Pharmacokinetic Analysis of Two N-Substituted Rosmarinic Acid Analogs in Rat Plasma by Liquid Chromatography-Tandem Mass Spectrometry.

This study reports the development of an effective high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for the quantification of two analogs of rosmarinic acid (RA) in rat plasma, namely methyl (E)-2-(3-(3,4-difluorophenyl)acrylamido)-3-(3,4-dihydroxyphenyl)propanoate (A11) and methyl (E)-3-(3,4-dihydroxyphenyl)-2-(3-(3,4-dihydroxyphenyl)acrylamido)propanoate (A2). These analogs, featuring N atoms instead of O atoms, exhibit enhanced bioavailability and distinct pharmacological activities compared with RA. The HPLC separation was carried out on a C18 column (1.9 µm, 2.1 mm × 100 mm) coupled with a security guard C18 column (5 µm, 2.1 mm × 10 mm). A triple-quadrupole mass spectrometer equipped with an electrospray ionization ion source was utilized for ion generation. Pseudoephedrine hydrochloride was utilized as a standard, and a single-step protein precipitation method using isopropanol:ethyl acetate (v/v, 20:80) was employed for sample pretreatment. The developed method demonstrated excellent linearity over the concentration range of 5-750 ng/ml for both A11 and A2, with relative standard deviations of <15% and relative errors within 15% during daily course analysis. The method allowed for the unambiguous quantification and identification of A11 and A2 in vivo. The results of this study provide a meaningful foundation for evaluating the clinical applications of these analogs.

Determination of rosmarinic acid and its N-substituted analog A1 in rat plasma using high-performance liquid chromatography-tandem mass spectrometry and its application in pharmacokinetics.

BACKGROUND: The aim of this study was to determine the concentration of rosmarinic acid (RA) and its analog (E)-3-(3,4-dihydroxyphenyl)-2-(3-(3,4-dihydroxyphenyl)acrylamido)propanoic acid (A1) in rat plasma following oral administration. The significance of this study lies in the development of a rapid, sensitive, and alternative method using liquid chromatography-tandem mass spectrometry for the accurate quantification and identification of RA and A1 in vivo. METHODS: Liquid chromatography-tandem mass spectrometry was employed to analyze RA and A1 in rat plasma. A C18 column (1.9 µm, 2.1 × 100 mm) with a C18 guard column (5 µm, 2.1 × 10 mm) and a triple-quadrupole mass spectrometer combined with an electrospray ionization source were utilized. Sample pretreatment involved a one-step protein precipitation using isopropanol:ethyl acetate (20:80, v/v) as the solvent. Pseudoephedrine hydrochloride served as a standard. RESULTS: The developed method exhibited a linear relationship within the concentration ranges of 5-750 ng/ml for both RA and A1. Relative standard deviations in daily courses were less than 15%, and the relative errors recorded were within 15%. This is the first study to concentrate on determining A1 and RA in rat plasma through oral administration. CONCLUSIONS: The liquid chromatography-tandem mass spectrometry method developed in this study offers a rapid, sensitive, and alternative approach for the accurate quantification and identification of RA and A1 in vivo. The findings serve as a significant foundation for evaluating the clinical applications of the medicine.

Ramulus mori (Sangzhi) alkaloids regulates gut microbiota disorder and its metabolism profiles in obese mice induced by a high-fat diet.

The imbalance of gut microbiota has been confirmed to have a close pathological and physiological correlation with obesity and metabolic syndrome. Ramulus Mori (Sangzhi) Alkaloids (SZ-A) derived from twigs of mulberry was approved by the National Medical Products Administration of China in 2020 for the treatment of type 2 diabetes mellitus. In addition to its hypoglycemic effect, previous studies have confirmed that SZ-A also alleviates high-fat diet-induced obesity and non-alcoholic fatty liver disease and ameliorates obesity-linked adipose tissue metabolism and inflammation, indicating the potential of SZ-A to regulate obesity and metabolic syndrome. However, whether SZ-A can improve obesity and metabolic syndrome by regulating gut microbiota and its metabolism profiles remains unclear. The purpose of this study was to assess the effect of SZ-A on gut microbiota in obese mice and to explore the association among changes in gut microbiota, obesity, and lipid metabolism. The results showed that oral administration of SZ-A could significantly reduce body weight, fat mass, and the level of total cholesterol and low-density lipoprotein in serum in obese mice induced by a high-fat diet. Interestingly, SZ-A also regulated gut microbiota and changed the fecal metabolite composition of obese mice. Compared with the high-fat diet group, the ratio of Firmicutes to Bacteroides changed at the phylum level and the abundance of Bifidobacterium and Akkermansia muciniphila significantly increased at the genus level in the SZ-A group. The gut microbiota of the SZ-A group was reshaped and the relative abundance of microbial genes in bile acid metabolism and fatty acid metabolism were altered, which was consistent with the metabolomics results. Additionally, SZ-A greatly enriched the number of goblet cells and reduced inflammatory colon injury and pro-inflammatory macrophage infiltration induced by a high-fat diet in obese mice. In conclusion, SZ-A can alleviate obesity and metabolic syndrome by improving the gut microbiota and its metabolism profiles of obese mice induced by a high-fat diet.

Hylocereus undatus flower extract suppresses OVA-induced allergic asthma in BALb/c mice by reducing airway inflammation and modulating gut microbiota.

Asthma is a chronic allergic respiratory disease with limited treatment options. Emerging findings indicate an important interaction between the gut microbiota and the lungs, and that the development of asthma causes changes in the gut environment. Hylocereus undatus flower (HUF) is a traditional Chinese medicine used in the treatment of pulmonary and intestinal diseases. Our previous studies have demonstrated significant anti-asthmatic and anti-inflammatory activity, but the exact mechanism has not been elucidated. In the current study, we validated the potential therapeutic asthma properties of HUF in vivo using an ovalbumin-induced allergic asthma mouse model. We found that HUF treatment significantly reduced the key features of allergic asthma, including an elevated respiratory rate, inflammatory cell accumulation, airway inflammation, and the expression of pro-inflammatory molecules. Histological analysis of mouse lungs showed that HUF attenuated lung inflammatory cell infiltration. Periodic acid-Schiff staining confirmed the reduced mucus secretion in lung mucosa, and Masson's staining confirmed the reduced collagen deposition in the lungs after HUF treatment. Western blot and immunohistochemistry confirmed that HUF increased lung SIRT1 and reduced p38MAPK, NF-κBp65, and caspase-1 proteins. 16 S rDNA sequencing showed that HUF improved the endostasis of the disrupted gut microbiota composition in asthmatic mice. Surprisingly, an inflammatory response was found in the gut of asthmatic mice, along with alterations in inflammation-associated SIRT1 and caspase-1 proteins, and HUF was able to ameliorate these lesions. In conclusion, these findings suggest that HUF may be a new drug candidate for the treatment of allergic asthma.

Erratum: Baicalin promotes the viability of Schwann cells in vitro by regulating neurotrophic factors.

[This corrects the article DOI: 10.3892/etm.2017.4524.].

Pharmacokinetic Analysis of Rosmarinic Acid and its Analog in Rat Plasma Using Liquid Chromatography-Tandem Mass Spectrometry.

The anticancer effects of rosmarinic acid (RA) are a hotspot of current research. In order to enhance its pharmacological activity, N-substituted RA was prepared, and it has been shown to exhibit notable antitumor effects. In order to elucidate the underlying mechanisms of action, pharmacokinetic analysis is necessary. In the present study, liquid chromatography-tandem mass spectrometric method, was used to determine the concentrations of RA and its analog, (E)-3-(3,4-dihydroxyphenyl)-2-(3-(3,4-dihydroxyphenyl)acrylamido)propanoic acid (A2) in plasma from rats. The analyses were divided into a C18 column (1.9 µm, 2.1 mm × 100 mm) with a security guard C18 column (5 µm, 2.1 mm × 10 mm) and a triple-quadrupole mass spectrometry with an electrospray ionization ion-source generates ions. The sample pretreatment is relevant to the one-step protein precipitation with isopropanol:ethyl acetate (v/v, 1:1) This method presented a linear association within ranges at the concentration of 5-2000 ng/mL for A2 and RA. Relative standard deviations in daily courses were <15% and the relative errors registered within 15%. The methods used in the present study make the unambiguous quantification and identification of RA and A2 possible in vivo. The present study is the first to focus on determining A2 and RA in rat plasma following oral administration. The results may provide a meaningful basis for the evaluation of the application of RA and its analog in clinical practice and also provide a reference method for the pharmacokinetic analysis of RA analogs.

Identification and structure-activity relationship exploration of uracil-based benzoic acid and ester derivatives as novel dipeptidyl Peptidase-4 inhibitors for the treatment of type 2 diabetes mellitus.

Our previously reported carboxyl-containing DPP-4 inhibitors were highly potent but were poorly bioavailable. Esters of the carboxyl analogs exhibited a significant DPP-4 potency loss albeit with enhanced oral absorption. Herein, we described identification and structure-activity relationship (SAR) exploration of a novel series of benzoic acid and ester derivatives as low single-digit nanomolar DPP-4 inhibitors. Importantly, the esters displayed comparable activities to the acids counterparts. Molecular simulation revealed that ester adopts a similar binding mode to acid. Moreover, the selected esters and acids demonstrated high selectivity and low cytotoxicity, as well as good metabolic stability. And more importantly, the esters possessed excellent pharmacokinetic profiles for oral administration. The best compound ester 19b demonstrated long DPP-4 inhibition in vivo, and robustly improved the glucose tolerance in normal and db/db mice while ensuring glucose-lowering potency in chronic treatment. Our results supported that the compound 19b can be served as a potential candidate for the treatment of type 2 diabetes.

Optimization of the benzamide fragment targeting the S2' site leads to potent dipeptidyl peptidase-IV inhibitors.

Our recently successful identification of benzoic acid-based DPP-4 inhibitors spurs the further quest for in-depth structure-activity relationships (SAR) study in S2' site DPP-4. Thus novel benzamide fragments were designed to target the S2' site to compromise lipophilicity and improve oral activity. Exploring SAR by introduction of a variety of amide and halogen on benzene ring led to identification of several compounds, exerting moderated to excellent DPP-4 activities, in which 4'-chlorine substituted methyl amide 17g showed most potent DPP-4 activity with the IC50 value of 1.6 nM. Its activity was superior to reference alogliptin. Docking study ideally verified and interpreted the obtained SAR of designed compounds. As a continuation, DPP-8/9 assays revealed the designed compounds exhibited good selectivity over DPP-8 and DPP-9. Subsequent cell-based test indicated compound 17g displayed low toxicity toward the LO2 cell line up to 100 µM. In vivo evaluation showed compound 17g robustly improved the glucose tolerance in normal mice. Importantly, 17g exhibited reasonable pharmacokinetic (PK) profiles for oral delivery. Overall, compound 17g has the potential to a safe and efficacious DPP-4 inhibitor for T2DM treatment.

Effects of rosmarinic acid on immunoregulatory activity and hepatocellular carcinoma cell apoptosis in H22 tumor-bearing mice.

Rosmarinic acid (RA) is a natural polyphenolic compound that exists in many medicinal species of Boraginaceae and Lamiaceae. The previous studies have revealed that RA had therapeutic effects on hepatocellular carcinoma (HCC) in the H22-xenograft models by inhibiting the inflammatory cytokines and NF-κB p65 pathway in the tumor microenvironment. However, its molecular mechanisms of immunoregulation and pro-apoptotic effect in HCC have not been fully explored. In the present study, RA at 75, 150, and 300 mg/kg was given to H22 tumor-bearing mice via gavage once a day for 10 days. The results showed that RA can effectively inhibit the tumor growth through regulating the ratio of CD4+/CD8+ and the secretion of interleukin (IL)-2 and interferon-γ, inhibiting the expressions of IL-6, IL-10 and signal transducer and activator of transcription 3, thereby up-regulating Bax and Caspase-3 and down-regulating Bcl-2. The underlying mechanisms involved regulation of immune response and induction of HCC cell apoptosis. These results may provide a more comprehensive perspective to clarify the anti-tumor mechanism of RA in HCC.

A population pharmacokinetic model of vancomycin for dose individualization based on serum cystatin C as a marker of renal function.

OBJECTIVES: This study aimed to establish a vancomycin population pharmacokinetics (PPK) model based on serum cystatin C and to optimize dosing for achieving targeted steady-state trough concentrations (Css ) of 10-15 and 15-20 mg/l. METHODS: Patients aged ≥18 years were prospectively enrolled. A vancomycin PPK model was built with glomerular filtration rate (GFR) as a renal covariate estimated by cystatin C. A new group of patients were used for external evaluation. PPK analysis and Monte Carlo simulations were performed using nonlinear mixed effect modelling programme. KEY FINDINGS: Two hundreds of patients with 514 samples were included. The final model was CL (L/h) = (5.07 × (GFR/105.5)0.524 × (AGE/48.5)-0.309 × (WT/60)0.491 ); V (l) = 46.3. Internal and external evaluations demonstrated good stability and predictability. The average probability of target attainment (PTA) of optimal dosing regimens for targeted Css achieving 10-15 and 15-20 mg/l were 51.2% and 40.6%, respectively. An average PTA ≥71% for targeted concentration of 10-20 mg/l was obtained. CONCLUSIONS: A vancomycin PPK model with cystatin C as the renal marker has good stability and predictability. The new proposed dosing regimens were predicted to achieve a good PTA.

Identification of novel uracil derivatives incorporating benzoic acid moieties as highly potent Dipeptidyl Peptidase-IV inhibitors.

Dipeptidyl Peptidase-IV (DPP-4) is a validated therapeutic target for type 2 diabetes. Aiming to interact with both residues Try629 and Lys554 in S2' site, a series of novel uracil derivatives 1a-l and 2a-i incorporating benzoic acid moieties at the N3 position were designed and evaluated for their DPP-4 inhibitory activity. Structure-activity relationships (SAR) study led to the identification of the optimal compound 2b as a potent and selective DPP-4 inhibitor (IC50 = 1.7 nM). Docking study revealed the additional salt bridge formed between the carboxylic acid and primary amine of Lys554 has a key role in the enhancement of the activity. Furthermore, compound 2b exhibited no cytotoxicity in human hepatocyte LO2 cells up to 50 µM. Subsequent in vivo evaluations revealed that the ester of 2b robustly improves the glucose tolerance in normal mice. The overall results have shown that compound 2b has the potential to a safe and efficacious treatment for T2DM.

Madecassoside prevents acute liver failure in LPS/D-GalN-induced mice by inhibiting p38/NF-κB and activating Nrf2/HO-1 signaling.

Madecassoside (MA), a triterpenoid saponin isolated from Centella asiatica, exerts various pharmacological activities including antioxidative and anti-inflammatory effects. The aim of this study was to explore the protective effect of MA in the treatment of lipopolysaccharide (LPS) and D-galactosamine (D-GalN)-induced acute liver failure(ALF) in mice. We hypothesized that MA administration may decrease the degree of liver injury caused by LPS/D-GalN. In this study, we investigated this hypothesis by treating a mouse model of LPS/D-GalN-induced liver injury with MA. Our study demonstrated that MA (20 mg/kg and 40 mg/kg) treatment for 10 days attenuated LPS/D-GalN-induced liver injury by protecting liver function, suppressing the production of inflammatory cytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)-1ß and IL-6, and recovering antioxidant enzyme activity. MA also significantly suppressed LPS-stimulated protein levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 by blocking the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and eukaryotic transcription factor nuclear factor-kappa B (NF-κB). In addition, MA treatment enhanced protein levels of heme oxygenase (HO)-1 and anti-oxidant enzymes (superoxide dismutase, catalase, and glutathione peroxidase) through the upregulation of nuclear factor E2-related factor 2 (Nrf2) in LPS-stimulated liver injury. These results suggest that MA is a promising agent for the treatment of LPS/D-GalN-induced liver injury that could serve as a candidate for the development of a hepatoprotective drug against ALF.

Baicalin promotes the viability of Schwann cells in vitro by regulating neurotrophic factors.

The proliferation and migration of Schwann cells (SCs) are key events in the process of peripheral nerve repair. This is required to promote the growth of SCs and is a challenge during the treatment of peripheral nerve injury. Baicalin is a natural herb-derived flavonoid compound, which has been reported to possess neuroprotective effects on rats with permanent brain ischemia and neuronal differentiation of neural stem cells. The association of baicalin with neuroprotection leads to the suggestion that baicalin may exert effects on the growth of SCs. In the present study, the effects of baicalin on SCs of RSC96 were investigated. RSC96 SCs were treated with various concentrations of baicalin (0, 5, 10 or 20 µM) for 2, 4 and 6 days. Cell attachment, viability and gene expression were monitored via the MTT assay and reverse transcription-quantitative polymerase chain reaction. The gene expression levels of several neurotrophic factors, such as glial cell-derived neurotrophic factor, brain-derived neurotrophic factor and ciliary neurotrophic factor, which are considered important factors in the process of never cell regeneration, were detected. The results indicated that baicalin was able to promote the viability of RSC96 SCs in a dose-dependent manner and the concentration of 20 µM of baicalin exhibited the greatest cell viability and gene expression of the studied neurotrophic factors. The present findings suggested that baicalin likely affects SCs metabolism, through modulating the expression of neurotrophic factors. To conclude, the present study indicates that baicalin may be potential therapeutic agent for treating peripheral nerve regeneration.

Rosmarinic acid inhibits inflammation and angiogenesis of hepatocellular carcinoma by suppression of NF-κB signaling in H22 tumor-bearing mice.

The aim of this study was to explore the anti-tumor effect and therapeutic potential of rosmarinic acid (RA) in the treatment of hepatocellular carcinoma (HCC). RA at 75, 150 and 300 mg/kg was given to H22 tumor-bearing mice by intragastric administration once daily for 10 consecutive days. Levels of inflammatory and angiogenic factors, including interleukin-1ß (IL-1ß), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), vascular endothelial growth factor (VEGF), and transforming growth factor-ß (TGF-ß) were measured by enzyme linked immunosorbent assays (ELISA). Protein levels of phosphorylated NF-κB p65 and p65 were detected by western blot. mRNA level of NF-κB p65 was analyzed by qRT-PCR. The results showed that RA could effectively suppress tumor growth with fewer toxic effects by regulating the secretion of cytokines associated with inflammation and angiogenesis, and suppressing the expression of NF-κB p65 in the xenograft microenvironment. Our findings unveil the possible anti-tumor mechanisms of RA and support RA as a potential drug for the treatment of HCC.

Synthesis of a temperature-sensitive matrine-imprinted polymer and its potential application for the selective extraction of matrine from radix Sophorae tonkinensis.

A temperature-sensitive matrine-imprinted polymer was prepared in chloroform by free-radical cross-linking copolymerization of methacrylic acid at 60 °C in the presence of ethylene glycol dimethacrylate as the cross-linker, N-isopropyl acrylamide as the temperature-responsive monomer and matrine as the template molecule. Binding experiments and Scatchard analyses revealed that two classes of binding sites were formed on molecular imprinted polymer (MIP) at 50 °C. Additionally, the thermoresponsive MIP was tested for its application as a sorbent material for the selective separation of matrine from Chinese medicinal plant radix Sophorae tonkinensis. It was shown that the thermoresponsive MIP displayed different efficiency in clean-up and enrichments using the SPE protocol at different temperatures.

Pharmacokinetic study of 14-(3-methylbenzyl)matrine and 14-(4-methylbenzyl)matrine in rat plasma using liquid chromatography-tandem mass spectrometry.

A rapid, sensitive and selective high-performance liquid chromatography-tandem mass spectrometric method (HPLC-MS) was developed and validated to determine the 14-(3-methylbenzyl)matrine (3MBM) and 14-(4-methylbenzyl)matrine (4MBM) levels in rat plasma in the present study. The analytes were separated using a C18 column (1.9 µm, 2.1 mm × 100 mm) equipped with a Security Guard C18 column (5 µm, 2.1 mm × 10 mm), followed by detection via triple-quadrupole mass spectrometry using an electrospray ionization (ESI) source. Sample pretreatment involved one-step protein precipitation with isopropanol:ethyl acetate (v/v, 25:75), and pseudoephedrine hydrochloride was used as an internal standard. The method was linear in the concentration range of 5-2000 ng/ml for both compounds. The intra-day and inter-day relative standard deviations (RSDs) were less than 15%, and all relative errors (REs) were within 15%. The proposed method enables the unambiguous identification and quantification of these two compounds in vivo. This study is the first to determine the 3MBM and 4MBM levels in rat plasma after oral administration of these compounds. These results provide a meaningful basis for evaluating the clinical applications of these medicines.

Simultaneous determination of 14-thienyl methylene matrine and matrine in rat plasma by high-performance liquid chromatography-tandem mass spectrometry and its application in a pharmacokinetic study.

A rapid, sensitive and selective high-performance liquid chromatography-tandem mass spectrometric method (HPLC-MS) has been developed and validated for the simultaneous determination of 14-thienyl methylene matrine (TMM) and matrine (MT) in rat plasma in the present study. The analytes were separated on a C18 column (1.9 µm, 2.1 mm × 100 mm) with a security guard C18 column (5 µm, 2.1 mm × 10 mm) and a triple-quadrupole mass spectrometry equipped with an electrospray ionization (ESI) source was applied for detection. With pseudoephedrine hydrochloride as internal standard, sample pretreatment involved in a one-step protein precipitation with isopropanol:ethyl acetate (v/v, 20:80). The method was linear over the concentration ranges of 5-1000 ng/ml for TMM and 10-2000 ng/ml for MT. The intra-day and inter-day relative standard deviations (RSD) were less than 15% and the relative errors (RE) were all within 15%. The proposed method enables unambiguous identification and quantification of TMM and MT in vivo. This was the first report on determination of the TMM and MT in rat plasma after oral administration of TMM. The results provided a meaningful basis for evaluating the clinical applications of the medicine.

Effects of l-dopa methyl ester on visual cortex injury induced by amblyopia and its underlying mechanism.

Amblyopia is related to the impairment of visual system, especially visual cortex. l-Dopa has been reported to treat the disease, but the effect of its derived methyl ester has not been investigated. Therefore this study assessed the anatomic and physiologic effects of 30 days' treatment of l-dopa methyl ester at different doses on visual cortex area 17 in feline model with amblyopia induced by monocular vision deprivation. Immunohistochemical staining and Western blot were used to examine the structural changes of nerve cells and the expression of nerve growth factor (NGF), respectively. Comparing to the control, l-dopa methyl ester intervention significantly increased the number and density of NGF-immunoreactive cells, elevated endogenous NGF expression in visual cortex and promoted neural regeneration. The results suggest that l-dopa methyl ester can effectively inhibit amblyopia process via a mechanism that may involve increasing endogenous NGF expression and promoting neural repairment in visual cortex area 17.

Simultaneous determination of L-dopa and its prodrug (S)-4-(2-acetamido-3-ethoxy-3-oxopropyl)-1,2-phenylene diacetate in rat plasma by high-performance liquid chromatography-tandem mass spectrometry and its application in a pharmacokinetic study.

A sensitive, simple and rapid HPLC-MS/MS method has been developed and validated for the simultaneous determination of L-dopa and its prodrug (S)-4-(2-acetamido-3-ethoxy-3-oxopropyl)-1,2-phenylene diacetate (AEPD) in rat plasma in the present study. The analytes were separated on a C(18) column (5 microm, 2.1 mm x 150 mm) with a security guard C(18) column (5 microm, 4 mm x 20 mm) and a triple-quadrupole mass spectrometry equipped with an electrospray ionization (ESI) source was applied for detection. With alpha-methyldopa as internal standard, sample pretreatment involved in a one-step protein precipitation with 0.4M perchloric acid. The method was linear over the concentration ranges of 50-5000 ng/ml for L-dopa and 12.5-2500 ng/ml for AEPD. The intra-day and inter-day relative standard deviations (RSD) were less than 15% and the relative errors (RE) were all within 15%. Finally, the method was successfully applied to support the pharmacokinetic study after L-dopa and its prodrug AEPD were orally administrated to the Sprague-Dawley rats, respectively.