Metabolic profile changes of kidney aging and protective effects of Polygonatum sibiricum polysaccharides on D-galactose-induced aging mice / 数字中医药（英文）

Objective@#To investigate the metabolic trajectory of kidney aging and the effects of Polygonatum sibiricum polysaccharides (PSP) against kidney aging in D-galactose (D-gal)-induced aging mice, based on ultra-performance liquid chromatography/Q-Exactive Orbitrap mass spectrometry (UPLC-Q-Exactive MS/MS). @*Methods@#A total of 36 C57 BL/6J mice were randomly allocated to six groups: control (CON), model (MOD), PSP low-dose (PSP-L), PSP medium-dose (PSP-M), PSP high-dose (PSP-H), and positive drug ascorbic acid (VC) groups. To create models of aging mice, D-gal was intraperitoneally administered to all other groups of mice except the CON group. After modeling, the appropriate Chinese medicine [PSP-L: 150 mg/(kg·d), PSP-M: 300 mg/(kg·d), PSP-H: 600 mg/(kg·d)] or positive drug [ascorbic acid, 300 mg/(kg·d)] was administered for intervention. Key markers of renal function in urine and serum of mice in each group, such as creatinine (Crea), urea nitrogen (BUN), and uric acid (UA) levels, as well as key indicators of oxidative stress in serum and kidney, including superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA), and glutathione peroxidase (GSH-Px) were determined to validate the successful establishment of kidney aging models and to estimate the effects of PSP. Hematoxylin and eosin (HE), periodic acid Schiff (PAS), and β-galactosidase staining were used to assess the renal pathological changes. The metabolic profiles of serum, kidney, and urine samples from CON, MOD, and PSP-H groups were analyzed by UPLC-Q-Exactive MS/MS, and pattern recognition methods were used to outline the metabolic trajectory of kidney aging and to identify the characteristic metabolites. @*Results@#Age-related alterations in renal histopathology and impaired renal function in mice were also associated with oxidative stress indicators. Following the injection of PSP [PSP-H: 600 mg/(kg·d)], the pathological indices associated with aging were adjusted to normal levels, renal function and oxidative stress were improved in aging mice, and renal pathological damage was markedly improved. Meanwhile, the potential biomarkers were identified by UPLC-Q-Exactive MS/MS analysis and were further analyzed to form related metabolic pathways, with P < 0.05 as a threshold. The results showed that purine, sphingolipid, glycerophospholipid, tryptophan, and riboflavin metabolisms were the main metabolic pathways associated with aging. After administration of PSP, these pathological indices returned to normal levels, and biomarkers related to the aging process, such as adenosine monophosphate (AMP), tryptophan, and 5-hydroxytryptophan, also demonstrated, to some degree, reverse regulation (promoting synthesis). @*Conclusion@#Metabolomics methods based on UPLC-Q-Exactive MS/MS and multivariate statistical analysis can be adopted to establish metabolic profiles in aging mice. PSP has been shown to protect against kidney aging by interfering with the purine, sphingolipid, glycerophospholipid, tryptophan, and riboflavin metabolisms in the kidney.

Analysis on Quality Attribute of Substance Benchmarks of Shentong Zhuyutang Based on Supramolecular "Imprinting Template" Theory / 中国实验方剂学杂志

ObjectiveBased on the supramolecular "imprinting template" theory， the autonomous action law of the component groups of Shentong Zhuyutang in the preparation process of medicinal materials-decoction pieces-formulas was studied to clarify the quantitative transfer law of its quality attributes. MethodUltra performance liquid chromatography（UPLC） fingerprint of Shentong Zhuyutang was established with mobile phase of 0.4% phosphoric acid aqueous solution（A）-acetonitrile（B） for gradient elution（0-2.5 min， 100%A； 2.5-6 min， 100%-96%A； 6-15 min， 96%-92%A； 15-25 min， 92%-88%A； 25-35 min， 88%-75%A； 35-50 min， 75%-65%A； 50-60 min， 65%-50%A； 60-65 min， 50%-30%A； 65-70 min， 100%A） and detection wavelength of 235 nm， and the total statistical moments， information entropy and primary feeding amount of fingerprint of medicinal materials， decoction pieces and benchmark samples were calculated. Dry extract rate of the benchmark samples， the transfer rates and the addition parameters of medicinal materials-decoction pieces-formulas were calculated. ResultSimilarities of the total statistical moments of UPLC fingerprint of 15 batches of medicinal materials and decoction pieces were>0.89， the relative standard deviations（RSDs） of information entropy of UPLC fingerprint of 12 medicinal materials and decoction pieces were<10%. RSDs of total first-order moment（MCRTT） and information entropy of Shentong Zhuyutang（medicinal materials） were 5.5% and 2.3%， while the RSDs of MCRTT and information entropy of Shentong Zhuyutang（decoction pieces） were 4.8% and 2.6%， respectively. The dry extract rate of 45 batches of Shentong Zhuyutang was 17.2%-20.2%. The transfer rate of medicinal materials to decoction pieces was within the range of data fluctuation， which was 70%-130% of the average value. The overall transfer rates of medicinal materials to decoction pieces and decoction pieces to benchmark samples were 101.8% and 83.0%， respectively. ConclusionThe quality properties of Shentong Zhuyutang benchmark samples can be studied by total statistical moment analysis and primary feeding amount analysis， which can confirm the supramolecular "imprinting template" theory to a certain extent.

Comparison of Effect of Chijian and Gastrodiae Rhizoma on Regulating Immune Function and Intestinal Microflora / 中国实验方剂学杂志

Objective:To compare the contents of adenosine， gastrodin， <italic>p</italic>-hydroxybenzyl alcohol， <italic>p</italic>-hydroxybenzaldehyde， parisinin B and parisinin A in Chijian （the aerial part of <italic>Gastrodia elata</italic>） and Gastrodiae Rhizoma， and compare their effects on immune function and intestinal microflora， evaluating whether it is necessary to study and develop Chijian. Method:The contents of these six constituents were determined by ultra performance liquid chromatography （UPLC）， the mobile phase was 0.1% formic acid aqueous solution （A）-acetonitrile （B） for gradient elution （0-4 min， 0.5%B； 4-5 min， 0.5%-2%B； 5-10 min， 2%-15%B； 10-12 min， 15%-20%B； 12-15 min， 20%-95%B； 15-17 min， 95%B； 17-17.5 min， 95%-0.5%B； 17.5-20 min， 0.5%B）， the flow rate was 0.5 mL·min^-1， the detection wavelength was 270 nm. The difference of pharmacological activity of water extracts of Chijian and Gastrodiae Rhizoma was compared， the clearance index， corrected clearance index and peripheral blood were measured in mice model with low immune function induced by cyclophosphamide， B lymphocyte proliferation was determined by lymphocyte transformation test <italic>in vitro</italic>， intestinal microflora was analyzed by 16S rDNA technology and bioinformatics was conducted. Result:The total contents of these six components in powder and ethanol extract of Chijian were higher than that of Gastrodiae Rhizoma， but the total contents of these six components in their water extract were similar， and the total contents of gastrodin and <italic>p</italic>-hydroxybenzyl alcohol met the requirements of the 2020 edition of <italic>Chinese Pharmacopoeia</italic>. Compared with the blank group， the clearance index of immunocompromised mice was significantly increased in the middle-dose （10 g·kg^-1） group of Chijian water extract， middle- and low-dose （10， 5 g·kg^-1） groups of Gastrodiae Rhizoma water extract （<italic>P</italic><0.05）， the levels of erythrocyte and hematocrit in peripheral blood were significantly increased in the high-dose （20 g·kg^-1） groups of water extracts of Chijian and Gastrodiae Rhizoma （<italic>P</italic><0.05， <italic>P</italic><0.01）， water extract of Gastrodiae Rhizoma with concentration of 400 g·L^-1 and the water extract of Chijian with the concentration of 100 g·L^-1 could promote the proliferation of B lymphocytes induced by lipopolysaccharide. Studies on intestinal microflora showed that compared with the blank group， at the phylum level， the water extracts of Chijian and Gastrodiae Rhizoma increased the relative abundance of Bacteroidetes and decreased the relative abundance of Firmicutes， at the genus level， they increased the relative abundance of <italic>Prevotellaceae</italic>_UCG-001 and <italic>Ruminococcaceae</italic>_UCG-005， and decreased the relative abundance of <italic>Anaerotruncus</italic>， unclassified_<italic>f</italic>_<italic>Erysipelotrichaceae</italic> and<italic> Candidatus</italic>_<italic>Stoquefichus</italic>.<italic> </italic>These intestinal bacteria were related to the immune system， cell proliferation， and metabolism regulation. Conclusion:The total contents of 6 components in the powder， the ethanol and the water extracts of Chijian are higher than or close to those of the corresponding samples of Gastrodiae Rhizoma， the pharmacological activity of Chijian water extract is similar to that of Gastrodiae Rhizoma water extract， indicating that Chijian is worthy of further research and development.

Comparison of UPLC Fingerprint and Determination of Index Components Before and After Processing of Cyperi Rhizoma Processed with Four Excipients / 中国实验方剂学杂志

Objective:To study the quality evaluation method of Cyperi Rhizoma processed with four excipients. Method:Ultra-performance liquid chromatography （UPLC） fingerprints of raw products and processed products with four excipients of Cyperi Rhizoma were established， and the changes of chemical components in the fingerprints before and after processing were compared by chemometric analysis. The mobile phase was consisted of methanol （A）-water （B） for gradient elution （0-10 min， 5%-40%A； 10-30 min， 40%-70%A； 30-40 min， 70%A） at a flow rate of 0.3 mL·min^-1. The injection volume was 3 μL， the column temperature was 35 ℃， and the detection wavelength was 280 nm. The content changes of main index components in Cyperi Rhizoma before and after processing were compared by UPLC. The mobile phase was methanol-water （75∶25） and the detection wavelength was 242 nm. Result:Processing with four excipients had a significant impact on the overall characteristics of chemical components in the fingerprint of Cyperi Rhizoma. A total of 28 characteristic peaks were identified in fingerprints of the raw and processed products. Among them， peaks 1， 2 and 4 were specific peaks of the processed products， peak 5 was characteristic peak of the raw products. Peak 2 was identified as 5-hydroxymethylfurfural， peak 24 as cyperenone and peak 27 as <italic>α</italic>-cyperone. The 5-hydroxymethylfurfural produced by the processing with four excipients came from rice vinegar， rice wine and Maillard reaction of polysaccharides in Cyperi Rhizoma. The results of determination showed that there was no significant difference in the content of cyperenone after processing， but the content of <italic>α</italic>-cyperone decreased significantly. Conclusion:In the process of Cyperi Rhizoma processed with four excipients， there are new components produced by structural transformation， which are accompanied by changes in the content of index components. In this study， the quality of raw and processed products of Cyperi Rhizoma can be rapidly and effectively evaluated from qualitative and quantitative aspects.

Establishment of UPLC Fingerprint and Quantitative Analysis of Multi-components of Shengyutang / 中国实验方剂学杂志

Objective:To establish ultra performance liquid chromatography （UPLC） fingerprint of Shengyutang and quantitative analysis method of 11 index components in this famous classical formula. Method:UPLC-diode array detector/evaporative light scattering detector （UPLC-PDA/ELSD） was used， two chromatographic conditions were established by different detectors according to the polarity of chemical components. Conditions of fingerprint 1 were as follows：ACQUITY UPLC HSS T₃ column （2.1 mm×100 mm， 1.8 µm） with the mobile phase of acetonitrile （A）-0.6% formic acid solution （C） for gradient elution （0-4 min， 0-4%A； 4-8 min， 4%A； 8-9 min， 4%-8%A； 9-14 min， 8%-9%A； 14-21 min， 9%-15%A； 21-26 min， 15%-17%A； 26-30 min， 17%-20%A； 30-35 min， 20%-32%A； 35-40 min， 32%-40%A； 40-50 min， 40%-80%A； 50-55 min， 80%A）， the flow rate of 0.3 mL·min^-1， PDA with detection wavelengths of 280 nm and 321 nm， the column temperature at 30 ℃. Conditions of fingerprint 2 were as follows：the CORTECS C₁₈ column （3.0 mm×100 mm， 2.7 µm） with the mobile phase of acetonitrile （A）-water （D） for gradient elution （0-11 min， 19%A； 11-16 min， 19%-25%A； 16-34 min， 25%-28%A； 34-47 min， 28%-47%A； 47-60 min， 47%-80%A）， the flow rate of 0.4 mL·min^-1， ELSD with drift tube temperature of 95 ℃， the carrier gas （air） flow rate of 2.0 L·min^-1， and the column temperature at 30 ℃. UPLC-PDA/ELSD fingerprints of 15 batches of Shengyutang were established， and the similarity was evaluated by similarity evaluation system of chromatographic fingerprint of traditional Chinese medicine （2012 edition） issued by the Chinese Pharmacopoeia Commission， and the contents of eleven index components in this famous classical formula were determined. Result:The similarities of UPLC-PDA/ELSD fingerprints of 15 batches of Shengyutang were >0.98 by comparing with the control fingerprint， 27 and 16 common peaks were identified in fingerprint 1， 2， respectively. It was tested and verified that the precision， repeatability， stability， linear relationship and other results of this method all met the requirements of the 2020 edition of <italic>Chinese Pharmacopoeia</italic>. The contents of chlorogenic acid， ferulic acid， calycosin glucoside， verbascoside， senkyunolide I， senkyunolide H， senkyunolide A， ginsenoside Rg₁， ginsenoside Re， ginsenoside Rb₁ and astragaloside A in 15 batches of Shengyutang were 0.063-0.193， 0.509-0.638， 0.160-0.318， 0.012-0.056， 0.394-0.519， 0.110-0.143， 0.031-0.097， 0.382-0.595， 0.292-0.505， 0.590-0.803， 0.142-0.367 mg·g^-1， respectively. Conclusion:The established detection method meets the requirements of the 2020 edition of <italic>Chinese Pharmacopoeia</italic>， which can characterize the overall characteristics of chemical components in Shengyutang， and provide experimental basis for the quality standard research of this famous classical formula.

Establishment of UPLC-PDA Fingerprint of Baoyuantang Substance Benchmark and Identification of Its Chemical Constituents / 中国实验方剂学杂志

Objective:To establish the fingerprint of Baoyuantang substance benchmark， and to analyze and identify the common peaks. Method:A total of 15 batches of Baoyuantang substance benchmark were prepared， ultra performance liquid chromatography-diode array detector method （UPLC-PDA） was used to establish the fingerprint of the substance benchmark， and the methodology was developed. The chromatographic conditions were as follows：ACQUITY UPLC BEH Shield C₁₈ column （2.1 mm×100 mm， 1.7 μm）， mobile phase of 0.05% formic acid solution （A） and 0.05% formic acid acetonitrile solution （ B） for gradient elution （0-0.5 min， 5%-19%B； 0.5-6 min， 19%B； 6-10 min， 19%-27%B； 10-20 min， 27%-45%B； 20-20.1 min， 45%-95%B； 20.1-23 min， 95%B）， the flow rate of 0.4 mL·min^-1， the column temperature of 30 ℃， the detection wavelength at 203 nm and 260 nm， and the injection volume of 2 μL. Similarity evaluation system of traditional Chinese medicine fingerprint （2012 edition） was used to establish the fingerprint and generate the control fingerprint. The chemical constituents of Baoyuantang substance benchmark were identified by comparison of standard substances and UPLC-electrospray ionization tandem mass spectrometry （UPLC-ESI-MS/MS） with full information tandem mass spectrometry （MS^E） and scanning range of <italic>m</italic>/<italic>z</italic> 50-1 200. Result:The similarities of 15 batches of Baoyuantang substance benchmark were above 0.90 by comparing with the control fingerprint. There were 37 common peaks， 22 of which were identified through UPLC-ESI-MS/MS， including liquiritin， violanthin， ginsenoside Rg₁， ginsenoside Rb₁， ginsenoside Re and so on. These components were all from Astragali Radix， Ginseng Radix et Rhizoma， Zingiberis Rhizoma Recens and Glycyrrhizae Radix et Rhizoma. Conclusion:This method is accurate， stable and reliable， which will basically reflect the overall chemical composition characteristics of Baoyuantang， and it provides experimental basis for development of the granules of this famous classical formulas.

Comparison of UPLC Fingerprint and Determination of Main Components Before and After Processing of Artemisiae Argyi Folium Processed with Four Excipients / 中国实验方剂学杂志

Objective:To establish the ultra-performance liquid chromatography （UPLC） fingerprints of Artemisiae Argyi Folium and Artemisiae Argyi Folium processed with four excipients， and quantitatively analyze the 8 phenolic acids and flavonoids contained in them， in order to explore the quality evaluation method of Artemisiae Argyi Folium processed with four excipients. Method:UPLC was used with Shim-pack XR-ODS C₁₈ column （2.0 mm×75 mm， 2.2 µm）， mobile phase of acetonitrile （A） -0.2% formic acid aqueous solution （B） for gradient elution （0-1 min， 10%A； 1-2 min， 10%-15%A； 2-17 min， 15%-18%A； 17-24 min， 18%-28%A； 24-36 min， 28%-38%A； 36-41 min， 38%-60%A； 41-45 min， 60%-100%A）， detection wavelength of 330 nm and flow rate of 0.2 mL·min^-1. The UPLC fingerprints of Artemisiae Argyi Folium before and after processing were established， and analyzed by chemometrics. Contents of 5-caffeoylquinic acid， 3-caffeoylquinic acid， 4-caffeoylquinic acid， 3，4-dicaffeoylquinic acid， 3，5-dicaffeoylquinic acid， 4，5-dicaffeoylquinic acid， jaceosidin and epuatilin in the decoction pieces were determined. Result:The fingerprints of Artemisiae Argyi Folium before and after processing were established， and the UPLC characteristic chromatograms of Artemisiae Argyi Folium before and after processing had good consistency， and the similarity was >0.94. Compared with Artemisiae Argyi Folium， the contents of 3-caffeoylquinic acid and 3，4-dicaffeoylquinic acid had no significant change after processing， the contents of jaceosidin and epuatilin decreased after processing， while the contents of 5-caffeoylquinic acid， 4-caffeoylquinic acid and 4，5-dicaffeoylquinic acid increased significantly （<italic>P<</italic>0.01）， their average increasing rates were 32.50%， 66.83%， 29.39%， respectively. And content of 3，5-dicaffeoylquinic acid was significantly decreased （<italic>P<</italic>0.01） ， and the average reduction rate was 51.25%. Conclusion:The contents of chemical components in Artemisiae Argyi Folium and Artemisiae Argyi Folium processed with four excipients have changed to a certain extent. Among them， 5-caffeoylquinic acid， 3，5-dicaffeoylquinic acid， 4-caffeoylquinic acid and 4，5-dicaffeoylquinic acid can be used as the key indicators for quality evaluation of Artemisiae Argyi Folium before and after processing.

Fingerprint Study and Primary Quality Evaluation of Niuhuang Qingwei Pills Based on UPLC and Reference Drug / 中国药学杂志

OBJECTIVE： To apply reference drug in the fingerprint study of Niuhuang qingwei pills, and perform primary evaluation of the quality grade of the samples. METHODS： Ultra performance liquid chromatography (UPLC) separation was performed on an AQUITY UPLC BEH C18 column with gradient elution using acetonitrile (containing 0.5% formic acid)-0.5% formic acid at a flow rate of 0.2 mL•min-1. The injection volume was 2 μL and the column temperature was maintained at 40 ℃. The detection wavelength was set at 254 nm. By comparing with reference substances and the reference herbal materials, characteristic peaks and their ascriptions were investigated. Using Niuhuang qingwei pills reference drug as the accompanying physical control, the similarities of the fingerprints of 49 batches of samples from 18 manufacturers were calculated, and the quality grades were evaluated. RESULTS： The similarities of all samples fell within the range of 0.76-0.98. The similarities of 49 samples were above 0.75 and met the second-grade limit. The similarity of 24 samples were above 0.90 and met the first-grade limit. CONCLUSION： The method is simple, accurate and rapid. It can be used for the quality control and grade evaluation of Niuhuang qingwei pills, which provides reference for the quality grade research of Chinese patent medicines.

Study on intestinal absorption of aqueous extract from Chaihu Shugan San / 药物评价研究

Objective To evaluate the intestinal absorption of main components (paeoniflorin,ferulic acid,narirutin,naringin,neohesperidin,and glycyrrhizic acid) in aqueous extract of Chaihu Shugan San (CSS).Methods UPLC method was established to simultaneously determine the concentrations of the six components in the test samples of everted gut sacs and in situ single pass intestinal perfusion (SPIP) models.Absorption kinetics parameters were calculated for describing their absorption characteristics.Results In SPIP model,the results indicated that ferulic acid was the well-absorbed ingredient in whole small intestine,while other ingredients presented moderate or poor absorption.In everted gut sacs model,paeoniflorin in jejunum,ferulic acid,naringin,and neohesperidin in duodenum,narirutin in duodenum and jejunum,had the best absorption,while there was no significant difference in absorption of glycyrrhizic acid in the intestine.Condusion In vivo model indicated that the main constituents in CSS could be absorbed in intestinal wall of rat,ferulic acid could be much more easily penetrated intestinal wall into the blood circulation than the other five components.Ex vivo model could further articulate that six index components could be absorbed selectively in different intestinal segments.

Study on intestinal absorption of aqueous extract from Chaihu Shugan San / 药物评价研究

Objective To evaluate the intestinal absorption of main components (paeoniflorin,ferulic acid,narirutin,naringin,neohesperidin,and glycyrrhizic acid) in aqueous extract of Chaihu Shugan San (CSS).Methods UPLC method was established to simultaneously determine the concentrations of the six components in the test samples of everted gut sacs and in situ single pass intestinal perfusion (SPIP) models.Absorption kinetics parameters were calculated for describing their absorption characteristics.Results In SPIP model,the results indicated that ferulic acid was the well-absorbed ingredient in whole small intestine,while other ingredients presented moderate or poor absorption.In everted gut sacs model,paeoniflorin in jejunum,ferulic acid,naringin,and neohesperidin in duodenum,narirutin in duodenum and jejunum,had the best absorption,while there was no significant difference in absorption of glycyrrhizic acid in the intestine.Condusion In vivo model indicated that the main constituents in CSS could be absorbed in intestinal wall of rat,ferulic acid could be much more easily penetrated intestinal wall into the blood circulation than the other five components.Ex vivo model could further articulate that six index components could be absorbed selectively in different intestinal segments.

Comparative study on extraction method of Zuojin Pills / 中草药

Objective: To study Zuojin Pills with different extraction methods and select the best one. Methods: To establish the fingerprints and determination the content of five alkaloids in Zuojin Pills by ultra performance liquid chromatography (UPLC), compare the water extraction, 70% ethanol extraction, HCl-methanol (1:100) extraction, and semi-bionic extraction according to the similarity of fingerprints and content of five alkaloids. Results: The appearance time of all chromatographic peaks of Zuojin Pills was within 8 min and 9 common chromatographic peaks were obtained. The linear is stable and peak form is better. The linear relationship and resolution of the five alkaloids are nice. The similarity of the fingerprints exceed 0.98; The contents of the five alkaloids are much more different, the content of the five alkaloids in the HCl-methanol sample is the most, however, the least is the water extraction sample. Conclusion: This study shows that the UPLC method is convenient with quick analysis rate. On account of indicated component quantitative analysis, it could be used to synthetically evaluate the extraction method. HCl-methanol (1:100) extraction method is the best one.

Determination of sildenafil citrate illegally mixed into traditional Chinese medicinal preparation by ultra performance liquid chromatography-tandem mass spectrometry / 中成药

AIM: To establish a quick,accurate,sensitive method for analysing sildenafil citrate illegally added into traditional Chinese medicinal preparation. METHODS: The ultra performance liquid chromatography-quadrupole mass spectrometry method was used.Electrospay ionization(ESI) source was applied and operated in the positive and negative ion mode.The compound of sildenafil citrate illegally added into traditional Chinese medicinal preparations were identified according to the spectrum,chromatographic behavior and mass spectral data by comparison with those of reference substance. RESULTS: Sildenafil citrate was found in two formulations of three. CONCLUSION: The method is selective and sensitive and can be used to detect the sildenafil citrate illegally added into traditional Chinese medicinal preparations.