A sensitive UPLC-MS/MS method for simultaneous determination of polyphenols and theaflavins in rat plasma: Application to a pharmacokinetic study of Da Hong Pao tea.

A method based on ultra-performance liquid chromatography-tandem mass spectrometry has been developed for the rapid and simultaneous determination of five catechins and four theaflavins in rat plasma using ethyl gallate as internal standard. The pharmacokinetic profiles of these compounds were compared after oral administration of five kinds of Da Hong Pao tea to rats. Biosamples processed with a mixture of ß-glucuronidase and sulfatase were extracted with ethyl acetate-isopropanol. Chromatographic separation was achieved by gradient elution using 10 mm HCOONH4 solution and methanol as the mobile phase. Analytes were detected using negative ion electrospray ionization in multiple reaction monitoring mode. The lower limits of quantification were 1.0, 0.74 and 0.5 ng/mL for theaflavins, two catechins and three catechins, respectively. The validation parameters were well within acceptable limits. The average half-lives (t1/2 ) in blood of the reference solution group was much shorter than those of tea samples. The values of AUC0-t and Cmax of the polyphenols and theaflavins exhibited linear pharmacokinetic characteristics which were related to the dose concentration.

Pharmacokinetic-pharmacodynamic modeling to study the anti-dysmenorrhea effect of Guizhi Fuling capsule on primary dysmenorrhea rats.

BACKGROUND: Primary dysmenorrhea (PDM) is one of the most common gynaecological disorders among women, which seriously affects women's life quality due to its high incidence rate. Guizhi Fuling capsule (GZFLC), a well-known traditional Chinese medical prescription, has been widely used to treat gynecological blood stasis syndromes such as PDM. However, its mechanisms of action and combination were still unknown. PURPOSE: The aim of this study was to develop a pharmacokinetic-pharmacodynamic (PK-PD) model to assess time-concentration-effect relationships for anti-dysmenorrhea effect of GZFLC and provide better understanding for mechanisms of action and combination of GZFLC. STUDY DESIGN AND METHODS: The PDM rats model was induced by oxytocin exposure following estradiol benzoate pretreatment. Gallic acid (GA), amygdalin (AMY), albiflorin (ALB), prunasin (PA) and cinnamic acid (CA) were evaluated as bioactive ingredients for investigating PK processes. GA, AMY, ALB and PA exhibited appropriate PK parameters and were selected as the PK markers to map the anti-dysmenorrhea effect of GZFLC. A PK-PD model was established on the basis of GA, AMY, ALB and PA plasma concentrations vs. the values of two ratios (PGE2/PGF2α and 6-Keto-PGF1α/TXB2), by a two-compartment PK model with a simple Emax model to explain the time delay between the drug plasma concentrations of PK markers and the anti-dysmenorrhea effect. RESULTS: The PDM rat model has been successfully established. Compared with the normal treated group, the bioactive ingredients in PDM treated group exhibited significant changing trends of PK behaviors, such as better absorption and distribution, slower elimination and delays in reaching the maximum concentration (Tmax). The analysis of PK-PD parameters indicated that the active metabolites and prototypes of bioactive ingredients in GZFLC were inclined to regulate the activity of prostacyclin synthetase and thromboxane synthetase to control the production of TXA2 and PGI2 so as to treat PDM. As the main effective medicinal materials for the treatment of PDM in GZFLC prescription Persicae Semen, Moutan Cortex and Paeonia lactiflora Pall, Persicae Semen played the most important role, while the role of Paeonia lactiflora Pall was the weakest. CONCLUSION: The PK-PD model results provided scientific basis for clarifying compatibility mechanisms of GZFLC prescription and a better understanding for biosynthetic mechanisms of four prostaglandins (PGE2, PGF2α, 6-Keto-PGF1α and TXB2) in the treatment of PDM by GZFLC. Investigations on the relationship between the effects and the bioactive ingredients are of benefit to explore the mechanisms of action and combination for traditional Chinese medical prescriptions (TCP) and facilitate the development of future clinical applications of TCP.

New alkylresorcinols from the fruits of Embelia ribes.

Nine new alkylresorcinols, designated as embelialkylresorcinols A-I (1-9), along with five known compounds (10-14) were isolated from the fruits of Embelia ribes. Their structures were elucidated by extensive spectroscopic analysis (1D, 2D NMR and HRESIMS), optical rotation data and modified Mosher method. Notably, embelialkylresorcinols A-H (1-8), possessing double aromatic rings linked with an aliphatic chain, are reported from the genus of Embelia (Myrsinaceae) for the first time. Most of the isolated compounds were evaluated for cytotoxic activity, and the results indicated that compounds 3, 5, 6 and 8 displayed moderate cytotoxicity against three human cancer cell lines (Hep3B, A549 and HCC1806) with IC50 values ranging from 23.06 to 41.49 µM.

Renoprotective Effects of Total Glucosides from Paeony against Nephrotoxicity Induced by Total Alkaloids from Semen Strychni.

Semen Strychni have been shown to have therapeutic effect in improving blood circulation, relieving rheumatic pain, and treating cancer. However, Semen Strychni could cause severe nephrotoxicity. The present study was designed to evaluate whether treatment with total glucosides from paeony (TGP) has renoprotective effect against nephrotoxicity induced by total alkaloids from Semen Strychni (TAS). The levels of blood urea nitrogen (BUN) and creatinine (Cr) were determined and histopathological changes were also examined to evaluate renal injury. Moreover, a HPLC-MS method was developed and validated to investigate the comparative toxicokinetics of strychnine and brucine in rats plasma after oral administration of TAS and pretreatment with TGP. Results demonstrated that the levels of BUN and Cr were significantly increased (p < 0.05) in TAS group, together with tubule epithelium cloudy swelling, degeneration, and glomerular atrophy in rats' kidneys. The TAS-induced kidney damage was alleviated after pretreatment with TGP. Besides, Tmax of strychnine and brucine were increased and T1/2 of strychnine and brucine were decreased after pretreatment with TGP. The toxicokinetics study showed that pretreatment with TGP could attenuate the absorption of strychnine and brucine, as well as accelerate their elimination. These results suggest that TGP possesses renoprotective effects.

Total glycosides of Paeony shows Neuroprotective effects against Semen Strychni-induced neurotoxicity by recovering secretion of hormones and improving brain energy metabolism.

In this study, we investigated the protective effect of total glycosides of paeony against Semen Strychni-induced neurotoxicity and discussed some probably mechanisms. Levels of estrone, estradiol, estriol and growth hormone in male rats' serum were determined by ELISA, levels of ATP and substances associated with energy metabolism in rats' brain were determined by HPLC and levels of progesterone was determined by a UPLC-MS/MS method. The results showed that neurotoxicity induced by Semen Strychni could cause a significant decrease (p < 0.05, compare to the blank group) in secretion of estrogens and GH and disorder brain energy metabolism at the same time. While, rats with total glycosides of paeony pre-protection (orally administrated with total glycosides of paeony for 15 days before administrating Semen Strychni extract) showed a much better condition in the secretion of hormones and brain energy metabolism, and showed no significant changes in most of those associated substances when comparing to the blank group. Our study indicated that total glycosides of paeony have neuroprotective effects on Semen Strychni-induced neurotoxicity. It could recover the disordered hormone secretion and improve the brain energy metabolism. Total glycosides of paeony is potential to be further used in clinic to protect against neurotoxicity induced by other reasons.

Monitoring the Consistency Quality and Antioxidant Activity of Da Hong Pao Teas by HPLC Fingerprinting.

In the present study, a high performance liquid chromatography (HPLC) method was developed and validated for fingerprint profiling to evaluate the quality consistency of the Da Hong Pao tea. The chromatographic separation was performed on a USA Waters C18 column (4.6 mm × 250 mm, 5 µm, Waters-XBridge Shield RP 18) with gradient elution. The column temperature was maintained at 30°C and the detection wavelength was set at 278 nm. The results demonstrated that the developed HPLC method achieved the desired linearity, precision, accuracy and limit of quantification. The fingerprints were developed and evaluated qualitatively using the ratio quantitative fingerprint method. The fingerprint analysis of the reference standards (caffeine, catechin, epicatechin, epigallocatechin, epicatechingallate, Epigallocatechingallate, theaflavin, theaflavin-3-gallate, theaflavin-3'-gallate and theaflavin-3, 3'-gallate) was found to reflect the content of the reference standards in the tea samples. Furthermore, the fingerprint-efficacy relationship among the components as well as the antioxidant activity in vitro were investigated. The selected samples showed good antioxidant activity based on the results. Our study demonstrated that the method of HPLC fingerprint profiling combine the antioxidant activity assay offering a reliable and efficient approach to quantitatively evaluate the quality consistency of the traditional Chinese medicine and other herbal preparations.

Development of quercetin-phospholipid complex to improve the bioavailability and protection effects against carbon tetrachloride-induced hepatotoxicity in SD rats.

Quercetin (QT) is a natural flavonoid with various biological activities and pharmacological actions. However, the bioavailability of QT is relatively low due to its low solubility which severely limits its use. In this study, we intended to improve the bioavailability of QT by preparing quercetin-phospholipid complex (QT-PC) and investigate the protective effect of QT-PC against carbon tetrachloride (CCl4) induced acute liver damage in Sprague-Dawley (SD) rats. The physicochemical properties of QT-PC were characterized in terms of infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), powder X-ray diffraction (XRPD) and water/n-octanol solubility. FTIR, DSC and XRPD data confirmed the formation of QT-PC. The water solubility of QT was improved significantly in the prepared complex, indicating its increased hydrophilicity. Oral bioavailability of QT and QT-PC was evaluated in SD rats, and the plasma QT was estimated by HPLC-MS. QT-PC exhibited higher Cmax (1.58±0.11 vs. 0.67±0.08µg/mL), increased AUC0-∞ (8.60±1.25 vs. 2.41±0.51mg/Lh) and t1/2z (7.76±1.09 vs. 4.81±0.87h) when compared to free QT. The greater absorption of QT-PC group suggested the improved bioavailability. Moreover, biochemical changes and histopathological observations revealed that QT-PC provided better protection to rat liver than free QT at the same dose. Thus, phospholipid complexation might be one of the suitable approaches to improve the oral bioavailability of QT and obtain better protective effects against CCl4 induced acute liver damage in SD rats than free QT at the same dose level.

Effects of phospholipid complexes of total flavonoids from Persimmon (Diospyros kaki L.) leaves on experimental atherosclerosis rats.

The total flavonoids from Persimmon leaves (PLF), extracted from the leaves of Diospyros kaki L. Dispryosl and Ebenaceae, is reported to possess many beneficial health effects. However, the oral bioavailability of PLF is relatively low due to its poor solubility. In the present study, the phospholipid complexes of total flavonoids from Persimmon leaves (PLF-PC) was prepared to enhance the oral bioavailability of PLF and to evaluate its antiatherosclerotic properties in atherosclerosis rats in comparison to PLF. A HPLC-MS method was developed and validated for the determination of quercetin and kaempferol in rats plasma to assess the oral bioavailability of PLF-PC. The effect of PLF (50mg/kg/d) and PLF-PC (equivalent to PLF 50mg/kg/d) on atherosclerosis rats induced by excessive administration of vitamin D (600,000IU/kg) and cholesterol (0.5g/kg/d) was assessed after orally administered for 4 weeks. The relative bioavailabilities of quercetin and kaempferol in PLF-PC relative to PLF were 242% and 337%, respectively. The levels of total cholesterol (TC), triglyceride (TG), low-density lipoprotein-cholesterol (LDL-C), high-density lipoprotein-cholesterol (HDL-C), apolipoprotein A1 (ApoA1) and apolipoprotein B (ApoB) in serum were measured by an automatic biochemistry analyzer. The morphological changes of aorta were observed with optical microscopy. According to the levels of biochemical parameters in serum and the morphological changes of aorta, PLF-PC showed better therapeutic efficacy compared to PLF. Thus, PLF-PC holds a promising potential for increasing the oral bioavailability of PLF. Moreover, PLF-PC exerts better therapeutic potential in the treatment of atherosclerotic disease than PLF.

Preparation and evaluation of kaempferol-phospholipid complex for pharmacokinetics and bioavailability in SD rats.

As one of the dietary flavonoids, kaempferol (KP) has been well known to show strong anti-oxidative effect along with other biological properties. However, the oral bioavailability of KP is relatively low due to its poor solubility. In this study, we intended to increase the solubility and bioavailability of KP by preparing kaempferol-phospholipid complex (KP-PC). The KP-PC's physicochemical properties were characterized in terms of infrared spectroscopy (IR), differential scanning calorimetry (DSC) and powder X-ray diffraction (PXRD), water/n-Octanol solubility and in vitro dissolution. KP-PC exhibited higher solubility and dissolution rate than KP, indicating a significant improvement in hydrophilicity. A UPLC-ESI-MS/MS method was developed and validated for the determination of KP in Sprague-Dawley (SD) rat plasma, so as to investigate the oral bioavailability of KP-PC versus KP. Results showed that Cmax and AUC(0-48 h) of KP from the complex (Cmax: 3.94 ± 0.83 µg/mL, AUC(0-48 h): 57.81 ± 9.43 mg/Lh) were higher than that of KP (Cmax: 1.43 ± 0.21 µg/mL, AUC(0-48 h): 13.65 ± 3.12 mg/Lh). This research indicated that phospholipid complex (PC) might be one of the suitable approachs to improve the oral bioavailability of KP and other poor-solubility flavonoids.

Evaluation of the indicative roles of seven potential biomarkers on hepato-nephrotoxicity induced by Genkwa Flos.

ETHNOPHARMACOLOGICAL RELEVANCE: Genkwa Flos, a classical traditional Chinese medicine, is used for the definite antitumor activity and tends to be taken overdose or long term in these years. While the excessive application can result in damage to liver and kidney. In this study, the indicative roles of seven potential biomarkers were evaluated to investigate hepato-nephrotoxicity in the early stages after oral administration of Genkwa Flos for 14 days. MATERIALS AND METHODS: Histopathology, serum biochemistry and seven potential biomarkers in serum or urine from male Sprague-Dawley rats were monitored. Hepatic and renal tissues were histopathologically examined to identify specific changes occurring. Routine serum biochemical parameters were tested by using standard clinical laboratory methods. Seven biomarkers including cholic acid, taurine, 5-oxoproline, hippuric acid, uric acid, 3-indoxyl sulfate and kynurenic acid were detected by a developed LC-MS method. RESULTS: The histopathological alterations and the increased levels of serum biochemistry were detected on the 8th day after Genkwa Flos treated. The seven analytes were also found significantly changed in Genkwa Flos treated group, especially cholic acid, taurine, 5-oxoproline and hippuric acid which were changed on the 2nd or 4th day. CONCLUSIONS: Although serum biochemistry and histopathology suggested that Genkwa Flos was responsible for the hepato-nephrotoxicity that occurred following the ingestion of this medicinal herb, evaluation of these biomarkers might be more beneficial for the early detection of liver and kidney injuries. This study could be further used in hepatic and renal failures caused by other reasons in the following research works.

Delta mean neutrophil volume (ΔMNV) is comparable to procalcitonin for predicting postsurgical bacterial infection.

BACKGROUND: The Coulter LH750 (Beckman Coulter, Brea, CA) analyzer can determine intrinsic biophysical properties of white blood cell (WBC), known as cell population data. Previous studies have shown that mean neutrophil volume (MNV) was significantly increased in postsurgical patients with bacterial infection. To further validate its potential clinical usefulness, we investigate the changes in MNV before and after surgery, called ΔMNV. We also compare the ΔMNV with procalcitonin (PCT) and C-reactive protein (CRP) in terms of diagnostic sensitivity and specificity for postsurgical bacterial infection. METHODS: Blood samples from 300 healthy controls, 219 cardiac surgical patients without postsurgical infection, and 31 cardiac surgical patients complicated with postsurgical bacterial infection were studied. RESULTS: There are no statistically significant differences for WBC count and neutrophil percentage prior to or after surgery between postsurgical noninfected and infected patients. However, the ΔMNV is significantly increased in postsurgical infected patients when compared with noninfected patients (P < 0.05). The receiver-operating characteristics analysis reveals the ΔMNV and PCT have largest areas under curves (0.92, 0.93 on the second day and 0.94, 0.99 on the third day postsurgery, respectively) compared to other parameters. CONCLUSION: ΔMNV shows comparable sensitivity and specificity to PCT and superior sensitivity and specificity to WBC or CRP for predicting postsurgical bacterial infection. The potential clinical application of this parameter merits further exploration in a larger prospective study.