Dissection of the potential pharmacological function of neohesperidin dihydrochalcone - a food additive - by in vivo substances profiling and network pharmacology.

Food additives are widely used in our daily life, and the side-effects caused by them have gained extensive attention around the world. Notably, constituent-oriented metabolites, in some sense, always contribute to pharmacological changes, inducing toxicity, therapeutic effects, etc. Characterization of the metabolites and their potential functions is of great importance to the practical applications. In this work, an integrated strategy by combining metabolite profiling and network pharmacology was applied to characterize the metabolic features and reveal pharmacological changes of neohesperidin dihydrochalcone (NHDC) in vivo to demonstrate its pharmacological mechanism and potential functions. As a result, a total of 19 metabolites (3 in plasma, 19 in urine, 8 in feces, 3 in heart, 5 in liver, 0 in spleen, 1 in lung, 2 in kidneys and 2 in brain) were screened and 18 of them were characterized for the first time. Phase I metabolic reactions of hydrolysis and phase II reactions of glucuronidation, sulfation, glutamylation, N-butyryl glycylation and lactylation were the main metabolic reactions of NHDC in vivo. Moreover, the results analyzed by network pharmacology revealed that, in addition to common pathways (steroid hormone biosynthesis) of NHDC, metabolites' targets were involved in pathways in cancer, ovarian steroidogenesis, proteoglycans in cancer, PI3K-Akt signaling pathway and progesterone-mediated oocyte maturation, indicating that these functional changes might result in potential novel functions or other side-effects, such as a disorder of steroid hormones. Our work provided the metabolic features and functional modifications of NHDC in vivo for the first time, and meaningful information for further pharmacological validations or potential functions is supplied.

Liquid chromatography tandem mass spectrometry method for determination of antidiabetic chalcones derivative S001-469 in rat plasma, urine and feces: application to pharmacokinetic study.

A sensitive and selective liquid chromatography tandem mass spectrometry assay was developed for quantitation of a novel antidiabetic chalcones derivative S001-469 in rat matrices. Plasma and urine samples were prepared by double liquid-liquid extraction with diethyl ether and feces by protein precipitation using acetonitrile. Chromatographic elution was carried on cyano guard column (30 mm × 4.6 mm i.d., 5 µm) in isocratic mode at a flow rate of 0.75 mL/min using mobile phase comprising of methanol: ammonium acetate buffer (pH 4.6, 10 mM) (90:10, v/v). Run time was 6 min. Detection was achieved by employing positive ionization mode on a triple-quadrupole LC-MS/MS system with an electrospray ionization (ESI) source. The calibration curves were linear over the range of 0.78-400 ng/mL for all 3 matrices. The method was validated and proved reliable through high and consistent intra- and inter- day accuracy and precision (<15%) values. Recoveries was >85% from spiked plasma, urine and feces samples. S001-469 was stable in plasma at room temperature till 8 h and at -60 °C for 30 d and 3 freeze-thaw cycles.

Bioavailability of dietary (poly)phenols: a study with ileostomists to discriminate between absorption in small and large intestine.

A feeding study was carried out in which six healthy ileostomists ingested a juice drink containing a diversity of dietary (poly)phenols derived from green tea, apples, grapes and citrus fruit. Ileal fluid and urine collected at intervals over the ensuing 24 h period were then analysed by HPLC-MS. Urinary excretions were compared with results obtained in an earlier study in which the juice drink was ingested by ten healthy control subjects with an intact colon. Some polyphenol components, such as (epi)catechins and (epi)gallocatechin(s), were excreted in urine in similar amounts in ileostomists and subjects with an intact colon, demonstrating that absorption took place principally in the small intestine. In the urine of ileostomists, there were reduced levels of other constituents, including hesperetin-7-O-rutinoside, 5-O-caffeoylquinic acid and dihydrochalcones, indicating their absorption in both the small and large intestine. Ileal fluid analysis revealed that even when absorption occurred in the small intestine, in subjects with a functioning colon a substantial proportion of the ingested components still pass from the small into the large intestine, where they may be either absorbed before or after catabolism by colonic bacteria.

Quantification of sofalcone in human plasma and urine by high performance liquid chromatography-mass spectrometry.

Sofalcone, isolated from the root of the Chinese medicinal plant Sophora subprostrata, is well known to be a mucosal protective agent for gastritis and peptic ulcer treatment. Although the LC-MS/MS and HPLC-DAD methods for assay of plasma concentration of sofalcone were reported before for the pharmacokinetic study, they were either too complicated or not sensitive enough for current pharmacokinetic study. In addition, no urinary assay method or pharmacokinetic information was available. Thus an improved high performance liquid chromatography-mass spectrometric method employing negative electrospray ionization was developed for the determination of sofalcone concentration in human plasma and urine sample. A liquid-liquid extraction method was utilized to extract sofalcone together with the indometacin (internal standards) from 0.5ml of human plasma or urine samples. Multiple reaction monitoring was used for quantification by monitoring the transition of m/z from 449.5 to 313.1 for sofalcone and 356.9 to 313.0 for IS. The validation of the method regarding to specificity, sensitivity, linearity, reproducibility, accuracy and stability was evaluated. The lower limit of quantification (LLOQ) of the developed assay method for sofalcone was 0.5ng/ml and the linear calibration curve was acquired with R(2)>0.99 between 0.5 and 500ng/ml for both plasma and urine samples. The intra- and inter-day variations of the current assay were evaluated with the relative standard deviation (RSD) within 13.77% at low concentration of quality control samples (QCs) and 8.71% for other QCs, whereas the mean accuracy ranged from 96.21 to 107.33%. The samples were found to be stable under the storage conditions at least for a month and other experimental conditions. This validated method was then utilized to test sofalcone concentration in clinical samples. Based on these data, the pharmacokinetic behavior of sofalcone in plasma as well as urine was described. As a conclusion, the present method proved to be a rapid and sensitive analytical tool for sofalcone in human plasma or urine samples and has been successfully applied to a clinical pharmacokinetic study of in healthy Chinese subjects.

Identification and quantification of metabolites of orally administered naringenin chalcone in rats.

Naringenin chalcone is the main active component of tomato skin extract, which has an antiallergic activity. In this study, naringenin chalcone was orally administered to rats, and the chemical structures and levels of the major metabolites in the plasma and urine of rats were determined. HPLC analysis indicated the presence of three major metabolites in the urine. LC-MS and NMR analyses tentatively identified these as naringenin chalcone-2'-O-beta-D-glucuronide, naringenin-7-O-beta-D-glucuronide, and naringenin-4'-O-beta-D-glucuronide. Naringenin chalcone-2'-O-beta-D-glucuronide was the only metabolite detected in the plasma, and its peak plasma level was observed 1 h after naringenin chalcone administration. Naringenin chalcone-2'-O-beta-D-glucuronide also inhibited histamine release from rat peritoneal mast cells stimulated with compound 48/80. This activity might contribute to the antiallergic activity of naringenin chalcone in vivo. To the best of the authors' knowledge, this study is the first to report determination of naringenin chalcone metabolites in rat plasma and urine.

Absorption, metabolism, and excretion of cider dihydrochalcones in healthy humans and subjects with an ileostomy.

The phloretin-O-glycosides, phloretin-2'-O-glucoside and phloretin-2'-O-(2''-O-xylosyl)glucoside, are thought to be unique to apples and apple products. To investigate the metabolism and bioavailability of these compounds, nine healthy and five ileostomy human subjects consumed 500 mL of Thatchers Redstreak apple cider containing 46 micromol of phloretin-O-glycosides. Over the ensuing 24 h period, plasma, urine, and ileal fluid were collected prior to analysis by high-performance liquid chromatography-mass spectrometry (HPLC-MS). The sole metabolite present in quantifiable amounts in plasma was phloretin-2'-O-glucuronide, which reached a peak concentration (C(max)) of 73 nmol/L and 0.6 h after ingestion (T(max)) with the healthy subjects, and statistically similar values were obtained with the ileostomy volunteers. Phloretin-2'-O-glucuronide was also detected in urine along with two additional phloretin-O-glucuronides and a phloretin-O-glucuronide-O-sulfate. The quantity of phloretin metabolites excreted in urine represented 5.0 + or - 0.9% of intake in healthy volunteers and 5.5 + or - 0.6% in ileostomy volunteers. The similarity in the excretion levels of the two groups and the rapid plasma T(max) indicate absorption of the dihydrochalcones in the small intestine. Of the two major phloretin-O-glycosides in cider, only phloretin-2'-O-(2''-O-xylosyl)glucoside was recovered in ileal fluid in quantities corresponding to 22% of intake. The absence of phloretin-2'-O-glucoside in ileal fluid suggests that it is more readily absorbed than phloretin-2'-O-(2''-O-xylosyl)glucoside. Phloretin-2'-O-glucuronide, two other phloretin-O-glucuronides, one phloretin-O-glucuronide-O-sulfate, two phloretin-O-sulfates, and the aglycone phloretin were also detected in the ileal fluid. This implies that the wall of the small intestine contains beta-glycosidase, sulfuryltransferase, and UDP-glucuronosyltransferase activities and that, as well as being absorbed, sizable amounts of the phloretin metabolites that are formed efflux back into the lumen of the gastrointestinal tract. The overall recovery of the dihydrochalcones and their metabolites in the ileal fluid was equivalent to 38.6% of intake.

Aspalathin, a flavonoid in Aspalathus linearis (rooibos), is absorbed by pig intestine as a C-glycoside.

Aspalathin, a dihydrochalcone and C-glycoside, is the most abundant flavonoid in rooibos (Aspalathus linearis), which is well known as an herbal tea in many countries. Aspalathin appears to have in vitro antioxidative and antimutagenic effects. To understand the effects of aspalathin in the body, research on the absorption in the intestinal tract, metabolism in the body, and identification of circulating metabolites in vivo is required. We investigated the metabolism of aspalathin to identify the parent compound and related metabolites in urine and plasma after orally administering a rooibos extract (16.3% aspalathin by 96 g rooibos extract, which equates to 1.1 kg dried rooibos material), produced from unfermented rooibos plant material, to pigs over a period of 11 days (oral dosage, 157-167 mg aspalathin per kg body weight daily). On days 7 and 11 of the study and days 1 and 2 after termination, urine was collected in 24-hour fractions, and plasma samples were collected at various time points. To our knowledge, this is the first time aspalathin metabolites have been identified in vivo, by presenting evidence of the absorption of aspalathin. Six substances identified in the urine by liquid chromatography-mass spectrometry were identified; these represent aspalathin and the metabolites methylated aspalathin, glucuronidated aspalathin glucuronidated and methylated aspalathin, a glucuronidated aglycone of aspalathin, as well as a metabolite of eriodictyol. The latter compound was methylated and contained 2 glucuronic acid moieties. This study showed that aspalathin can be absorbed by the intestine as C-glycoside as well as being cleaved in an aglycone and sugar moiety. The major metabolite in the enzymatically treated samples was methylated aspalathin. Between 0.1% and 0.9% of the administered dose of aspalathin could be detected in the urine on days 7 and 11 of the feeding study. No metabolites or aspalathin were found in plasma samples. The identification of the metabolites in vivo enables investigations to determine the biological potential of rooibos extracts.