Optimization of SARS-CoV-2 spike protein receptor binding domain expression in Pichia pastoris and evaluation of its immunogenicity / 中华微生物学和免疫学杂志

Objective:To effectively express the receptor binding domain (RBD) of SARS-CoV-2 spike protein in Pichia pastoris and to evaluate its immunogenicity. Methods:The gene encoding the RBD protein was synthesized and cloned into the pPICZαA plasmid. After linearization, the plasmid was transferred and integrated into the genome of Pichia pastoris. The expressed RBD protein in culture supernatant was analyzed by Western blot and Biolayer interferometry. After screening, a single clone expressing the RBD protein was selected. The high-level expression of RBD protein was achieved by optimizing the fermentation process, including the salt concentration adjusting of the medium and induction condition optimization (pH, temperature and duration). The immunogenicity of the expressed RBD protein was evaluated in a mouse model. Results:A single clone with a high expression level of RBD protein was obtained and named RBD-X33. The expression level of RBD protein in the fermentation supernatant reached up to 240 mg/L after optimization of the induction condition (HBSM medium, pH=6.5±0.3, 22℃ and 120 h). In the mouse experiment, the recombinant RBD protein was formulated with Alum+ CpG dual adjuvant and injected into mice. The binding IgG antibody levels were up to 2.7×10 6 tested by ELISA and the neutralizing antibody levels were up to 726.8 tested by live virus neutralizing antibody assay (prototype). Conclusions:The RBD protein could be efficiently expressed in Pichia pastoris and induce stronger immune response in animals. This study suggested that the recombinant SARS-CoV-2 RBD protein expressed in Pichia pastoris could serve as a candidate antigen in the development of SARS-CoV-2 vaccine.

Protective effect of okra seed oil on acute gastric ulcer in mice / 中国药科大学学报

To investigate the protective effect of the seed oil of Abelmoschus esculentus on gastric ulcer,two acute gastric ulcer mice models were established by intragastric administration of aspirin or absolute ethanol,respectively.Clinical index of ulcer area,ulcer index,gastric volume,gastric pH value,free acidity,total acidity,and histopathological assessment were measured to evaluate the injuries of gastric ulcer and the protective effect of okra seed oil In order to comprehensively uncover the possible underlying mechanism,a series of biochemical assays were also performed,including serum TNF-α,IL-6,IL-10 and Tbil,NO,MPO and SOD in the stomach included.Moreover,the ALT,AST and ALP in the liver of mice were also tested to evaluate the possible hepatic toxicity of the seed oil.The results indicated that the seed oil of A.esculentus exerted protective effect in ethanol-induced gastric ulcer mice by reducing the ulcer area and ulcer index,declining the free and total acidity,and increasing the pH value of gastric content.Histopathological observation showed the gastric mucosa of the acute gastric ulcer mice induced by alcohol was incomplete and severely damaged,with submucosal edema and nuclear pyknosis,as well as glandular structure disappearing,compared with that of normal mice.What's more,a number of inflammatory cell infiltration occured in the gastric mucosa of alcohol-model mice,with messes of neutrophils,lymphocytes,eosinophils and plasma cells.Okra seed oil could improve the damaged structure of the gastric mucosa and gland caused by ethanol,but could not ameliorate the condensation of nucleus and infiltration of inflammatory cells.Biochemical analysis revealed that the seed oil of A.esculentus could counteract the damage induced by ethanol via decreasing Tbil and TNF-α in serum,decreasing NO and myeloperoxidase,and increasing SOD in stomach.Meanwhile,okra seed oil exhibited protective effect in aspirin-induced gastric ulcer mice by increasing the gastric content pH,and reducing free and total acidity.Compared with the control group,the gastric mucosa of aspirin-model group showed multifocal coagulation necrosis,sheet edema and infiltration of inflammatory cells by histopathological assessment.Compared with the aspirin-model group,the soybean oil group and okra seed oil group could ameliorate the inflammatory cell infiltration.Biochemical analysis revealed that okra seed oil could counteract the injury induced by aspirin via decreasing TNF-α and IL-6,and increasing IL-1O in serum,decreasing NO and MPO and increasing SOD in stomach.In a word,the okra seed oil exerted protective effect on acute gastric ulcer by anti-inflammation,anti-oxidation and hepatocyte protection.The okra seed oil deserves further development and utilization.

The action mechanisms of Morus alba leaves extract for the treatment of diabetes based on plasma metabolomics / 药学学报

In order to evaluate the effect and mechanism of the mulberry leaf alkaloid, flavones, and polysaccharide intervention on diabetes, the overall metabolite profiling characteristics for the plasma of diabetic mouse was performed by using an ultra-performance liquid chromatography/electrospray-tandem mass spectrometry (UPLC-ESI-MS). The 8 potential biomarkers were found in diabetic mice plasma based on the data of MS/MS characteristics obtained from the UPLC-OrbitrapMS analysis, which mainly involved in sphingolipids, amino acid metabolic pathway. The principal component analysis showed that the normal group and model group were obviously distinguished and implied that metabolic disturbance was happened in diabetic mice plasma. The extracts of mulberry leaf flavonoids, polysaccharide, alkaloid had exhibited the effects of callback function for diabetic mice through regulating the amino acid metabolism and sphingolipid metabolism.

Analysis and identification of water soluble components of water buffalo horn / 药学学报

This study is to analyze and identify the water soluble components of water buffalo horn (Bubali Cornu, WBH), and also establish a method for investigating these components. Shotgun proteomic analysis identified proteins in WBH aqueous extraction: keratin, collagen, desmoglein, etc. Ultrafiltration and LC-MS/MS were used to separate and identify the peptides in WBH aqueous extract, as a result, identified peptides were mainly derived from nonspecific degradation products of keratin and collagen, which including C-terminal peptides and non C-terminal peptides. Hypoxanthine, uridine, guanosine, and adenosine were identified by comparing with the standards. The strategy in present study could be used in analyzing water soluble components of animal horn derived TCM. It provides a reference for investigation of the material basis of animal horn derived TCM.

Strategies on the Utilization of Castoff from Process in Chinese Materia Medica Resource Industrialization based on Microbial Transformation / 世界科学技术-中医药现代化

Based on the research methods and technologies of science of Chinese materia medica, chemistry and mi-crobiology, the resource material conversion of functional material, biomass energy, nutrients in castoff from Chinese materia medica industrialization is promoted by microbial transformation. This will help enhance the resource utiliza-tion value, extend resource industry chains, and realize Chinese materia medica resource industry with the best use of everything, resource-saving and environment-friendly recycling economy development.

The interaction between ononin and human intestinal bacteria / 药学学报

The study aims to screen the ability of the bacteria to metabolize ononin and assess the effect of ononin on the intestinal bacteria. Fresh human fecal sample was obtained from a healthy volunteer, diluted serially in sterile water and sixty-nine different bacterial colonies were picked out ultimately. UPLC-Q-TOF/MS with automated data analysis software (MetaboLynx) was applied to fast analysis of ononin metabolites. Furthermore, an E(max) precision microplate reader was employed to determine the growth situation of Enterococcous sp., Enterobacter sp., Lactobacilli sp., and Bifidobacteria sp. Results indicated that hydrogenation, demethylation, hydroxylation and deglycosylation were the major metabolic pathways of ononin by human intestinal bacteria in vitro. Ononin can inhibit the growth of pathogen such as Enterococcus sp., Enterobacter sp. and can promote the growth of probiotics such as Bifidobacteria sp. and Lactobacilli sp. This study suggested that intestinal bacteria have the metabolic effects of ononin and the biotransformation was completed by different bacteria. And ononin can affect the balance of intestinal flora and the degree of influence varies depending on the bacterial species and the concentration of ononin.

Metabolism of naringin produced by intestinal bacteria / 药学学报

Naringin has been reported to possess a wild range of biological activities. However, the route and metabolites of naringin produced by intestinal bacteria are not well understood. In this paper, different bacteria were isolated from human feces and their abilities to convert naringin to different metabolites were studied. Ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) with automated data analysis software (MetaboLynx) was applied to fast analysis of naringin metabolites. Using MSE and mass defect filter techniques, three metabolites were detected and tentatively identified. The results indicated that acetylation, hydrolyzation and hydrolyzation with hydrogenation were the major metabolic pathways of naringin in vitro. Then, we studied the gene sequence of the 16S rRNA of the bacteria by extraction of genomic DNA of the strain, PCR amplification and clone of the 16S rRNA. The consequence proved that Enterococcus sp.30, Bacillus sp.46, Escherichia sp.54 and Escherichia sp.63 have the peculiar metabolism characteristic of naringin.

In vitro transdermal delivery of the active fraction of xiangfusiwu decoction based on principal component analysis / 药学学报

The objective of the present study was to establish a method based on principal component analysis (PCA) for the study of transdermal delivery of multiple components in Chinese medicine, and to choose the best penetration enhancers for the active fraction of Xiangfusiwu decoction (BW) with this method. Improved Franz diffusion cells with isolated rat abdomen skins were carried out to experiment on the transdermal delivery of six active components, including ferulic acid, paeoniflorin, albiflorin, protopine, tetrahydropalmatine and tetrahydrocolumbamine. The concentrations of these components were determined by LC-MS/MS, then the total factor scores of the concentrations at different times were calculated using PCA and were employed instead of the concentrations to compute the cumulative amounts and steady fluxes, the latter of which were considered as the indexes for optimizing penetration enhancers. The results showed that compared to the control group, the steady fluxes of the other groups increased significantly and furthermore, 4% azone with 1% propylene glycol manifested the best effect. The six components could penetrate through skin well under the action of penetration enhancers. The method established in this study has been proved to be suitable for the study of transdermal delivery of multiple components, and it provided a scientific basis for preparation research of Xiangfusiwu decoction and moreover, it could be a reference for Chinese medicine research.

Metabolomic study of the action mechanism of nourishing blood effect of fo-shou-san on blood deficiency mice / 药学学报

The metabolic effect of Fo-Shou-San on blood deficiency mice was studied by using metabolomic method. UPLC-QTOF/MS was used to analyze the plasma metabolome in blood deficiency mice. MS data were processed by MarkerLynx software. With multivariate statistical analysis of plasma metabolite profiles, a clear separation among control, blood deficiency model, and Fo-Shou-San groups was achieved. Potential biomarkers were selected according to the parameters of variable importance in the projection (VIP) and identified according to MS information and database retrieval. The metabolic network of blood deficiency was predicted via MetPA database. Twenty-two potential biomarkers were identified and used to explain the thiamine metabolism, arachidonic acid metabolism, sphingolipid metabolism, glyoxylate and dicarboxylate metabolism, histidine metabolism, nicotinate and nicotinamide metabolism, cysteine and methionine metabolism, tryptophan metabolism, starch and sucrose metabolism, tyrosine metabolism and citrate cycle (TCA cycle). Those metabolic pathways were disturbed in blood deficiency mice, but which could be regulated nearly to normal state after Fo-Shou-San administration. In this study, the metabolomics of blood deficiency mice and the action mechanism of nourishing blood effect of Fo-Shou-San were evaluated. The physiological and metabolic state of the organism could be represented comprehensively by using metabolomics. And metabolomics can be used to evaluate the pharmacodynamics and related mechanisms of Chinese medicine and formulae.

Study on metabolites of ferulic acid and gallic acid compatible with danggui and chishao in rat plasma and urine / 中国中药杂志

<p><b>OBJECTIVE</b>To study the major metabolites of ferulic acid and gallic acid compatible with Danggui Chishaoyao in rat plasma and urine.</p><p><b>METHOD</b>The metabolites of ferulic acid and gallic acid in rat plasma and urine were analyzed after oral administration of compatible Danggui Chishaoyao using UPLC-Q-TOF-MS.</p><p><b>RESULT</b>On the basis of the mass information, it was inferred that in vivo metabolites of ferulic acid were be in the form of methylation products, sulfate conjugation products and glucuronidation conjugation products and so on; meanwhile, gallic acid was mainly transformed into eduction products and methylation products.</p><p><b>CONCLUSION</b>There are kinds of phase I and phase II metabolites of ferulic acid and gallic acid in rat plasma and urine, which provide a basis for its efficacious materials and action mechanism.</p>

Influence of different solutions on extracting of active components in Danggui Chishao drug pair / 中国中药杂志

<p><b>OBJECTIVE</b>To study the dissolution rate of active components of different extracting solvents of Danggui Chishao drug pair.</p><p><b>METHOD</b>The dissolution rates of phenolic acids (ferulic acid, vanillic acid and gallic acid), monoterpenes (gallic acid, peoniflorin, albiflorin, hydroxypeoniflorin and galloylpaeoniflorin) and phthalates (senkyunolide and ligustilide) contained in Danggui Chishao drug pair were determined by quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF-MS) and ultra high performance liquid chromatography in combination with triple-quadrupole mass spectrometry (UPLC-TQ-MS).</p><p><b>RESULT</b>The dissolution rates of phenolic acids and monoterpenes decreased with the increase in alcohol concentration, on the contrary the rates of phthalates increased. The relative dissolution rates of most active components were relatively high in water and low concentration alcohol than other solvents.</p><p><b>CONCLUSION</b>Liquid chromatography-mass spectrometry is practical for comprehensive multi-component assessment on traditional Chinese medicine preparation processes and can provide reference for optimization of processing parameters.</p>

Research on patulin biosynthesis and infection of Chinese medicinal materials by its producing strains / 中国中药杂志

<p><b>OBJECTIVE</b>To investigate the effect of patulin producing strains on the different Chinese medicinal materials and the toxin biosynthesis mechanism.</p><p><b>METHOD</b>Microbiology and HPLC analytical methods were adopted in this paper.</p><p><b>RESULT</b>It was showed that the materials rich in starch and other polysaccharides were easily polluted by the patulin producing strain. This strain grew well and produced more toxins under 25 degrees C, 95% moisture content and bulk package. And the effect of low illumination intensity on the strain growth and toxin biosynthesis was not notable. Sample stability, precision, repeatability and rate of recovery were studied. HPLC analytic method was established and it revealed that the test method was suitable.</p><p><b>CONCLUSION</b>The pollution of Chinese medicinal materials by toxin producing microbes will be effectively controlled through establishing the suitable storage methods. So the study on the growing characteristics and toxin biosynthesis mechanism of toxin producing strains will be an important practical significance for controlling the toxin pollution of herbal medicines and contribute to establish the evaluation system of Chinese medicine safety.</p>

Enriching blood effect comparison in three kinds of blood deficiency model after oral administration of drug pair of Angelicae Sinensis Radix and Chuanxiong Rhizoma and each single herb / 中国中药杂志

<p><b>OBJECTIVE</b>Through establishing different blood deficiency animal model, to evaluate enriching blood effect changes of the drug pair of Angelicae Sinensis Radix and Chuanxiong Rhizoma and each single herb, and to explore the effect characteristics of their compatibility.</p><p><b>METHOD</b>Three different methods of acetyl phenylhydrazine (APH) hemolytic method, cyclophosphamide (CTX) chemical damage method, APH-CTX complex method were used respectively to copy different blood deficiency model mice. Changes of orbit blood routine, thymus index, spleen index and ATPase activity of red cell membrane of model mice were tested.</p><p><b>RESULT</b>Compared with normal group, all indexes had significant differences in three model mice. The drug pair and each single herb had significant impact on most indexes of the APH-CTX complex model mice, and on the individual indexes of APH hemolytic model mice and CTX chemical damage model mice. Therefore, APH and CTX complex blood deficiency model was more suitable for the enriching blood mechanism study of the drug pair of Angelicae Sinensis Radix and Chuanxiong Rhizoma. Compared with the single herb of Angelicae Sinensis Radix and Chuanxiong Rhizoma, the drug pair of them had presented enriching blood effect at different extent with strengthening trend in regulating the invigorating blood indexes, immune organs and energy metabolic enzymes.</p><p><b>CONCLUSION</b>The results of this research have provided scientific basis for revealing the mutual promotive composition law of the drug pair of Angelicae Sinensis Radix and Chuanxiong Rhizoma, and responded effectively the mult-link and mult-target effect characteristics of Chinese medicine bio-effect, to offer reference for the bio-effect research of the complicated substance group of Chinese medicine and traditional Chinese medicine formulae, and to supply demonstrative reference for researching the formulae compatibility law which takes the single drug-drug pair-formulae as main line.</p>

Studies on influence of Siwu decoction and its composite drugs on chemical-induced blood-deficiency model mice / 中国中药杂志

<p><b>OBJECTIVE</b>To investigate Siwu decoction and its composite drugs on the blood-deficiency model mice induced by acetylphenyhydrazine and cyclophosphamide.</p><p><b>METHOD</b>Acetylphenyhydrazine and cyclophosphamide were used to copy the blood-deficiency model mice. Automatic hematology analyzer was used to test the peripheral hemogram. Weighting method was used to test the liver index and spleen index; Kits for ATPase test was used to test the activities of Na+ - K+ - ATPase/Ca2+ - Mg2+ - ATPase in erythrocyte membrane. Flow cytometry was used to test the bone marrow cells' cell cycle.</p><p><b>RESULT</b>Angelicar Sinensis Radix and Paeoniae Radix Alba had the most effective activity on the peripheral hemogram. Paeoniae Radix Alba, the drug pair including Angelicar Sinensis Radix and the drug- group including Paeoniae Radix Alba had the most effective activity on the liver index. All the drugs, drug-pairs, drug-groups and the formula had effect on the spleen index. To the activity of Na+ - K+ - ATPase in erythrocyte membrane, Rehmanniae Radix Praeparata, the drug-pairs and drug-groups including Angelicar Sinensis Radix exhibited the most effective activity. All the drugs, drug-pairs, drug-groups and the formula had the protective effect on the damaged bone marrow cells.</p><p><b>CONCLUSION</b>Siwu decoction and its composite drugs all had effect on the blood-deficiency model mice, but the action intensity was different. Angelicar Sinensis Radix and Paeoniae Radix Alba exhibited the most effective activity to the protection of the blood-deficiency model mice.</p>

Identification of the metabolites of Sinisan extract in rat plasma, urine, feces and bile after intragastric administration / 药学学报

Sinisan is a widely used traditional Chinese medicine (TCM) in treating various diseases; however, the in vivo metabolic profile of its multiple components remains unknown. In this paper, ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) was applied to identify the metabolites of Sinisan extract in rat plasma, urine, feces and bile after intragastric administration. Using MS(E) and mass defect filter techniques, 41 metabolites of 10 parent compounds (naringin, naringenin, hesperidin, neohesperidin, liquiritin, liquiritigenin, glycyrrhizic acid, glycyrrhetinic acid, saikosaponin a and saikosaponin d) were detected and tentatively identified. It was shown by our results that these compounds was metabolized to the forms of hydroxylation, glucuronidation, sulfation, glucuronidation with sulfation and glucuronidation with hydroxylation in vivo.

Absorption of flavonoids from Abelmoschus manihot extract by in situ intestinal perfusion / 药学学报

To explore the mechanism of the absorption of flavonoids from Abelmoschus manihot flowers, in situ intestinal recirculation was performed to study the effect of the absorption at different concentrations and different intestinal regions. To evaluate the conditions of the absorption of six flavonoids from Abelmoschus manihot flowers, the concentrations of Abelmoschus manihot in the perfusion solution were determined by HPLC at predesigned time. And we have investigated the inhibitory effect of six flavonoids from Abelmoschus manihot flowers on P-glycoprotein (P-gp) drug efflux pump. The results demonstrated that the absorption rates of flavonoids from Abelmoschus manihot flowers are not significantly different (P > 0.05) at various drug concentrations, the absorption of flavonoids from Abelmoschus manihot flowers is a first-order process with the passive diffusion mechanism. The absorption rates of each of flavonoids are significantly different. The absorption rate of flavonoid glycoside was lower than that of aglycone; the flavonoids from Abelmoschus manihot flowers could be absorbed in all of the intestinal segments. The best parts of intestine to absorb hyperoside and myricetin are jejunum and duodenum, separately. Verapamil could enhance the absorption of isoquercitrin, hyperoside, myricetin and quercetin-3'-O-glucoside by inhibiting P-glycoprotein (P-gp) drug efflux pump.

Analysis on competitive interaction between arenobufagin and verapamil hydrochloride with serum albumin / 中西医结合学报

The present study was performed to investigate competitive interaction between arenobufagin and verapamil hydrochloride with serum albumin.

Discussion on quality changes mechanism of Chinese medicinal materials during storage / 中国中药杂志

Storage of Chinese medicinal materials was an important part in the circulation process. During storage, medicinal herbs, the factors in the outside world and their own physical and chemical properties of the interaction, gradually occurring physical, chemical and biological changes. Phenomenon such as mold, moth-eaten, color change, taste change and "Fan You" would be seen. The intrinsic quality changes related to the mechanism: oxidation, polymerization or decomposition reaction, Maillard reaction, enzymatic reaction and transformation reactions. It resulted to increasing or decreasing of the active ingredient content in medicinal materials, losing of volatile components, forming of biological metabolites and transformation products of damage insects.

UPLC-Q-TOF-MS analysis of naringin and naringenin and its metabolites in rat plasma after intragastrical administration of alcohol extract of exocarpium Citri grandis / 中国中药杂志

<p><b>OBJECTIVE</b>To analyze naringin, naringenin and its metabolites in rat plasma after intragastric administration of exocarpium Citri grandis alcohol extract.</p><p><b>METHOD</b>Rat blood samples were collected 1.0 hour after oral administration of 50 g x kg(-1) exocarpium Citri grandis alcohol extract and analyzed by UPLC-Q-TOF with MS(E) function. The post-acquisition data were processed using Metabolynx.</p><p><b>RESULT</b>Naringin (M1), naringenin (M2), naringin-5-O-glucuronide (M3), naringin-4-O-glucuronide (M4), glucuronide conjugate of naringenin (M5), naringin-4-O-sulfate (M6), methylated conjugate of hydroxylated naringenin (M7), glucuronide and sulfate conjugate of naringenin (M8), glucuronide conjugate of hydroxylated naringenin (M9) in rat plasma were detected. M3, M4, M6 were first reported as the metabolites of naringin. M7, M9 were first reported as the metabolites of naringenin.</p><p><b>CONCLUSION</b>The results indicated that naringin, naringenin can be metabolited as the forms of glucuronidation, sulfation and naringenin can also be metabolited as the forms of methylation with hydroxylation and glucuronidation with hydroxylation in vivo after administration.</p>

UPLC-Q-TOF/MS analysis of naringin and naringenin and its metabolites in rat urine and feces after intragastric administration of alcohol extract of Exocarpium Citri grandis / 药学学报

To analyze naringin, naringenin and its metabolites in rat urine and feces after intragastric administration of alcohol extract of Exocarpium Citri Grandis, healthy SD rats were fed with alcohol extract of Exocarpium Citri Grandis for 3 days. On the last day, 0-24 h feces and 0-4 h, 4-8 h, 8-24 h urine were collected and analyzed by UPLC-Q-TOF/MS. The post-acquisition data were processed using Metabolynx The result is that naringin and its 6 metabolites, naringenin and its 4 metabolites were detected in the urine of rat. Meanwhile, naringin and its 3 metabolites, naringenin and its 2 metabolites were detected in the feces of rat.