1.Aspirin alleviates OGD/R-induced injury in mouse hippocampal neurons by regulating ferroptosis
Yujiao HU ; Shan CONG ; Lei ZHAO ; Chunxue DONG ; Dongmei WANG ; Nannan WANG ; Ying MAO
Chinese Journal of Geriatric Heart Brain and Vessel Diseases 2024;26(8):960-964
Objective To investigate the effect of aspirin on oxygen glucose deprivation/reoxygen-ation(OGD/R)-induced injury in mouse hippocampal neuron HT22 cells by regulating ferropto-sis.Methods HT22 cells were randomly divided into control group,model group,low-,medium-and high-dose groups(n=3).Cellular OGD/R injury model was established in the other 4 groups except the control group.Aspirin of 100,200 and 400 μg/ml was used to treat the cells from the above 3 treatment groups,respectively.Cell viability was detected by CCK-8 assay.The contents of TNF-α,IL-1β and IL-6 were detected by ELISA.The contents of SOD,catalase,glutathione,reac-tive oxygen species(ROS),lactate dehydrogenase(LDH),Fe2+and MDA were detected by the corresponding reagent kits.Western blot analysis was performed to determine the expression of solute carrier family 7 members 11(SLC7A11),glutathione peroxidase 4(GPX4)and Acyl-CoA synthase long chain member 4(ACSL4).Results The model group had significantly lower cell vi-ability than the control group(0.49±0.07 vs 1.00±0.12,P<0.01),but the viability of the low-,medium-and high-dose groups were higher than that of the model group(0.72±0.10 vs 0.49±0.07,P<0.05;0.87±0.10 vs 0.49±0.07,P<0.01;0.93±0.07 vs 0.49±0.07,P<0.01).Compared with the control group,the contents of TNF-α,IL-1β,IL-6,ROS,LDH,Fe2 and MDA and the protein expression of ACSL4 were significantly increased,while the contents of SOD,catalase,glutathione and protein levels of SLC7A11 and GPX4 were obviously decreased in the model group(P<0.01).Compared with model group,aspirin treatment reversed all above indicators no matter its doses(P<0.05,P<0.01).Conclusion Aspirin alleviates OGD/R-induced neuronal in-jury through regulating ferroptosis in mouse neuron HT22 cells in a dose-dependent manner.
2.Exploration on the pharmacological basis of Lycopi Herba as alternative of Alismatis Rhizoma for the treatment of heart failure based on network pharmacology and molecular docking techniques
Siyu LIU ; Yujiao SHI ; Yongcheng LIU ; Xiaoyu LIANG ; Chenguang YANG ; Wenbo QIAO ; Guoju DONG
International Journal of Traditional Chinese Medicine 2024;46(8):1045-1052
Objective:To investigate whether Lycopi Herba can serve as a viable alternative to Alismatis Rhizoma in the treatment of heart failure (HF) through network pharmacology and molecular docking techniques.Methods:TCMSP database was used to filter active components of Lycopi Herba and Alismatis Rhizoma. SwissTargetPrediction database was used to predict potential targets. HF-related targets were collected from databases such as GeneCards, OMIM, and DisGeNET. Venny 2.1.0 was used to draw a Venn diagram illustrating the intersection of targets between Lycopi Herba and Alismatis Rhizoma and HF. A protein-protein interaction (PPI) network was established using the String database, and key targets for the treatment of HF with Lycopi Herba and Alismatis Rhizoma were selected using Cytoscape 3.9.1 software to construct a component-intersection target network. The intersection targets were then analyzed for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways using Metascape. Molecular docking techniques were used to evaluate the affinity between active components and key targets.Results:Lycopi Herba primarily targeted pivotal proteins such as HMGCR and CYP27B1, while Alismatis Rhizoma had a broader target spectrum, including PPARA, JAK2, among others. Shared key targets between the two included HMGCR and ESR1, which were primarily involved in cholesterol synthesis and steroid hormone biosynthesis. Enrichment pathway analysis showed similarities in steroid metabolism between the two; Alismatis Rhizoma, however, was more likely to act through protein phosphorylation regulation and modulating the PI3K-Akt signaling pathway for HF treatment. A unique target for Lycopi Herba in treating HF was CHRM4, indicating its potential for blood pressure regulation and myocardial protection.Conclusions:Both Lycopi Herba and Alismatis Rhizoma exhibit certain commonalities in the treatment of HF, but Alismatis Rhizoma has a wider range of targets and signaling pathways, implying more extensive therapeutic potential. However, considering the nephrotoxicity of Alismatis Rhizoma, Lycopi Herba could be considered as an alternative treatment for HF, especially in patients with renal insufficiency or in the early stages of HF.
3.Effect of ganoderic acid A on glycolysis and its key rate-limiting enzymes of non-small cell lung cancer PC9 cells
Aihua REN ; Yanbo DONG ; Runzhi MIAO ; Yujiao LYU ; Yanfeng LIU
Journal of Jilin University(Medicine Edition) 2024;50(3):682-688
Objective:To discuss the effects of different doses of ganoderic acid A(GAA)on the biological activities,glycolysis,and the expression of rate-limiting enzymes of non-small cell lung cancer(NSCLC)PC9 cells,and to clarify the mechanism.Methods:The NSCLC PC9 cells were cultured in vitro and divided into blank control group,low dose(25 μmol·L-1)of GAA group,medium dose(50 μmol·L-1)of GAA group,and high dose(100 μmol·L-1)of GAA group.The methylthiazolydiphenyltetrazolium(MTT)assay was used to detect the survival rates of the PC9 cells in various groups;Transwell chamber assay was used to detect the number of migration cells of the PC9 cells in various groups;the glucose uptakes of the PC9 cells in various groups were detected by glucose assay kit;the levels of ATP in the PC9 cells in various groups were detected by ATP assay kit;the levels of lactic acid in the PC9 cells in various groups were detected by lactate assay kit;the expression levels of hexokinase 2(HK2)and pyruvate kinase M2(PKM2)mRNA in the PC9 cells in various groups were detected by real-time fluorescence quantitative PCR(RT-qPCR)method;the expression levels of HK2 and PKM2 proteins in the PC9 cells in various groups were detected by Western blotting method.Results:The MTT assay results showed that,at 24,48,and 72 h of culture,compared with blank control group,the survival rates of the cells in medium and high doses of GAA groups were significantly decreased(P<0.05).At 72 h of culture,compared with low dose of GAA group,the survival rate of the cells in high dose of GAA group was significantly decreased(P<0.05).The Transwell chamber assay results showed that compared with blank control group,the numbers of migration cells in medium and high doses of GAA groups were significantly decreased(P<0.05);compared with low doses of GAA group,the number of migration cells in high dose of GAA group was significantly decreased(P<0.05).Compared with blank control group,the glucose uptakes and levels of lactic acid in the cells in medium and high dose of GAA groups were significantly decreased(P<0.05),and the level of ATP in the cells in high dose of GAA group was significantly decreased(P<0.05).The RT-qPCR results showed that compared with blank control group,the expression levels of HK2 and PKM2 mRNA in the cells in medium and high doses of GAA groups were significantly decreased(P<0.05).The Western blotting results showed that compared with blank control group,the expression levels of HK2 and PKM2 proteins in the cells in medium and high doses of GAA groups were significantly decreased(P<0.05).Conclusion:Medium and high doses of GAA can inhibit the biological activities of proliferation and migration of the PC9 cells,reduce the glycolysis,and its mechanism may be related to the inhibition of the expressions of the key rate-limiting enzymes HK2 and PKM2.
4.Establishment and evaluation of a rat model of heart failure with a preserved ejection fraction induced by combined factors
Yujiao SHI ; Chenguang YANG ; Wenbo QIAO ; Yongcheng LIU ; Siyu LIU ; Guoju DONG
Acta Laboratorium Animalis Scientia Sinica 2024;32(3):275-285
Objective To evaluate the characteristics of a rat model of heart failure with a preserved ejection fraction(HFpEF)induced by combined factors,and to investigate the correlation of myocardial strain parameters to myocardial hypertrophy and fibrosis.Methods Eight WKY rats and eight spontaneously hypertensive rats(SHR)served as control groups and were fed normal feed until the end of the experiment.Thirty-two SHR rats were equally divided into SHR+S,SHR+F,SHR+SF,and SHR+Combined groups,and fed high-salt,high-fat,high-salt-fat,or high-salt-fat-sugar feed,respectively,in combination with intraperitoneal injection of streptozotocin for 30 weeks.After modeling,the heart weight/body weight(HW/BW)ratio,systolic blood pressure(SBP),and diastolic blood pressure(DBP)were measured.Echocardiography was performed to measure the left ventricular(LV)end-diastolic internal diameter(LVIDd),LV anterior wall thickness(LVAWd),LV posterior wall thickness(LVPWd),LV ejection fraction(LVEF),isovolumetric diastolic time(IVRT),and peak early diastolic passive filling velocity(E)/early diastolic mitral annular velocity(e').Speckle tracking echocardiography was conducted to determine the global longitudinal strain(GLS)and strain rate(GLSr),global radial strain(GRS)and strain rate(GRSr),as well as the global circumferential strain(GCS)and strain rate(GCSr).Serum was collected and analyzed for triglycerides(TG),total cholesterol(TC),low-density lipoprotein cholesterol(LDL-C),glucose(GLU),and glycated serum protein(GSP).ELISA were used to measure serum B-type brain natriuretic peptide(BNP),angiotensin Ⅱ(AngⅡ),and galectin-3(Gal-3).Myocardial tissue was subjected to HE and Masson staining for cardiomyocytes and myocardial fibrosis,and the cardiomyocyte cross-sectional area(CSA)and collagen volume fraction(CVF)were calculated.Additionally,the correlation of myocardial strain parameters to CSA and CVF was analyzed.Results Compared with the control group,in model groups,especially the SHR+combined group,HW/BW,SBP,DBP,serum indexes(TC,TG,LDL-C,GLU,GSP,BNP,AngⅡ,and Gal-3)and echocardiographic parameters(LVIDd,LVAWd,LVPWd,IVRT,and E/e')were significantly up-regulated.Absolute values of speckle-tracking echocardiographic parameters(GLS,GLSr,GRS,GRSr,GCS,and GCSr)were decreased considerably.HE and Masson staining of myocardial tissues suggested marked cardiomyocyte hypertrophy and fibrosis,and significant increases were observed in CSA and CVF(P<0.05).Correlation analysis showed that GLSr,GCS,and GCSr were strongly linked to CSA,and GLS,GLSr,and GCSr were strongly linked to CVF(P<0.01).Conclusions A rat model of HFpEF induced by hypertension and dysregulation of glucolipid metabolism replicated the basic characteristics of HFpEF in terms of etiology,clinical features,and myocardial pathological changes,and might be a reliable animal model of metabolic syndrome-related HFpEF.Moreover,myocardial strain indices were closely related to myocardial hypertrophy and fibrosis and might indirectly reflect subtle myocardial lesions and dysfunction.
5.A pilote study of Chaiqi Ningshen Anmian Decoction combined with wrist ankle acupuncture in chronic insomnia patients with heart and spleen deficiency
Yujiao LI ; Shan REN ; Zhizhi DONG ; Na DUAN ; Wenwen TIAN ; Zhigang CHEN
China Pharmacist 2024;27(6):1055-1062
Objective To explore the clinical efficacy of Chaiqi Ningshen Anmian Decoction(CNAD)combined with wrist ankle acupuncture(WAA)in treating chronic insomnia(CI)patients with heart and spleen deficiency.Methods CI patients diagnosed and treated at the Beijing Huairou Hospital of Traditional Chinese Medicine from April 2022 to April 2023 were selected,and patients were randomly divided into the Eszolam(ET)group and the combination group(CNAD combined with WAA)according to the random number table method,with 50 cases in each group.The primary outcome was clinical efficacy[evaluated by the Traditional Chinese Medicine Syndrome Integral(TCMSI)].Secondary outcomes included changes in cognitive function[assessed by the Montreal Cognitive Assessment(MoCA)],anxiety level[assessed by the Hamilton Anxiety Scale(HAMA)],sleep quality[assessed by the Pittsburgh Sleep Quality Index(PSQI)],glycated serum albumin(GA),serotonin(5-HT),interleukin-1 β(IL-1β),and C-reactive protein(CRP)before and after treatment in both groups.Results Before treatment,there was no significant differences in total TCMSI between the two groups(P>0.05).After treatment,the total TCMSI of the combination group was significantly lower than that of the ET group(P<0.05),and the treatment effectiveness rate was significantly higher in the combination group(P<0.05).Before treatment,there were no significant differences in MoCA score,HAMA score,PSQI score,serum GA level,serum 5-HT level,serum IL-1 level and serum CRP level between the groups(P>0.05).After treatment,the MoCA score,serum GA level,and serum 5-HT level in the combination group were significantly higher than those in the ET group(P<0.05),while the HAMA score,PSQI score,serum IL-1β level,and serum CRP level were significantly lower in the combination group(P<0.05).Conclusion Compared with ET,CNAD combined with WAA significantly improves insomnia symptoms in CI patients with heart and spleen deficiency,enhances cognitive function and sleep quality,and reduces anxiety levels.This may be related to the upregulation of serum 5-HT,IL-1 β and the inhibition of the inflammatory response.
6.Material Basis and Molecular Mechanism of Linggui Qihua Prescription Against Myocardial Fibrosis in Heart Failure with Preserved Ejection Fraction
Yujiao SHI ; Lin YANG ; Chunqiu LIU ; Chenguang YANG ; Wenbo QIAO ; Yongcheng LIU ; Siyu LIU ; Jiangang LIU ; Guoju DONG
Chinese Journal of Experimental Traditional Medical Formulae 2023;29(20):20-29
ObjectiveTo explore the material basis and molecular mechanism of Linggui Qihua prescription (LGQH) against myocardial fibrosis in heart failure with preserved ejection fraction (HFpEF). MethodLiquid chromatography-mass spectrometry (LC-MS) was used to qualitatively analyze the active components of LGQH. AutoDock software was employed for molecular docking between the active components of LGQH and target proteins including α-smooth muscle actin (α-SMA), type Ⅰ collagen (ColⅠ), type Ⅲ collagen (ColⅢ), matrix metalloproteinase-9 (MMP-9), and tissue inhibitor of metalloproteinase-1 (TIMP-1). In vivo experiments were conducted on 40 spontaneously hypertensive rats (SHRs) aged 4 weeks, which were divided into an HFpEF group, an Entresto group (0.018 g·kg-1), and low- and high-dose LGQH groups (3.87, 7.74 g·kg-1). A high-fat, high-salt, and high-sugar diet was administered for 16 weeks along with intraperitoneal injection of streptozotocin solution for 8 weeks to establish an HFpEF model in rats. The blank group consisted of 10 Wistar Kyoto (WKY) rats and 10 SHRs. After successful modeling, the WKY, SHR, and HFpEF groups were given equal volumes of normal saline, while the other three groups received predetermined interventions. Daily oral gavage was performed for 6 weeks. After intervention, echocardiography was conducted to measure left ventricular (LV) anterior wall thickness (LVAWd), LV posterior wall thickness (LVPWd), LV internal diameter at end-diastole (LVIDd), LV ejection fraction (LVEF), isovolumic relaxation time (IVRT), early diastolic peak velocity of mitral valve inflow (E), and early diastolic mitral annular velocity (e'). The E/e' ratio was calculated. Enzyme-linked immunosorbent assay (ELISA) was used to detect serum atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP), and galectin-3 (Gal-3). Myocardial fibrosis was observed through Masson staining of pathological sections, and collagen volume fraction (CVF) and perivascular fibrosis ratio (PFR) were calculated. Real-time polymerase chain reaction (PCR) and Western blot were employed to detect LV myocardial mRNA and protein expression of α-SMA, ColⅠ, ColⅢ, MMP-9, and TIMP-1. ResultLC-MS identified 13 active components in LGQH. Molecular docking indicated stable binding of the 13 compounds with five target proteins. In vivo experiments showed that compared with the blank group, the HFpEF group had significantly increased LVAWd, LVPWd, LVIDd, IVRT, E/e', ANP, BNP, Gal-3, CVF, and PFR. LV myocardial α-SMA, ColⅠ, and ColⅢ mRNA and protein expression was significantly upregulated, while MMP-9/TIMP-1 mRNA and protein ratios were significantly downregulated (P<0.05, P<0.01). Compared with the HFpEF group, LGQH might dose-dependently reduce LVAWd, LVPWd, LVIDd, IVRT, E/e', ANP, BNP, Gal-3, CVF, and PFR, downregulated myocardial α-SMA, ColⅠ, ColⅢ mRNA expression, α-SMA, and ColⅠ protein expression, and upregulated MMP-9/TIMP-1 mRNA and protein expression (P<0.05, P<0.01). ConclusionLGQH contains multiple active components and may inhibit myocardial fibrosis in HFpEF rats. It may further alleviate LV hypertrophy, dilation, and diastolic dysfunction, making it an effective Chinese medicinal prescription for treating HFpEF.
7.Effect of VIT1/VIT2 overexpression on Fe and Cd accumulation in rice endosperm.
Yiqi HE ; Dong LIU ; Yujiao SHI ; Bao GUO ; Lin ZHOU ; Jinsong LUO ; Zhenhua ZHANG
Chinese Journal of Biotechnology 2023;39(2):713-723
Iron (Fe) deficiency and excess cadmium (Cd) in rice grain are important problems to be solved in agricultural production. Previous studies have shown that OsVIT1 and OsVIT2 are vacuolar iron transporters. In this study, wild-type ZH11 was selected as the background material and OsVIT1 and OsVIT2 were overexpressed in endosperm by using endosperm specific promoter Glb-1. Field experiments were conducted to study the effect of OsVIT1 and OsVIT2 overexpression on Fe and Cd accumulation in different parts of rice. The results showed that OsVIT1 overexpression in endosperm significantly reduced Fe content in grain by about 50%, while significantly increased zinc (Zn) and copper (Cu) contents in straw and Cu content in grain. OsVIT2 overexpression in endosperm significantly decreased Fe and Cd contents in grain by about 50%, and significantly increased Fe content in straw by 45%-120%. Overexpression of OsVIT1 and OsVIT2 in endosperm did not affect the agronomic traits of rice. In conclusion, OsVIT1 and OsVIT2 overexpression in endosperm reduced Fe accumulation in rice grain, which did not achieve the expected effect. OsVIT2 overexpression in endosperm also decreased Cd accumulation in grain and increased Fe accumulation in straw, which provided reference for iron biofortification and cadmium reduction in rice.
Cadmium
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Endosperm/chemistry*
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Oryza/genetics*
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Iron
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Zinc
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Edible Grain
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Soil Pollutants
8.Analysis of ABO and RhD blood group detection results among voluntary blood donors in Huainan of Anhui province
Hui ZHANG ; Tao ZHOU ; Xing CHENG ; Yujiao SHUI ; Xun FANG ; Xuerong LIU ; Dong LI
Chinese Journal of Primary Medicine and Pharmacy 2023;30(8):1216-1219
Objective:To analyze the distributions of ABO and RhD blood groups by analyzing the basic data of blood group detection among voluntary blood donors in Huainan in 2021, to provide data support for blood recruitment, clinical use of blood, and emergency guarantee of rare groups of blood.Methods:ABO and RhD blood groups of 24 484 voluntary blood donors eligible for blood donation in 2021 were detected using the Metis150-8 automatic blood group analyzer, manual saline method, antihuman globulin method, and manual polybrene test. The blood group results were statistically analyzed.Results:Among 24 484 voluntary blood donors in Huainan in 2021, A blood group accounted for the highest proportion (7 463 cases, 30.48%), followed by O blood group (7 444 cases, 30.40%) and B blood group (7 056 cases, 28.82%), and the last was AB blood group (2 521 cases, 10.30%). A total of 143 cases of RhD-negative blood were detected, and the negative frequency of RhD was 0.58% (143/24 484). Among the RhD-negative blood samples, 43 cases of type A, 41 cases of type B, 46 cases of type O, and 13 cases of type AB were RhD-negative, accounting for 30.07%, 28.67%, 32.17%, and 9.09%, respectively. There was no statistical difference in the detection rate of Rh-negative blood among different ABO blood groups in Huainan ( χ2 = 0.36, P = 0.948). The ABO blood group distribution of voluntary blood donors in Huainan in 2021 was not identical to those of voluntary blood donors in Guangzhou, Yueyang, Xinjiang Bozhou, Zhangzhou, and Liuzhou. The proportion of type A blood donors in Yueyang was higher than those in other regions. The proportions of type B blood donors in Huainan, Xinjiang Bozhou, and Zhangzhou were higher than those in other regions. The proportion of type O blood donors in Liuzhou was higher than those in other regions. The proportions of type AB blood donors in Huainan and Xinjiang Bozhou were higher than those in other regions. Conclusion:The distributions of ABO and RhD blood groups among voluntary blood donors in Huainan region have certain regional characteristics. Central blood banks and medical institutions should reasonably store and supply blood according to the blood collection from voluntary blood donations and the needs of clinical transfusion, to prevent the occurrence of situations such as blood expiration and waste.
9.Ameliorative effect of Abeliophyllum distichum Nakai on benign prostatic hyperplasia in vitro and in vivo
Young-Jin CHOI ; Meiqi FAN ; Yujiao TANG ; Sangho MOON ; Seung-Hyun LEE ; Bokyung LEE ; Sung Mun BAE ; Sang Moo LEE ; Eun-Kyung KIM
Nutrition Research and Practice 2022;16(4):419-434
BACKGROUND/OBJECTIVES:
Benign prostatic hyperplasia (BPH) is the most common prostate disease and one of the most common chronic diseases caused by aging in men. On the other hand, there has been no research on BPH using Abeliophyllum distichum Nakai (A.distichum). Therefore, this study investigated the effects of A. distichum on BPH.MATERIALS/METHODS: A. distichum leaves were extracted with distilled water, 70% ethanol, and 95% hexane as solvents. Subsequently, the inhibitory effects of each A. distichum extract on androgen receptor (AR) signaling were evaluated in vitro. The testosterone-induced BPH model was then used to confirm the efficacy of A. distichum leaves in 70% ethanol extract (ADLE).
RESULTS:
ADLE had the strongest inhibitory effect on AR signaling. A comparison of the activity of ADLE by harvest time showed that the leaves of A. distichum harvested in autumn had a superior inhibitory effect on AR signaling to those harvested at other times. In the BPH rat model, the administration of ADLE reduced the prostate size and prostate epithelial cell thickness significantly and inhibited AR signaling. Subsequently, the administration of ADLE also reduced the expression of growth factors, thereby inactivating the PI3K/AKT pathway.
CONCLUSIONS
An analysis of the efficacy of ADLE to relieve BPH showed that the ethanol extract grown in autumn exhibited the highest inhibitory ability of the androgen-signaling related factors in vitro. ADLE also inhibited the expression of growth factors by inhibiting the expression of the androgen-signaling related factors in vivo. Overall, ADLE is proposed as a functional food that is effective in preventing BPH.
10.Study on integrated technology of producing area processing and decoction pieces processing of Curcuma longa
Mingyue AO ; Ying PENG ; Baohua DONG ; Yunxiu JIANG ; Yujiao LIAO ; Lingying YU ; Zhimin CHEN ; Changjiang HU
China Pharmacy 2022;33(2):172-178
OBJECTIVE To op timize the i ntegrated technology of producing area processing and decoction pieces processing of Curcuma longa (hereinafter refer to “integrated technology ”). METHODS The content of ethanol-soluble extract in C. longa was determined by hot leaching method ;the contents of curcumin ,demethoxycurcumin and bisdemethoxycurcumin were determined by high performance liquid chromatography. On the basis of identification of producing area processing technology , Using overall desirability (OD) value of the contents of ethanol-soluble extract , curcumin, demethoxycurcumin and bisdemethoxycurcumin as evaluation indexes ,moisture content ,slice thickness and drying temperature as factors ,the integrated technology of C. longa was optimized by single factor tests combined with central composite design-response surface method ,and the validation tests were conducted. At the same time ,prepared product was compared with traditional decoction pieces prepared according to 2020 edition of Chinese Pharmacopoeia (part Ⅰ). RESULTS The best integrated technology was that the fresh C. longa was boiled in boiling water for 5 min,dried at 50 ℃ to 40% water content ,cut into 2 mm thin slices ,and dried at 50 ℃ until moisture content not exceeding 15.0%. After validation ,The deviation between the average OD value (0.811 3,RSD=2.13%) and the predicted value (0.848 1)of the contents of ethanol-soluble extract ,curcumin,demethoxycurcumin and bisdemethoxycurcumin was 4.34%. OD value of the contents of ethanol-soluble extract ,curcumin,demethoxycurcumin and bisdemethoxycurcumin in decoction pieces prepared by integrated technology were all higher than those prepared by traditional technology. CONCLUSIONS The process optimized in this study is simple ,stable and feasible.

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