A Pharmacokinetic Study of Mix-160 by LC-MS/MS: Oral Bioavailability of a Dosage Form of Citroflavonoids Mixture

This study was performed to evaluate and compare the pharmacokinetic parameters between two dosage formulations of hesperidin and naringenin: mixture and tablet. Our objective was to determine that the flavonoid tablet does not significantly modify the pharmacokinetic parameters compared with the mixture. For this study, we administered 161 mg/kg of either mixture (Mix-160) or tablet composed of hesperidin and by intragastric administration. Blood microsamples were collected from tail vein up to 24 h. Serum flavonoid extraction was performed by solid phase extraction and analyzed by LC-MS/MS of triple quadrupole (QqQ). Serum concentration vs. time plot showed that data fitted for a first-order model. The pharmacokinetic parameters were calculated by a noncompartmental model. The results showed that the absorption constant is higher than the elimination constant. The first concentration was found at five minutes, and minimal concentration at 24 h after administration, suggesting a enterohepatic recirculation phenomena and regulation of liver cytochromes’ activity. We did not find meaningful differences between the pharmacokinetic parameters of both samples. We concluded that tablet form did not interfere with the bioavailability of hesperidin and naringenin, and it could be a suitable candidate for developing a drug product.

Secoisolariciresinol Diglucoside Improves Ovarian Reserve in Aging Mouse by Inhibiting Oxidative Stress

Ovarian reserve is a key factor in the reproductive function of the ovaries. Ovarian aging is characterized by a gradual decline in the quantity and quality of follicles. The underlying mechanism of ovarian aging is complex and age-related oxidative stress is considered one of the most likely factors. Secoisolariciresinol diglucoside (SDG) has been shown to have good scavenging ability against reactive oxygen species (ROS) which slowly accumulates in ovarian tissues. However, it is unknown whether SDG had beneficial effects on aging ovaries. In this study, we used 37-week-old female C57BL/6J mouse as a natural reproductive aging model to evaluate the role of SDG in ovarian aging. SDG (7 and 70 mg/kg) intragastric administration was performed in the mice daily. After 8 weeks, the effects of SDG on aging ovaries were evaluated by counting the number of follicles and the expression of follicle-stimulating hormone receptors (FSHR) in the ovary. The mechanism of SDG on the aging ovaries was further explored through ovarian metabolomics. It was found that SDG can effectively increase the number of growing follicles and increase the expression of the FSHR protein. The metabolomics results showed that the ovaries in the SDG intervention group achieved better uptake and transport of nutrients, including amino acids and glucose that are necessary for the development of oocytes. At the same time, the ovaries of the SDG intervention group showed that the drug reduced ROS generation. Additionally, we found that ovarian telomere length and ovarian mitochondrial DNA copy number that are highly susceptible to ROS damage and are also related to aging. The results showed that SDG can significantly increase mitochondrial DNA copy number and slow down the process of telomere shortening. These data indicate that SDG improves ovarian reserve by inhibiting oxidative stress.

Pharmacokinetics Study of Rabdosia Rubescens Drop Pills Based on UPLC-MS/MS

Background: Rabdosia rubescens drop pills have the effects of clearing away heat and toxin, detumescence, relieving pain. Objective: A simple and sensitive method for simultaneous determination of oridonin, ponicidin, and rosmarinic acid in rat plasma was developed based on ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Methods: Chromatographic separation was performed on a Waters ACQUITY UPLC BEH C18 column (2.1 × 100 mm, 1.7 µm) with a mobile phase consisting of water containing 0.2% formic acid (mobile phase A) and methanol (mobile phase B) at a flow rate of 0.3 mL/min over a total run time of 3.8 min. All analytes were measured with optimized multiple reaction monitoring (MRM) in positive and negative ion ESI mode. Results: The transitions of oridonin, ponicidin, rosmarinic acid, diphenhydramine, and chloramphenicol were 365.3→347.3, 363.3→345.2, 359.0→160.9, 256.0→167.2, and 321.1→151.9, respectively. The linear ranges were 1-256 ng/mL for ponicidin and rosmarinic acid and 2-512 ng/mL for oridonin. The validated method wasstable and reliable. There was no significant difference in the half-life (t1/2) of the three analytes at three doses. The area under the curve (AUC0-t) and peak concentration (Cmax) of the three analytes decreased linearly in each dose range, and the linear correlation R2 of each analyte under the three doses was greater than 0.95. Conclusion: This method was successfully applied to pharmacokinetic studies of oridonin, ponicidin, and rosmarinic acid in rat plasma after intragastric administration of Rabdosia rubescens drop pills.

Change of Embryotoxic Effects of Metal Citrates Depending on the Duration of Their Introduction

Given the increase in cadmium in the environment (air, soil, water) through modern industrial processes, the absorption of significant amounts from cigarette smoke is relevant to studying the effect of cadmium compounds on embryogenesis. The purpose of the study: experimental study and comparison of embryolethality and embryotoxicity of metal citrates depending on the duration of their intragastric administration (during 13 and 20 days of gestation). Materials and methods. The study was performed on 120 white adult female Wistar rats that weigh 170-200 g. They were divided into 6 groups due to the intragastric administration of solutions of the studied metals – rats treated with citrates: cadmium at a dose of 1.0 mg/kg – 1st group (nfemale = 20, nemb = 166); cadmium at a dose of 1.0 mg/kg and cerium (1.3 mg/kg) – 2nd group (nfemale = 20, nemb = 185); cadmium in a dose of 1.0 mg/kg and germanium (0.1 mg/kg) – 3rd group (nfemale = 20, nemb = 184); cadmium at a dose of 1.0 mg/kg and zinc (1.5 mg/kg) – 4th group (nfemale = 20, nemb = 179); cadmium at a dose of 1.0 mg/kg and nanocomposite (iodine + sulfur + selenium) at a dose of 2.0 mg/kg – 5th group (nfemale = 20, nemb = 180), 6th group – control (nfemale = 20, nemb = 212) – proportional volume of sterile saline in the same way. Females were divided into 2 subgroups of 10 animals each, depending on the duration of administration of test substances. The embryotoxic and embryolethal effects of the test substances were evaluated according to generally accepted criteria, which were calculated according to well-known formulas. Results and discussion. Cadmium compounds harm the embryogenesis of rats in the experimental groups and increase the rates of embryolethality. The most pronounced differences in these parameters concerning the control were found in animals with isolated exposure to cadmium citrate: the rates of total embryonic mortality increased by 4.0 times in both study periods of embryogenesis and 20% from the 13th to the 20th day; increased pre-implantation mortality by 6.0 times with an increase in post-implantation mortality by 3 times on the 13th day and by 15.0 times and 2.8 times on the 20th day of embryogenesis, respectively. Depending on the duration of introduction, the indicators of pre-implantation mortality increased by 25.0% with an increase in post-implantation mortality by 22.2% in the same period. The experimental group of exposure to cadmium citrate at a dose of 1.0 mg/kg recorded the lowest indicators of the number of live fetuses and intrauterine survival with the highest resorption rate studied of embryonic development. At the same time, in the groups of combined exposure to cadmium citrate with metal citrates, a decrease in the indicators of total embryonic mortality was 50.0% - 30.0%, pre-implantation mortality – 50.0% - 25.0%, post-implantation mortality – 60.0% - 44.4 % and increase in the number of fetuses per female – 12.7% - 25.3%. Conclusion. Analysis of the results shows a pronounced embryotoxic effect of cadmium citrate at a dose of 1.0 mg/kg on the processes of embryogenesis, which is a significant increase in overall embryonic mortality, preimplantation, and postimplantation mortality compared with the control group in all studied terms. In the groups of combined action of cadmium citrate with metal citrates, the data obtained indicate a decrease in the accumulation of cadmium under the influence of the studied citrates, which allows them to be considered as potential bioantagonists of cadmium citrate

Dimethyl Phthalate Induces Blood Immunotoxicity Through Oxidative Damage And Caspase-Dependent Apoptosis

Dimethyl phthalate (DMP), a low molecular weight phthalate ester, is present in ectoparasiticides, plastics, and insect repellants, has been linked to neurotoxic, reproductive, and endocrine disruptive responses. However, its blood immunotoxic effects and mechanism remain poorly understood. In this study, rats were exposed to graded concentrations of DMP through intragastric administration to assess the blood immunotoxic effects using a combination assay of biomarker, cytometry, and transcriptomics. DMP treatment altered the redox status of rats, causing that oxidative damage. Significantly decreased blood cell counts and disordered antibody and cytokine secretion were observed, suggesting the suppressed immune defense and destructed inflammatory regulation. Flow cytometry showed for lymphocytes, especially CD3+CD4+ T cells, apoptosis/necrosis occurred positively related to DMP exposure level. Transcriptomics revealed responses that were in line with oxidative damage effects. Overexpression of the Bcl-2 family genes and activation of the Fas/FasL pathway trigger downstream caspase cascade, causing reactive oxygen species signaling mediated apoptosis/necrosis. This is the first report on immunotoxic effects of low molecular weight phthalate esters.

Lactobacillus plantarum MA2 Ameliorates Methionine and Choline-Deficient Diet Induced Non-Alcoholic Fatty Liver Disease in Rats by Improving the Intestinal Microecology and Mucosal Barrier

Non-alcoholic fatty liver disease (NAFLD) has become a highly concerned health issue in modern society. Due to the attentions of probiotics in the prevention of NAFLD, it is necessary to further clarify their roles. In this study, the methionine and choline-deficient (MCD) diet induced NAFLD rats model were constructed and treated with strain L. plantarum MA2 by intragastric administration once a day at a dose of 1 × 108 cfu/g.bw. After 56 days of the therapeutic intervention, the lipid metabolism and the liver pathological damage of the NAFLD rats were significantly improved. The content of total cholesterol (TC) and total triglyceride (TG) in serum were significantly lower than that in the NAFLD group (p < 0.05). Meanwhile, the intestinal mucosal barrier and the structure of intestinal microbiota were also improved. The villi length and the expression of claudin-1 was significantly higher than that in the NAFLD group (p < 0.05). Then, by detecting the content of LPS in the serum and the LPS-TLR4 pathway in the liver, we can conclude that Lactobacillus plantarum MA2 could reduce the LPS by regulating the gut microecology, thereby inhibit the activation of LPS-TLR4 and it downstream inflammatory signaling pathways. Therefore, our studies on rats showed that L. plantarum MA2 has the potential application in the alleviation of NAFLD. Moreover, based on the application of the strain in food industry, this study is of great significance to the development of new therapeutic strategy for NAFLD.

Pharmacokinetics, Bioavailability and Tissue Distribution of Chitobiose and Chitotriose in Rats

Chitooligosaccharides (COSs) have various physiological activities and broad application prospects; however, their pharmacokinetics and tissue distribution remain unclear. In this study, a sensitive and selective ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) method for determining chitobiose (COS 2) and chitotriose (COS 3) in rat serum and tissues was developed. This method was successfully validated based on FDA guidelines in terms of selectivity, calibration curves (lower limit of quantification was 0.002 µg/mL for COS 2 and 0.02 µg/mL for COS 3), precision (intra-day relative standard deviation of 0.04–3.55% and inter-day relative standard deviation of 1.94–11.63%), accuracy (intra-day relative error of -1.81–11.06% and inter-day relative error of -9.41–8.63%), matrix effects, recovery (97.10–101.29%), stability, dilution integrity, and carry-over effects. Then, the method was successfully applied to the pharmacokinetics and tissue distribution study of COS 2 and COS 3 after intragastric and intravenous administration. After intragastric administration, COS 2 and COS 3 were rapidly absorbed, reached peak concentrations in the serum after approximately 0.45 h, and showed rapid elimination with clearances greater than 18.82 L/h/kg and half-lives lower than 6 h. The absolute oral bioavailability of COS 2 and COS 3 was 0.32–0.52%. COS 2 and COS 3 were widely distributed in Wistar rat tissues and could penetrated the blood-brain barrier without tissue accumulation.

Qingjie Fuzheng Granule Inhibits EMT and Induces Autophagy in Colorectal Cancer via mTOR Signaling Pathways

Qingjie Fuzheng granule (QFG) is a traditional Chinese medicinal formula used extensively as an alternative medicine for cancer treatment, including colorectal cancer (CRC). But its pathological mechanism in CRC is unclear. To study antitumor treatment effects and mechanisms of QFG, we established a CRC HCT-116 xenograft mouse model and assessed QFG on EMT and autophagy progression in vivo. The mice were randomly divided into 2 groups (n = 10 each group) and treated with intragastric administration of 1 g/kg of QFG or saline 6 days a week for 28 days (4 weeks). Body weight was measured every other day with electronic balance. At the end of the treatment, the tumor weight was measured. Immunohistochemical (IHC) and western blot (WB) assay were used to detect the expression level of E-cadherin, N-cadherin, vimentin, and TWIST1 to evaluate the effect of QFG on tumor cell EMT progression. IHC and WB assay were also used to detect the expression level of beclin-1, LC3-II, and p62 to evaluate the effect of QFG on tumor cell autophagy progression. Furthermore, the expression level of relative proteins in mTOR pathway was detected by WB assay to investigate the mechanism of QFG effect on CRC. We discovered that QFG inhibited the rise of tumor weight while it had no effect on mice body weight, which proved that QFG could inhibit CRC growth progression without significant side effects in vivo. In addition, QFG treatment inhibited EMT and induced autophagy progression in CRC tumor cells, including that QFG upregulated the expression of E-cadherin, beclin-1, and LC3-II, but downregulated the expression of N-cadherin, vimentin, TWIST1, and p62. And, QFG decreased the ratio of p-PI3K/PI3K, p-AKT/AKT, and p-mTOR/mTOR, but increased the ratio of p-AMPK/AMPK. All findings from this research proved that QFG can induce autophagy and inhibit EMT progression in CRC via regulating the mTOR signaling pathway.

Integrated Strategy From In Vitro, In Situ, In Vivo to In Silico for Predicting Active Constituents and Exploring Molecular Mechanisms of Tongfengding Capsule for Treating Gout by Inhibiting Inflammatory Responses

The study of screening active constituents from traditional Chinese medicine (TCM) is important for explicating the mechanism of action of TCM and further evaluating the safety and efficacy effectively. However, detecting and identifying the active constituents from complicated biological samples still remain a challenge. Here, a practical, quick, and novel integrated strategy from in vitro, in situ, in vivo to in silico for rapidly screening the active constituents was developed. Firstly, the chemical profile of TCM in vitro was identified using UPLC-Q Exactive-Orbitrap HRMS. Secondly, the in situ intestinal perfusion with venous sampling (IPVS) method was used to investigate the intestinal absorption components. Thirdly, after intragastric administration of the TCM extract, the in vivo absorbed prototype components were detected and identified. Finally, the target network pharmacology approach was applied to explore the potential targets and possible mechanisms of the absorbed components from TCM. The reliability and availability of this approach was demonstrated using Tongfengding capsule (TFDC) as an example of herbal medicine. A total of 141 compounds were detected and identified in TFDC, and among them, 64 components were absorbed into the plasma. Then, a total of 35 absorbed bioactive components and 50 related targets shared commonly by compounds and gout were integrated via target network pharmacology analysis. Ultimately, the effects of the absorbed components on metabolism pathways were verified by experiments. These results demonstrated that this original method may provide a practical tool for screening bioactive compounds from TCM treating particular diseases. Furthermore, it also can clarify the potential mechanism of action of TCM and rationalize the application of TFDC as an effective herbal therapy for gout.

Preclinical evaluation of the veterinary drug “Amoxidev 15” for its use in surgical and obstetric practice

Suspension for injection “Amoxidev 15” is prescribed to fur-bearing animals (mink, fox), dogs and cats for the treatment of respiratory diseases (tonsillitis, tracheitis, pneumonia, bronchitis, rhinitis, sinusitis, bronchopneumonia), digestive (gastritis, enteritis, enteritis). genitourinary systems (nephritis, urethritis, urocystitis, mastitis, metritis, agalactia), musculoskeletal system (arthritis, osteoarthritis, joint injuries, tendonitis, hoof lesions), skin and soft tissues (eczema, dermatitis) caused by sensitive drug by microorganisms, including colibacillosis, streptococcus, bronchopneumonia, etc. Toxicological evaluation of the veterinary drug “Amoxidev 15” under the conditions of acute and subacute toxicological experiments on a model of white rats. According to the results of an acute toxicological experiment with intragastric administration of the drug “Amoxidev 15” white rats DL50 could not be calculated because the death of laboratory animals was not detected within 14 days after administration. The maximum administered dose (in absolute weight of the drug) was 20000.0 mg/kg body weight, which allows to refer the drug to class VI toxicity of relatively harmless substances (DL50 > 15000 mg/kg body weight), and the degree of safety to class IV – low-hazard substances (DL50 > 5000 mg/kg). According to the results of an acute toxicological experiment with subcutaneous administration of the drug “Amoxidev 15” white rats DL50 could not be calculated because the death of laboratory animals was not detected within 14 days after administration, the maximum dose was 5000.0 mg/kg body weight, therefore, the drug “Amoxidev 15” when administered subcutaneously by toxicity can be classified as class VI substances relatively harmless (DL50 Subcut > 4500.0 mg/kg). When administered subcutaneously to white rats, the drug “Amoxidev 15” under conditions of subacute toxicological experiment in doses of 0.1–1.0 ml/kg does not cause hemo-, hepato- and nephrotoxic effects on the body of laboratory animals, although 3-day administration of the drug in a dose 1.0 ml/kg body weight caused an increase in the activity of hepatospecific enzymes ALT and AST by 12.5 and 11.1 % (P < 0.05), respectively, relative to the control, which was restored to the control level 7 days after cessation.