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Objective To investigate the effect of ginkgo diterpene lactones on hypoxia-induced apoptosis and angiogenesis of human umbilical vein endothelial cells (HUVECs) and the related molecular mechanisms. Methods HUVECs were cultured under hypoxia for 24 h, and then treated with ginkgo diterpene lactones (low-dose: 6.25 mg/L and high-dose: 25.00 mg/L). MTT assay was used to detect the cell activity. Flow cytometry was used to detect the apoptosis of HUVECs. Transwell assay was employed to detect the migration of HUVECs. Quantitative real-time polymerase chain reaction and Western blotting were used to test the expression levels of mRNA and protein of hypoxia-inducible factor 1α (HIF-1α), apoptosis-related genes (B-cell lymphoma 2［Bcl-2］and B-cell lymphoma 2-related X protein［Bax］), and angiogenesis-related genes (vascular endothelial growth factor［VEGF］and transforming growth factor β［TGF-β］). Results Compared with the normal group, the HUVEC activity was significantly decreased after exposed to hypoxia for 24 h (P＜0.05), and the apoptosis rate of HUVECs and mRNA and protein expression levels of HIF-1α were significantly higher (all P＜0.05). The cell activity and migration ability of HUVECs were significantly higher in the low- and high-dose ginkgo diterpene lactones groups than those in the hypoxia group (all P＜0.05), and the apoptosis rates of HUVECs were significantly lower than those in the hypoxia group (both P＜0.05). Meanwhile, the cell activity and migration ability were significantly higher in the high-dose group than those in the low-dose group (both P＜0.05), and the apoptosis rate was significantly lower than that in the low-dose group (P＜0.05). The mRNA and protein expression levels of HIF-1α and Bax were lower in the low- and high-dose ginkgo diterpene lactones groups than those in the hypoxia group, and the mRNA and protein expression levels of Bcl-2, VEGF and TGF-β were higher than those in the hypoxia group; and the expression changes of the above genes were more significant in the high-dose group. Conclusion Ginkgo diterpene lactones can improve hypoxia-induced apoptosis and angiogenesis of HUVECs by regulating the expression levels of HIF-1α and apoptosis- and angiogenesis-related genes, and it might be used as a new agent to treat anoxic vascular diseases.
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To solve the problems of the poor resolution of chromatographic separation,the weak durability of the relative correction factors,and the low accuracy of content determination results in the quantitative analysis of multi-components by single-marker( QAMS) method with andrographolide as the internal reference substance in the existing research of Andrographis Herba,a new QAMS method using dehydroandrographolide as the internal reference substance was established for the first time in this study. This new method can be used to simultaneously determine four diterpene lactones,including andrographolide( A),neoandrographolide( B),14-deoxyandrographolide( C),and dehydroandrographolide( S) through the optimization of chromatographic conditions and systematic investigation of methodology. At the present HPLC chromatographic conditions,four components could be well separated( R > 1. 5),and the methodology validations could satisfy the requirement of quantitative analysis. The relative correction factors( RCFs) of fA/S,fB/S,fC/S were determined as 0. 65,0. 54,0. 78,respectively. The relative standard deviations( RSDs) of their RCFs ranged between 1. 3%-5. 1%,0. 25%-0. 33%,0. 070%-0. 15%,0. 070%-0. 22%,respectively with three brands of HPLC instruments,five brands of C18 column,different flow rates( 0. 9,1. 0,1. 1 m L·min~(-1)),and different column temperatures( 25,30,35 ℃),indicating good durability of the RCFs. The relative retention value( RRV) method was used to locate the chromatographic peak of the components to be determined.The RRVs of rA/S,rB/S,and rC/Swere 0. 44,0. 86,0. 97,respectively. The RSDs of the RRVs ranged between 0. 030%-1. 6% with different HPLC instruments and columns,showing accurate peak location. The present QAMS method and the external standard method( ESM)were both used to determine the contents of four diterpene lactones from Andrographis Herba( 6 batches of medicinal materials and 18 batches of cut crude drugs). The relative errors of the determined content results between two methods were less than 2. 0%. It demonstrated that there was no significant difference in content results between these two methods,indicating good accuracy of the present QAMS method. Therefore,in this study,an accurate and highly durable QAMS method using dehydroandrographolide as the internal reference substance was established for simultaneous determination of four diterpene lactones. This method could be used to effectively control the quality of Andrographis Herba and provide technical basis for the formulation of traditional Chinese medicine industry standard and improvement of the Chinese Pharmacopoeia standard of Andrographis Herba.
Subject(s)
Andrographis , Chromatography, High Pressure Liquid , Diterpenes , Drugs, Chinese Herbal , Quality ControlABSTRACT
Objective: Taking electronic-eye (visual analyzer) technique,based on the powder color of Andrographis Herba,to investigate the applicability of electronic-eye technique and evaluate the quality of Andrographis Herba with different commercial specifications. Method: HPLC was employed to determine contents of andrographolide,dehydroandrographolide,14-deoxyandrographolide,neoandrographolide in 50 batches of Andrographis Herba with different commercial specifications(stems,leaves and aerial parts).Color of these samples were measured by electronic-eye technique.The data were analyzed by principal component analysis(PCA) and Pearson correlation analysis.The ability of electronic-eye to distinguish the different commercial specifications of Andrographis Herba was investigated and the correlation of chroma space system parameters (L*,a*,b*) with active components was investigated. Result: There was remarkable difference in contents of 4 diterpenoids in Andrographis Herba from different parts,their contents in leaves was the highest,followed by the aerial parts(mixture of stems and leaves),and their contents in stems was the lowest.The results of PCA was divided into two classes,namely the stem part,leaf and aerial parts,indicating that electronic-eye could be used to distinguish the quality of Andrographis Herba.The correlation results showed that there were significant negative correlation(PL*(lightness value) and the contents of andrographolide,dehydroandrographolide,14-deoxyandrographolide,neoandrographolide and the total content of these 4 components.In addition,L* of samples that did not conform to the lower limit of determination in the 2015 edition of Chinese Pharmacopeia was ≥ 69.5,and the L* of more than 90% of the samples in accordance with the requirements was Conclusion: Electronic-eye technique provides a new method and idea for the quality evaluation of Andrographis Herba.
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Objective To understand the relationship between isozyme activities and diterpene lactone biosynthesis of Andrographis paniculata. Methods Plants were collected during ontogeny from seeding to seed maturity, and separated into leaves and stems for determination. Morphological and yield parameters were used to describe plant growing states. Isozyme changes were tested by polyacrylamide gel electrophoresis. HPLC was used to develop the fingerprints as well as to determine the diterpene lactone content. Results Significant increases were observed in the activities of isozymes, such as aspartate aminotransferase (AST), malate dehydrogenase (MDH), peroxidase (POD), and catalase (CAT), around the early stage of bud in leaves, and the activities of these four kinds of isozymes increased gradually as time progressed in stems. The content changes of diterpene lactones in leaves and stem were various. In the leaves, andrographolide (1) was recorded the highest [(23.63 ± 1.06) mg/g] at the early stage of bud, whereas deoxyandrographolide (2) was the lowest [(6.78 ± 0.27) mg/g] at this period and it reached the highest level at the seeding stage [(26.05 ± 1.04) mg/g]. Dehydroandrographolide (3) and neoandrographolide (4) fluctuated during growing stages. Meanwhile, the HPLC fingerprint showed that the content changes of two unknown compounds were related to that of dehydroandrographolide in leaves. In stems, andrographolide had increased gradually until the bud stage [(8.26 ± 0.33) mg/g], and other three diterpene lactones showed a trend of fluctuation. The yield of total diterpene lactones in aerial part reached the highest at the first flowering stage (806.71 mg/plant). Conclusion These results lay the foundation for the future research on the relationship of isozymes and diterpene lactones, and for determining the most favorable time for harvesting A. paniculata.
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Ginkgo diterpene lactones meglumine injection (GDLI) is a commercially available product used for neuroprotection. However, the pharmacokinetic properties of the prototypes and hydrolyzed carboxylic forms of the primary components in GDLI, i.e., ginkgolide A (GA), ginkgolide B (GB), and ginkgolide K (GK), have never been fully evaluated in beagle dogs. In this work, a simple, sensitive, and reliable method based on ultra-fast liquid chromatography-tandem mass spectrometry (UFLC-MS/MS) was developed, and the prototypes and total amounts of GA, GB, and GK were determined in beagle dog plasma. The plasma concentrations of the hydrolyzed carboxylic forms were calculated by subtracting the prototype concentrations from the total lactone concentrations. For the first time, the pharmacokinetics of GA, GB, and GK were fully assessed in three forms, i.e., the prototypes, the hydrolyzed carboxylic forms, and the total amounts, after intravenous administration of GDLI in beagle dogs. It was shown that ginkgolides primarily existed in the hydrolyzed form in plasma, and the ratio of hydrolysates to prototype forms of GA and GB decreased gradually to a homeostatic ratio. All of the three forms of the three ginkgolides showed linear exposure of AUC to the dosages. GA, GB, and GK showed a constant half-life approximately 2.7, 3.4, and 1.2 h, respectively, which were consistent for the forms at three dose levels (0.3, 1.0, and 3.0 mg·kg) and after a consecutive injection of GDLI for 7 days (1.0 mg·kg).
Subject(s)
Animals , Dogs , Ginkgo biloba , Chemistry , Ginkgolides , Pharmacokinetics , Lactones , Pharmacokinetics , Plant Extracts , Pharmacokinetics , Tandem Mass SpectrometryABSTRACT
This study was aimed to develop the method for determination of mannitol and meglumine in diterpene lactones of Ginkgo Injection. The HPLC-ELSD was used to determine mannitol and meglumine in diterpene lactones of Ginkgo Injection. The analysis was carried out on Waters XBridge Amide (4.6 mm í 150 mm, 3.5 μm) column. The mobile phase was the mixture of acetonitrile, and 50 mmol·mL-1 ammonium acetate with a linear gradient elu-tion at a flow rate of 1.0 mL·min-1. The column temperature wad maintained at 30℃. The operating conditions of the ELSD were a nebulizer nitrogen with the flow rate of 2.0 L·min-1 and an evaporator tube at the temperature of 90℃. The results showed that mannitol had a good linear relationship in a range of 1.9665 μg - 19.665 μg. The average recovery rate was 100.57% (RSD = 0.92%, n = 9). The meglumine showed a good linear relationship in a range of 0.4838 μg - 4.638 μg. The average recovery rate was 100.67% (RSD = 0.72%, n = 9). It was concluded that this method was simple and accurate with good reproducibility. It met with the requirement of the determination of the contents of mannitol and meglumine in diterpene lactones of Ginkgo Injection.
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Objective: To obtain the monoclonal antibodies against the protein of ginkgo diterpene lactones meglumine injection. Methods: After total proteins of ginkgo leaf running two-dimensional electrophoresis, the protein spots were digged and analyzed by mass spectrometry (MS). According to the result of MS, the polypeptide was synthesized and used to immunize the BALB/c mice. The indirect ELISA was utilized to develop monoclonal antibody after cell fusion between SP2/0 cells and spleen cells from the immunized BALB/c mice. Results: The immunoglobulin subtypes of five monoclonal antibodies were IgM and light chain was Kappa identified with a commercial capture-ELISA kit. Western blotting analysis also indicated that the monoclonal antibodies were specific to the protein of ginkgo leaf. Conclusion: The experiment would be helpful for the establishment of detection method specific to the proteins of ginkgo diterpene lactones meglumine injection in the future study.
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Objective To prepare gastric floating sustained-release tablets of Triperygium wilfordii and study its behavior of floating and release characteristics. Methods The sustained-release tablets were prepared by direct powder compared to tablets technique. The floating and release of total diterpene-lactones were used as indicators to evaluate and optimize the formulation. Then the formulation was optimized by influential factors and orthogonal design test. Results The gastric floating sustained-release tablets which was taken orally twice one day, were prepared with HPMCK4M as matrix, cetyl alcohol as floating assistant, sodium bicarbonate as gas-producer, PVP as poremaking. The tablets released 30% in 2 h, 60% in 6 h, exceeding 90% in 12 h, the release behavior of the tablets was fitted to Higuchi equation,and it was properly characterized by the drug diffusion and bulk erosion mechanism. Conclusion Gastric floating sustained-release tablets of T. wilfordii prepared has good behavior of floating and release characteristics.