiting cell proliferation and inducing apoptosis in HCT116. Furthermore, the in vivo study confirmed that HB4 and HQD have similar pharmacological activity and could both enhance the antitumor effect of CPT-11 in HCT116 xenograft model. Meanwhile, HB4 could also reduce the CPT-11 induced GI toxicity.

Quantification of phospholipid fatty acids by chemical isotope labeling coupled with atmospheric pressure gas chromatography quadrupole- time-of-flight mass spectrometry (APGC/Q-TOF MS).

Phospholipid fatty acids play the crucial role in biophysical properties and the function of cellular membranes. In the present study, an accurate and sensitive method was developed to quantify phospholipid fatty acids in biological samples by using chemical isotope labeling coupled with atmospheric pressure gas chromatography quadrupole-time-of-flight mass spectrometry (APGC/Q-TOF MS). APGC, a soft ionization source, was operated under proton-transfer condition by introducing methanol into the ionization source as a modifier, which provided high quantifiable molecular ion peaks to substantially enhance the sensitivity. Fatty acid standards were methylated with methanol-d4 to yield FAMEs-d3 that were used as one-to-one internal standards to ensure accurate quantification. Thirty fatty acids in phospholipids were accurately quantified in wide linear range with limit of quantification ranging from 84.6 to 113.2 pg/mL. The newly developed method was successfully applied to quantify phospholipid fatty acids in brain and liver tissues from both fat-1 and WT mice. This method might be expanded to quantify free fatty acids or other conjugated fatty acids in biological samples or other matrices.

Quantitative Evaluation of the Compatibility Effects of Huangqin Decoction on the Treatment of Irinotecan-Induced Gastrointestinal Toxicity Using Untargeted Metabolomics.

Huangqin decoction (HQD), a traditional Chinese medicine (TCM), has been widely used to treat gastrointestinal syndrome in China for thousands of years. Chemotherapy drug irinotecan (CPT-11) is used clinically to treat various kinds of cancers but limited by its side effects, especially delayed diarrhea. Nowadays, HQD has been proved to be effective in attenuating the intestinal toxicity induced by CPT-11. HQD consists of four medicinal herbs including Scutellaria baicalensis Georgi, Glycyrrhiza uralensis Fisch, Paeonia lactiflora Pall, and Ziziphus jujuba Mill. Due to its complexity, the role of each herb and the multi-herb synergistic effects of the formula are poorly understood. In order to quantitatively assess the compatibility effects of HQD, mass spectrometry-based untargeted metabolomics studies were performed. The serum metabolic profiles of rats administered with HQD, single S. baicalensis decoction, S. baicalensis-free decoction and baicalin/baicalein combination were compared. A time-dependent trajectory upon principal component analysis was firstly used to visualize the overall differences. Then metabolites deregulation score and relative area under the curve were calculated and used as parameters to quantitatively evaluate the compatibility effects of HQD from the aspect of global metabolic profile and the specifically altered metabolites, respectively. The collective results indicated that S. baicalensis played a crucial role in the therapeutic effect of HQD on irinotecan-induced diarrhea. Both HQD and SS decoction regulated glycine, serine and threonine pathway. This study demonstrated that metabolomics was a promising tool to elucidate the compatibility effects of TCM or combinatorial drugs.

Quantitation of camellianin A in HepG2 cells using a high performance liquid chromatography-electrospray ionization tandem mass spectrometric method.

The present study was designed to develop a sensitive and selective high performance liquid chromatography-tandem mass spectrometric method for the determination of Camellianin A in HepG2 cells. The extraction of Camellianin A was achieved using 15% trichloroacetic acid and then separated on a C18 column interfaced with a triple quadrupole tandem mass spectrometer in multiple reaction monitoring mode. The mobile phase was consisted of methanol-water (0.1% formic acid) (55 : 45, V/V). The total run time was 5.0 min. The method was linear in the concentration range of 0.25-250.0 ng·mL-1. The lower limit of quantification was 0.25 ng·mL-1. The intra- and inter-day relative standard deviations of entire concentration range were less than 9.3%. The proposed HPLC-MS/MS method was successfully applied to detect the intracellular concentration of Camellianin A in HepG2 cells.

Renal Medulla is More Sensitive to Cisplatin than Cortex Revealed by Untargeted Mass Spectrometry-Based Metabolomics in Rats.

Nephrotoxicity has long been the most severe and life-threatening side-effect of cisplatin, whose anticancer effect is therefore restricted. Previous pathological studies have shown that both renal cortex and medulla could be injured by cisplatin. Our TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling) assay results further uncovered that medulla subjected more severe injury than cortex. In order to depict the underlying metabolic mechanism of spatial difference in response to cisplatin, in the present study, mass spectrometry-based untargeted metabolomics approach was applied to profile renal cortex and medulla metabolites of rat after receiving a single dose of cisplatin (2.5, 5 or 10 mg/kg). Eventually, 53 and 55 differential metabolites in cortex and medulla were screened out, respectively. Random forest, orthogonal partial least squares-discriminant analysis and metabolic cumulative fold change analysis revealed that metabolic changes in medulla were more obviously dose-dependent than those in cortex, which confirmed the conclusion that medulla was more sensitive to cisplatin exposure. Furthermore, 29 intermediates were recognized as the most contributive metabolites for the sensitivity difference. Metabolic pathways interrupted by cisplatin mainly included amino acid, energy, lipid, pyrimidine, purine, and creatine metabolism. Our findings provide new insight into the mechanism study of cisplatin-induced nephrotoxicity.

Jiawei Erzhiwan improves menopausal metabolic syndrome by enhancing insulin secretion in pancreatic ß cells.

Menopausal metabolic syndrome (MMS) is a series of syndrome caused by ovarian function decline and hormone insufficiency, and is a high risk factor for cardiovascular diseases (CVD) and type II diabetes mellitus (T2DM). Erzhiwan (EZW), composed of Herba Ecliptae and Fructus Ligustri Lucidi, is a traditional Chinese herbal formula that has been used to treat menopausal syndrome for many years. We added Herba Epimedii, Radix Rehmanniae, and Fructus Corni into EZW, to prepare a new formula, termed Jiawei Erzhiwan (JE). The present study was designed to determine the anti-MMS effects of JE using ovariectomized (OVX) adult female rats that were treated with JE for 4 weeks, and ß-tc-6 cells and INS cells were used to detected the protect effectiveness of JE. Our results showed JE could increase insulin sensitivity and ameliorated hyperlipidemia. Metabolomics analysis showed that the serum levels of branched and aromatic amino acids were down-regulated in serum by JE administration. Moreover, JE enhanced the function of islet ß cells INS-1 and ß-tc-6, through increasing the glucose stimulated insulin secretion (GSIS), which was abolished by estrogen receptor (ER) antagonist, indicating that JE functions were mediated by ER signaling. Additionally, JE did not induce tumorigenesis in rat mammary tissue or promoted proliferation of MCF-7 and Hela cells. In conclusion, our work demonstrated that JE ameliorated OVX-induced glucose and lipid metabolism disorder through activating estrogen receptor pathway and promoting GSIS in islet ß cells, thus indicating that JE could be a safe and effective medication for MMS therapy.

Effect of Phyllanthus amarus Extract on 5-Fluorouracil-Induced Perturbations in Ribonucleotide and Deoxyribonucleotide Pools in HepG2 Cell Line.

The aim of this study was to investigate the antitumor activities of Phyllanthus amarus (PHA) and its potential of herb-drug interactions with 5-Fluorouracil (5-FU). Cell viability, ribonucleotides (RNs) and deoxyribonucleotides (dRNs) levels, cell cycle distribution, and expression of thymidylate synthase (TS) and ribonucleotide reductase (RR) proteins were measured with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, high performance liquid chromatography tandem mass spectrometry (HPLC/MS/MS) method, flow cytometry and Western blot analysis, respectively. Our standardized PHA extract showed toxicity to HepG2 cells at high concentrations after 72 h exposure and induced G2/M cell cycle arrest. Combined use of 5-FU with PHA resulted in significant decreases in ATP, CTP, GTP, UTP and dTTP levels, while AMP, CMP, GMP and dUMP levels increased significantly compared with use of 5-FU alone. Further, PHA could increase the role of cell cycle arrest at S phase induced by 5-FU. Although PHA alone had no direct impact on TS and RR, PHA could change the levels of RNs and dRNs when combined with 5-FU. This may be due to cell cycle arrest or regulation of key enzyme steps in intracellular RNs and dRNs metabolism.

Discrimination of Citrus reticulata Blanco and Citrus reticulata 'Chachi' by gas chromatograph-mass spectrometry based metabolomics approach.

Citri Reticulatae Pericarpium, mainly including the pericarp of Citrus reticulata Blanco and the pericarp of Citrus reticulata 'Chachi', has been consumed daily as food and dietary supplement for centuries. In this study, GC-MS based metabolomics was employed to compare comprehensively the volatile constituents in Citrus reticulata Blanco and Citrus reticulata 'Chachi'. Principal component analysis and orthogonal partial least squares discrimination analysis indicated that samples could be distinguished effectively from one another. Fifteen metabolites were finally identified for use as chemical markers in discrimination of Citri Reticulatae Pericarpium samples. The antimicrobial activity against Gram-negative and Gram-positive bacteria of the volatile oil from Citrus reticulata Blanco and Citrus reticulata 'Chachi' was investigated preliminarily.