Objective To develop a reversed-phase high-performance liquid chromatography(RP-HPLC) method for the determination of residual phenylmethanesulfonyl fluoride(PMSF) content in recombinant virus-like particle(VLP) vaccine stock solution,and to verify the method for the determination of PMSF residues in recombinant VLP vaccine stock solution.Methods A RP-HPLC method for the determination of PMSF residues was developed by screening detection wavelength,flow rate,injection amount and chromatographic column.The specificity,linearity and range,limit of quantitation(LOQ),limit of detection(LOD),accuracy,precision and durability of the method were verified.The developed method was used to detect PMSF residues in three batches of recombinant hepatitis E vaccine stock solution.Results The RP-HPLC method was developed,with ChromCore 300 C18(4.6 mm × 250 mm,5 μm) as the chromatographic column at the column temperature of 25 ℃,the detection wavelength of 210 nm,the injection amount of 100 μL,0.1% trifluoroacetic acid(TFA)/water and0.1% TFA/acetonitrile as mobile phase at a flow rate of 1 mL/min,and the detection time of 18 min.The absorption peaks of PMSF control and vaccine stock solution containing PMSF appeared around 5.0 min,while the vaccine stock solution and stock solution buffer showed no absorption peak.There was a good linear relationship between the concentration of PMSF control and peak area in the range of 0-5.0 μg/mL,with R~2 of 0.999.The LOQ and LOD of this method was 0.250 μg/mL and 0.125 μg/mL,respectively.The recovery rates of PMSF control with high,medium and low concentrations were all between 90% and 110%.The relative standard deviations(RSDs) of retention time,peak height and peak area for the determination of instrument repeatability,sample repeatability and intermediate precision were all less than 5%.The RSDs of retention time,peak height and peak area were all less than 5% under the TFA concentration of 0.09%,0.1% and 0.11% at column temperature of 20,25 and 30 ℃.PMSF was not detected in the three batches of recombinant hepatitis E vaccine stock solution.Conclusion The developed RP-HPLC method has good specificity,linearity and durability,high accuracy and precision,and can be used to detect residual PMSF content in recombinant VLP vaccine stock solution.
Phenylmethanesulfonyl fluoride(PMSF) ; Reversed-phase high-performance liquid chromatography(RP-HPLC) ; Virus-like particle(VLP) vaccine

OBJECTIVE

This study aimed to determine and quantify the presence of the active components in Thai propolis extracts using high performance liquid chromatography (HPLC). Moreover, the anti-caries potential of Thai propolis extract and its active ingredients were tested.

Fifty milligrams of Thai propolis were extracted using either 100%, 90%, 80%, or 70% ethanol and subsequently analyzed using HPLC with a mobile phase gradient system of 10-100% acetonitrile in 0.05% aqueous ortho-phosphoric acid, flow rate of 0.8 mL/min, and detection wavelength of 280 nm. Varying concentrations of Thai propolis extracts as well as four active ingredients were subjected to agar well diffusion test against the growth of Streptococcus mutans (S. mutans) or Lactobacillus caseii (L. caseii).

The concentrations of the four active ingredients: vicenin-2, vitexin, apigenin, and cinnamic acid, were significantly affected by ethanolic concentrations. The chromatographic peaks of all active ingredients from 70% and 80% ethanolic extracts appeared more defined, as compared to those which used higher concentrations of ethanol for extraction. Except for the absolute ethanolic extract, all of the examined propolis extracts, as well as its active ingredients inhibited both S. mutans and L. caseii.

Thai propolis extracts contain vicenin-2, vitexin, apigenin, and cinnamic acid as part of its active ingredients. These were found to be significantly affected by the increase in ethanol during its extraction. The presence of these active ingredients might have contributed to the anti-caries potential of Thai propolis extracts.

Objectives: The aim of this study is to establish a Reversed Phase – High Performance Liquid Chromatographic (RP-HPLC) method for the quantification of Rhein from Cassia fistula L. leaves. Methods: A Shimadzu system equipped with a C18 Column (150 x 4.6 mm, 5 μm) with an isocratic elution of Acetonitrile (solvent A) and 0.1% trifluoroacetic acid aqueous solution (solvent B) (Merck, 1.08178.0050) with a 55:45 ratio, respectively and a flow rate of 1.0 mL/min and sample injection of 10 μL detection was done at 230 nm. Standard solution of Rhein (Chengdu Biopurify) was prepared for method development. This study was validated using the guidelines set under “ICH Topic Q2 R2 or the Validation of Analytical Procedures”. Procedures for linearity, precision, accuracy, limit of detection, and limit of quantitation were performed. Results: The retention time of Rhein standard was determined at 5.10 minutes. LOD and LOQ were determined to be 1.278 mcg/mL and 3.872 mcg/mL, respectively with good linearity (R2 ≥0.996) with a linear range of 2.5-20 ug/mL of the Rhein standard. The accuracy of the method was determined based on % recovery method and ranged from 94.75%-100.32% (intraday, n=3) with %RSD of 0.71. The intraday precision %RSD was 2.92 (n=6) while interday precision %RSD was 3.75 (n=3). The method was able to check the Rhein quantity among 10 samples of Cassia fistula L. leaves from different locations in the Philippines. Conclusion The method was found to be sensitive and accurate for the quantification of Rhein. The method was found to be useful for the quantification of the amount of Rhein and can be used as a Quality Control tool for the assessment of Cassia fistula.
Cassia ; Chromatography, High Pressure Liquid

Male ; Humans ; Liquid Chromatography-Mass Spectrometry ; Chromatography, Liquid ; Tandem Mass Spectrometry ; Varicocele/diagnosis* ; Biomarkers

Objective: This study aimed to determine and quantify the presence of the active components in Thai propolis extracts using high performance liquid chromatography (HPLC). Moreover, the anti-caries potential of Thai propolis extract and its active ingredients were tested. Methods: Fifty milligrams of Thai propolis were extracted using either 100%, 90%, 80%, or 70% ethanol and subsequently analyzed using HPLC with a mobile phase gradient system of 10-100% acetonitrile in 0.05% aqueous ortho-phosphoric acid, flow rate of 0.8 mL/min, and detection wavelength of 280 nm. Varying concentrations of Thai propolis extracts as well as four active ingredients were subjected to agar well diffusion test against the growth of Streptococcus mutans (S. mutans) or Lactobacillus caseii (L. caseii). Results: The concentrations of the four active ingredients: vicenin-2, vitexin, apigenin, and cinnamic acid, were significantly affected by ethanolic concentrations. The chromatographic peaks of all active ingredients from 70% and 80% ethanolic extracts appeared more defined, as compared to those which used higher concentrations of ethanol for extraction. Except for the absolute ethanolic extract, all of the examined propolis extracts, as well as its active ingredients inhibited both S. mutans and L. caseii. Conclusions Thai propolis extracts contain vicenin-2, vitexin, apigenin, and cinnamic acid as part of its active ingredients. These were found to be significantly affected by the increase in ethanol during its extraction. The presence of these active ingredients might have contributed to the anti-caries potential of Thai propolis extracts.
flavonoids ; chromatography, high performance liquid

INTRODUCTION

One of the novel strategies in cancer treatment is the combination of conventional chemotherapeutic drugs and natural products. In a previous study, co-treatment of the anti-cancer drug cyclophosphamide (CP) with honey from giant honey bee (Apis dorsata) resulted to a dose-dependent increase in its cytotoxic effect in human lung carcinoma (A549) cells. However, the molecular mechanism of this combinatorial effect remains unknown.

In this study, the effect of A. dorsata honey on the expression of selected CYP450 genes at the mRNA level, as well as the proapoptotic gene CASP8 and antiapoptotic gene BCL2 was investigated in CP-treated A549 cells.

MTT Assay was performed to determine the cell viability of A549 cells after treatment with CP with or without A. dorsata honey, as well as the EC50 of CP with honey thereafter. RT-qPCR was then performed to study the effect of A. dorsata honey on the expression of selected CYP450 genes as well as CASP8 and BCL2 genes in CPtreated A549 cells. LC-MS was carried out to screen for putative compounds in A. dorsata honey which may possibly have anti-cancer activity.

Honey in the lowest concentration (0.6% v/v) most effectively enhanced the cytotoxic effect of CP. CYP2J2 and CYP1B1 indicated a 2.38-fold and 1.49-fold upregulation, respectively as compared to untreated cells. This cytotoxic effect is further enhanced by upregulation of CASP8 that is paralleled by a downregulation of BCL2. Phytosphingosine and sphinganine are honey constituents which may be linked to the increased cytotoxicity of CP observed in A549 cells.

This study provides further knowledge on the molecular basis by which A. dorsata honey potentiates the cytotoxic effect of cyclophosphamide in A549 cells.

OBJECTIVES

The aim of this study is to establish a Reversed Phase – High Performance Liquid Chromatographic (RP-HPLC) method for the quantification of Rhein from Cassia fistula L. leaves.

A Shimadzu system equipped with a C18 Column (150 x 4.6 mm, 5 μm) with an isocratic elution of Acetonitrile (solvent A) and 0.1% trifluoroacetic acid aqueous solution (solvent B) (Merck, 1.08178.0050) with a 55:45 ratio, respectively and a flow rate of 1.0 mL/min and sample injection of 10 μL detection was done at 230 nm. Standard solution of Rhein (Chengdu Biopurify) was prepared for method development. This study was validated using the guidelines set under “ICH Topic Q2 R2 or the Validation of Analytical Procedures”. Procedures for linearity, precision, accuracy, limit of detection, and limit of quantitation were performed.

The retention time of Rhein standard was determined at 5.10 minutes. LOD and LOQ were determined to be 1.278 mcg/mL and 3.872 mcg/mL, respectively with good linearity (R2 ≥0.996) with a linear range of 2.5-20 ug/mL of the Rhein standard. The accuracy of the method was determined based on % recovery method and ranged from 94.75%-100.32% (intraday, n=3) with %RSD of 0.71. The intraday precision %RSD was 2.92 (n=6) while interday precision %RSD was 3.75 (n=3). The method was able to check the Rhein quantity among 10 samples of Cassia fistula L. leaves from different locations in the Philippines.

The method was found to be sensitive and accurate for the quantification of Rhein. The method was found to be useful for the quantification of the amount of Rhein and can be used as a Quality Control tool for the assessment of Cassia fistula.

Mice ; Animals ; Tandem Mass Spectrometry ; Alpha-Amanitin ; Chromatography, Liquid ; Metabolomics ; Chromatography, High Pressure Liquid/methods*

To investigate the formation of polystyrene nanoplastic-plant protein corona and its potential impact on plants, three differently modified polystyrene nanoplastics with an average particle size of 200 nm were taken to interact with the leaf proteins of Impatiens hawkeri for 2 h, 4 h, 8 h, 16 h, 24 h, and 36 h, respectively. The morphological changes were observed by scanning electron microscopy (SEM), the surface roughness was determined by atomic force microscopy (AFM), the hydrated particle size and zeta potential were determined by nanoparticle size and zeta potential analyzer, and the protein composition of the protein corona was identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The proteins were classified in terms of biological processes, cellular components, and molecular functions to study the adsorption selection of nanoplastics to proteins, investigate the formation and characteristics of polystyrene nanoplastic-plant protein corona and predict the potential impact of protein corona on plants. The results showed that the morphological changes of the nanoplastics became clearer as the reaction time extends, as evidenced by the increase in size and roughness and the enhancement of stability, thus demonstrating the formation of protein corona. In addition, the transformation rate from soft to hard protein corona was basically the same for the three polystyrene nanoplastics in the formation of protein corona with leaf proteins under the same protein concentration conditions. Moreover, in the reaction with leaf proteins, the selective adsorption of the three nanoplastics to proteins with different isoelectric points and molecular weights differed, and the particle size and stability of the final formed protein corona also differed. Since a large portion of the protein fraction in protein corona is involved in photosynthesis, it is hypothesized that the formation of the protein corona may affect photosynthesis in I. hawkeri.
Polystyrenes/chemistry* ; Protein Corona/chemistry* ; Microplastics ; Plant Proteins ; Chromatography, Liquid ; Tandem Mass Spectrometry ; Nanoparticles/chemistry*

This research established a high-performance liquid chromatography(HPLC) method for simultaneous determination of isoorientin, orientin, vitexin, and isovitexin in Commelina communis to conduct content difference analysis and quality evaluation of 62 batches of C. communis from different origins. The HPLC content determination was performed on a Dikma Platisil ODS chromatographic column(4.6 mm×250 mm, 5 μm), with acetonitrile-0.1% formic acid(14∶86) as the mobile phase. The detection wavelength was set at 348 nm, the flow rate was 1.0 mL·min~(-1), and the column temperature was 35 ℃. The differences in origins and quality of 62 batches of C. communis were studied by chemometrics. The results showed that the determination of four components mani-fested a good linear relationship in the range of mass concentration(r>0.999 9), and the average recovery rate was 96.17%-103.0%. The relative standard deviations(RSDs) of precision, stability, and repeatability were all less than 2.0%. The content of four components from high to low was isoorientin>isovitexin>orientin>vitexin. Forty-seven batches of C. communis with clear origins were classified into six categories by chemometrics. C. communis from different origins had different qualities. Generally, C. communis from Western China, Central China, and South of China had superior qualities. The HPLC method established in this study is specific, simple, and efficient, which provides references for the comprehensive evaluation of the quality of C. communis. The chemometrics shows that the qualities of C. communis from different origins are largely different. Isoorientin can be used as an index to determine the content of C. communis, and its content limit should be set no less than 0.023%.
Commelina ; Chemometrics ; Drugs, Chinese Herbal/chemistry* ; China ; Chromatography, High Pressure Liquid/methods*