1.Comprehensive Application of AHP-CRITIC Hybrid Weighting Method, Grey Correlation Analysis and BP-ANN in Optimization of Extraction Process of Qizhi Prescription
Qun LAN ; Yi CHENG ; Zian LI ; Bingyu WU ; Jinyu WANG ; Dewen LIU ; Yan TONG
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(8):176-186
ObjectiveBased on analytic hierarchy process(AHP)-criteria importance through intercriteria correlation(CRITIC) hybrid weighting method, grey relational analysis and backpropagation artificial neural network(BP-ANN), to optimize the water extraction process of Qizhi prescription, so as to provide an experimental basis for optimization of the preparation process of this prescription and the establishment of quality standards. MethodsL9(34) orthogonal test was employed, and the AHP-CRITIC hybrid weighting method was utilized to determine the weight coefficients of the quality fractions of various components, including astragaloside Ⅳ, polygalaxanthone Ⅲ, calycosin-7-O-β-D-glucoside, tenuifolin, and 3,6′-disinapoylsucrose, as well as the dry extract yield. The comprehensive score of each factor level combination in the orthogonal test were calculated as evaluation indicator to select the optimal extraction process parameters. The effects of extraction times, extraction time, and solvent dosage on the aqueous extraction process of the formula were investigated through intuitive analysis, variance analysis, and grey relational analysis. Meanwhile, a BP-ANN model was established to reverse-predict the optimal extraction process parameters of Qizhi prescription, and the optimized process parameters were validated. ResultsThe weight coefficients of the five index components(astragaloside Ⅳ, tenuifolin, calycosin-7-O-β-D-glucoside, polygalaxanthone Ⅲ, and 3,6′-disinapoylsucrose) and dry extract yield were 25.7%, 20.82%, 16.41%, 12.45%, 15.96% and 8.67%, respectively. The optimized extraction process parameters were extracted 3 times with 8, 6, 6 times the amount of water, each time for 1 h. The network prediction results of BP-ANN test samples were consistent with the orthogonal test results, and the mean square error(MSE) of the predicted and measured values of the network was <1%. The water extraction process of Qizhi prescription analyzed and predicted by relevant mathematical models was stable and feasible, which could effectively improve the extraction efficiency of the active ingredients of Astragali Radix and Polygalae Radix, and the average comprehensive score of the validation test was 90.85 with the relative standard deviation(RSD) of 1.55%. ConclusionThis study establishes a water extraction process for compound Qizhi granules, and the optimized extraction process can effectively improve the extraction efficiency of active ingredients, which provides useful references for the optimization of preparation process and the establishment of quality standards for other clinical experience formulas.
2.Research progress of metabolomics in age-related macular degeneration
Feng WANG ; Chenghong LAN ; Yiling LIU ; Yi SHAO
International Eye Science 2025;25(5):760-764
Age-related macular degeneration(ARMD)is a common multifactorial disease among the elderly, which may lead to irreversible vision loss; however, the pathogenesis of ARMD is still unclear. Metabolomics is a relatively new “omics” technique that can provide qualitative and quantitative information about low molecular weight metabolites that make up biological systems, thereby revealing the physiological or pathological state of cell or tissue samples at specific time points. In recent years, increasing evidence suggests that metabolic dysfunction plays an important role in the development and progression of ARMD. Metabolic pathway dysregulation involves lipid metabolism, nucleotide metabolism, amino acid metabolism, and energy metabolism, which may play important roles in the occurrence and development of ARMD. The retina is one of the most metabolically active tissues in the human body, so using metabolomics techniques to measure molecular changes in ARMD will further enhance our understanding of the pathogenesis. This will provide important insights for the prevention and treatment of ARMD, This article reviews the application of metabolomics in ARMD.
3.Role and mechanism of caffeic acid in a mouse model of severe acute pancreatitis
Siyu XU ; Tao LIU ; Lulu LAN ; Yining XUE ; Wei WEI ; Yi HAN ; Sucheng MU ; Haiyan SONG ; Shilin DU
Journal of Clinical Hepatology 2025;41(4):722-730
ObjectiveTo investigate the effect and potential mechanism of caffeic acid (CA) on severe acute pancreatitis (SAP) induced by caerulein combined with lipopolysaccharide (LPS), and to provide a basis for the research on novel drugs for the treatment of SAP. MethodsC57BL/6J mice, aged 6 weeks, were divided into control group, model group, CA group, and octreotide acetate (OA) group, with 6 mice in each group. The mice in the control group were given injection of normal saline, and those in the other groups were given intraperitoneal injection of caerulein combined with LPS to establish a mouse model of SAP. At 1 hour after the first injection of caerulein, the mice in the CA group and the OA group were given intraperitoneal injection of CA or subcutaneous injection of OA at an interval of 8 hours. The general status of the mice was observed after 24 hours of modeling, and serum, pancreas, lung, and colon samples were collected. HE staining was used to observe the histopathological changes of the pancreas and lungs, and the serum levels of α-amylase, lipase, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), alanine aminotransferase, aspartate aminotransferase, and creatinine were measured. RT-PCR was used to measure the expression of proinflammatory factors in the pancreas and lungs; myeloperoxidase (MPO) immunohistochemistry was used to observe the degree of neutrophil infiltration; Western blot was used to measure the activation of nuclear factor-kappa B (NF-κB) and the level of citrullinated histone H3 (CitH3), a marker for the formation of neutrophil extracellular traps (NETs), in the pancreas and lungs, as well as the expression level of ZO-1 in colon tissue. A one-way analysis of variance was used for comparison of continuous data between multiple groups, and the Dunnett’s t-test was used for further comparison between two groups. ResultsCompared with the control group, the model group had severe injury in the pancreas and lungs and significant increases in the activity of serum α- amylase and lipase and the levels of the proinflammatory cytokines IL-6, interleukin-1β (IL-1β), and TNF-α in serum and lung tissue (all P<0.05), as well as significant increases in NF-κB activation, neutrophil infiltration, and the formation of NETs in the pancreas and lungs (all P<0.05). Compared with the model group, the CA group had alleviated pathological injury of the pancreas and lungs and significant reductions in the activity of serum α-amylase and the levels of the proinflammatory cytokines IL-6, IL-1β, and TNF-α in serum and lung tissue (all P<0.05), as well as significant reductions in NF-κB activation, neutrophil infiltration, and the formation of NETs in the pancreas and lungs (all P<0.05). ConclusionCA can alleviate SAP induced by caerulein combined with LPS in mice, possibly by inhibiting neutrophil recruitment and the formation of NETs.
4.Determination method of clopidogrel and its metabolites in rat plasma and its pharmacokinetic study
Huan YI ; Lan MIAO ; Changying REN ; Li LIN ; Mingqian SUN ; Qing PENG ; Ying ZHANG ; Jianxun LIU
China Pharmacy 2025;36(13):1599-1603
OBJECTIVE To establish a method for determining the contents of clopidogrel (CLP), clopidogrel carboxylate (CLP-C), clopidogrel acyl-β-D-glucuronide (CLP-G) and contents of clopidogrel active metabolite (CAM) in rat plasma, and to investigate their in vivo pharmacokinetic characteristics. METHODS The Shisedo CAPCELL ADME column was used with a mobile phase consisting of water and acetonitrile (both containing 0.1% formic acid) in a gradient elution. The flow rate was 0.4 mL/min, and the column temperature was maintained at 20 ℃. The injection volume was 2 μL. The analysis was performed in positive ion mode using electrospray ionization with multiple reaction monitoring. The ion pairs for quantitative analysis were m/z 322.1→211.9 (for CLP), m/z 308.1→197.9 (for CLP-C), m/z 322.1→154.8 (for CLP-G), m/z 504.1→154.9 [for racemic CAM derivative (CAMD)]. Six rats were administered a single intragastric dose of CLP (10 mg/kg). Blood samples were collected before medication and at 0.08, 0.33, 0.66, 1, 2, 4, 6, 10, 23 and 35 hours after medication. The established method was used to detect the serum contents of various components in rats. Pharmacokinetic parameters were then calculated using WinNonlin 6.1 software. RESULTS The linear ranges for CLP, CLP-C and CAMD were 0.08-20.00, 205.00-8 000.00, and 0.04-25.00 ng/mL, respectively (r≥0.990). The relative standard deviations for both intra-day and inter-day precision tests were all less than 15%, and the relative errors for accuracy ranged from -11.68% to 14.40%. The coefficients of variation for the matrix factors were all less than 15%, meeting the requirements for bioanalytical method validation. The results of the pharmacokinetic study revealed that, following a single intagastric administration of CLP in rats, the exposure to the parent CLP in plasma was extremely low. Both the area under the drug concentration-time curve (AUC0-35 h) and the peak concentration of the parent CLP were lower than those of its metabolites. The AUC0-35 h of the active metabolite CAM was approximately 43 times that of CLP, though it had a shorter half-life (2.53 h). The inactive metabolite CLP-C exhibited the highest exposure level, but it reached its peak concentration the latest and was eliminated slowly. The AUC0-35 h of CLP-G was about four times that of CAM, and its half-life was similar to that of CLP-C. CONCLUSIONS This study successfully established an liquid chromatography-tandem mass spectrometry method for the determination of CLP and its three metabolites, and revealed their pharmacokinetic characteristics in rats. Specifically, the parent drug CLP was rapidly eliminated, while the inactive metabolites CLP-C and CLP-G exhibited long half-lives, and active metabolite CAM displayed a transient exposure pattern.
5.Feixin Decoction Treats Hypoxic Pulmonary Hypertension by Regulating Pyroptosis in PASMCs via PPARγ/NF-κB/NLRP3 Signaling Pathway
Junlan TAN ; Xianya CAO ; Runxiu ZHENG ; Wen ZHANG ; Chao ZHANG ; Jian YI ; Feiying WANG ; Xia LI ; Jianmin FAN ; Hui LIU ; Lan SONG ; Aiguo DAI
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(18):1-9
ObjectiveTo investigate the mechanism by which Feixin decoction treats hypoxic pulmonary hypertension (HPH) by regulating the peroxisome proliferator-activated receptor gamma (PPARγ)/nuclear factor-kappa B (NF-κB)/NOD-like receptor pyrin domain containing 3 (NLRP3) signaling pathway. MethodsForty-eight male SD rats were randomly allocated into normal, hypoxia, and low-, medium- and high-dose (5.85, 11.7, 23.4 g·kg-1, respectively) Feixin decoction groups, with 8 rats in each group. Except the normal group, the remaining five groups were placed in a hypoxia chamber with an oxygen concentration of (10.0±0.5)% for 8 h per day, 28 days, and administrated with corresponding drugs during the modeling process. After 4 weeks of treatment, echocardiographic parameters [pulmonary artery acceleration time (PAT), pulmonary artery ejection time (PET), right ventricular anterior wall thickness (RVAWd), and tricuspid annular plane systolic excursion (TAPSE)] were measured for each group. The right ventricular systolic pressure (RVSP) was measured by the right heart catheterization method, and the right ventricular hypertrophy index (RVHI) was calculated by weighing the heart. The pathological changes in pulmonary arterioles were observed by hematoxylin-eosin staining. The co-localization of α-smooth muscle actin (α-SMA) with NLRP3, N-terminal gasdermin D (N-GSDMD), and cysteinyl aspartate-specific proteinase-1 (Caspase-1) in pulmonary arteries was detected by immunofluorescence. The protein levels of PPARγ, NF-κB, NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), N-GSDMD, interleukin-1β (IL-1β), interleukin-18(IL-18), and cleaved Caspase-1 in the lung tissue was determined by Western blot. The ultrastructural changes in pulmonary artery smooth muscle cells (PASMCs) were observed by transmission electron microscopy. ResultsCompared with the normal group, the hypoxia group showed increased RVSP and RVHI (P<0.01), decreased right heart function (P<0.01), increased pulmonary vascular remodeling (P<0.01), increased co-localization of α-SMA with NLRP3, N-GSDMD, and Caspase-1 in pulmonary arterioles (P<0.01), up-regulated protein levels of NF-κB, NLRP3, ASC, N-GSDMD, IL-1β, IL-18, and cleaved Caspase-1 in the lung tissue (P<0.05, P<0.01), a down-regulated protein level of PPARγ (P<0.05, P<0.01), and pyroptosis in PASMCs. Compared with the hypoxia group, Feixin decoction reduced RVSP and RVHI, improved the right heart function and ameliorated pulmonary vascular remodeling (P<0.05, P<0.01), decreased the co-localization of α-SMA with NLRP3, N-GSDMD, and Caspase-1 (P<0.05, P<0.01), down-regulated the protein levels of NF-κB, NLRP3, ASC, N-GSDMD, IL-1β, IL-18, and cleaved Caspase-1 in the lung tissue (P<0.05, P<0.01), up-regulated the protein level of PPARγ (P<0.05, P<0.01), and alleviated pyroptosis in PASMCs. ConclusionFeixin decoction can ameliorate pulmonary vascular remodeling and right heart dysfunction in chronically induced HPH rats by regulating pyroptosis in PASMCs through the PPARγ/NF-κB/NLRP3 pathway.
6.Key Challenges and Mitigation Strategies for Animal Pregnancy in Non-clinical Reproductive Toxicity Testing of Drugs
Kun LIU ; Qing LAN ; Bing YI ; Xiaojie XIE
Laboratory Animal and Comparative Medicine 2025;45(4):449-456
Non-clinical reproductive toxicity studies typically employ mammals like rats, rabbits, and cynomolgus monkeys, with animal pregnancy being a key challenge in such testing. This article focuses on the difficulties encountered in the animal pregnancy process and potential countermeasures. Rats can be used for fertility and early embryonic development toxicity studies (Segment Ⅰ), embryo-fetal development toxicity studies (Segment Ⅱ), and perinatal toxicity studies (Segment Ⅲ). The estrous cycle of female rats can be determined by vaginal smear, and mating behavior is confirmed through copulatory plug checks the following day after pairing one female with one male in the same cage. Rabbits are commonly used in embryo-fetal development toxicity studies (Segment Ⅱ). Mating behavior between male rabbits and estrous females is observed to determine the time of conception. However, challenges such as atypical estrus of female rabbits, large variations in estrus between batches, and mating failure often occur in reproductive toxicity testing, which may be addressed through prolonged light exposure, increased protein supplementation, optimized mating strategies, and environmental modifications like female and male rabbits are raised adjacent to each other. Non-human primates (NHPs) are typically employed in perinatal toxicity studies (Segment Ⅲ), where one of the key challenges lies in accurately determining sexual maturity in males - a critical factor for reproductive toxicity testing, which can be assessed through comprehensive evaluation of age, body weight, and testicular volume. Generally, male macaques are considered sexually mature when they meet the following criteria: age >4.5 years, body weight >4.5 kg, single testis volume >10 mL, and combined testicular volume >20 mL. For pregnancy confirmation, ultrasound examination demonstrating visible gestational sacs is required, though this necessitates experienced veterinary clinicians to establish standardized ultrasound examination protocols. In conclusion, reproductive toxicity studies should employ species-appropriate detection methods and evaluation criteria based on anatomical characteristics of the reproductive system to ensure successful mating and proper study execution.
7.Modified Lianpoyin Formula Treats Hp-associated Gastritis by Regulating Mitochondrial Autophagy and NLRP3 Inflammasome Signaling Pathway
Siyi ZHANG ; Haopeng DANG ; Wenliang LYU ; Wentao ZHOU ; Wei GUO ; Lin LIU ; Lan ZENG ; Yujie SUN ; Luming LIANG ; Yi ZHAO
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(21):178-187
ObjectiveTo explore the effect of modified Lianpoyin formula (LPYJWF) in the treatment of Helicobacter pylori (Hp)-associated gastric mucosal damage based on mitochondrial autophagy and NLRP3 inflammasome signaling pathway. MethodsA total of 60 eight-week-old Balb/c male mice were assigned via the random number table method into control, model, high-dose LPYJWF (LPYJWF-H, 27.3 g·kg-1·d-1), medium-dose LPYJWF (LPYJWF-M, 13.65 g·kg-1·d-1), low-dose LPYJWF (LPYJWF-L, 6.83 g·kg-1·d-1), and quadruple therapy groups. Except the control group, other groups were modeled for Hp infection. Mice were administrated with LPYJWF at corresponding doses by gavage. Quadruple therapy group was given omeprazole (6.06 mg·kg-1·d-1) + amoxicillin (303 mg·kg-1·d-1) + clarithromycin (151.67 mg·kg-1·d-1) + colloidal pectin capsules (30.3 mg·kg-1·d-1) by gavage. The control group was given an equal volume of 0.9% NaCl for 14 days. Hematoxylin-eosin (HE) staining was used to observe the pathological changes of gastric mucosa, and Warthin-Starry (W-S) silver staining was used to detect Hp colonization. Transmission electron microscopy was employed to observe the mitochondrial ultrastructure of the gastric tissue, and immunofluorescence co-localization assay was adopted to detect the expression of mitochondrial transcription factor A (TFAM) and translocase of the outer mitochondrial membrane member 20 (TOMM20). The water-soluble tetrazolium salt method and thiobarbituric acid method were used to determine the levels of superoxide dismutase (SOD) and malondialdehyde (MDA), respectively, in the gastric tissue. Western blot was employed to measure the protein levels of PTEN-induced kinase 1 (PINK1), Parkin, p62, microtubule-associated protein 1 light chain 3 (LC3), NOD-like receptor protein 3 (NLRP3), apoptosis-associated speck-like protein containing a CARD (ASC), interleukin-1β (IL-1β), and interleukin-18 (IL-18). Real-time quantitative PCR was employed to assess the mRNA levels of PINK1, Parkin, p62, and LC3. ResultsCompared with the control group, the model group presented obvious gastric mucosal damage, colonization of a large number of Hp, severe mitochondrial damage, vacuolated structures due to excessive autophagy, reduced TOMM20 and TFAM co-expression in the gastric mucosal tissue, and reduced SOD and increased MDA (P<0.01). In addition, the gastric tissue in the model group showed up-regulated protein and mRNA levels of PINK1, Parkin, and LC3 and down-regulated protein and mRNA levels of p62 (P<0.01, as well as increased expression of inflammasome-associated proteins NLRP3, ASC, IL-1β, and IL-18 (P<0.01). Compared with the model group, the LPYJWF and quadruple therapy groups showed alleviated pathological damage of gastric mucosa, reduced Hp colonization, mitigated mitochondrial damage, and increased co-expression of TOMM20 and TFAM. The SOD level was elevated in the LPYJWF-L group (P<0.01), and the MDA levels became lowered in the LPYJWF and quadruple therapy groups (P<0.05, P<0.01). Furthermore, the LPYJWF and quadruple therapy groups showed down-regulated mRNA levels of PINK1, Parkin, and LC3 and protein levels of PINK1 and Parkin, and up-regulated mRNA level of p62 (P<0.01). The LPYJWF-M, LPYJWF-H, and quadruple therapy groups showcased down-regulated LC3 Ⅱ/LC3 Ⅰ level (P<0.05, P<0.01) and up-regulated protein level of p62 (P<0.01). The expression of inflammasome-associated proteins NLRP3, ASC, IL-1β, and IL-18 were reduced in the LPYJWF and quadruple therapy groups (P<0.05, P<0.01). ConclusionLPYJWF ameliorates gastric mucosal damage and exerts mucosa-protective effects in Hp-infected mice, which may be related to the inhibition of excessive mitochondrial autophagy, thereby inhibiting the activation of the NLRP3 inflammasome pathway.
8.Modified Lianpoyin Formula Treats Hp-associated Gastritis by Regulating Mitochondrial Autophagy and NLRP3 Inflammasome Signaling Pathway
Siyi ZHANG ; Haopeng DANG ; Wenliang LYU ; Wentao ZHOU ; Wei GUO ; Lin LIU ; Lan ZENG ; Yujie SUN ; Luming LIANG ; Yi ZHAO
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(21):178-187
ObjectiveTo explore the effect of modified Lianpoyin formula (LPYJWF) in the treatment of Helicobacter pylori (Hp)-associated gastric mucosal damage based on mitochondrial autophagy and NLRP3 inflammasome signaling pathway. MethodsA total of 60 eight-week-old Balb/c male mice were assigned via the random number table method into control, model, high-dose LPYJWF (LPYJWF-H, 27.3 g·kg-1·d-1), medium-dose LPYJWF (LPYJWF-M, 13.65 g·kg-1·d-1), low-dose LPYJWF (LPYJWF-L, 6.83 g·kg-1·d-1), and quadruple therapy groups. Except the control group, other groups were modeled for Hp infection. Mice were administrated with LPYJWF at corresponding doses by gavage. Quadruple therapy group was given omeprazole (6.06 mg·kg-1·d-1) + amoxicillin (303 mg·kg-1·d-1) + clarithromycin (151.67 mg·kg-1·d-1) + colloidal pectin capsules (30.3 mg·kg-1·d-1) by gavage. The control group was given an equal volume of 0.9% NaCl for 14 days. Hematoxylin-eosin (HE) staining was used to observe the pathological changes of gastric mucosa, and Warthin-Starry (W-S) silver staining was used to detect Hp colonization. Transmission electron microscopy was employed to observe the mitochondrial ultrastructure of the gastric tissue, and immunofluorescence co-localization assay was adopted to detect the expression of mitochondrial transcription factor A (TFAM) and translocase of the outer mitochondrial membrane member 20 (TOMM20). The water-soluble tetrazolium salt method and thiobarbituric acid method were used to determine the levels of superoxide dismutase (SOD) and malondialdehyde (MDA), respectively, in the gastric tissue. Western blot was employed to measure the protein levels of PTEN-induced kinase 1 (PINK1), Parkin, p62, microtubule-associated protein 1 light chain 3 (LC3), NOD-like receptor protein 3 (NLRP3), apoptosis-associated speck-like protein containing a CARD (ASC), interleukin-1β (IL-1β), and interleukin-18 (IL-18). Real-time quantitative PCR was employed to assess the mRNA levels of PINK1, Parkin, p62, and LC3. ResultsCompared with the control group, the model group presented obvious gastric mucosal damage, colonization of a large number of Hp, severe mitochondrial damage, vacuolated structures due to excessive autophagy, reduced TOMM20 and TFAM co-expression in the gastric mucosal tissue, and reduced SOD and increased MDA (P<0.01). In addition, the gastric tissue in the model group showed up-regulated protein and mRNA levels of PINK1, Parkin, and LC3 and down-regulated protein and mRNA levels of p62 (P<0.01, as well as increased expression of inflammasome-associated proteins NLRP3, ASC, IL-1β, and IL-18 (P<0.01). Compared with the model group, the LPYJWF and quadruple therapy groups showed alleviated pathological damage of gastric mucosa, reduced Hp colonization, mitigated mitochondrial damage, and increased co-expression of TOMM20 and TFAM. The SOD level was elevated in the LPYJWF-L group (P<0.01), and the MDA levels became lowered in the LPYJWF and quadruple therapy groups (P<0.05, P<0.01). Furthermore, the LPYJWF and quadruple therapy groups showed down-regulated mRNA levels of PINK1, Parkin, and LC3 and protein levels of PINK1 and Parkin, and up-regulated mRNA level of p62 (P<0.01). The LPYJWF-M, LPYJWF-H, and quadruple therapy groups showcased down-regulated LC3 Ⅱ/LC3 Ⅰ level (P<0.05, P<0.01) and up-regulated protein level of p62 (P<0.01). The expression of inflammasome-associated proteins NLRP3, ASC, IL-1β, and IL-18 were reduced in the LPYJWF and quadruple therapy groups (P<0.05, P<0.01). ConclusionLPYJWF ameliorates gastric mucosal damage and exerts mucosa-protective effects in Hp-infected mice, which may be related to the inhibition of excessive mitochondrial autophagy, thereby inhibiting the activation of the NLRP3 inflammasome pathway.
9.Analysis of Brain Absorption Components and Their Distribution of Tianyuan Zhitong Prescription Based on UPLC-Q-TOF-MS and DESI-MSI
Yi CHENG ; Qun LAN ; Bingyu WU ; Jinyu WANG ; Dewen LIU ; Yan TONG
Chinese Journal of Experimental Traditional Medical Formulae 2024;30(12):166-172
ObjectiveTo investigate the brain absorption components of Tianyuan Zhitong prescription and their distribution based on ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry(UPLC-Q-TOF-MS), desorption electrospray ionization mass spectrometry imaging(DESI-MSI) and hyperspectral imaging techniques. MethodTen BALB/c mice were randomly divided into blank group(n=3) and administration group(n=7), the administration group was gavaged with 0.3 mL of Tianyuan Zhitong prescription liquid at a concentration of about 5 g·mL-1 of the raw material, and the blank group was gavaged with an equal volume of normal saline, and the whole brain of the mice were taken for the preparation of tissue homogenates and frozen sections, respectively. The tissue homogenates were qualitatively analyzed by UPLC-Q-TOF-MS for the brain absorption components in positive and negative ion modes, frozen sections were used for imaging to observe the distribution of these components in the brain. Cytoviva dark-field enhancement microscope was used to perform hyperspectral imaging scanning on the brain sections of mice from each group, and the scattered light data of at least 1 000 pixels in the visible-near-infrared(400-1 000 nm) band in the microscopic field of view were collected and average spectrum were created, which were used to compare the components in the brain tissues of mice from the blank and administration groups. ResultA total of 27 brain absorption components of Tianyuan Zhitong prescription were identified by UPLC-Q-TOF-MS, including 10 organic acids, 5 glycosides, 4 alkaloids, 1 phenol, 4 flavonoids, 2 phthalides and 1 other compound, which were mainly derived from Gastrodiae Rhizoma, Chuanxiong Rhizoma, vinegar-processed Corydalis Rhizoma, Ziziphi Spinosae Semen and processed Morindae Officinalis Radix. A total of 14 components were identified by mass spectrometry imaging, of which ferulic acid, tetrahydropalmatine and N-methyl dehydroberberine were mainly distributed in the cerebral cortex, vitamin B5, vemonoic acid and ricinoleic acid were mainly distributed in the hypothalamus, elemicin, octadecenic acid and octadecanoic acid were mainly distributed in the cortex and hypothalamus, while senkyunolide B, ligustilide, linoleic acid, 9,12-octadecadienoyl ethyl ester and spinosin were distributed in most regions of the brain tissues. Hyperspectral imaging showed that in the visible-near-infrared band range, the average spectrum of the brain tissues of mice in the administration group was significantly red-shifted, indicating that there were differences in the physical properties or contents of the chemical components in the brain between mice in the blank group and those in the administration group, and further verified the results of mass spectrometry imaging. ConclusionThrough the combination of UPLC-Q-TOF-MS and imaging techniques, the pharmacodynamic components of Tianyuan Zhitong prescription in the treatment of headache and the regional characteristics in brain tissue are clarified, which can provide reference for the selection of the index components of the research on the quality standard of this prescription and the research on the mechanism of the pharmacological effect.
10.Detection of Amantadine by Label-free Fluorescence Method Based on Truncated Aptamer and Molybdenum Disulfide Nanosheet Signal Enhancement Strategy
Yi-Feng LAN ; Bo-Ya HOU ; Zhi-Wen WEI ; Wen LIU ; Chao ZHANG ; Ya-Hui ZUO ; Ke-Ming YUN
Chinese Journal of Analytical Chemistry 2024;52(2):208-219,中插4-中插7
Amantadine(AMD)residue can accumulate in organisms through the food chain and cause serious harm to human body.AMD can specifically bind to AMD specific aptamer and cause its conformation to change from a random single strand to a stem-loop structure.To avoid the influence of excess nucleotides on binding of aptamer to AMD,the truncation of the AMD original aptamer J was optimized by retaining an appropriate stem-loop structure,and a new type of truncation aptamers was developed in this work.By comparing the truncated aptamer with the original aptamer,it was found that the truncated aptamer J-7 had better affinity and specificity with AMD.The detection limit of AMD was 0.11 ng/mL by using J-7 as specific recognition element and molybdenum disulfide nanosheet(MoS2Ns)as signal amplification element.The developed method base on truncated aptamer J-7 was used for detection of AMD in milk,yogurt and SD rat serum samples for the first time with recoveries of 86.6%-108.2%.This study provided a reference for truncating other long sequence aptamers and provided a more sensitive detection method for monitoring AMD residues in food.

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