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ObjectiveBased on response surface methodology combined with principal component analysis（PCA）， the optimal decocting process of Moringa oleifera leaf standard decoction was optimized， and its multi-index quality evaluation system was established， in order to provide scientific basis for the quality control of this standard decoction. MethodResponse surface methodology and PCA were used to optimize the decoction process by taking the relative peak areas of 8 characteristic peaks and dry extract yield as indexes. Based on this， the quality of 15 batches of the standard decoction was evaluated by high performance liquid chromatography（HPLC） characteristic chromatogram， determination of major components（neochlorogenic acid， L-tryptophan， cryptochlorogenic acid， vicenin-2， isoquercetin， astragalin）， determination of active parts（total flavonoids， total organic acids， total polysaccharides， total α-amino acids， total sinapine）， dry extract yield， specific gravity and pH. ResultThe optimal decocting process was to soak M. oleifera leaves（100.00 g） for 30 min and decoct twice with the first decoction of 12 times the amount of water for 30 min and the second decoction of 10 times the amount of water for 20 min. Standard decoction containing 0.2 g·mL-1 of crude drug was defined by x¯±30%， the specific gravity was 0.722-1.340， pH was 3.86-7.16， dry extract yield was 23.1%-42.9%， and the alcohol-soluble extract content was 8.26%-15.34%. Calculated according to the dried products of the standard decoction， the contents of neochlorogenic acid， L-tryptophan， cryptochlorogenic acid， vicenin-2， isoquercetin and astragalin were 1.99-3.69， 1.20-2.22， 1.44-2.67， 0.53-0.99， 2.45-4.55， 1.22-2.26 mg·g-1， the relative transfer rates relative to the herbs were 34.37%-63.83%， 62.43%-115.94%， 64.65%-120.06%， 56.98%-105.82%， 37.46%-69.57%， 41.81%-77.64%， respectively. The contents of total flavonoids， total organic acids， total polysaccharides， total α-amino acids， total sinapine were 10.19-18.92， 11.82-21.96， 94.07-174.71， 42.69-79.27， 9.55-17.73 mg·g-1， the relative transfer rates for herbs were 25.72%-47.77%， 41.78%-77.59%， 64.90%-120.54%， 42.30%-78.57%， 34.99%-64.99%， respectively. ConclusionThe optimized decocting technology of M. oleifera leaf standard decoction is stable and feasible， and the established multi-indicator quality evaluation system can lay the foundation for the quality control of this standard decoction.
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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.
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@#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.
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OBJECTIVE To establish the characteristic chromatogram of Chaenomeles sinensis， determine the contents of rutin， hyperin and quercitrin， and to identify C. sinensis and C. speciosa. METHODS HPLC method was performed on Agilent 5 TC-C18 column， with acetonitrile-0.2% formic acid solution as the mobile phase for gradient elution， at the flow rate of 1.0 mL/min. The column temperature was 30 ℃ . The detection wavelength was 330 nm in characteristic chromatogram and 350 nm in content determination. The characteristic chromatogram of C. sinensis was established and similarity was evaluated by the Similarity Evaluation System for Chromatographic Fingerprint of TCM （2012 edition）. Hierarchical cluster analysis of 15 batches of C. sinensis （S1-S15） was performed by using SPSS 23.0 software. The contents of 3 flavones in 15 batches of C. sinensis and 7 batches of C. speciosa （S16-S22） were determined， while their characteristic chromatograms were compared. RESULTS The similarities of the characteristic chromatogram for 15 batches of C. sinensis ranged from 0.783 to 0.969， and 11 characteristic peaks were confirmed. Four constituents were identified as chlorogenic acid， rutin， hyperin and quercitrin. The medicinal materials in 15 batches of C. sinensis could be divided into 2 categories： S5-S8 were one category， and the others belonged to one category. The characteristic chromatogram of C. sinensis was obviously different from C. speciosa. The contents of rutin， hyperin and quercitrin in 15 batches of C. sinensis were 48.99-294.45， 3.49-102.55， 31.98-149.49 μg/g， respectively. The content of rutin in C. speciosa was lower than that in C. sinensis. None of hyperin （except for S20） and quercitrin were detected in C. speciosa. CONCLUSIONS The characteristic chromatogram and the method for content determination of 3 flavones in C. sinensis are established successfully and can be used for the quality control of C. sinensis and its identification from C. speciosa.
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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.
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Qualitative analysis of the ingredients absorbed into blood and their metabolites of Xihuang pill (XHP) were conducted using high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (HPLC-Q-TOF-MS/MS) technology. Network pharmacology was used to explore the potential anticancer mechanisms of the ingredients against glioma, and their specific mechanisms were validated through molecular docking and experimental verification. SD rats were intragastrically administered with XHP, and rat serum samples were collected. Ingredients absorbed into blood and their metabolites were identified based on the retention time of chromatographic peaks, accurate molecular mass, characteristic fragment ions, and comparisons with reference substances and literature data. PharmMapper and SwissTarget Prediction databases were used to obtain the targets of the XHP-medicated serum, while GeneCards, OMIM, PharmGKB, TTD, and DrugBank databases were used to obtain glioma disease targets. The "component-target" network relationship diagram was constructed using Cytoscape 3.9.1 software. The protein-protein interaction (PPI) network diagram was constructed using the STRING database, and the targets were analyzed using GO and KEGG analyses. Molecular docking was used to verify the binding ability of core targets with their corresponding compounds in XHP-medicated serum. The potential mechanism of the anti-glioma effect of 11-keto-β-boswellic acid (KBA), a representative component of XHP-medicated serum, was verified using CCK-8 and Western blot assays. A total of 40 compounds were identified in the XHP-medicated serum, including 28 prototype components and 12 metabolites. The network pharmacology results showed that elemonic acid, 3-acetyl-β-boswellic acid, KBA, α-boswellic acid, and other 5 compounds might be the active ingredients of XHP-medicated serum in the treatment of glioma. Glutathione reductase (GSR), glucose-6-phosphate dehydrogenase (G6PD), ATP-citrate lyase (ACLY), aldo-keto reductase family 1 member B1 (AKR1B1) and glutaredoxin (GLRX) were identified as key targets, involving pathways such as glutathione metabolism and the pentose phosphate pathway. Further cell experiments showed that KBA significantly inhibited the proliferation of T98G cells with an IC50 of 30.96 μmol·L-1, and KBA (30 μmol·L-1) significantly downregulated the protein expression levels of GSR in T98G cells. In summary, XHP-medicated serum may exert its anti-glioma effect by regulating GSR and G6PD-targeted pathways involved in glutathione metabolism. These results provide valuable evidence for further investigating the mechanism of XHP in treating glioma. The animal welfare and experimental procedures were approved by the Ethical Committee of Laboratory Animals at Nanjing University of Chinese Medicine (approval No. ACU221001).
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AIM: To establish a method for quantitation of cefepime and avibactam in M-H broth, and applicated in the in vitro dynamic PK/PD model. METHODS: The cefepime was also determined using the high-performance liquid chromatography method (HPLC), the avibactam was also determined using the liquid chromatography-mass spectrometry (LC-MS/MS), an in vitro dynamic PK/PD model was established to study the PK/PD relationship of cefepime/avibactam against carbapenem resistant Klebsiella pneumoniae (CRKP). RESULTS: The linear ranges of cefepime and avibactam were good at (0.5-20) and (0.1-25) μg/mL (r=0.999), and the lower limit concentrations were 0.5 and 0.1 μg/mL. The extraction recoveries of cefepime and avibactam in M-H broth were 88.0%-101.7% and 90.9%-95.2%, the relative standard deviation of intra-day precision and inter-day precision were less than 5.2%. The concentration-time curves were well simulated by the PK/PD model. All observed concentrations in each experiment were in the range of 20% of the targeted values. For the CRKP of MIC=8 μg/mL and MIC=16 μg/mL, the colony decreased to 2.783Log10 CFU/mL and 1.325Log10 CFU/mL at the cefepime/avibactam 2.5 g q8 h administration after 24 h. CONCLUSION: The determination method of cefepime and avibactam in broth established in this study has high sensitivity and good stability. For the CRKP with MIC≤8 μg/mL,cefepime/avibactam showed that good anti-CRKP activity under routine administration in vitro dynamic PK/PD model.
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Aim To explore the possible mechanism of "component-target-pathway" of Radix Hedysari against target organ damage caused by radiotherapy and chemotherapy, and to verify the " dose-effect" relationship of the main active components. Methods TCMSP, Uniprot, Swiss Target Prediction, GeneCards, Cytoscape, Omicshare and other platforms were used for network pharmacology analysis. Autodock, Pymol and Ligplot were used for molecular docking. The water extract of Radix Hedysari was used for animal experiment verification. The contents of eight main components were determined by HPLC. Results Four active components, eight key targets and four key pathways of Radix Hedysari were identified to resist the damage of target organs caused by radiotherapy and chemotherapy. Molecular docking showed that formononetin and quercetin had good binding activity with HSP90AA1, naringenin and MAPK3, and ursolic acid and TP53. Animal experiments showed that gastrointestinal factors MTL and VIP increased significantly, liver and kidney factors Cr, BUN, AST and ALT decreased significantly, inflammatory factor IL-10 increased significantly and TNF-a decreased significantly. The content of ononm was the highest (2 . 884 8 µg • g "
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OBJECTIVE To establish an HPLC fingerprint of Xiao’er resuqing oral liquid， and to determine the contents of twelve index components. METHODS HPLC method was adopted. The determination was performed on Venusil MP C18 column with mobile phase consisting of acetonitrile-0.1% phosphate aqueous solution （gradient elution） at a flow rate of 1.0 mL/min. The detection wavelength was set at 210 nm， the column temperature was 30 ℃， the injection volume was 10 μL. HPLC fingerprint of Xiao’er resuqing oral liquid was established by using the Similarity Evaluation System of Chromatographic Fingerprint of TCM （2012 edition） to evaluate the similarity. The contents of 12 components were determined， including （R， S）-goitrin， 3，5-O-dicaffeoyl quinic acid， puerarin， forsythin， forsythoside A， chlorogenic acid， baicalin， saikosaponins d， wogonoside， baicalein， emodin and chrysophanol. RESULTS The similarity of HPLC fingerprints of 13 batches of Xiao’er resuqing oral liquid was greater than 0.97， and 14 common peaks were confirmed. The contents of the above 12 index components in 13 batches of Xiao’er resuqing oral liquid were as follows： 0.078-0.172， 1.564-2.736， 1.338-2.578， 0.426-0.872， 1.477-2.628， 1.396-2.447， 4.052-9.146， 0.367- 0.692， 1.974-4.674， 1.274-2.969， 0.085-0.167 and 0.155-0.307 mg/mL. CONCLUSIONS The established HPLC fingerprint and content determination methods have high accuracy and high specificity， which can be used for the quality evaluation of Xiao’er resuqing oral liquid.
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@#Objective To develop and verify a cation exchange high performance liquid chromatography(CEX-HPLC)method for the detection of charge variants of pembrolizumab.Methods Pembrolizumab was bound to the exchange column matrix by using MabPac SCX-10 column,and the variants with different charges were eluted by gradually increasing the salt concentration of the mobile phase.The specificity,precision,linear range,accuracy and durability of the method were verified,and the charge variants of three batches of pembrolizumab finished products were detected by using the developed method.Results The resolution of the last acidic isomer peak and the first basic isomer peak of pembrolizumab from the main peak were 1.28 and 1.42,respectively.The mobile phase A and preparation buffer had no obvious interference peaks at the peak of the sample;The RSD values of the precision verification were all less than 2.0%;The total peak area,main peak area,acidic isomer peak area and basic isomer peak area of the standard all exhibited good linear relationship with the theoretical dilution concentration with each R~2 of 1.00;The recovery rates of the total peak area and main peak area of the standard at three concentrations were between 96.81% and 106.07%;When pH value of the mobile phase was within the range of 6.30±0.10,the RSD values of the total peak area and main peak area percentage of the standard were1.5% and 1.9%,and when the column temperature was within the range of(35±4) ℃,the RSD values of the total peak area and main peak area percentage of the standard were 0.4% and 0.3%,respectively.The RSD value of the main peak retention time of the three batches of finished products was 0.Conclusion The developed CEX-HPLC method can effectively separate the acidic isomers,main peaks and basic isomers of pembrolizumab with good specificity,precision and accuracy,which can be used for the follow-up research and development of pembrolizumab,the process verification of expanding production and the stability research.
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ObjectiveTo improve the quality standard of Yuanhu Zhitong oral liquid in order to strengthen the quality control of this oral liquid. MethodThin layer chromatography（TLC） was used for the qualitative identification of Corydalis Rhizoma and Angelicae Dahuricae Radix in Yuanhu Zhitong oral liquid by taking tetrahydropalmatine， corydaline reference substances and Corydalis Rhizoma reference medicinal materials as reference， and cyclohexane-trichloromethane-methanol（5∶3∶0.5） as developing solvent， Corydalis Rhizoma was identified using GF254 glass thin layer plate under ultraviolet light（365 nm）. And taking petroleum ether（60-90 ℃） -ether-formic acid（10∶10∶1） as developing solvent， Angelicae Dahuricae Radix was identified using a silica gel G TLC plate under ultraviolet light（305 nm）. High performance liquid chromatography（HPLC） was performed on a Waters XSelect HSS T3 column（4.6 mm×250 mm， 5 μm） with acetonitrile（A）-0.1% glacial acetic acid solution（adjusted pH to 6.1 by triethylamine）（B） as the mobile phase for gradient elution（0-10 min， 20%-30%A； 10-25 min， 30%-40%A； 25-40 min， 40%-50%A； 40-60 min， 50%-60%A）， the detection wavelength was set at 280 nm， then the fingerprint of Yuanhu Zhitong oral liquid was established， and the contents of tetrahydropalmatine and corydaline were determined. ResultIn the thin layer chromatograms， the corresponding spots of Yuanhu Zhitong oral liquid， the reference substances and reference medicinal materials were clear， with good separation and strong specificity. A total of 12 common peaks were identified in 10 batches of Yuanhu Zhitong oral liquid samples， and the peaks of berberine hydrochloride， dehydrocorydaline， glaucine， tetrahydropalmatine and corydaline. The similarities between the 10 batches of samples and the control fingerprint were all >0.90. The results of determination showed that the concentrations of corydaline and tetrahydropalmatine had good linearity with paek area in the range of 0.038 6-0.193 0， 0.034 0-0.170 0 g·L-1， respectively. The methodological investigation was qualified， and the contents of corydaline and tetrahydropalmatine in 10 batches of Yuanhu Zhitong oral liquid samples were 0.077 5-0.142 9、0.126 1-0.178 2 g·L-1， respectively. ConclusionThe established TLC， fingerprint and determination are simple， specific and reproducible， which can be used to improve the quality control standard of Yuanhu Zhitong oral liquid.
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Objective To establish a method for the simultaneous determination of DOX·HCl and LND. Methods HPLC was performed on Agilent 5 HC-C18（2） （4.6 mm × 250 mm, 5 µm） column. The mobile phase was methanol-0.1% TFA aqueous solution, and the gradient elution procedure were: 0 to 3 min, 65% methanol; 3 to 7 min, 65%→90% methanol; 7 to 13 min, 90% methanol; 13 to 15 min, 90%→65% methanol; 15 to 20 min, 65% methanol. The collection time was 20 min, the balance time was 3 min, the UV detection wavelengths were 205 nm and 253 nm. The flow rate was 1.0 ml/min and the column temperature was 35℃. The amount of inlet was 10 µl. Results The method was highly specific, and both DOX·HCl and LND exhibited good linearity in the concentration range of 1-40 µg/ml and 6-240 µg/ml, respectively. The two compounds’ precision, stability, and recovery satisfied the requirements of the method. Conclusion This study established a HPLC method that was suitable for the simultaneous detection of DOX·HCl and LND. This method’s high level of specificity, accuracy, and reliability .
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OBJECTIVE@#Based on metabonomics technology of high-performance liquid chromatography-mass spectrometry (HPLC-MS/MS) and hydrogen nuclear magnetic resonance spectroscopy (1H NMR), the pharmacokinetic characteristics and therapeutic mechanism of Rhei Radix et Rhizoma (RhRR, Dahuang in Chinese), Eupolyphaga Steleophaga (EuS, Tubiechong in Chinese) combined with RhRR acting on acute liver injury were explored.@*METHODS@#Models of acute liver injury were established, and the pharmacokinetic methods of five components of RhRR-EuS in rats were found by HPLC-MS/MS. The liver tissues of different groups of mice were analyzed by 1H NMR spectroscopy combined with multivariate statistical analysis to investigate the metabolomics of RhRR-EuS and RhRR.@*RESULTS@#Pharmacokinetic results showed there were different levels of bimodal phenomenon in different groups, and the absorption of free anthraquinone in RhRR increased after compatibility with EuS. In addition, the pathological state of acute liver injury in rats can selectively promote the absorption of emodin, chrysophanol, physcion and aloe emodin. Through 15 differential metabolites in the liver tissue of acute liver injury mice, it was revealed that RhRR-EuS and RhRR could protect the liver injury by regulating the metabolism of glutamine and glutamic acid, alanine, aspartic acid and glutamic acid, and phosphoinositide. However, the regulation of RhRR was weaker than that of RhRR-EuS.@*CONCLUSION@#For the first time, we studied the pharmacokinetics and metabolomics differences of RhRR-EuS and RhRR in rats and mice with acute liver injury, in order to provide theoretical reference for clinical treatment of liver disease by DHZCP.
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Abstract Lactobacilli are probiotics with Aflatoxin (AF) detoxification ability, found in fermented products, GIT of animals and environment. Purpose of this study was to investigate the ability of broiler isolates of Lactobacillus against Aflatoxin B1 (AFB1). For this purpose, 5 isolates of Lactobacillus from broiler gut were incubated with 100 ppb AFB1 in aqueous environment and effect of different parameters (cell fractions, time, temperature, pH) on detoxification was determined by HPLC. The ameliorative effect of Lactobacillus salivarius (LS) against AFB1 was studied in broiler. The results revealed that LS (CR. 4) showed the best results (in vitro) as compared to other isolates (L. salivarius (CR. 3, CR, 4), L. agilis (CE. 2.1, CE. 3.1) and L. crispatus (CE. 28). Cell debris of CR. 4 showed significantly higher detoxification (P 0.05). Maximum amount of AFB1 was detoxified at 30°C (97%), pH 4.0 (99%) and 6 h (99.97%). In vivo study showed that AFB1 decreased weight gain (1,269 ± 0.04 gm/ bird), feed consumed (2,161 ± 0.08 gm/ bird), serum total protein (2.42 ± 0.34 gm/ dl), serum albumin (0.5 ± 0.2 2 gm/dl) and antibody titer (4.2 ± 0.83). Liver function enzymes were found (alanine transaminase (ALT): 32 ± 10.7 U/L) and aspartate transaminase (AST): 314.8 ± 27 U/L) elevated in AFB1 fed broilers. Treatment with 1% LS not only decreased the toxic effects of AFB1 (group D) but also improved the overall health of broilers due to its probiotic effects (p 0.05) as compared to control negative (group A). The detoxification ability of LS was better than commercial binder (CB) (0.2% Protmyc). It was concluded that detoxification of AFB1 by Lactobacillus was strain, temperature, pH and time dependent. LS has detoxification ability against AFB1 in vivo.
Resumo Os lactobacilos são probióticos com capacidade de desintoxicação da Aflatoxina (AF), encontrados em produtos fermentados, TGI de animais e meio ambiente. O objetivo deste estudo foi investigar a capacidade de isolados de frango de corte de Lactobacillus contra a Aflatoxina B1 (AFB1). Para tanto, 5 isolados de Lactobacillus de intestino de frango foram incubados com 100 ppb AFB1 em meio aquoso, e o efeito de diferentes parâmetros (frações celulares, tempo, temperatura, pH) na desintoxicação foi determinado por CLAE. O efeito melhorador de Lactobacillus salivarius (LS) contra AFB1 foi estudado em frangos de corte. Os resultados revelaram que LS (CR. 4) apresentou os melhores resultados (in vitro) em comparação com outros isolados [L. salivarius (CR. 3, CR. 4), L. agilis (CE. 2.1, CE. 3.1) e L. crispatus (CE. 28)]. Detritos celulares de CR. 4 mostraram desintoxicação significativamente maior (P 0.05). A quantidade máxima de AFB1 foi desintoxicada a 30 °C (97%), pH 4.0 (99%) e 6 h (99,97%). O estudo in vivo mostrou que AFB1 diminuiu o ganho de peso (1,269 ± 0.04 g / ave), alimento consumido (2,161 ± 0.08 g / ave), proteína total sérica (2.42 ± 0.34 g / dl), albumina sérica (0.5 ± 0.22 gm / dl) e título de anticorpo (4.2 ± 0.83). As enzimas da função hepática foram encontradas (alanina transaminase (ALT): 32 ± 10.7 U / L) e aspartato transaminase (AST): 314.8 ± 27 U / L) elevadas em AFB1 alimentados com frangos. O tratamento com 1% LS não só diminuiu os efeitos tóxicos de AFB1 (grupo D), mas também melhorou a saúde geral dos frangos devido aos seus efeitos probióticos (p 0.05) em comparação com o controle negativo (grupo A). A capacidade de desintoxicação do LS foi melhor do que o aglutinante comercial (CB) (0.2% Protmyc). Concluiu-se que a desintoxicação de AFB1 por Lactobacillus foi dependente da cepa, temperatura, pH e tempo. LS tem capacidade de desintoxicação contra AFB1 in vivo.
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Abstract We developed poly-ε-caprolactone (PCL)-based nanoparticles containing D-α-tocopherol polyethylene glycol-1000 succinate (TPGS) or Poloxamer 407 as stabilizers to efficiently encapsulate genistein (GN). Two formulations, referred to as PNTPGS and PNPol, were prepared using nanoprecipitation. They were characterized by size and PDI distribution, zeta potential, nanoparticle tracking analysis (NTA), GN association (AE%), infrared spectroscopy (FT-IR), and differential scanning calorimetry (DSC). PNTPGS-GN exhibited a particle size of 141.2 nm, a PDI of 0.189, a zeta potential of -32.9 mV, and an AE% of 77.95%. PNPol-GN had a size of 146.3 nm, a better PDI than PNTPGS-GN (0.150), a less negative zeta potential (-21.0 mV), and an AE% of 68.73%. Thermal and spectrometric analyses indicated that no new compounds were formed, and there was no incompatibility detected in the formulations. Cellular studies revealed that Poloxamer 407 conferred less toxicity to PCL nanoparticles. However, the percentage of uptake decreased compared to the use of TPGS, which exhibited almost 80% cellular uptake. This study contributes to the investigation of stabilizers capable of conferring stability to PCL nanoparticles efficiently encapsulating GN. Thus, the PCL nanoparticle proposed here is an innovative nanomedicine for melanoma therapy and represents a strong candidate for specific pre-clinical and in vivo studies.
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Abstract Lactobacilli are probiotics with Aflatoxin (AF) detoxification ability, found in fermented products, GIT of animals and environment. Purpose of this study was to investigate the ability of broiler isolates of Lactobacillus against Aflatoxin B1 (AFB1). For this purpose, 5 isolates of Lactobacillus from broiler gut were incubated with 100 ppb AFB1 in aqueous environment and effect of different parameters (cell fractions, time, temperature, pH) on detoxification was determined by HPLC. The ameliorative effect of Lactobacillus salivarius (LS) against AFB1 was studied in broiler. The results revealed that LS (CR. 4) showed the best results (in vitro) as compared to other isolates (L. salivarius (CR. 3, CR, 4), L. agilis (CE. 2.1, CE. 3.1) and L. crispatus (CE. 28). Cell debris of CR. 4 showed significantly higher detoxification (P<0.05). Maximum amount of AFB1 was detoxified at 30°C (97%), pH 4.0 (99%) and 6 h (99.97%). In vivo study showed that AFB1 decreased weight gain (1,269 ± 0.04 gm/ bird), feed consumed (2,161 ± 0.08 gm/ bird), serum total protein (2.42 ± 0.34 gm/ dl), serum albumin (0.5 ± 0.2 2 gm/dl) and antibody titer (4.2 ± 0.83). Liver function enzymes were found (alanine transaminase (ALT): 32 ± 10.7 U/L) and aspartate transaminase (AST): 314.8 ± 27 U/L) elevated in AFB1 fed broilers. Treatment with 1% LS not only decreased the toxic effects of AFB1 (group D) but also improved the overall health of broilers due to its probiotic effects (p<0.05) as compared to control negative (group A). The detoxification ability of LS was better than commercial binder (CB) (0.2% Protmyc). It was concluded that detoxification of AFB1 by Lactobacillus was strain, temperature, pH and time dependent. LS has detoxification ability against AFB1 in vivo.
Resumo Os lactobacilos são probióticos com capacidade de desintoxicação da Aflatoxina (AF), encontrados em produtos fermentados, TGI de animais e meio ambiente. O objetivo deste estudo foi investigar a capacidade de isolados de frango de corte de Lactobacillus contra a Aflatoxina B1 (AFB1). Para tanto, 5 isolados de Lactobacillus de intestino de frango foram incubados com 100 ppb AFB1 em meio aquoso, e o efeito de diferentes parâmetros (frações celulares, tempo, temperatura, pH) na desintoxicação foi determinado por CLAE. O efeito melhorador de Lactobacillus salivarius (LS) contra AFB1 foi estudado em frangos de corte. Os resultados revelaram que LS (CR. 4) apresentou os melhores resultados (in vitro) em comparação com outros isolados [L. salivarius (CR. 3, CR. 4), L. agilis (CE. 2.1, CE. 3.1) e L. crispatus (CE. 28)]. Detritos celulares de CR. 4 mostraram desintoxicação significativamente maior (P < 0.05). A quantidade máxima de AFB1 foi desintoxicada a 30 °C (97%), pH 4.0 (99%) e 6 h (99,97%). O estudo in vivo mostrou que AFB1 diminuiu o ganho de peso (1,269 ± 0.04 g / ave), alimento consumido (2,161 ± 0.08 g / ave), proteína total sérica (2.42 ± 0.34 g / dl), albumina sérica (0.5 ± 0.22 gm / dl) e título de anticorpo (4.2 ± 0.83). As enzimas da função hepática foram encontradas (alanina transaminase (ALT): 32 ± 10.7 U / L) e aspartato transaminase (AST): 314.8 ± 27 U / L) elevadas em AFB1 alimentados com frangos. O tratamento com 1% LS não só diminuiu os efeitos tóxicos de AFB1 (grupo D), mas também melhorou a saúde geral dos frangos devido aos seus efeitos probióticos (p < 0.05) em comparação com o controle negativo (grupo A). A capacidade de desintoxicação do LS foi melhor do que o aglutinante comercial (CB) (0.2% Protmyc). Concluiu-se que a desintoxicação de AFB1 por Lactobacillus foi dependente da cepa, temperatura, pH e tempo. LS tem capacidade de desintoxicação contra AFB1 in vivo.
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Animais , Aflatoxina B1/análise , Aflatoxina B1/toxicidade , Probióticos , Galinhas , Lactobacillus , Ração Animal/análiseRESUMO
Resumen Objetivo: Detectar y cuantificar residuos de glifosato en cereales de desayuno comercializados en la ciudad de Saltillo, Coahuila. Materiales y Métodos: Se analizaron tres muestras de 12 marcas de cereal distintas. Para la extracción de plaguicidas se utilizó un equipo Soxhlet y la cuantificación se realizó por medio de Cromatografía Líquida de Alta Eficiencia (HPLC), utilizando un equipo marca Agillent modelo 1100 Series acoplado a un detector UV-Vis. Resultados: Se detectó glifosato en el 100% de las muestras analizadas, se obtuvieron concentraciones de entre 170 a 2 400 mg/kg, las muestras que presentaron mayores concentraciones fueron las de marcas internacionales. Conclusiones: Se detectaron residuos de glifosato en las 24 muestras analizadas de cereales de desayuno, las cuales sobrepasaron los LMR establecidos en el CODEX ALIMENTARIUS, por lo que es importante realizar más estudios con el fin de monitorear y asegurar la inocuidad de los productos procesados que son consumidos.
Abstract Objective: To detect and quantify glyphosate residues in breakfast cereals marketed in the city of Saltillo, Coahuila. Materials and Methods: Two samples of 12 different cereal brands were analyzed. A Soxhlet apparatus was used for pesticide extraction and quantification was performed by High Performance Liquid Chromatography (HPLC) using an Agillent model 1100 Series equipment coupled to a UV- Vis detector. Results: Glyphosate was detected in 100% of the samples analyzed, with concentrations ranging from 170 to 2 400 mg/kg; the samples with the highest concentrations were those of international brands. Conclusions: All the concentrations detected in the commercial cereal samples exceeded the MRLs established in CODEX ALIMENTARIUS, so it is important to carry out more studies in order to monitor and ensure the safety of the processed products that are consumed.
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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
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Objective To prepare and optimize the formulation of Albumin nanoparticles loading PROTAC molecule and observe the inhibition effect of nanoparticles on the proliferation and NAD+ metabolism of glioma cells. Methods Albumin nanoparticles loading NPT-B2 were prepared and characterized with a thermal driving method, and the prescription was optimized. An HPLC method was established to determine the content of NPT-B2. The proliferation inhibition of NPT-B2 and B2-BSA-NPs on U251 cells were investigated by the CCK8 method, and the degradation effects of NPT-B2 and B2-BSA-NPs on NAMPT in glioma cells were investigated by western blotting. Results The HPLC method was stable, with good linearity, precision, and recovery rate. The nanoparticles had a particle size of about 55.48 nm, a potential of about −12.9 mV, an encapsulation rate of about 94.74%, and a drug loading amount of about 8.61%. The IC50 of NPT-B2 on glioma cells was 61.16 nmol/L, which had a degradation effect on NAMPT. The IC50 of B2-BSA-NPs on glioma was 41.21 nmol/L, which had a very significant degradation effect on NAMPT. Conclusion Albumin nanoparticles loading PROTAC molecules were constructed. The prescription was optimized to improve the drug encapsulation rate, and the low water solubility of PROTAC molecule was improved, which had a significant inhibitory effect on the proliferation and NAD+ energy metabolism of glioma cells.
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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.