1.Preparation,characterization,and in vitro antitumor activity of Gambogic acid-loaded intelligent responsive liposome-hydrogel nanopreparation
Yu CHEN ; Shengnan HUANG ; Ziang WANG ; Yunlong ZHAO ; Gaojian WEI ; Sinan WU ; Yanbin GUAN ; Xiali ZHU
China Pharmacy 2026;37(5):613-619
OBJECTIVE To prepare an intelligent responsive liposome-hydrogel nanopreparation co-loaded with gambogic acid (GA), and characterize its antitumor activity in vitro . METHODS GA-ICG-Lip-gel was prepared by ethanol injection and cold dissolution, incorporating GA and the photosensitizer indocyanine green (ICG). The appearance and microscopic morphology of GA-ICG-Lip-gel were observed, its encapsulation efficiency and drug loading capacity were measured, and its photothermal conversion performance, photothermal stability, and infrared imaging properties were investigated, along with the determination of its in vitro release profile. Human breast cancer MCF-7 cells were used as objects to investigate the effects of GA-ICG-Lip-gel (or with near-infrared light irradiation) on cell viability, migration ability, and the cellular uptake capacity of GA-ICG-Lip-gel. RESULTS GA-ICG-Lip-gel existed in a solution state at room temperature and transformed into a gel state at 37 ℃. Its microstructure was dense with small pores, and its encapsulation efficiency and drug loading were (96.07±0.86) % and (6.28±1.16) %, respectively. After exposure to near-infrared light, the temperature of GA-ICG-Lip-gel rose above 42 ℃, with no significant attenuation observed in the heating curve. The heating efficiency was dependent on both the irradiation time and drug concentration. Compared to media without gelatinase, the cumulative release rate of GA-ICG-Lip-gel increased in media containing gelatinase. In vitro studies showed that GA-ICG-Lip-gel could be efficiently taken up by MCF-7 cells; GA-ICG-Lip-gel significantly inhibited the viability and migration ability of MCF-7 cells ( P <0.05), and this inhibitory effect was further enhanced under near-infrared light irradiation. CONCLUSIONS This study successfully prepares GA-ICG-Lip-gel, which exhibits favorable photothermal conversion properties and temperature/enzyme dual-responsive drug release characteristics, and demonstrates significant inhibitory effects on the proliferation and migration of breast cancer cells.
2.Preparation of Triptolide-Chuanxiong Rhizoma Extract Ethanol Transfersomes and Analysis on Its in Vitro Anti-inflammatory Mechanism
Ling TAO ; Zhiyan WAN ; Yidan LIU ; Zhe LI ; Zhenzhong ZANG ; Weifeng ZHU ; Yongmei GUAN
Chinese Journal of Experimental Traditional Medical Formulae 2026;32(10):210-218
ObjectiveTo prepare triptolide-Chuanxiong Rhizoma extract ethanol transfersomes(TP-CX@TESs), conduct its quality evaluation, and investigate its in vitro anti-inflammatory efficacy and the underlying mechanisms. MethodsTP-CX@TESs was prepared via the ultrasonic injection method. With encapsulation efficiency and particle size as evaluation indicators, Box-Behnken design-response surface methodology(BBD-RSM) was employed to optimize the formulation process. The TP-CX@TESs prepared under the optimal process was characterized and evaluated for in vitro transdermal performance. A lipopolysaccharide(LPS)-induced RAW264.7 cell inflammation model was established. After 24 h of drug intervention, the levels of inflammatory factors such as nitric oxide(NO), interleukin-6(IL-6), and tumor necrosis factor-α(TNF-α) in the cell supernatant were detected. Western blot was used to determine the protein expression levels of Janus kinase 2(JAK2), signal transducer and activator of transcription 3(STAT3), and α7 nicotinic acetylcholine receptor(α7nAChR), and real-time fluorescence quantitative polymerase chain reaction(Real-time PCR) was applied to measure the mRNA expression levels of JAK2, STAT3, the encoding gene of α7nAChR(CHRNA7), and nuclear transcription factor-κB(NF-κB). ResultsResults of BBD-RSM showed that the optimal formulation for preparing TP-CX@TESs was as follows:egg yolk lecithin content of 2.3%, ethanol volume fraction of 30%, and ratio of polysorbate-80 to egg yolk lecithin of 2∶5. Microscopic characterization revealed that TP-CX@TESs exhibited a spherical-like structure with a particle size of (105.60±3.85) nm, a polydispersity index of 0.19±0.03, and a Zeta potential of (-15.89±0.98) mV. The encapsulation efficiencies of triptolide, ferulic acid, and ligustilide were (76.88±4.40)%, (78.84±4.40)%, and (65.88±0.06)%, respectively. Both in vitro release and transdermal penetration of triptolide, ferulic acid, and ligustilide in TP-CX@TESs all followed the first-order kinetic model, showing a certain sustained-release property. Experimental results in RAW264.7 cells indicated that TP-CX@TESs significantly inhibited the release of NO, TNF-α, and IL-6(P<0.01), remarkably upregulated the protein expression levels of STAT3 and α7nAChR(P<0.01), increased the mRNA expression level of CHRNA7, and significantly downregulated the mRNA expression level of NF-κB(P<0.05, P<0.01). ConclusionThe optimized formulation process of TP-CX@TESs is simple and feasible, along with favorable in vitro release property, good transdermal permeability, and excellent in vitro anti-inflammatory activity, the mechanism is related to the inhibition of NF-κB.
3.Preparation of Triptolide-Chuanxiong Rhizoma Extract Ethanol Transfersomes and Analysis on Its in Vitro Anti-inflammatory Mechanism
Ling TAO ; Zhiyan WAN ; Yidan LIU ; Zhe LI ; Zhenzhong ZANG ; Weifeng ZHU ; Yongmei GUAN
Chinese Journal of Experimental Traditional Medical Formulae 2026;32(10):210-218
ObjectiveTo prepare triptolide-Chuanxiong Rhizoma extract ethanol transfersomes(TP-CX@TESs), conduct its quality evaluation, and investigate its in vitro anti-inflammatory efficacy and the underlying mechanisms. MethodsTP-CX@TESs was prepared via the ultrasonic injection method. With encapsulation efficiency and particle size as evaluation indicators, Box-Behnken design-response surface methodology(BBD-RSM) was employed to optimize the formulation process. The TP-CX@TESs prepared under the optimal process was characterized and evaluated for in vitro transdermal performance. A lipopolysaccharide(LPS)-induced RAW264.7 cell inflammation model was established. After 24 h of drug intervention, the levels of inflammatory factors such as nitric oxide(NO), interleukin-6(IL-6), and tumor necrosis factor-α(TNF-α) in the cell supernatant were detected. Western blot was used to determine the protein expression levels of Janus kinase 2(JAK2), signal transducer and activator of transcription 3(STAT3), and α7 nicotinic acetylcholine receptor(α7nAChR), and real-time fluorescence quantitative polymerase chain reaction(Real-time PCR) was applied to measure the mRNA expression levels of JAK2, STAT3, the encoding gene of α7nAChR(CHRNA7), and nuclear transcription factor-κB(NF-κB). ResultsResults of BBD-RSM showed that the optimal formulation for preparing TP-CX@TESs was as follows:egg yolk lecithin content of 2.3%, ethanol volume fraction of 30%, and ratio of polysorbate-80 to egg yolk lecithin of 2∶5. Microscopic characterization revealed that TP-CX@TESs exhibited a spherical-like structure with a particle size of (105.60±3.85) nm, a polydispersity index of 0.19±0.03, and a Zeta potential of (-15.89±0.98) mV. The encapsulation efficiencies of triptolide, ferulic acid, and ligustilide were (76.88±4.40)%, (78.84±4.40)%, and (65.88±0.06)%, respectively. Both in vitro release and transdermal penetration of triptolide, ferulic acid, and ligustilide in TP-CX@TESs all followed the first-order kinetic model, showing a certain sustained-release property. Experimental results in RAW264.7 cells indicated that TP-CX@TESs significantly inhibited the release of NO, TNF-α, and IL-6(P<0.01), remarkably upregulated the protein expression levels of STAT3 and α7nAChR(P<0.01), increased the mRNA expression level of CHRNA7, and significantly downregulated the mRNA expression level of NF-κB(P<0.05, P<0.01). ConclusionThe optimized formulation process of TP-CX@TESs is simple and feasible, along with favorable in vitro release property, good transdermal permeability, and excellent in vitro anti-inflammatory activity, the mechanism is related to the inhibition of NF-κB.
4.Effect of fluoride exposure on endoplasmic reticulum-mitochondrial calcium transfer and apoptosis in primary nerve cells
Yongheng LU ; Shuang ZHU ; Feiyan ZHAO ; Fujun AI ; Yanjie LIU ; Yangting DONG ; Zhizhong GUAN ; Na WEI
Chinese Journal of Tissue Engineering Research 2026;30(1):111-119
BACKGROUND:Previous studies have found that neuronal damage caused by continuous excessive fluoride exposure is related to Ca2+overload,but the mechanism of Ca2+flow conversion between intracellular calcium stores and cell apoptosis damage is still unclear.OBJECTIVE:To investigate the effect of fluoride exposure on Ca2+transport channel proteins and apoptosis levels in the mitochondria-associated endoplasmic reticulum membrane of primary cultured neural cells.METHODS:Primary nerve cells of neonatal SD rats were cultured in vitro and identified by immunofluorescence staining with neuronal nucleus-specific antibody up to day 7.The nerve cells were divided into control group(containing 0 mmol/L sodium fluoride),low fluoride group(containing 0.5 mmol/L sodium fluoride),and high fluoride group(containing 1 mmol/L sodium fluoride).The cell morphological changes were observed by light microscope 24 hours after fluorine exposure.The expression levels of apoptosis-related protein BAX/BCL-2 and calcium transfer-related pathways VDAC1,GRP 75,and IP3R were detected using western blot assay.The expression levels of VDAC1,GRP 75,and IP3R mRNA were detected by RT-PCR.Ca2+levels were detected by Rhood-2AM Ca2+probe.Mitochondrial membrane potential detection kit was used to detect the change in mitochondrial membrane potential.The level of apoptosis was determined by flow cytometry and TUNEL staining.RESULTS AND CONCLUSION:(1)The purity of neurons cultured on day 7 had been determined to be over 90%,with few impurities,good growth status,and tight cell network connections,meeting the requirements of subsequent experiments.(2)Compared with the control group,growth of neural cell clusters in the low-fluoride group and the high-fluoride group increased;the processes were broken;the cell body was rounded,and the connection network between cells was destroyed.Compared with the low-fluoride group,the cell damage changes in the high-fluoride group were more obvious.(3)Compared with the control group,the protein expressions of VDAC1,GRP75,and IP3R were increased in the low-fluoride group and the high-fluoride group(P<0.05),and the ratio of apoptosis-related protein BAX/BCL-2 was increased(P<0.05).Compared with the control group,the expression of VDAC1 and GRP75 mRNA in the low-fluoride group was significantly increased(P<0.05);the expression levels of VDAC1,GRP75,and IP3R mRNA in the high-fluoride group were significantly increased(P<0.01).(4)The level of cell apoptosis increased significantly after fluoride exposure,and the high-fluoride group was significantly higher than the control and low-fluoride groups(P<0.01).(5)After fluoride exposure,the concentration of mitochondrial Ca2+in nerve cells increased significantly(P<0.05),the mitochondrial membrane potential decreased(P<0.01),and the degree of damage in the high-fluoride group was more obvious(P<0.05).The results show that fluoride exposure impairs the morphological structure of primary neural cells,resulting in upregulation of Ca2+transfer pathway protein expression between the endoplasmic reticulum and mitochondria,mitochondrial Ca2+overload,mitochondrial damage,and increased levels of apoptosis.
5.Effect of fluoride exposure on endoplasmic reticulum-mitochondrial calcium transfer and apoptosis in primary nerve cells
Yongheng LU ; Shuang ZHU ; Feiyan ZHAO ; Fujun AI ; Yanjie LIU ; Yangting DONG ; Zhizhong GUAN ; Na WEI
Chinese Journal of Tissue Engineering Research 2026;30(1):111-119
BACKGROUND:Previous studies have found that neuronal damage caused by continuous excessive fluoride exposure is related to Ca2+overload,but the mechanism of Ca2+flow conversion between intracellular calcium stores and cell apoptosis damage is still unclear.OBJECTIVE:To investigate the effect of fluoride exposure on Ca2+transport channel proteins and apoptosis levels in the mitochondria-associated endoplasmic reticulum membrane of primary cultured neural cells.METHODS:Primary nerve cells of neonatal SD rats were cultured in vitro and identified by immunofluorescence staining with neuronal nucleus-specific antibody up to day 7.The nerve cells were divided into control group(containing 0 mmol/L sodium fluoride),low fluoride group(containing 0.5 mmol/L sodium fluoride),and high fluoride group(containing 1 mmol/L sodium fluoride).The cell morphological changes were observed by light microscope 24 hours after fluorine exposure.The expression levels of apoptosis-related protein BAX/BCL-2 and calcium transfer-related pathways VDAC1,GRP 75,and IP3R were detected using western blot assay.The expression levels of VDAC1,GRP 75,and IP3R mRNA were detected by RT-PCR.Ca2+levels were detected by Rhood-2AM Ca2+probe.Mitochondrial membrane potential detection kit was used to detect the change in mitochondrial membrane potential.The level of apoptosis was determined by flow cytometry and TUNEL staining.RESULTS AND CONCLUSION:(1)The purity of neurons cultured on day 7 had been determined to be over 90%,with few impurities,good growth status,and tight cell network connections,meeting the requirements of subsequent experiments.(2)Compared with the control group,growth of neural cell clusters in the low-fluoride group and the high-fluoride group increased;the processes were broken;the cell body was rounded,and the connection network between cells was destroyed.Compared with the low-fluoride group,the cell damage changes in the high-fluoride group were more obvious.(3)Compared with the control group,the protein expressions of VDAC1,GRP75,and IP3R were increased in the low-fluoride group and the high-fluoride group(P<0.05),and the ratio of apoptosis-related protein BAX/BCL-2 was increased(P<0.05).Compared with the control group,the expression of VDAC1 and GRP75 mRNA in the low-fluoride group was significantly increased(P<0.05);the expression levels of VDAC1,GRP75,and IP3R mRNA in the high-fluoride group were significantly increased(P<0.01).(4)The level of cell apoptosis increased significantly after fluoride exposure,and the high-fluoride group was significantly higher than the control and low-fluoride groups(P<0.01).(5)After fluoride exposure,the concentration of mitochondrial Ca2+in nerve cells increased significantly(P<0.05),the mitochondrial membrane potential decreased(P<0.01),and the degree of damage in the high-fluoride group was more obvious(P<0.05).The results show that fluoride exposure impairs the morphological structure of primary neural cells,resulting in upregulation of Ca2+transfer pathway protein expression between the endoplasmic reticulum and mitochondria,mitochondrial Ca2+overload,mitochondrial damage,and increased levels of apoptosis.
6.Impact of hepatic steatosis on viral load and hepatic fibrosis progression in chronic hepatitis B
Shi YIN ; Lianxin ZHU ; Guanghui REN ; Guiqun HUANG ; Yunpeng GUAN ; Ying ZHU
Journal of Clinical Hepatology 2026;42(6):1279-1286
ObjectiveTo investigate the association of hepatic steatosis with the level of viral replication and the progression of hepatic fibrosis in patients with chronic hepatitis B (CHB) through a real-world study, and to determine the independent influencing factors for hepatic fibrosis. MethodsA total of 887 CHB patients who attended the outpatient service and inpatient ward of Hepatology in The First Affiliated Hospital of Dalian Medical University from July 2021 to March 2025 were enrolled as subjects, and according to the presence or absence of metabolic dysfunction-associated steatotic liver disease (MASLD), they were divided into CHB group with 560 patients and CHB+MASLD group with 327 patients. The association between hepatic steatosis and HBV load in CHB patients was analyzed, as well as the impact of varying degrees of hepatic steatosis on hepatic fibrosis. The t-test or the Mann-Whitney U test was used for comparison of continuous data between groups; the chi-square test or the Fisher’s exact test was used for comparison of categorical data between groups; a Spearman correlation analysis was performed; a Logistic regression analysis was used to investigate influencing factors. ResultsThe patients enrolled received antiviral therapy according to viral load; of all patients, 275 (31%) received nucleos(t)ide analogue combined with pegylated interferon-α, 532 (60%) received nucleos(t)ide analogue monotherapy, and 80 (9%) did not receive antiviral therapy. A total of 44 patients (5.0%) who were receiving PEG-IFN-α therapy and had marked thrombocytopenia were excluded, and finally 843 patients were included for comparison of aspartate aminotransferase-to-platelet ratio index (APRI) and fibrosis-4 index (FIB-4). Compared with the CHB group, the CHB+MASLD group had a significantly higher proportion of male patients (χ2=5.917, P<0.05) and a significant increase in CAP value (t=21.646, P<0.05). The median values of liver function parameters for the population enrolled were within the normal range, and no significant inflammatory activity was observed. The virological analysis showed that compared with the CHB group, the CHB+MASLD group had significantly lower serum level of pregenomic RNA (Z=-2.894, P<0.05) and HBeAg positivity rate (χ2=8.725, P<0.05), and CAP was significantly negatively correlated with pgRNA (r=-0.117, P<0.05). In the HBeAg-negative subgroup, compared with the CHB group, the CHB+MASLD group had a significantly lower level of hepatitis B surface antigen (Z=-0.765, P<0.05); in the HBeAg-positive subgroup, the CHB+MASLD group had a significantly higher level of HBV DNA than the CHB group (Z=-2.509, P<0.05). The analysis of hepatic fibrosis showed that the CHB+MASLD group had significantly lower values of APRI (Z=-3.418, P<0.05) and FIB-4 (Z=-6.237, P<0.05). Compared with the CHB group, the CHB+MASLD S1 group had a significantly higher proportion of patients without fibrosis and a significantly lower proportion patients with liver cirrhosis (χ2=7.935, P<0.05); in the CHB+MASLD S2 group, there was no significant difference in the proportion of patients with different fibrosis stages; the CHB+MASLD S3 group had a significantly lower proportion of patients without fibrosis and a significantly higher proportion of patients with early-stage fibrosis (χ2=9.101, P<0.05). The Logistic regression analysis showed that an increase in CAP (odds ratio [OR]=1.08, 95% confidence interval [CI]: 1.03 — 1.13, P<0.05), an increase in age (OR=1.02, 95%CI: 1.01 — 1.04, P<0.05), male sex (OR=1.77, 95%CI: 1.19 — 2.64, P<0.05), an increase in HBV DNA (OR=1.35, 95%CI: 1.15 — 1.57, P<0.001), an increase in aspartate aminotransferase (OR=1.02, 95%CI: 1.01 — 1.03, P<0.001), and an increase in gamma-glutamyl transpeptidase (OR=1.00, 95%CI: 1.00 — 1.01, P=0.028) were independent influencing factors for hepatic fibrosis. The risk of hepatic fibrosis in S3 patients was higher than that in patients without steatosis (OR=5.05, 95%CI: 2.48 — 10.29, P<0.001). ConclusionThere is a lower level of HBV replication in CHB patients comorbid with MASLD. Hepatic steatosis is associated with HBV-related virological markers and the progression of hepatic fibrosis, and severe fatty liver disease may promote the progression of hepatic fibrosis in CHB patients.
7.Structural and Spatial Analysis of The Recognition Relationship Between Influenza A Virus Neuraminidase Antigenic Epitopes and Antibodies
Zheng ZHU ; Zheng-Shan CHEN ; Guan-Ying ZHANG ; Ting FANG ; Pu FAN ; Lei BI ; Yue CUI ; Ze-Ya LI ; Chun-Yi SU ; Xiang-Yang CHI ; Chang-Ming YU
Progress in Biochemistry and Biophysics 2025;52(4):957-969
ObjectiveThis study leverages structural data from antigen-antibody complexes of the influenza A virus neuraminidase (NA) protein to investigate the spatial recognition relationship between the antigenic epitopes and antibody paratopes. MethodsStructural data on NA protein antigen-antibody complexes were comprehensively collected from the SAbDab database, and processed to obtain the amino acid sequences and spatial distribution information on antigenic epitopes and corresponding antibody paratopes. Statistical analysis was conducted on the antibody sequences, frequency of use of genes, amino acid preferences, and the lengths of complementarity determining regions (CDR). Epitope hotspots for antibody binding were analyzed, and the spatial structural similarity of antibody paratopes was calculated and subjected to clustering, which allowed for a comprehensively exploration of the spatial recognition relationship between antigenic epitopes and antibodies. The specificity of antibodies targeting different antigenic epitope clusters was further validated through bio-layer interferometry (BLI) experiments. ResultsThe collected data revealed that the antigen-antibody complex structure data of influenza A virus NA protein in SAbDab database were mainly from H3N2, H7N9 and H1N1 subtypes. The hotspot regions of antigen epitopes were primarily located around the catalytic active site. The antibodies used for structural analysis were primarily derived from human and murine sources. Among murine antibodies, the most frequently used V-J gene combination was IGHV1-12*01/IGHJ2*01, while for human antibodies, the most common combination was IGHV1-69*01/IGHJ6*01. There were significant differences in the lengths and usage preferences of heavy chain CDR amino acids between antibodies that bind within the catalytic active site and those that bind to regions outside the catalytic active site. The results revealed that structurally similar antibodies could recognize the same epitopes, indicating a specific spatial recognition between antibody and antigen epitopes. Structural overlap in the binding regions was observed for antibodies with similar paratope structures, and the competitive binding of these antibodies to the epitope was confirmed through BLI experiments. ConclusionThe antigen epitopes of NA protein mainly ditributed around the catalytic active site and its surrounding loops. Spatial complementarity and electrostatic interactions play crucial roles in the recognition and binding of antibodies to antigenic epitopes in the catalytic region. There existed a spatial recognition relationship between antigens and antibodies that was independent of the uniqueness of antibody sequences, which means that antibodies with different sequences could potentially form similar local spatial structures and recognize the same epitopes.
8.Effect of Different Fermentation Conditions on Fungal Community and Chemical Composition of Aurantii Fructus
Zhihong YAN ; Xiumei LIU ; Qiuyan GUAN ; Yonggui SONG ; Zhifu AI ; Genhua ZHU ; Yuhui PING ; Ming YANG ; Qin ZHENG ; Huanhua XU ; Dan SU
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(11):254-262
ObjectiveTo investigate the effects of different fermentation methods and times on the fungal flora and chemical composition of Aurantii Fructus, in order to obtain the optimal fermentation conditions and flora structure, and to ensure the stability and controllability of the fermented varieties. MethodsScanning electron microscopy was used to observe and analyze the colony characteristics on the surface of Aurantii Fructus under different fermentation conditions. Internal transcribed spacer 2(ITS2) high-throughput sequencing, combined with fungal community diversity analysis and fungal community structure analysis, were used to obtain the fungal flora microbial categories of Aurantii Fructus under the conditions of traditional pressure-shelf fermentation and non-pressure-shelf natural fermentation for 7, 14, 21 d(numbered Y1-Y3 for the former, and numbered F1-F3 for the latter), respectively. At the same time, the chemical components in the fermentation process were detected by ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry(UPLC-Q-TOF-MS/MS), combined with principal component analysis(PCA), partial least squares-discriminant analysis(PLS-DA) and compound retention time, parent ions, characteristic fragment ions and other information, the differential compounds between the different fermentation samples were screened and identified. ResultsThe analysis of fungal community diversity showed that the dominant flora did not change at different fermentation time points in the traditional pressure-shelf fermentation method, while in the non-pressure-shelf natural fermentation method, there was a significant difference with the fermentation process, and at the genus level, the dominant genus of samples Y1, Y2, Y3 and F2 was Aspergillus, while the dominant genera of samples F1 and F3 were both Rhizopus. This indicated that the microbial growth environment provided by the traditional fermentation method was more stable, and the microbial community structure was more stable, which was more conducive to the stable and controllable fermentation process and fermented products. A total of 155 compounds were identified by compositional analysis, including 70 flavonoids, 38 coumarins, 10 alkaloids, 34 organic acids and 3 other compounds. After fermentation, two new components of ribalinine and pranferin were produced. Different fermentation conditions also brought about differences in chemical composition, multivariate statistical analysis obtained 26 differential compounds under two different fermentation methods, mainly including flavonoids, organic acids and coumarins. Comprehensively, the microbial community structure of samples fermented by the traditional pressure-shelf method of Aurantii Fructus for 14 d was stable, the species richness was high and the overall content of differential compounds was high, which was the optimal processing condition. ConclusionCompared with non-pressure-shelf natural fermentation, the traditional method has obvious advantages in terms of the stability of the microbial community structure and the content of chemical compounds, and the optimal condition is 14 days of fermentation. This study is helpful to promote the quality stability and fermentation bioavailability of fermented products of Aurantii Fructus, as well as to provide an experimental basis for the further improvement of the quality control methods of this variety.
9.Effect of Different Fermentation Conditions on Fungal Community and Chemical Composition of Aurantii Fructus
Zhihong YAN ; Xiumei LIU ; Qiuyan GUAN ; Yonggui SONG ; Zhifu AI ; Genhua ZHU ; Yuhui PING ; Ming YANG ; Qin ZHENG ; Huanhua XU ; Dan SU
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(11):254-262
ObjectiveTo investigate the effects of different fermentation methods and times on the fungal flora and chemical composition of Aurantii Fructus, in order to obtain the optimal fermentation conditions and flora structure, and to ensure the stability and controllability of the fermented varieties. MethodsScanning electron microscopy was used to observe and analyze the colony characteristics on the surface of Aurantii Fructus under different fermentation conditions. Internal transcribed spacer 2(ITS2) high-throughput sequencing, combined with fungal community diversity analysis and fungal community structure analysis, were used to obtain the fungal flora microbial categories of Aurantii Fructus under the conditions of traditional pressure-shelf fermentation and non-pressure-shelf natural fermentation for 7, 14, 21 d(numbered Y1-Y3 for the former, and numbered F1-F3 for the latter), respectively. At the same time, the chemical components in the fermentation process were detected by ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry(UPLC-Q-TOF-MS/MS), combined with principal component analysis(PCA), partial least squares-discriminant analysis(PLS-DA) and compound retention time, parent ions, characteristic fragment ions and other information, the differential compounds between the different fermentation samples were screened and identified. ResultsThe analysis of fungal community diversity showed that the dominant flora did not change at different fermentation time points in the traditional pressure-shelf fermentation method, while in the non-pressure-shelf natural fermentation method, there was a significant difference with the fermentation process, and at the genus level, the dominant genus of samples Y1, Y2, Y3 and F2 was Aspergillus, while the dominant genera of samples F1 and F3 were both Rhizopus. This indicated that the microbial growth environment provided by the traditional fermentation method was more stable, and the microbial community structure was more stable, which was more conducive to the stable and controllable fermentation process and fermented products. A total of 155 compounds were identified by compositional analysis, including 70 flavonoids, 38 coumarins, 10 alkaloids, 34 organic acids and 3 other compounds. After fermentation, two new components of ribalinine and pranferin were produced. Different fermentation conditions also brought about differences in chemical composition, multivariate statistical analysis obtained 26 differential compounds under two different fermentation methods, mainly including flavonoids, organic acids and coumarins. Comprehensively, the microbial community structure of samples fermented by the traditional pressure-shelf method of Aurantii Fructus for 14 d was stable, the species richness was high and the overall content of differential compounds was high, which was the optimal processing condition. ConclusionCompared with non-pressure-shelf natural fermentation, the traditional method has obvious advantages in terms of the stability of the microbial community structure and the content of chemical compounds, and the optimal condition is 14 days of fermentation. This study is helpful to promote the quality stability and fermentation bioavailability of fermented products of Aurantii Fructus, as well as to provide an experimental basis for the further improvement of the quality control methods of this variety.
10.Clinical Research on Guizhi Fulingwan in Treatment of Ovarian Cancer: A Review
Muxin GUAN ; Jiaxing FENG ; Mengyi ZHU ; Yu WANG ; Xiaoke WU
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(12):296-303
Ovarian cancer (OC) is a common gynecological malignant tumor in clinical practice. In the early stage,it is often asymptomatic,while in the late stage,it mainly presents with non-specific symptoms such as abdominal distension,poor appetite,and dull abdominal pain. Some patients may also have cachexia such as weight loss and anemia. Early diagnosis is difficult,and the mortality rate ranks first among gynecological malignant tumors,making OC a major challenge in clinical treatment. The classic Chinese medicine formula Guizhi Fulingwan comes from the Jingui Yaolue and has the effects of promoting blood circulation,removing blood stasis,and reducing abdominal lumps. In recent years,it has been widely used to treat OC with good results. This article summarized the clinical application of Guizhi Fulingwan in the treatment of OC from two aspects:The analysis of its basic prescriptions and clinical research. In terms of basic prescriptions,the formula has the ability to promote blood circulation,remove blood stasis,and reduce abdominal lumps. It can exert therapeutic effects considering both water and blood aspects and reduce abdominal lumps, with characteristics of simultaneous Yang warming and heat clearing and parallel supplementation and elimination. Through the methods of "circulation" and "supplementation", it strengthens the body,dispels evil,and eliminates underlying symptoms. In clinical studies,Guizhi Fulingwan can be applied to various stages of patients with OC,which not only promotes the recovery of the body after OC surgery but also can be combined with chemotherapy and immunotherapy to synergistically treat advanced OC and enhance treatment efficacy. In addition,the formula can also alleviate various adverse reactions caused by chemotherapy,with high safety,improve patients' quality of life,prolong survival,and optimize tumor control effects. Based on the above analysis,this article elaborated on the current clinical research status of Guizhi Fulingwan combined with Western medicine in the treatment of OC and proposed suggestions and improvements to address the shortcomings in current clinical research,so as to provide reference for the clinical application of this formula in the treatment of OC and the construction of a combined traditional Chinese and Western medicine treatment model.

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