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On the basis of the previous work of the research group, the orthogonal design method was further used to optimize the processing technology for reducing toxicity of fried Tripterygium wilfordii in Lysimachia christinae Decoction. A total of 9 processed products of T.wilfordii in L.christinae decoction were prepared by four factors and three levels orthogonal design table. The contents of triptolide in T.wilfordii were determined by high performance liquid chromatography(HPLC) before and after processing: 4.27, 3.92, 3.57, 2.75, 2.42, 2.66, 3.51, 1.87, 1.75, 2.03 μg·g~(-1). On this basis, the above processed products were orally given to mice for 28 days. 12 hours after the last administration, food fasting except water was provided, and 24 hours later, the eyeballs were taken for blood and liver tissue. Serum biochemical indexes, liver lipid peroxidation and antioxidant related indexes were detected by kit method. Twenty-eight days after oral administration of raw T.wilfordii, the levels of serum alanine aminotransferase(AST), alanine aminotransferase(ALT), alkaline phosphatase(ALP) and liver malondialdehyde(MDA) in mice increased by 91%(P<0.01), 46%(P<0.05), 73%(P<0.01) and 99%(P<0.01), while the liver antioxidant indexes such as superoxide dismutase(SOD), glutathione(GSH), glutathione peroxidase(GPX) and glutathione-S transferase(GST) significantly decreased(P<0.01). After administration of the processed products, the above indexes were significantly reversed(P<0.01 or P<0.05). Especially, the processing conditions of A_3B_2C_1D_3 had the best detoxification effect on T.wilfordii, which decreased the high levels of AST, ALT, ALP and MDA by 49%(P<0.01), 32%(P<0.01), 42%(P<0.01), and 17%(P<0.05). Therefore, the best processing conditions for T.wilfordii in L.christinae decoction were A_3B_2C_1D_3, namely "15% mass fraction of L.christinae, 1 h moistening time, 160 ℃ frying temperature, and 9 min frying time".
Subject(s)
Animals , Mice , Antioxidants , Liver , Primulaceae , Technology , TripterygiumABSTRACT
OBJECTIVE：To e stablish UPLC characteristics fingerprints of Lysimachia christinae ，and to simultaneously determine 3 effective components and to comprehensively evaluate the quality of L. christinae from different production areas. METHODS：UPLC method was adopted to establish characteristics fingerprint of the whole plant ，stem and leaves of 10 batches of L. christinae ，and determine the contents of kaemperfol- 3-O-rutinoside，quercetin，kaemperfol. The determination was performed on Waters CORTECS UPLC T 3 column with mobile phase consisted of acetonitrile- 0.2％ phosphoric acid aqueous solution （gradient elution ）at the flow rate of 0.2 mL/min. The detection wavelength was set at 364 nm，and column temperature was 30 ℃. The sample size was 1 µL. Similarity Evaluation System for TCM Chromatographic Fingerprint （2012 edition）was adopted to evaluate its similarity ， and common peaks were confirmed. Using the contents of kaemperfol- 3-O-rutinoside， quercetin， kaemperfol，total ash ，acid-insoluble ash and sulfur dioxide residue ，the ethanol-soluble extract as index ，entropy weight TOPSIS was used to evaluate the overall quality of L. christinae comprehensively. RESULTS ：There were 7 common peaks in the whole plant，stem and leaves of 10 batches of L. christinae ，among which 3 peaks were identified as kaemperfol- 3-O-rutinoside， quercetin and kaemperfol. The similarity of same part in the whole plant of L. christinae from different batches were not lower than 0.830. The similarity between stem and leaves of L. christinae in same batch was 0.504-0.859； the similarity between whole plant and stem was 0.593-0.904；the similarity between whole plant and leaves was 0.885-0.995. The linear ranges were 0.392 0-39.197 0 μg/mL for kaempferol-3-O-rutinoside， 0.397 0- 39.703 4 μg/mL for quercetin，0.380 9-38.093 0 μg/mL for kaempferol（r＞0.999 0）. RSDs of precision ，stability and repeatability tests were all lower than 2％. The recoveries were 96.43％（RSD＝0.63％，n＝9），100.32％（RSD＝0.46％，n＝9）， 101.80％（RSD＝0.32％，n＝9），respectively. The content range of above components in L. christinae were 0.006 3％-0.041 1％， 0.002 9％-0.008 6％，0.004 4％-0.017 5％（stem）；0.024 8％-0.290 5％，0.000 9％-0.009 0％，0.001 3％-0.012 4％（leaves）； 0.007 9％-0.118 0％，0.001 5％-0.008 8％，0.002 8％-0.012 5％（whole plant ）. There was no significant difference in the contents of 3 components in L. christinae among different producing areas （P＞0.05）. The order of the contents of kaempferol- 3-O- rutinoside in different parts of L. christinae was leaves ＞whole plant ＞stem. The contents of quercetin and kaempferol were high relatively in the stem. Results of entropy weight TOPSIS method showed that mean values of Ci for L. christinae from Zhongjiang county and Shuangliu county of Sichuan province ，Shizhu county of Chongqing city were 0.446，0.512，0.287. CONCLUSIONS ： Established fingerprint and content determination method are stable and feasible ，and multi-index evaluation model constructed by characteristic chromatogram combined with entropy weight TOPSIS analysis method can be used for comprehensive quality evaluation of L. christinae . The quality of L. christinae from Sichuan province is better.
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OBJECTIVE： To explore the mechanism of Lysimachia christinae in the treatment of cholecyst related diseases by network pharmacology. METHODS： The active ingredients of L. christinae were screened through TCMSP database with “Lipinski rule” and “Oral bioavailability ＞30％” rules， and their related targets were predicated correspondingly， then compound-target network were constructed by Cytoscape 3.2.1 software. Disease related targets were predicted by searching TTD database， OMIM database， PharmGKB database， DrugBank database and GAD database with “cholelithiasis” “gallstones” “cholecystitis” and “cholangitis” as keywords. Then， the network of disease-target was constructed and merged with active ingredient target to obtain therapeutic target. After pathway enrichment analysis of therapeutic target were performed by utilizing the DAVID database， molecular docking between target and active ingredient was also conducted in order to screen the main active ingredients of L. christinae. RESULTS： Twenty-seven active ingredients with good oral absorption and drug-like properties were screened from L. christinae. Thirty-three targets were attained after constructing and merging the network. Seven pathways， mainly related to cancer pathway and ABC transporter pathway were achieved. Top 4 active ingredients of L. christinae in the list of docking score were kaempferin， acacia， hesperetin and isorhamnetin， which acted on ABCC3， ABCB1， ABCC2， ABCB4 target. CONCLUSIONS： L. christinae treat cholecyst related diseases through ABC transporter pathway.
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Objective To isolate and identify anti-tumor constituents from Lysimachia christinae Hance.Methods The compounds were isolated and purified by chromategraphy on AB-8 macroporous resin, silica gel, Sephadex LH-20, ODS and preparative HPLC.Their structures were elucidated using chemical and spectroscopic methods.All thecompounds obtained were evaluated on their growth inhibitory effects in vitro using the MTT method.Results Eleven compounds were isolated from the whole plant of L.christinae Hance and were identified as 3,3'-dimethoxy-6,6'-di[(Z)-pentadec-10-en-1-yl]-[1,1'-bi(cyclohexane)]-3,3',6,6'-tetraene-2,2',5,5'-tetraone(1),physcion(2),munjistin(3), 9,16-dioxooctadec-10,12,14-trienoic acid(4),cyclamiretinA-3-O-{β-D-xylopyranosyl-(1→2)-β-D-glucopyranosyl-(1→2)-[β-D-glucopyranosyl-(1→4)]-α-L-arabinopyranoside(5),lysikoianoside(6),quercetin-3-O-glucopyranoside(7),isorhamnetin3-O-β-D-galactopyranoside(8),3',4',5',5,7-pentahydroxyflavone(9), rutin(10), and quercetin(11).The screening results of antitumor activity in vitro showed that IC50 of compound 5 for human cervical carcinoma cells HeLa, human osteosarcoma cells U20S, human lung adenocarcinoma cells PC-9,and human colorectal carcinoma cells CT-26 was 2.29, 1.22, 1.43, and 1.29 μmol/L respectively.Conclusion Compound 1 is a new compound and compounds 2-4 are isolated from the plants of Lysimachia for the first time.Compound 5 shows obvious inhibitory effects on tumor cell growth in vitro.
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Objective: The differences in the compounds between Lysimachia christinae and Desmodium styracifolium were compared in order to provide the references for pharmacodynamic differences of them. Methods: The RP-HPLC-UV and RP-HPLC-ELSD fingerprints of L. christinae and D. styracifolium were established, and the common peaks of two kinds of fingerprints were compared. Results: The HPLC-UV fingerprints of L. christinae were obviously different from the HPLC-ELSD fingerprints, and the strong peaks detected in UV were not the same as what detected in ELSD, so were those of D. styracifolium. There were 14 and 5 common peaks, respectively in the HPLC-UV and HPLC-ELSD fingerprints of L. christinae, while 28 and 18 common peaks, respectively in the HPLC-UV and HPLC-ELSD fingerprints of D. styracifolium. The similarities of chromatograms of D. styracifolium were better than those of L. christinae. Four common peaks were identified in the HPLC-UV fingerprints of L. christinae and D. styracifolium, and two common peaks in their HPLC-ELSD fingerprints. Conclusion: It is better using HPLC-ESLD to establish the fingerprints of L. christinae and D. styracifolium. The chemical constituents of L. christinae were significantly different between the different batches. However, little variation was found in the chromatograms between the different batches of D. styracifolium. The two plants may contain two same compounds with high content, which may have the function as the substantial bases of their treatments for the same indication. However, they may have their special efficacies.
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OBJECTIVE: To analyze the genetic diversity of Lysimachia christinae Hance germplasm resources from 36 different places of origin with ISSR DNA markers. METHODS: Ten primers screened from 45 ISSR primers wrea mplified with PCR and the amplification products were examined by 1.5% agarose gel electrophoresis. POPGENE32 software was used for statistical analysis of genetic parameters and UPGMA method (NTSYS2.10 software) for cluster analysis and dendrogram constructing. RESULTS: A total of 91 sites were amplified from the 10 ISSR primers, of which 80 sites were polymorphic loci. The percentage of total polymorphic loci (PBB) was 87.91%. The number of observed alleles (Na) was 1.879 1 and the number of effective alleles was number (Ne) 1.381 7. The Nei's genetic diversity index (He) was 0.239 0 and the Shannon information index (I) was 0.374 5. The genetic similarity coefficient of the 36 germplasm resources varied from 0.69 to 0.97 and the 36 materials couldbe divided into six categories at the similarity coefficient of 0.76. Clustering results showed that the samples within small geographic range clustered together in the dendrogram, indicating the nearer genetic relationships of them. Meanwhile, the samples from different provinces or cities could not be differentiated from each other through cluster analysis. CONCLUSION: The germplasm resources of Lysimachia christinae have rich genetic diversities. The genetic diversities of Lysimachia christinae have no significant correlation with their geographic distribution.
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OBJECTIVE: To investigate the hepatoprotective effects of Lysimachia christinae Hance against acute liver injury induced by tripterygium glycosides (TG) in vivo and the related mechanism for the first time. METHODS: The mice were administered ethanol extract of L. christinae (JE), water extract of L. christinae (JW), and bifendate by ig for seven consecutive days. 12 h after the last dose, the mice were orally given TG 270 mg · kg-1 except those in the normal group. Serum and liver tissue samples were collected at 18 h after TG treatment. Besides, the amounts of quercetin and kaempferol in JE and JW were analyzed by high-performance liquid chromatography (HPLC). RESULTS: TG-induced elevated serum alanine transferase(ALT) and aspartate transaminase (AST) activities were significantly reduced by JE(100, 200 mg · kg-1) in dose-dependent manners, but not by JW. Further analysis demonstrated that lipid peroxidation(LPO) level significantly decreased, while superoxide dismutase(SOD) and catalase(CAT) activities increased in livers of JE-treated mice. Besides, the amounts of quercetin and kaempferol were 9.8 and 8.9 mg · g-1 in JE, and 2.8 and 1.9 mg · g-1 in JW, respectively. CONCLUSION: This study for the first time demonstrates that L. christinae can protect against TG-induced acute liver injury in dose-dependent manners in vivo, the potential mechanism may be related to inhibiting liver oxidative stress injury, and the hepatoprotective activity may be correlated with the contents of quercetin and kaempferol. Copyright 2013 by the Chinese Pharmaceutical Association.