[Potential pharmacodynamic substances of Laportea bulbifera in treatment of rheumatoid arthritis based on serum pharmacochemistry and pharmacology].

The present study investigated the pharmacodynamic material basis of Laportea bulbifera in the treatment of rheumatoid arthritis. Firstly, human rheumatoid arthritis fibroblast-like synoviocyte line MH7A was cultured in vitro and treated with tumor necrosis factor alpha(TNF-α, 50 ng·mL~(-1)). The proliferation and the levels of inflammatory cytokines such as prostaglandin E2(PGE2), interleukin-1ß(IL-1ß), and interleukin-6(IL-6) of the MH7A cells exposed to the serum containing L. bulbifera were determined to evaluate the anti-rheumatoid arthritis effects of the serum. Furthermore, the ultra-performance liquid chromatography tandem mass spectrometry fingerprints of the L. bulbifera crude extract, the drug-containing serum, and the drug-free serum were compared to identify the compounds newly generated in the serum after oral administration of the extract. According to the peak areas of common peaks and the results of anti-rheumatoid arthritis effect test, the active components were identified. The serum containing L. bulbifera significantly inhibited the proliferation of the MH7A cells activated by TNF-α and the expression of PGE2, IL-6, and IL-1ß. Thirty newly generated compounds were detected in the drug-containing serum. Among them, neochlorogenic acid, cryptochlorogenic acid, chlorogenic acid, rutin, isoquercitrin, luteoloside, kaempferol-3-O-rutinoside, and quercitrin were also present in the crude extract. Twelve characteristic peaks(3, 7, 8, 14, 18, 19, 21, 23, 24, m6, m7, and m15) were significantly correlated with the pharmaceutical effect. According to the correlations, neochlorogenic acid, cryptochlorogenic acid, and chlorogenic acid had great contributions to the anti-rheumatoid arthritis activity. This study preliminarily clarified the potential pharmacodynamic substances of L. bulbifera in the treatment of rheumatoid arthritis, which laid a theoretical and experimental foundation for further development and application of the medicinal plant.

[Study on metabolites of Laportea bulbifera extract in rat feces based on UHPLC-Q-TOF-MS~E technique].

This project is to study the metabolites of Laportea bulbifera extract in rat feces. After the SD rats were gavaged with the extract(136 g·kg~(-1), according to the crude drug dose), the metabolites in their feces were detected by UHPLC-Q-TOF-MS~E technique, and the obtained mass spectrometry data was combined with UNIFI software for prediction. The prototype components and metabolites in rat feces were identified with reference materials and related literature. A total of 43 metabolites were identified(including 8 prototype components and 35 metabolites). The metabolic pathways mainly include monocaffeoylquinic acid(hydrogenation reduction, ring-opening cracking, sulfation, hydroxylation, glucuronidation), quercetin(O-C2 bond ring-opening cleavage, C2-C3 double bond reduction, rutin carbonylation) and so on. The metabolites and metabolic process of L. bulbifera extract in rat feces were clarified, which provided a basis for the study of the active substances and its mechanism of action.

[Differences in intestinal absorption characteristics of Laportea bulbifera extract in normal and rheumatoid arthritis model rats by isolated everted intestine model].

This work aimed to investigate the intestinal absorption characteristics of Laportea bulbifera extract in normal and rheumatoid arthritis model rats. The contents of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, rutin, kaempferol-3-O-rutinoside, galuteolin, quercetin and isoquercetin in intestinal absorption solution samples were detected by UPLC-MS/MS with 5.0 g·L~(-1) as the absorption concentration. The cumulative absorption(Q) and absorption rate constant(K_a) were calculated, and the absorption characteristics of different components of L. bulbifera in intestinal absorption solution of normal rats and rheumatoid arthritis rats were compared. The results showed that all the eight index components in the extract of L. bulbifera could be absorbed into the intestinal capsule, the cumulative absorption-time curve of each component showed an upward trend without saturation, and the correlation regression coefficient(R~2) was greater than 0.92, which is consistent with the zero-order absorption rate process. It was speculated that the possible absorption mode of each component was passive diffusion. In normal condition, the absorption of ileum was the best(except chlorogenic acid), and in pathological condition, duodenum was the best. The total absorption of 8 components in each intestinal segment of RA rats was better than that of normal rats, which speculated that rheumatoid arthritis may change the specific site of drug absorption. The experimental results showed that rheumatoid arthritis could change the intestinal absorption of the extract of L. bulbifera, and its mechanism needs further study.

[Determination of plasma protein binding rates of nine compounds of Inula cappa extraction based on method of equilibrium dialysis].

To determine the plasma protein binding rate of the nine compounds in Inula cappa extraction by the method of equilibrium dialysis. The proteins in plasma samples were precipitated by methanol, and the ultra-performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS) was developed for determination of the concentrations of the nine active compounds, namely chlorogenic acid, scopolin, neochlorogenic acid, cryptochlorogenic acid, 1,3-O-dicaffeoylquinic acid, galuteolin, 3,4-O-dicaffeoylquinic acid, 3,5-O-dicaffeoylquinic acid, 4,5-O-dicaffeoylquinic acid, with the internal standard of puerarin. We found that all components have a good linearity(r≥0.999), and accuracy, precision, extraction recovery and stability conformed to the requirements of determination, without endogenous compounds disturbing within the range of optimum concentration. This suggested that the method was stable and reliable, and could be used for the determination of the plasma protein binding rates of the nine active compounds in rat and human plasma of I. cappa. The plasma protein binding rates of the nine active compounds in rat and human plasma respectively were(41.07±0.046)%-(94.95±0.008)%, and(37.66±0.043)%-(97.46±0.013)%. According to the results, there were differences in the plasma protein binding rates of the nine compounds in I. cappa extraction between rat and human.

In Vivo and In Vitro Anti-Arthritic Effects of Cardenolide-Rich and Caffeoylquinic Acid-Rich Fractions of Periploca forrestii.

Periploca forrestii Schltr. (P. forrestii) is a species used in Traditional Chinese Medicine (TCM) known as "Miao medicine", and has a long history of use in the treatment of rheumatism, rheumatoid arthritis (RA), and joint pain. The present study aimed to evaluate the anti-arthritis effects of the cardenolide-rich and caffeoylquinic acid-rich fractions (CDLFs and CQAFs) of P. forrestii in collagen-induced arthritic (CIA) rats, and defined the mechanisms of therapeutic action in MH7A cells treated with TNF-α. Serum rheumatoid factor (RF), TNF-α, IL-6, IL-1ß, PGE2, NO, SOD, and MDA were determined by ELISA or other commercially assay kits. Histopathological changes in ankle joint tissues were examined. The mRNA expressions of IL-1ß, IL-6, COX-2, and iNOS in MH7A cells were measured by qRT-PCR assays. In addition, the expressions of iNOS, COX-2, and p65 proteins, and the phosphorylation of IκBα, p38, ERK1/2, and JNK proteins in MH7A cells were analyzed by Western blot. The results showed that CDLF and CQAF could suppress the paw swelling in CIA rats at different doses (125 mg/kg, 250 mg/kg, and 500 mg/kg). Histopathological examination suggests that the CDLF and CQAF significantly relieved the damage of the structure of the ankle joint in CIA rats. In addition, serum RF, TNF-α, IL-6, IL-1ß, PGE2, NO, and MDA were decreased, along with increased activity of serum SOD. Furthermore, CDLF and CQAF downregulated the expressions of IL-1ß, IL-6, COX-2, iNOS, and p65, and inhibited the phosphorylation of IκBα, p38, ERK1/2, and JNK in MH7A cells treated with TNF-α. These findings demonstrated that both CDLF and CQAF exhibited anti-arthritic activity, which might be associated with their inhibitory effects on the NF-κB and MAPK signaling pathways.

[Protective effect of Polygonum orientale flower extract on H2O2-induced oxidative damage of HUVEC cells].

To investigate the protective effects and mechanism of Polygonum orientale flower extract on H2O2-induced oxidative damage of human umbilical vein endothelial cells (HUVEC), H2O2 was used to induce the oxidativestress damage on HUVEC cells and efforts were made to screen the low, medium and high drug concentrations of P.orientale flower extract. Cell viability was detected by the MTS assay. The content of lactate dehydrogenase (LDH) and malondialdehyde (MDA), and the activities of superoxidedimutase (SOD) and catalase (CAT) were detected by biochemical kits. The mRNA and protein levels of Bax, Bcl-2 were detected respectively by quantitative real time polymerase chain reaction (qRT-PCR) and Western blot. The protein level of cleaved caspase-3 was detected by Western blot. According to the results, the viability of HUVEC cells was reduced to around 55% after being treated with 120 µmol·L⁻¹ H2O2 for 0.5 h. Treatment of H2O2 also could increase LDH leakage rate and MDA content and attenuate the activities of SOD and CAT, up-regulate the expression level of Bax and cleaved caspase-3, and down-regulate the expression level of Bcl-2. As compared with H2O2 model group, P.orientale flower extract of 50-200 mg·L⁻¹ could increase the viability of HUVEC cells, reduce LDH release and MDA content, enhance the activities of SOD and CAT, down-regulate pro-apoptotic protein cleaved caspase-3 and Bax, and up-regulate apoptosis inhibitory protein Bcl-2. In summary, P.orientale flower extract showed a protective effect on H2O2-induced HUVEC cells injury, which may result from enhancing the cell capability of clearing the oxygen free radial, decreasing the production of lipid peroxidation and inhibiting apoptosis.

[Absorption of Inula cappa extract based on everted intestinal sac method].

To investigate the absorptive characteristics of Inula cappa extract based on the rat everted intestinal sac method in vitro. Nine representative ingredients in I. cappa extract were selected as the study objects. An UPLC-MS/MS method was established to determine and detect their cumulative absorption amount for expounding the absorptive characteristics of ingredients in different intestinal sections. According to the results， the transport mechanism of 8 compounds showed passive diffusion by the reverted gut sac method. And scopolin was actively transported in the intestine. The best absorption site of chlorogenic acid was duodenum. The best absorption site of cryptochlorogenic acid， 1，3-O-dicaffeoylquinic acid， luteolin-7-glucoside and 3，4-O-dicaffeoylquinic acid were jejunum. The best absorption site of neochlorogenic acid， scopolin， 4，5-O-dicaffeoylquinic acid and 3，5-O-dicaffeoylquinic acid was ileum. The absorption of all the compounds was affected by pH and bile. All of the nine ingredients in I. cappa extract could be absorbed in intestines， but with differences in the absorption rate， the best absorptive site and mechanism， indicating that the intestinal absorption of I. cappa extract was selective.

A UPLC-MS/MS Method for Simultaneous Determination of Free and Total Forms of a Phenolic Acid and Two Flavonoids in Rat Plasma and Its Application to Comparative Pharmacokinetic Studies of Polygonum capitatum Extract in Rats.

The principal active constituents of Polygonum capitatum are phenolic acids and flavonoids, such as gallic acid, quercitrin, and quercetin. The aim of this study was to develop and validate a method to determine the three constituents and the corresponding conjugated metabolites of Polygonum capitatum in vivo and to conduct pharmacokinetic studies on the herb, a well-known Miao medicinal plant in China. Gallic acid, quercitrin, and quercetin were analysed by ultra-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS). Protein precipitation in plasma samples was performed using methanol. For the determination of total forms of analytes, an additional process of hydrolysis was conducted using ß-glucuronidase and sulphatase. The analytes were separated on a BEH C18 column (50 mm × 2.1 mm; i.d., 1.7 µm) and quantified by multiple reaction monitoring (MRM) mode. The linear regression showed high linearity over a 729-fold dynamic range for the three analytes. The relative standard deviations of intra- and inter-day measurements were less than 9.5%, and the method was accurate to within -11.1% to 12.5%. The extraction recoveries for gallic acid, quercitrin, and quercetin were 94.3%-98.8%, 88.9%-98.8%, and 95.7%-98.5%, respectively. All samples were stable under short- and long-term storage conditions. The validated method was successfully applied to a comparative pharmacokinetic study of gallic acid, quercitrin, and quercetin in their free and total forms in rat plasma. The study revealed significantly higher exposure of the constituents in total forms for gallic acid and quercetin, while quercitrin was detected mainly in its corresponding free form in vivo. The established method was rapid and sensitive for the simultaneous quantification of free and total forms of multiple constituents of Polygonum capitatum extract in plasma.

[Metabolism of Inula cappa extract by rat intestinal bacteria in vitro].

To investigate the metabolism of major components in Inula cappa by rat intestinal bacteria in vitro. I. cappa extract was incubated for 24 h with rat intestinal bacteria under anaerobic environment. After the samples were precipitated by n-butanol, UPLC-Q-TOF-MS/MS was applied for the qualitative analysis of the metabolites, combined with data software such as Metabolite Tools, Data Analysis and so on. The potential metabolites in rat intestinal bacteria were analyzed by comparing the total ion current of the test samples and blank samples and analyzing the quasi-molecular ion and fragment ion of all chromatograms. The results injected that fourteen metabolites were detected in rat intestinal flora. Various types of metabolic reactions happen to caffeoylquinic acid in intestinal flora, including isomerization, hydrolyzation, there were also methylation, hydrogenation and acetylation of caffeic acid. At the same time, a methylate of dicaffeoylquinic acid was also detected. Presumably, caffeoylquinic acids were gradually transformed into more hydrophobic metabolites with smaller molecular mass, which were better absorbed by the intestinal tract.

[Protective Effect of Polygonum orientale Flower Extract with Different Preparation Methods on Acute Myocardial Ischemia in Mongrel Dogs].

OBJECTIVE: To observe the effect of Polygonum orientale flower extract with different preparation methods on acute myocardial ischemia in mongrel dogs. METHODS: The acute myocardial ischemia model of dog was set up though ligation of anterior descending branch of coronary artery in dogs, then the epicardium electrocardiogram was used to measure the degree of myocardial ischemia and the quantitative histological assay was adopted to determine the area of myocardial ischemia. At the same time, the lactic dehydrogense (LDH) and creatine kinase(CK) levels in serum were analyzed. All of them were used to evaluate the protective effect of Polygonum orientale flower with different preparation methods on acute myocardial ischemia in mongrel dog. RESULTS: Compared with the model group, the sample whose preparation methods was extracted by n-butanol after water extracting and alcohol precipitation showed obvious reduction in the area of myocardial ischemia,with decline in ∑ST and NST. Moreover,the LDH and CK levels in serum were decreased (P <0. 05 or P <0. 01). CONCLUSION: The sample which is extracted by n-butanol after water extracting and alcohol precipitation has a protective effect on injury induced by acute myocardial ischemia in mongrel dog.

[Researching on fingerprint of Inulacappa by HPLC].

OBJECTIVE: This study is to establish the fingerprint and find out the common chromatographic peaks of Inula cappa by HPLC. METHOD: The HPLC analysis was performed on an Agilent Eclipse Plus C18 column (2.1 mm x 150 mm, 1.8 µm) with 0.1% fomic acid aqueous solution-0.1% fomic acid acetonitrile solution as mobile phase at a flow rate of 0.3 · mL(-1) · min(-1); The detective wavelength is 325 nm; The column temperature is 45 °C. RESULT: The results indicated that 5 of 17 common peaks were identified . The similarity about 10 groups of Inulacappais is over 0.95. CONCLUSION: This method is able to be a scientific basis of quality assessment according to its convenient and reliable.

Report: structures and hepatocytotoxicity of co-occurring substances in oleanolic acid tablets.

Tablets of oleanolic acid (OA) have been approved by SFDA in China as an adjuvant therapy for acute and chronic hepatitis. Co-occurring substances present in the tablets of OA and their hepatocytotoxicity have not yet been reported. In the current investigation, the crude OA drug was separated by repeated column chromatography. The structures of the isolated compounds were characterized by spectral analysis and the cytotoxicity of each compound was evaluated in vitro against the human normal liver cell L02 at concentrations from 0.125 to 1000 µmol/L using the MTT method. As a result, OA and its 11 co-occurring trace compounds including one new triterpenoid, 3-O- (4-oxo-pentanoyl)-olean-12- en-28-oic acid, were isolated and structurally characterized. Cytotoxicity tests indicated that these compounds were all non-toxic at concentrations up to 50µmol/L. Clear structure-activity relationship (SAR) was also observed. The results suggested that OA tablets of similar origin might not cause obvious cytotoxicity to the normal liver cell. The work may facilitate further SAR studies of OA-type triterpenoids.

UPLC-PDA-ESI-MS/MS analysis of compounds extracted by cardiac h9c2 cell from Polygonum orientale.

INTRODUCTION: A flavonoid-enriched extract (FEE) of Polygonum orientale was reported to show cardioprotective effect but only very few compounds were reported to contribute to the effect. Identification of compounds interacting with the target cardiac cell is important for the understanding of active compounds. OBJECTIVE: To develop an efficient method for the screening of potential active compounds directly acting on the target cardiac cell in FEE and to structurally characterise these compounds. METHODOLOGY: Flavonoid-enriched extract was prepared by extraction of the plant with water, addition of ethanol to the solution to remove polysaccharides and proteins, and removal of tannins by a polyamide column chromatography. Cell extraction was conducted on a cardiac h9c2 cell and the solution containing compounds released from the cell were desalted by solid phase extraction. Compounds present in the cell extract were detected by ultra-performance liquid chromatography (UPLC) and targeted multi-reaction monitoring (MRM), while their structures were characterised by UPLC-photodiodide array (PDA)-electrospray ion source (ESI)-MS/MS investigations of the FEE. RESULTS: Twenty-three potentially active phenolics including ten flavonoid C-glycosides and six flavonoid O-glycosides have been identified from the 40 compounds screened in the cell extract. Among these compounds, three were new and nine were identified from this plant for the first time. Strategies for the structural characterisation of flavonoid glycosides were also discussed. CONCLUSION: The study has shown that FEE contains the flavonoid as its major principles and the coupling of UPLC-PDA-ESI-MS/MS and targeted UPLC-MRM with target cell extraction is an efficient method for the screening and structural characterisation of potential active compounds.

Identification and characterisation of phenolics in Polygonum capitatum by ultrahigh-performance liquid chromatography with photodiode array detection and tandem mass spectrometry.

INTRODUCTION: Polygonum capitatum is a well-known Chinese medicinal plant widely used by the Miao people for the treatment of various urologic disorders. Previous investigations have shown the presence of various types of phenolics. Our ultrahigh-performance liquid chromatography with photodiode array detection and mass spectrometry (UPLC-PDA-MS) analysis indicated that flavonoid glycosides and polyphenolic glycosides were its major constituents and quite a number of phenolic compounds have not yet been identified. Identification or characterisation of the major compounds of this plant will contribute to the scientific understanding of the medicinal plant and the authentication of the plant material and its pharmaceutical preparations. OBJECTIVE: To develop an efficient method for the identification and structural characterisation of the major compounds present in P. capitatum. METHODS: Elution of the 70% ethanol extract of P. capitatum by 80% ethanol on a D101 macroporous resin column afforded a phenolics-enriched fraction, separation of which by Sephadex LH-20 column chromatography eluted with absolute ethanol gave a tannin-free phenolic fraction (TFPF). Compounds present in TFPF were identified and structurally characterised by UPLC-PDA-ESI-MS/MS. RESULTS: An amino acid and 40 phenolics including a number of flavonoid glycosides and polyphenolic glycosides were identified or structurally characterised in TFPF. Among these compounds, four were new and 19 were described in the plant for the first time. CONCLUSION: The study showed that TFPF of P. capitatum contained flavonoid glycosides and polyphenolic glycosides as its major principles. Polyphenolic glycosides were perhaps the most typical chemical markers of P. capitatum.

Electrospray ionization and collision-induced dissociation tandem mass spectrometric discrimination of polyphenolic glycosides: exact acylation site determination of the O-acylated monosaccharide residues.

RATIONALE: Acylated monosaccharide residues are structural subunits of natural products or synthetic intermediates that have received much attention in past years. Determination of the acylation sites of these residues still relies heavily on the comparison of their characteristic NMR signals with those of known standards and synthesized acylated glycosides. It is important to develop a rapid analytical method for determining the acylation sites for these compounds, and this is described in this study. METHODS: Six known polyphenolic glycosides were used for the electrospray ionization and collision-induced dissociation tandem mass spectrometry (ESI-CID-MS/MS) discrimination of the acylated monosaccharide residues with different acylation sites. A combination of ESI-CID-MS/MS, using a triple quadrupole mass spectrometer, with ultra-performance liquid chromatography (UPLC) and photo-diode array (PDA) detection (UPLC-PDA) has been applied to the identification or characterization of polyphenolic glycosides in Polygonum capitatum that possess an acylated monosaccharide residue. RESULTS: An ESI-MS and CID-MS/MS method has been developed for the determination of the acylation sites of polyphenolic glycosides that possess an acylated monosaccharide residue. Twelve polyphenolic glycosides including four new ones have been identified or characterized in P. capitatum. Eight (including the new ones) of the twelve glycosides were reported for the first time from this plant. CONCLUSIONS: The developed ESI-MS and CID-MS/MS method provided a very useful strategy for the determination of the sites of polyphenolic glycosides that possess an acylated monosaccharide residue. The acylation site could be determined by the characteristic product ion spectra of the in-source CID-generated O-acyl monosaccharide ion [B(1)](+). The presented work may facilitate the structural characterization of these types of compounds.

[Design, synthesis and anti-oxidative evaluation of L-amino acid prodrugs of scutellarein].

To design and synthesize a series of novel scutellarein 4'-L-amino acid prodrugs with more potent anti-oxidative activity and improved physicochemical properties. Scutellarein was used as lead compound, according to successful experience of improving bioavailability of oral administration drugs by active transport mechanism, principle of hybridization was used to introducing L-amino acid structural fragments at 4'-position of scutellarein to design and synthesize target scutellarein 4'-L-amino acid prodrugs. The synthetic compounds were tested on their physicochemical properties and in vitro anti-oxidative activity against H202 induced oxidative damage in PC12 cells. Five compounds were found to have more potent anti-oxidative activity than positive control VE. Moreover the physicochemical properties of synthesized compounds were evaluated, and the results revealed that L-amino acid ether derivatives are more stable (t1/2 9-92 h) than their corresponding ester derivatives (t1/2 0.5 h). Water solubility of scutellarein 4'-L-amino acid ester and ether derivatives were 1 796-4 100 microg.mL-1 and 27.7-81.1 microg.mL-1 respectively, in comparison with scutellarin, the solubility of compounds 18, 19 and 22, 24-27 increased about 120-280 fold and 2-6 fold respectively. All these results suggested that L-amino acid prodrug strategy has significant potential in scutellarein prodrug design.

Rapid screening and identification of caffeic acid and its esters in Erigeron breviscapus by ultra-performance liquid chromatography/tandem mass spectrometry.

Caffeic acid and its esters (CAEs) are widely distributed in the plant kingdom and have been reported to elicit a wide range of exceptional biological activities. Present methods for screening and characterization of CAEs normally need the use of liquid chromatography diode-array detection/multistage mass spectrometry (LC-DAD/MS(n)). In this report, a rapid and efficient method coupling ultra-performance liquid chromatography (UPLC) with fragment-targeted multi-reaction monitoring (MRM) has been developed for screening CAEs in a crude extract of Erigeron breviscapus, while a UPLC/quasi-MS(n) method has been applied in the structural identification of these compounds. Furthermore, a simple quasi-UPLC/MS/MS method based on in-source collision-induced dissociation (CID) has been proposed for rapid identification of the CAEs. As a result, a total of more than 34 CAEs were detected and their structures characterized. Nine of them were reported from E. breviscapus for the first time. Applications of these strategies in the chemical investigation of an injection of E. breviscapus resulted in the identifications of 16 CAEs. These strategies, if appropriate modifications are made, will be very useful in screening and characterization not only of CAEs, but of other structural types of compounds in various complex matrices.

Two new phenolic glycosides from Inula cappa.

Two new phenolic glycosides, syringic acid-4-O-α-L-rhamnoside (1) and (-)-hydnocarpin-7-O-ß-D-glucoside (2), were isolated from the traditional Chinese medicinal herb Inula cappa. The structures of the new compounds were elucidated by means of spectroscopic methods such as 1D, 2D NMR, and HR-ESI-MS.

[Design, synthesis and anti-hBV evaluation of adefovir mono-L-amino acid ester, mono non-steroidal anti-inflammatory drugs carboxylic ester prodrugs].

A series of adefovir mono-L-amino acid esters, mono non-steroidal anti-inflammatory drugs carboxylic ester prodrugs with more potent anti-HBV activity and lower nephrotoxicity were designed and synthesized. Adefovir bis (L-amino acid) ester was used as lead compound, according to pathological and pharmacological findings that non-steroidal anti-inflammatory drugs can effectively inhibit the organic anion transporter 1 (hOAT1)-mediated adefovir phosphonic acid pairs of anion transport across tubular basement membrane thereby reducing the nephrotoxicity of adefovir. Flatten design principle was used to introducing non-steroidal anti-inflammatory drugs structural fragments to design and synthesize target adefovir mixture ester prodrugs. HepG2 2.2.15 cell line was used as in vitro anti-HBV activity evaluation model. Five compounds exhibited antiviral activity, and compound 18 showed the most potent anti-HBV activity and relatively high selective index (EC50 3.92 micromol L(-1), SI 9.97). HK-2 cell line was used as in vitro model to evaluate nephrotoxicity. Results suggested the target compounds have lower cytotoxicity than the positive control. Moreover, by analyzing the primary structure and activity relationship of these compounds, it could suggest that mono-L-amino acid ester, mono non-steroidal anti-inflammatory drugs carboxylic ester prodrugs strategy has significant potential in the acyclic nucleoside phosphonates prodrug design.

Simultaneous Determination of Three Bioactive Constituents from Bletilla striata by UPLC-MS/MS and Application of the Technique to Pharmacokinetic Analyses.

Bletilla striata has been widely used as a valuable hemostatic agent for thousands of years due to the high levels of bioactive constituents it contains. Here, we used a sensitive ultrahigh-performance liquid chromatography-tandem mass spectroscopy (UPLC-MS/MS) method for the simultaneous determination of three major active ingredients of the B. striata extract, namely, α-isobutylmalic acid, gymnoside I, and militarine in rat plasma. The three major active ingredients were determined using the multiple reaction monitoring (MRM) mode at m/z 189 ⟶ 129 for α-isobutylmalic acid, m/z 457.2 ⟶ 285.1 for gymnoside I, m/z 725.3 ⟶ 457.2 for militarine, and m/z 417.0 ⟶ 267.0 for the IS puerarin. All calibration curves showed good linearity (R 2 ≥ 0.999) over the concentration range with the lower limit of quantification between 0.015 and 0.029 µg/mL. The relative standard deviations of intraday and interday measurements were less than 15%, and the method was accurate within 93.3-100.4%. The extraction recovery was 92.65-100.98%, and no matrix effect was observed. The results indicated that after oral administration of B. striata in rats, the T max of α-isobutylmalic acid was significantly longer than that of gymnoside I and militarine and the mean residence time and area under the curve of α-isobutylmalic acid and gymnoside I in rats were significantly higher than those of militarine. Moreover, the blood concentration-time curve of α-isobutylmalic acid showed double peaks, suggesting that α-isobutylmalic acid could exhibit the phenomenon of enterohepatic circulation or metabolic conversion. We also explored some of the pharmacokinetic characteristics of three ingredients from B. striata extract in vivo, and the data obtained may provide a basis for the further investigation of B. striata.