Antioxidant activity, cytotoxicity, phenolic content, and phytochemical composition of Pithecellobium Diversifolium benth: a pilot study / Atividade antioxidante, citotóxica, conteúdo fenólico e composição fitoquímica de Pithecellobium Diversifolium benth: um estudo piloto / Actividad antioxidante, citotóxica, contenido fenólico y composición fitoquímica de Pithecellobium Diversifolium benth: un estudio piloto

Species of Pithecellobium (Fabaceae) are used in traditional medicine to treat diabetes, cough, bronchitis, and inflammation. This study aims to evaluate the content and determine the antioxidant activity, phenolic compounds content, and cytotoxicity of the extract and the fractions of Pithecellobium diversifolium. This is unprecedented research with an exotic species from the Caatinga, northeastern Brazil, using High-performance Liquid Chromatography-Electrospray Ionization-Mass Spectrometry (HPLC-ESI-MS). The MeOH fractions of leaves and stem barks showed a high content of flavonoids (198.1 ± 106.50 and 542.7 ± 2.52 mg EqQ/g). The CH2Cl2 fraction of peels showed a high content of total phenolic compounds (516.7 ± 3.00 mg EqAG /g). The DPPH test showed that the CH2Cl2 fraction (leaves) held an EC50 of 0.08 ± 0.02, a higher value than that observed for the standards used in the test­Butylated hydroxyanisole (BHA), Butylated hydroxytoluene (BHT), and ascorbic acid. The AcOEt and MeOH fractions of peels presented moderate cytotoxicity with values below 500 µg/mL. The MeOH fraction of leaves showed seven major compounds: myricetin, quercetin, quercetin-arabinofuranoside, apigenin-triglycosides, and apigenin-diglucoside, being the last three unpublished in studies involving the genus. The tests conducted in this study show the potential of P. diversifolium as a promising source of biomolecules with therapeutic applicability.

Espécies de Pithecellobium (Fabaceae) são usadas na medicina tradicional para tratar diabetes, tosse, bronquite e inflamação. Este estudo teve como objetivo avaliar o teor e determinar a atividade antioxidante, o teor de compostos fenólicos e a citotoxicidade do extrato e das frações de Pithecellobium diversifolium, uma pesquisa inédita com uma espécie exótica da Caatinga do Nordeste do Brasil, utilizando a instrumentação Clae-IES. As frações MeOH das folhas e cascas do caule apresentaram alto teor de flavonoides (198,1 ± 106,50 e 542,7 ± 2,52 mg EqQ/g). A fração CH2Cl2 das cascas apresentou um elevado teor de compostos fenólicos totais (516,7 ± 3,00 mg EqAG/g). O teste DPPH mostrou que a fração CH2Cl2 (folhas) apresentou um EC50 de 0,08 ± 0,02, valor superior ao observado para os padrões utilizados no teste ­ Butil hidroxianisol (BHA), Butil hidroxitolueno (BHT) e ácido ascórbico. As frações AcOEt e MeOH das cascas apresentaram citotoxicidade moderada com valores inferiores a 500 µg/mL. A fração MeOH das folhas apresentou sete compostos majoritários: miricetina, quercetina, quercetina-arabinofuranosídeo, apigenina-triglicosídeos e apigenina-diglucosídeo, sendo os três últimos inéditos em estudos envolvendo o gênero. Os testes realizados demonstram o potencial de P. diversifolium, uma promissora fonte de biomoléculas com aplicabilidade terapêutica.

Las especies de Pithecellobium (Fabaceae) se utilizan en la medicina tradicional para tratar diabetes, tos, bronquitis e inflamación. Este estudio tuvo como objetivo evaluar el contenido y determinar la actividad antioxidante, el contenido de compuestos fenólicos y la citotoxicidad del extracto y de las fracciones de Pithecellobium diversifolium, un estudio inédito con una especie exótica de la Caatinga de la región Nordeste de Brasil, que utilizó la instrumentación HPLC-ESI. Las fracciones MeOH de hojas y cortezas de tallo mostraron un alto contenido de flavonoides (198,1 ± 106,50 y 542,7 ± 2,52 mg EqQ/g). La fracción CH2Cl2 de las cortezas presentó un alto contenido de compuestos fenólicos totales (516,7 ± 3,00 mg EqAG/g). El ensayo DPPH mostró que la fracción CH2Cl2 (hojas) tenía EC50 de 0,08 ± 0,02, valor superior a lo observado para los estándares utilizados en el ensayo ­Butilhidroxianisol (BHA), butilhidroxitolueno (BHT) y ácido ascórbico. Las fracciones AcOEt y MeOH de las cortezas presentaron una citotoxicidad moderada con valores inferiores a 500 µ g/mL. La fracción MeOH de las hojas contiene siete compuestos principales: miricetina, quercetina, quercetina-arabinofuranosido, apigenina-triglucósidos y apigenina-diglucósido, de los cuales los tres últimos son inéditos en estudios sobre el género. Las pruebas realizadas demuestran el potencial de P. diversifolium, una fuente prometedora de biomoléculas con aplicabilidad terapéutica.

A rapid and sensitive High-Performance Liquid Chromatography method with fluorescence detection for quantification of melatonin in small volume rat plasma samples: application to a preclinical study to determine the oral pharmacokinetics of melatonin under gestational conditions

Abstract A novel, simple and sensitive high-performance liquid chromatography with fluorescence detection method was developed and validated for the characterization of the preclinical pharmacokinetics of melatonin under pregnant conditions. Plasma samples (25 µL) were treated with 30 µL of ethanol absolute (containing the internal standard, IS). After a centrifugation process, aliquots of supernant (5 µL) were injected into the chromatographic system. Compounds were eluted on a Xbridge C18 (150 mm x 4.6 mm i.d., 5 µm particle size) maintained at 30°C. The mobile phase consisted in a mixture of aqueous solution of 0.4% phosphoric acid and acetonitrile (70:30 v/v). The wavelengths were set at 305 nm (excitation) and 408 nm (emission) and the total analysis time was 8 min/sample. All validation tests were obtained with accuracy and precision, according to FDA guidelines, over the concentration range of 0.005-20 µg/mL. Pharmacokinetic study showed that melatonin systemic exposure increased from day 14, with a significant difference at 19 days of gestation compared to the control group. Our findings suggest a decreased metabolism of melatonin as result of temporary physiological changes that occur throughout pregnancy. However, other maternal physiological changes cannot be ruled out.

Comparison of Quality Change During Processing Process of Achyranthis Bidentatae Radix from Different Origins Based on Color-component Correlation Analysis / 中国实验方剂学杂志

ObjectiveTaking Achyranthis Bidentatae Radix（ABR） from different origins as samples， to quantitatively analyze the chemical composition and chromaticity of ABR with different processing degrees， and clarify the correlation and change law between color and composition in the processing process of ABR， so as to provide reference for the quality evaluation of processed products of ABR. MethodThe colorimeter is used to measure the chromaticity values of three kinds of processing degrees of ABR in different origins to show the color value change trend during the processing process， and the color parameters of wine-processed and salt-processed products of ABR with different processing degrees were analyzed by principal component analysis（PCA）， orthogonal partial least squares-discriminant analysis（OPLS-DA） and other analysis methods. The contents of eight representative components of ABR were measured by high performance liquid chromatography（HPLC）， the correlation between chromaticity and each representative component was analyzed by Pearson correlation analysis， and the applicability of the selected eight representative components was further verified by Fisher linear discriminant analysis， and the wine-processed and salt-processed products of ABR with different processing degrees were grouped according to the degree of processing， and 48 samples of wine-processed and salt-processed products with different processing degrees were used as training samples. Taking the contents of 5-hydroxymethylfurfural， polypodine B， β-ecdysterone， 25R-inokosterone， 25S-inokosterone， ginsenoside Ro， chikusetsusaponin Ⅳa and polysaccharides as variables， the discriminant function was established respectively， and 12 samples of wine-processed and salt-processed products of ABR with different processing degrees were back-tested to verify the discriminant function and test the reliability of the function. ResultPCA and OPLS-DA results showed that ABR samples with different processing degrees were classified into clusters， and the results could significantly distinguish different processed products. During the process of wine and salt processing， the contents of 5-hydroxymethylfurfural， ginsenoside Ro， and chikusetsusaponin Ⅳa gradually increased with the deepening of the processing degree， while the contents of polypodine B， β-ecdysterone， 25R-inokosterone， 25S-inokosterone and polysaccharides showed a gradual decreasing trend， indicating these 8 components increased and decreased to different degrees in the process of wine and salt processing. The results of Pearson correlation analysis showed that the 5-hydroxymethylfurfural content of the samples with different processing degrees of wine-processed and salt-processed products were negatively correlated with the brightness value（L*） and the total color difference value（E*ab）（P<0.01）， and positively correlated with the red-green value（a*） and the yellow-blue value（b*）（P<0.01）， and that the content of polypodine B and polysaccharides were positively correlated with L* and E*ab（P<0.01）. The discriminant functions of wine-processed and salt-processed products of ABR were established by Fisher linear discriminant analysis， and their accuracy rates in the training samples were 93.75% and 95.83%， respectively. Twelve test samples of wine-processed and salt-processed products with different processing degree were back substitution， and the correct rate was 100%. ConclusionThe trend of composition and color changes of ABR with different processing degrees in different production areas is relatively consistent， and the color value can better distinguish ABR with different processing degrees， and the color of ABR is related to some representative components in the processing process， indicating that the color can provide reference for the identification of the processing degree of ABR and the prediction of component content.

Determination and Mechanism Prediction of Potential Active Ingredients in Erdongtang Based on UHPLC-QqQ-MS and Network Pharmacology / 中国实验方剂学杂志

ObjectiveTo establish an ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometry（UHPLC-QqQ-MS） for determination of the active ingredients in Erdongtang， and to predict the targets and pathways of anti-insulin resistance action of this formula. MethodThe analysis was performed on an ACQUITY UPLC BEH C18 column（2.1 mm×100 mm， 1.7 μm） with the mobile phase of 0.1% formic acid aqueous solution（A）-acetonitrile（B） for gradient elution（0-3 min， 90%-87%A； 3-6 min， 87%-86%A； 6-9 min， 86%-83%A； 9-11 min， 83%-75%A； 11-18 min， 75%-70%A； 18-19 min， 70%-52%A； 19-22 min， 52%A； 22-25 min， 52%-5%A； 25-27 min， 5%-90%A； 27-30 min， 90%A）. The contents of active ingredients in Erdongtang was detected by electrospray ionization（ESI） and multiple reaction monitoring（MRM） mode under positive and negative ion modes. On this basis， network pharmacology was applied to predict the targets and pathways of Erdongtang exerting anti-insulin resistance effect. ResultThe 20 active ingredients in Erdongtang showed good linear relationships within a certain mass concentration range， and the precision， stability， repeatability and recovery rate were good. The results of determination showed that the ingredients with high content in 15 batches of samples were baicalein（1 259.39-1 635.78 mg·L-1）， baicalin（1 078.37-1 411.52 mg·L-1）， the ingredients with medium content were mangiferin（148.59-217.04 mg·L-1）， timosaponin BⅡ（245.10-604.89 mg·L-1）， quercetin-3-O-glucuronide（89.30-423.26 mg·L-1）， rutin（46.91-1 553.61 mg·L-1）， glycyrrhizic acid（55.97-391.47 mg·L-1）， neomangiferin（37.45-127.03 mg·L-1）， nuciferine（0.89-63.48 mg·L-1）， hyperoside（6.96-136.78 mg·L-1）， liquiritin（30.89-122.78 mg·L-1）， liquiritigenin（26.64-110.67 mg·L-1）， protodioscin（58.57-284.26 mg·L-1）， the ingredients with low content were wogonin（7.16-20.74 mg·L-1）， pseudoprotodioscin（5.49-22.96 mg·L-1）， ginsenoside Rb1（7.31-23.87 mg·L-1）， ginsenoside Rg1（10.78-28.33 mg·L-1）， ginsenoside Re（7.78-24.76 mg·L-1）， ophiopogonin D（2.08-4.29 mg·L-1）， methylophiopogonanone A（0.74-1.67 mg·L-1）. The results of network pharmacology indicated that the mechanism of anti-insulin resistance exerted by Erdongtang might be related to the phosphatidylinositol 3-kinase/protein kinase B（PI3K/Akt） signaling pathway. ConclusionThe established UHPLC-QqQ-MS has the advantages of simple sample processing， strong exclusivity and high sensitivity， and can simultaneously determine the contents of the main ingredients from seven herbs in Erdongtang， which can lay the foundation for the development of Erdongtang compound preparations. The results of the network pharmacology can provide a reference for the mechanism study of Erdongtang in the treatment of type 2 diabetes mellitus.

Development and verification of a reversed phase high-performance liquid chromatography method for purity determination of recombinant Mycobacterium tuberculosis Ag85b protein stock solution / 中国生物制品学杂志

@#Objective To develop and verify a reversed phase high-performance liquid chromatography method for the determination of the purity of recombinant Mycobacterium tuberculosis(Mtb)Ag85b protein stock solution.Methods Fourfactor,three-level orthogonal test was designed,with the area,trailing factor,peak area and peak area RSD as the evaluation indexes to explore the optimal detection conditions. The methodology verification of specificity,linear range,precision and durability was conducted in accordance with the general principles of Chinese Pharmacopoeia(Volume Ⅳ,2020 edition)9101.Results The results of all the evaluation indexes were good when the elution ratio of organic phase was30% ～ 95%,the detection temperature was 35 ℃,the sample volume was 3 μg,and the elution time of 95% organic phase was 15 min. The method had the linear correlation coefficient(R2)of 0. 998 5,the linear range of 1. 8 ～ 4. 2 μg,the reproducibility RSD of 0. 01%,and the intermediate precision RSD of 0. 16%,with good durability under slight changes of column temperature and flow rate.Conclusion The reversed phase high-performance liquid chromatography method for the purity determination of recombinant Mtb Ag85b protein stock solution was developed,which has good specificity,precision and durability,and can be used for the quality control of recombinant Mtb Ag85b protein stock solution.

Development and verification of a high performance liquid chromatography method for determination of aluminium adjuvant content in vaccine / 中国生物制品学杂志

@#Objective To develop a high performance liquid chromatography(HPLC)method for determination of aluminium adjuvant content in vaccine,and verify and preliminarily apply the method.Methods The 8-hydroxyquinoline derivatization method was used for determination. The chromatographic column was phenyl-hexyl column[Luna 5u PhenylHexyl(250 mm × 4. 6 mm)],and the mobile phase was composed of ammonium acetate solution-acetonitrile(with 8-hydroxyquinoline)(60 ∶ 40)containing 20 mg/L ascorbic acid,while eluted at a flow rate of 1. 0 mL/min with the isocratic eluent. The excitation wavelength and the emission wavelength of the fluorescence detector were 380 nm and 520 nm respectively. The column temperature was 40 ℃,and the sample injection was 50 μL. The developed method was verified for the specificity,linear range,accuracy,repeatability,stability and durability,and used to determine the aluminum content in 12 batches of vaccines. The results were compared with those determined by titration in general principle 3106of Chinese Pharmacopoeia(VolumeⅢ,2020 edition).Results No interference peaks appeared in the sample chromatogram,and the non-aluminum adjuvant vaccine components and phosphate buffer had no interference with the determination. The linearity of aluminum standard was good in the concentration range of 6. 25 ～ 100 μg/mL,r = 0. 999 6. The average results of spike recoveries of aluminum content in inactivated hepatitis A vaccine,recombinant hepatitis B vaccine,adsorbed acellular DTP vaccine and inactivated enterovirus 71 vaccine were 98. 32%,100. 85%,101. 09% and 99. 31%,respectively in the verification for accuracy. The relative standard deviations(RSDs) of the determination results of aluminum content in the solution of six samples of the four vaccines in the same batch were 1. 09%,1. 42%,0. 97% and1. 30%,respectively. The RSDs of aluminum content of four vaccine samples stored at room tempe-rature for 0,2,4,6 and8 h were 0. 82%,0. 73%,0. 40% and 0. 48%,respectively. When the ratio of ammonium acetate solution to 8-hydroxyquinoline acetonitrile solution in mobile phase changed within 5%,the fluctuation range of aluminum content of four vaccines was less than 2%. There was no significant difference between the developed HPLC method and the titration method of Chinese Pharmacopoeia(VolumeⅢ,2020 edition)for determination of aluminum content in the 12 batches of vaccine samples.Conclusion A HPLC method for determination of aluminum adjuvant content in vaccines has been successfully established with good specificity,linearity,accuracy,repeatability,stability and durability,simple operation,high degree of automation and less interference of manual factors. It can realize the determination of aluminium content in single dose,which provides an effective means for the rapid and large-scale determination of aluminum content in vaccine products and monitoring the dispensing of semi-final products in the production process.

Research on Material Basis of Homologous and Heterogeneous Effect of Aurantii Fructus Immaturus and Aurantii Fructus Based on Total Statistical Moment Analysis and Molecular Connectivity Index / 中国实验方剂学杂志

ObjectiveTo investigate the material basis of homologous and heterogeneous effect of Aurantii Fructus Immaturus（AFI） and Aurantii Fructus（AF） based on the total statistical moment analysis and molecular connectivity index（MCI）. MethodRelevant literature at home and abroad and Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform（TCMSP） were consulted to establish the chemical composition database of AFI and AF， and set up their fingerprints by ultra-high performance liquid chromatography（UPLC）， and the total statistical moments and similarity parameters of the fingerprint were calculated. According to MCI， all components of AFI and AF were divided into different component groups， the average values of 0-8th order（0χ-8χ） MCI of the common component groups of AFI and AF were calculated. ResultThe values of total zero-order moment（AUCT） of AFI and AF were （10.57±2.45）×106， （5.09±0.89）×106 μV·s， the values of total first-order moment（MCRTT） were （11.57±1.58）， （12.10±1.29） min， the values of total second-order moments（VCRTT） were（24.49±2.30）， （26.49±2.54） min2， respectively. It showed that qualitative and quantitative parameters of AFI and AF were significantly different. The components with high similarity such as neohesperidin， hesperidin and narirutin were screened as the common potential pharmacodynamic components of AFI and AF. The non-common components of AFI， such as alysifolinone and imperatorin， and the non-common components of AF， such as neoeriocitrin and isosakuranin， with high similarity were screened out as potential heterogeneous components of AFI and AF. The composition groups of AFI and AF were classified into six categories， and the similarities between the composition groups of AFI and AF and the total constituents were 0.872-0.979 and 0.918-0.997， the average values of 0χ-8χ MCI of alkaloids in AFI and AF were 3.65 and 3.14， the average values of 0χ-8χ MCI of flavonoids were 8.47 and 8.47， the average values of 0χ-8χ MCI of volatile oils were 2.71 and 3.48， respectively. It showed that there were some differences in MCI of chemical constituents（groups） between AFI and AF. ConclusionThe chemical constituents（groups） of AFI and AF not only differ in content and species， but also in structural characteristics and structure-activity relationship， which can provide a basis for further explaining the scientific connotation of homologous and heterogeneous effect of AFI and AF.

In vivo Pharmacokinetic Analysis of Shenbai Nanosuspension in Rats Based on Overall Composition of Chinese Materia Medica / 中国实验方剂学杂志

ObjectiveTo clarify the scientific validity of in vivo pharmacokinetic determination of the whole drug composition in Shenbai nanosuspension in rats， and to provide methodological guidance and theoretical basis for the in vivo study of multi-component complex system of traditional Chinese medicine（TCM） preparations. MethodThe concentration of the overall components， mainly total saponins and total polysaccharides in Shenbai decoction and Shenbai nanosuspension， was determined in rat plasma at different times by area under the absorbance-wavelength curve method（AUAWC）， and the concentration of individual ginsenoside Rg1 was determined by high performance liquid chromatography（HPLC）， and the methodology was verified. The pharmacokinetic parameters of the whole component were compared with those of ginsenoside Rg1 to evaluate the in vivo operational characteristics of the two preparations. ResultThe methodological investigations of AUAWC and HPLC were in accordance with the requirements. AUAWC analysis showed that the overall components in both the decoction group and the nanosuspension group showed a one-compartment model， with half-life（t1/2） of 2.43 h and 2.04 h， respectively. The relative bioavailability of Shenbai nanosuspension was 138.99%. HPLC assay showed that ginsenoside Rg1 in the decoction group and the nanosuspension group showed a two-compartment model， with distribution half-life（t1/2α） of 0.13 h and 2.55 h， and elimination half-life（t1/2β） were 14.28 h and 3.85 h， respectively. The relative bioavailability of Shenbai nanosuspension was 127.49%. Compared with Shenbai decoction， the time to peak（tmax）， peak concentration（Cmax） and area under the drug-time curve（AUC） of the overall components and ginsenoside Rg1 in Shenbai nanosuspension were increased. ConclusionThe established AUAWC can be used for the pharmacokinetic study of the overall components of TCM preparations， which is complementary to the results of individual components measured by HPLC， and can provide useful reference for the in vivo study of new dosage forms of TCM.

Analysis on Quality of Sojae Semen Praeparatum Based on Traditional Quality Evaluation / 中国实验方剂学杂志

ObjectiveBased on the quality evaluation experience of "it is better to have a fragrant and strong aroma" summarized by materia medica of past dynasties， the chemical components of Sojae Semen Nigrum（SSN） and Sojae Semen Praeparatum（SSP） were systematically compared and analyzed， and the main fermentation products in different fermentation time were quantitatively analyzed， so as to clarify the transformation law of internal components in the processing process and provide scientific basis for the modern quality control of SSP. MethodUltra performance liquid chromatography-quadrupole tandem time-of-flight mass spectrometry（UPLC-Q-TOF-MS） was used for the structural identification of the chemical constituents of SSN and SSP， and with the aid of Progenesis QI v2.3 software， the negative ion mode was employed for principal component analysis（PCA） pattern recognition， and the data were analyzed with the aid of orthogonal partial least squares-discriminant analysis（OPLS-DA） for two-dimensional data to obtain S-plot， and components with |P|>0.1 were selected as the differential constituents. The contents of isoflavonoids in SSP during fermentation was determined by UPLC， and the samples were taken every 8 h in the pre-fermentation period and every 2 d in the post-fermentation period， and the dynamic changes of isoflavonoid contents in different fermentation stages were analyzed. The contents of amino acids and nucleosides in SSP and SSN from different fermentation stages were quantitatively analyzed by phenyl isothiocyanate（PITC） pre-column derivatization and high performance liquid chromatography（HPLC） gradient elution， and the contribution of flavor substances to the "delicious" taste of SSP was discussed by taste intensity value（TAV）. ResultA total of 19 kinds of differential components were screened out， mainly soybean saponins and isoflavones， and their contents decreased significantly or even disappeared after fermentation. In the pre-fermentation process of SSP， glycoside bond hydrolysis mainly occurred， and isoflavone glycosides in SSN were degraded and converted into the corresponding aglycones， the content of flavor substances such as amino acids increased gradually. In the post-fermentation process， protein degradation mainly occurred， after 8 d of post-fermentation， the content of isoflavones was basically stable， while the total content of amino acids increased by 8-40 times on average. Different amino acids form the special flavor of SSP， such as the TAV of glutamate is always ahead of other flavor substances， and sweet substances such as alanine and valine have made relatively great contributions to SSP. ConclusionBased on the law of constituent transformation， combined with the traditional evaluation index of "fragrant and strong"， it is difficult to control the fermentation degree of SSP by the existing standards in the 2020 edition of Chinese Pharmacopoeia. It is suggested that description of the characteristics of SSP be refined and changed to "fragrant， delicious and slightly sweet"， and at the same time， the post-fermentation index compounds such as glutamic acid， alanine and valine should be added as the quality control indicators of SSP， so as to standardize the production process and improve the quality of SSP.

STUDY ACCLIMATIZED SEED SAMPLE SAPOSHNIKOVIA DIVARICATA (TURCZ) SCHISCHK IN VIETNAM

In this study, we evaluated several agronomic characteristics of S. divaricata when grown in various regions in Vietnam, including Northeast, Northwest, and Central Highlands. Among the three regions, S. divaricata grown in Northwestern Vietnam has the highest yield reaching 6.21 tons/ha, with a total active ingredient content of 0.655% (Prim-O-glucosylcimifugin 0.383% and 5-O-methylvisamminoside 0.272%). This is followed by the Central Highlands of which S. divaricata yielded 4.12 tons/ha, with a total active ingredient content is 0.543% (Prim-O-glucosylcimifugin 0.292% and 5-O-methylvisamminoside 0.251%). The lowest yield of S. divaricata was recorded in Northeast with 3.13 tons/ha, of which a total active ingredient content was 0.394% with 0.253% for Prim-O-glucosylcimifugin and 0.141% for 5-O- methylvisamminoside. With the applied analytical conditions, HPLC - DAD chromatograms are obtained with sharp peaks of prim-O-glucosylcimifugin (tR = 7.51 min) and 5-O-methylvisamminoside (tR = 20.23 min) , balanced, clear on the background of the medicinal plants Saposhnikovia divaricata (Turcz.) Schischk. In particular, the applicable analytical conditions allow for simultaneous qualitative and quantitative analysis of both prim-O-glucosylcimifugin and 5- O-methylvisaminoside. The results of building the standard curve prim-O-glucosylcimifugin Y = (16146)X – 4020.3, R2 = 0.9992, standard curve 5-O- methylvisamminoside Y = (20490)X – 6921.8, R2 = 0.9999. The quantitative results of prim-O-glucosylcimifugin and 5-O- methylvisamminoside in all regions were slightly higher than that of the pharmacopoeias of Vietnam, China and Hong Kong with the total active ingredient content not less than 0.24%. Thus, The study concluded that Vietnam is a country that can develop medicinal plants Saposhnikovia divaricata (Turcz.) Schischk

Effect of megaloblastic anemia on hemoglobin A2 and diagnosis of ?-thalassemia trait

Context: ?-thalassemia trait is usually diagnosed by raised hemoglobin A2 (HbA2). The presence of megaloblastic anemia can cause an increase in HbA2 and create a diagnostic dilemma. Here, we have analyzed the effect of vitamin B12 and folic acid supplementation on HbA2 and diagnosis of ?-thalassemia trait in cases of megaloblastic anemia with raised HbA2. Materials and Methods: Cases of megaloblastic anemia with raised HbA2 on high-performance liquid chromatography (HPLC) were supplemented with vitamin B12 and folic acid. Post-treatment evaluation was done after 2 months. Cases showing adequate hematological response were subjected to statistical analysis. Based on post-treatment HbA2 value, the cases were diagnosed as normal, borderline raised HbA2, or ?-thalassemia trait. Pre- and post-treatment values of red cell parameters and HbA2 were analyzed. Results: There was a significant decrease in HbA2 value after vitamin B12 and folic acid supplementation. The diagnosis was changed in 70.97% of the cases after treatment. The chance of inconclusive diagnosis was decreased from more than 50% to less than 10%. Pre-treatment mean corpuscular volume (MCV) and HbA2% showed a significant difference between the thalassemic and normal groups. Conclusions: Megaloblastic anemia can lead to false-positive diagnosis of ?-thalassemia trait on HPLC. Repeat HPLC should be done after adequate supplementation of vitamin B12 and folic acid in cases of megaloblastic anemia with raised HbA2. Red cell parameters are not helpful to suspect ?-thalassemia trait in presence of megaloblastic anemia. However, HbA2% on HPLC can be a useful parameter to suspect or exclude ?-thalassemia trait in cases of megaloblastic anemia.

Comparative Analysis of the Elementary and Amino Acid Composition of Nepeta olgae Regel L. Plants Growing in Uzbekistan

This article for the first time presents the results of the study of qualitative and quantitative elemental and amino acid composition of the aboveground part of the plant Nepeta olgae Regel (L.) taken in the territory of Chust and Kosonsai districts (from the slopes of Gova and Kosonsai mountains) of Namangan region during the period before and during flowering (May-June, 2021-2022). The use of instrumental analysis of high-throughput energy dispersive X-ray fluorescence spectrometry, allowed to establish 20 mineral elements in the plant Nepeta olgae Regel (L.), among which to vital 9 elements and 3 to conditionally necessary. The amino acid composition of the plant Nepeta olgae Regel (L.) was studied by high performance liquid chromatography (HPLC) and 17 compounds were identified. Of these, 8 were substitutable and 9 essential amino acids.

Comparison of HPLC fingerprints and determination of main components of Viticis Fructus from different species / 中国中药杂志

In order to comprehensively evaluate the quality of Viticis Fructus, this study established HPLC fingerprints and evaluated the quality of 24 batches of Viticis Fructus samples from different species by similarity evaluation and multivariate statistical analysis(PCA, HCA, PLS-DA). On this basis, an HPLC method was established to compare the content differences of the main components, including casticin, agnuside, homoorientin, and p-hydroxybenzoic acid. The analysis was performed on the chromatographic column(Waters Symmetry C_(18)) with a gradient mobile phase of acetonitrile(A)-0.05% phosphoric acid solution(B) at the flow rate of 1 mL·min~(-1) and detection wavelength of 258 nm. The column temperature was 30 ℃ and the injection volume was 10 μL. The HPLC fingerprint of 24 batches of Viticis Fructus samples was established with 21 common peaks, and nine peaks were identified. Similarity analysis was carried out based on chromatographic data of 24 batches of chromatographic data of Viticis Fructus, and the results showed that except for DYMJ-16, the similarity of Vitex trifolia var. simplicifolia was ≥0.900, while that of V. trifolia was ≤0.864. In addition, the similarity analysis of two different species showed that the similarity of 16 batches of V. trifolia var. simplicifolia was 0.894-0.997 and that of the eight batches of V. trifolia was between 0.990 and 0.997. The results showed that the similarity of fingerprints of these two species was different, but the similarity between the same species was good. The results of the three multivariate statistical analyses were consistent, which could distinguish the two different species. The VIP analysis results of PLS-DA showed that casticin and agnuside contributed the most to the distinction. The content determination results showed that there was no significant difference in the content of homoorientin and p-hydroxybenzoic acid in Viticis Fructus from different species, but the content of casticin and agnuside was significantly different in different species(P<0.01). The content of casticin was higher in V. trifolia var. simplicifolia, while agnuside was higher in V. trifolia. The findings of this study show that there are differences in fingerprint similarity and component content of Viticis Fructus from different species, which can provide references for the in-depth study of the quality and clinical application of Viticis Fructus.

Site-directed mutagenesis enhances the activity of benzylidene acetone synthase of polyketide synthase from Polygonum cuspidatum / 生物工程学报

Polygonum cuspidatum polyketide synthase 1 (PcPKS1) has the catalytic activity of chalcone synthase (CHS) and benzylidene acetone synthase (BAS), which can catalyze the production of polyketides naringenin chalcone and benzylidene acetone, and then catalyze the synthesis of flavonoids or benzylidene acetone. In this study, three amino acid sites (Thr133, Ser134, Ser33) that may affect the function of PcPKS1 were identified by analyzing the sequences of PcPKS1, the BAS from Rheum palmatum and the CHS from Arabidopsis thaliana, as well as the conformation of the catalytic site of the enzyme. Molecular modification of PcPKS1 was carried out by site-directed mutagenesis, and two mutants were successfully obtained. The in vitro enzymatic reactions were carried out, and the differences in activity were detected by high performance liquid chromatography (HPLC). Finally, mutants T133LS134A and S339V with bifunctional activity were obtained. In addition to bifunctional activities of BAS and CHS, the modified PcPKS1 had much higher BAS activity than that of the wild type PcPKS1 under the conditions of pH 7.0 and pH 9.0, respectively. It provides a theoretical basis for future use of PcPKS1 in genetic engineering to regulate the biosynthesis of flavonoids and raspberry ketones.

Implementation and effect evaluation of virtual simulation teaching of high performance liquid chromatography / 中华医学教育探索杂志

Objective:To study the teaching effect of traditional teaching combined with virtual simulation teaching in pharmaceutical content analysis using high performance liquid chromatography (HPLC).Methods:The students in two parallel classes of grade 2016 were divided into experimental group (44 students) and control group (38 students). Taking the content analysis and its related analytical methodology of Metronidazole by HPLC as an example, the experimental group adopted the traditional offline teaching combines with virtual simulation teaching, and the control group adopted the traditional offline teaching. The teaching effects of the two groups were compared. SPSS 22.0 was used to conduct independent-samples t-test. Results:According to the mid-term test results, the experimental group was superior to the control group in practical operation assessment [(17.98±6.75) vs. (14.03±5.92)], and the difference was statistically significant ( P < 0.05). But there were no differences between the two groups in the scores of theory, discussion report and presentation. There was no significant difference in the final exam scores between the two groups. Conclusion:The ability of practical training and the teaching effect can be improved by the teaching method of traditional offline teaching combined with virtual simulation. Through the training of this project, students have mastered the operation of HPLC, the content determination of pharmaceutical preparations, the verification of analytical methods, data processing, and the writing of analytical reports, and have certain practical analysis ability.

Simultaneous determination of six aniline compounds in workplace air by high performance liquid chromatography / 中国职业医学

Objective To establish a high performance liquid chromatography (HPLC) method for simultaneous determination of six aniline compounds (ADs) in workplace air. Methods GDH-1 air sampling tube was used to collect six co-existing ADs such as aniline, o-toluidine, N-methylaniline, m-methylaniline, p-methylaniline and N,N-dimethylaniline in the vapor and aerosol of workplace air. The samples were desorbed and eluted using a methanol solution containing 1.00% ammonia water, followed by separation on a C18 chromatographic column and detection using a diode array detector. Results The quantification range of the method was 0.19 -253.50 mg/L, with the correlation coefficient of 0.999 9 for all six ADs. The minimum detection range was 0.02-0.06 mg/m3, and the minimum quantitation range was 0.04-0.19 mg/m3 [both calculated for a 15.0 L sample with a desorption (elution) solution volume of 3.00 mL]. The average desorption and elution efficiencies were 92.15%-104.41% (silica gel) and 94.29%-104.29% (filter membrane). The intra-assay relative standard deviation (RSD) ranged from 0.90%-9.72% (silica gel) and 0.57%-6.96% (filter membrane). The inter-assay RSD ranged from 2.03%-9.78% (silica gel) and 2.50%-8.62% (filter membrane). The samples were stable at room temperature for seven days. Conclusion This method can be used for the simultaneous determination of six ADs in workplace air.

Establishment and application of detection methods of dicamba in drinking water / 环境与职业医学

Background Dicamba is widely used in agricultural production in China, but it is extremely soluble in water and can be harmful to human health when it enters the body via water drinking. It is necessary to establish an accurate, sensitive, and rapid detection method to determine the residues of dicamba in domestic drinking water. Objective To establish two methods for the determination of dicamba residues in drinking water by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) and gas chromatography-tandem mass spectrometry (GC-MS/MS) respectively. Methods The conditions of the proposed method using HPLC-MS/MS included CAPCELL PAK ST chromatographic column, ammonium formate water solution and methanol as the mobile phase, and isocratic elution. The system was operated under multiple reaction monitoring mode and electrospray negative ionization mode. Trimethylsilylated diazomethane was used as a derivatizing agent for GC-MS/MS, and an external standard curve was used to evaluate the system. The residues of dicamba in seven water samples of tap water or secondary water supply from six regions in Chengdu were detected by the established systems to evaluate their applicability and to understand the status quo of dicamba residues in drinking water. Results For the HPLC-MS/MS, the linear range of dicamba was 1.00-100 μg·L−1, the regression equation was \begin{document}$\hat Y $\end{document}=1250.9X+2681.5, the correlation coefficient was 0.9988, the relative standard deviations were 1.23%-26.3%, the limit of detection was 0.95 μg·L−1, and the spiked recoveries were 91.8%-111%. For the GC-MS/MS, the linear range of dicamba was 0.200-10.0 μg·L−1, the regression equation was \begin{document}$\hat Y $\end{document}=190597X+40911, the correlation coefficient was 0.9993, the relative standard deviations were 0.64%-3.90%, the limit of detection was 0.18 μg·L−1, and the spiked recoveries were 97.3%-105%. No dicamba residue was identified in the seven water samples of tap water or secondary water supply from six regions in Chengdu by the proposed methods. Conclusion The two detection methods established in this study are sensitive and rapid, meet the requirements from the detection of dicamba residues in drinking water, and provide an experimental basis for subsequent research on the detection of dicamba residues. In the future, it is necessary to continue to pay attention to the pollution of dicamba in drinking water in Chengdu.

Establishment and clinical application of ibrutinib blood concentration assay / 中国药房

OBJECTIVE To establish a method to detect the blood concentration of ibrutinib and apply it to the clinic. METHODS Using zanubrutinib as internal standard， the concentration of ibrutinib was detected by high performance liquid chromatography （HPLC） after plasma samples were processed by solid-phase extraction. The separation was performed on an Agilent 5 TC-C18（2） column with acetonitrile-0.5% potassium dihydrogen phosphate solution （43∶57， V/V） as the mobile phase at a flow rate of 1 mL/min， a detection wavelength of 260 nm， a column temperature of 40 ℃ ， a sample size of 20 μL， and a run time of 25 min. The concentration of ibrutinib was measured in the plasma of 9 patients with non-Hodgkin’s lymphoma 2 h after drug administration on the 30th day by the above method. RESULTS The linear range of the assayed mass concentration of ibrutinib was 10-500 ng/mL （R 2＝0.998 9）， the lower limit of quantification was 10 ng/mL， and the RSDs of the intra-batch and inter-batch precision tests were not higher than 12.77%. The recoveries of the extraction were 74.80% and 97.70%， with both RSDs＜2.90%， and the RSDs of the stability tests were not higher than 7.10%. The peak plasma concentrations of 9 patients were 15.341-279.628 ng/mL. CONCLUSIONS The established HPLC method is simple and rapid， and can be used for the determination of ibrutinib concentration in plasma samples.

Comparative Analysis of Serum Pharmacochemistry of Alismatis Rhizoma Before and After Salt Processing Based on UPLC-Q-TOF-MS / 中国实验方剂学杂志

ObjectiveBased on serum pharmacochemistry and ultra performance liquid chromatography-quadrupole-time-of-flight mass spectrometry（UPLC-Q-TOF-MS） the transitional components in the serum of rats after intragastric administration of water extract of Alismatis Rhizoma（AR）and salt-processed Alismatis Rhizoma（SAR） were compared. MethodSD rats were randomly divided into blank group, AR group（10 g·kg-1） and SAR group（10 g·kg-1）, 3 rats in each group, the administration groups were given AR and SAR aqueous extracts by gavage, respectively, and the blank group was given an equal volume of drinking water by gavage once in the morning and once in the evening, for 3 consecutive days. Sixty minutes after the last administration, blood was collected from the eye orbits, and the serum samples were prepared. The serum samples were prepared on an ACQUITY UPLC BEH C18 column（2.1 mm×50 mm, 1.7 μm） with the mobile phase of acetonitrile（A）-0.1% formic acid aqueous solution（B） in a gradient elution（0-10 min, 10%-50% A; 10-27 min, 50%-95%A; 27-27.1 min, 95%-10% A; 27.1-30 min, 10%A）, the data were collected at a flow rate of 0.3 mL·min-1 in positive ion mode with a scanning range of m/z 100-1 200. Based on the self-constructed chemical composition library of AR, the total ion flow diagrams and secondary MS fragmentation information of the aqueous extracts of AR and SAR, as well as the administered serum and the blank serum, were compared with each other by UNIFI 1.9.2, so as to deduce the possible blood-migrating constituents and their cleavage patterns in the aqueous extracts, and the response intensity ratios of each chemical component were calculated before and after processing. ResultA total of 20 components, including 5 prototypical components and 15 metabolites, were analyzed and deduced from the serum of rats given aqueous extract of AR. And 14 components, including 5 prototypical components and 9 metabolites, were analyzed and deduced from the serum of rats given aqueous extract of SAR. Of these, 13 components were common to both of them, including 5 prototypical components and 8 metabolites. The 5 prototypical components were 16-oxoalisol A, alisol A 24-acetate, alisol A, alisol B and alisol C. The metabolites were mainly involved in phase Ⅰ metabolism（oxidation） and phase Ⅱ metabolism（glucuronidation）. There was a big change in the intensity of response of the common components before and after salt-processing, and the response intensities of the prototypical components, 16-oxoalisol A, alisol B and alisol C, were elevated, while the type and response intensity of metabolites were generally decreased, and it was hypothesized that the metabolic rate of terpenoids might be slowed down after salt-processing of AR, so that the blood-migrating constituents could participate in the metabolism of the body more in the form of prototypes. ConclusionSalt-processing of AR may promote the absorption of prototypical components into the blood by slowing down the metabolic rate of terpenoids, which can provide support for the research on material basis of AR and SAR.

Quality Evaluation of Aurantii Fructus from Sichuan by Specific Chromatogram Combined with Chemometrics and Grey Correlation Analysis / 中国实验方剂学杂志

ObjectiveHigh performance liquid chromatography （HPLC） was used to establish the specific chromatograms of Aurantii Fructus from different origins， and the quality variability of Aurantii Fructus from Sichuan was analyzed and evaluated by combining entropy weighting method and grey correlation method. MethodHPLC was performed on an Agilent Eclipse Plus C18 column （4.6 mm×250 mm， 5 μm） with a gradient elution of methanol （A）-0.1% phosphoric acid aqueous solution as the mobile phase （0-12 min， 25%-33%A； 12-21 min， 33%-41%A； 21-30 min， 41%-42%A； 30-40 min， 42%-59%A； 40-53 min， 59%-72%A； 53-60 min， 72%A； 60-65 min， 72%-100%A； 65-70 min， 100%A； 70~71 min， 100%-25%A； 71-80 min， 25% A） at a flow rate of 1.0 mL·min-1， the injection volume was 10 μL and the detection wavelength was 330 nm. Fifty batches of Aurantii Fructus samples from different origins （Sichuan， Chongqing， Jiangxi and Hunan） were tested， and the similarity evaluation software is used to generate characteristic profiles and compare them with control profile for peak identification， and then to evaluate the similarity of the samples. IBM SPSS 19.0 and SIMCA 14.1 were used to perform multivariate statistical analysis on the results of the samples， and then the entropy weighting method and grey correlation were used to calculate the overall quality score of samples from Sichuan. ResultHPLC specific chromatogram of Aurantii Fructus was established， and 14 common peaks were identified as eriocitrin， neoeriocitrin， narirutin， naringin， hesperidin， neohesperidin， meranzin hydrate， poncirin， meranzin， marmin， nobiletin， 3，3′，4′，5，6，7，8-heptamethoxyflavone， tangeretin and auraptene. And the similarities between the samples from Sichuan and the control chromatogram were all above 0.980. The samples could be classified into four categories according to their main origins by chemical pattern recognition， and the results of cluster analysis， principal component analysis and orthogonal partial least squares-discriminant analysis were all able to discriminate the samples of different main origins effectively. The comprehensive evaluation results of entropy weighting method combined with grey correlation showed that the quality of Aurantii Fructus from Sichuan varied greatly among different origins， and the quality of Aurantii Fructus from Sichuan was ranked as Bazhong>Luzhou>Chongqing>Neijiang. ConclusionIn this study， the characteristic mapping of Aurantii Fructus from Sichuan is established， and combined with the analytical methods of chemometrics and grey correlation， the quality of samples from different origins can be effectively differentiated， which can provide a reference for the comprehensive evaluation and control of the quality of Aurantii Fructus from Sichuan.