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Objective:To observe the protective effect of forsythiaside A on acute lung injury (ALI) in septic rats.Methods:Male Sprague-Dawley (SD) rats were randomly divided into normal control group, sham operation group, sepsis model group, and forsythiaside A intervention group, with 10 rats in each group. The rats in the normal control group did not receive any intervention; the rats in the sham operation group only underwent abdominal surgery; and those in the model group and forsythiaside A intervention group received cecal ligation and puncture (CLP) to establish the sepsis rat model. The rats in the forsythiaside A intervention group were given 75 mL/kg of forsythiaside A within 0.5 hour after operation, and repeated after 6 hours. The rats in the sham operation group and model group were given the same amount of normal saline at the same time points. The lung tissues were collected for pathological examination 12 hours after operation. The lung homogenate was prepared, and enzyme-linked immunosorbent assay (ELISA) was used to detect tumor necrosis factor-α (TNF-α), interleukins (IL-1β, IL-6). The activity of superoxide dismutase (SOD) was detected by xanthine oxidase method, and the content of malonaldehyde (MDA) was detected by colorimetry. The expression of nuclear factor-κB p65 (NF-κB p65) was detected by Western blotting.Results:There was no significant pathological change of lung tissue in both normal control group and sham operation group, and there was no significant difference in each parameter between the two groups. The rats in the model group had interstitial infiltration of inflammatory cells, alveolar structure destruction, alveolar septum thicken, extensive alveolar hemorrhage, telangiectasia; the levels of TNF-α, IL-1β, IL-6, MDA and NF-κB p65 protein expression in lung tissue were significantly higher than those in the normal control group and sham operation group [TNF-α (ng/L): 132.81±16.15 vs. 45.08±5.98, 46.10±6.72, IL-1β (ng/L): 137.32±15.22 vs. 51.03±7.89, 50.92±8.13; IL-6 (ng/L): 138.39±14.28 vs. 51.68±7.03, 52.48±7.36; MDA (kU/g): 1.79±0.13 vs. 0.96±0.05, 0.97±0.05; NF-κB p65 protein (NF-κB p65/GAPDH): 2.82±0.23 vs. 1.76±0.12, 1.82±0.13; all P < 0.05], the activity of SOD decreased significantly (kU/g: 45.90±5.46 vs. 92.11±10.13, 93.36±10.56, both P < 0.05). The changes in lung histopathology in the forsythiaside A intervention group were obviously improved as compared with the model group, which showed less inflammatory cell infiltration, less alveolar septum thickening, less bleeding and more intact structures; the levels of TNF-α, IL-1β, IL-6, MDA and the expression of NF-κB p65 protein in lung tissue were significantly lower than those in the model group [TNF-α (ng/L): 72.48±9.78 vs. 132.81±16.15, IL-1β (ng/L): 83.85±12.46 vs. 137.32±15.22, IL-6 (ng/L): 81.88±11.89 vs. 138.39±14.28, MDA (kU/L): 1.29±0.09 vs. 1.79±0.13, NF-κB p65 protein (NF-κB p65/GAPDH): 2.29±0.19 vs. 2.82±0.23, all P < 0.05], SOD activity increased significantly (kU/g: 66.03±7.98 vs. 45.90±5.46, P < 0.05). Conclusions:Forsythiaside A can effectively alleviate ALI in septic rats. The mechanism may be related to down-regulate the expression of NF-κB p65 and reduce the level of inflammatory factors and free radicals in lung tissue, thereby against acute lung injury in septic rats.
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A drug delivery system of forsythoside A-loaded exosomes(FTA-Exos) with high biocompatibility and low immunogenicity was established to investigate its impact on the migration of human lung epithelial adenocarcinoma A549 cells. The exosomes from A549 cells were extracted and purified by ultra-high speed centrifugation and ultrafiltration. FTA-Exos were prepared by ultrasonic incubation, and characterized by particle size analysis, transmission electron microscopy, and Western blot assay. The uptake of FTA-Exos by A549 cells was observed under the laser confocal microscope, and the impact of FTA-Exos on the migration of A549 cells was investigated by cell scratch assay. The results showed that the average particle size of the prepared FTA-Exos was(138.90±2.37) nm, which increased slightly after drug loading. The PDI was 0.291±0.013, and the average potential was(-10.1±0.66) mV. The FTA-Exos were spheroidal in appearance as observed by transmission electron microscope, with an obvious saucer-like double-layer membrane. Western blot assay indicated that the specific proteins CD63 and Alix were both expressed in exosomes. The laser confocal microscopy suggested that FTA-Exos were taken up by A549 cells and stably maintained in the cell for 4-8 h, and the fluorescence was significantly enhanced at 4 h. The scratch assay showed that the inhibitory effect of FTA-Exos on the migration of A549 cells was significantly stronger than that of forsythoside A(P < 0.05). In conclusion, the drug delivery system of FTA-Exos established in this study had good stability, reliable preparation process, and potent inhibitory effect on the migration of A549 cells in vitro, which can provide an important reference for subsequent in-depth research and application.
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Humans , Exosomes , GlycosidesABSTRACT
Objective:To observe the effect of forsythiaside A on gastrointestinal motility disorder induced by chemotherapy in mice， and explore the mechanism of forsythiaside A regulating gastrointestinal motility. Method:The 60 KM mice were randomly divided into normal group， model group， metoclopramide group （5 mg·kg-1） and forsythiaside A low， medium and high-dose groups （30， 60， 120 mg·kg-1）， 10 for each group， which include half male and half female. The above dose was given once a day for 4 consecutive days， which the intragastric volume was 10 mL·kg-1. One hour after 1rd day administration， equal volume of saline was intraperitoneally injected to the normal group， 2 mg·kg-1 cisplatin was intraperitoneally injected to the other groups with daily for 4 consecutive days. Observing the effects of forsythiaside A on gastric emptying and small intestinal propulsion on mice models， serum gastrin （GAS） and somatostatin （SS）， motilin （MTL）， vasoactive intestinal peptide （VIP） levels were examined by enzyme-linked immunosorbent assay （ELISA）. Activities of acetylcholinesterase （AChE） and total nitric oxide synthase （tNOS） in gastric antrum and ileum were detected by ELISA. The expression of AChE and inducible nitric oxide synthase （iNOS） in gastric antrum and ileum were detected by Western blot. Result:Compared with normal group， the gastric retention rate and small intestinal propulsion rate of the model group were significantly increased （P<0.01）， serum levels of MTL， GAS， SS and VIP， the AChE activity in the homogenate of ileum in the model group were significantly reduced （P<0.05，P<0.01）， while the tNOS activities in gastric antrum and ileum were significantly increased （P<0.05，P<0.01）. Protein expression of AChE in gastric antrum and ileum were significantly decreased （P<0.05）， and the expression level of iNOS protein was significantly increased in the model group （P<0.05）. Compared with model group， different doses of forsythiaside A can reduce the gastric residual rate and small intestinal propulsion rate of mice to varying degrees. Meanwhile forsythiaside A can increase the serum levels of MTL， GAS， SS， and VIP， and the AChE activity and protein expression levels in gastric antrum and ileum tissues were also increased， while tNOS activity and iNOS protein expression were decreased in gastric antrum and ileum （P<0.05，P<0.01）. Conclusion:Forsythiaside A can significantly ameliorate the delayed gastric emptying and small intestine hyperfunction induced by cisplatin in mice. Its mechanism to ameliorate gastrointestinal dysfunction caused by chemotherapy is related to the regulation of gastrointestinal AChE and NOS activity in gastric antrum and ileum and the regulation of gastrointestinal hormone levels.
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OBJECTIVE： To investigate the changes of extraction rates of forsythiaside A and forsythin in Forsythia suspensa compatible with other medicinal material of Menshi huwei formula before and after decoction. METHODS： HPLC method was used to determine the extraction amounts and to calculate the extraction rates of forsythiaside A and forsythin in F. suspensa （5 g×7 doses）， F. suspensa （5 g×7 doses） compatible with Pinelliae rhizoma praeparatum cum zingibere et alumine （PRZA）， Menshi huwei formula [including 6 ingredients as F. suspense （5 g×7 doses）， PRZA] after decocted with water. The determination was performed on Diamonsil C18 column with mobile phase consisted of acetonitrile-0.2％ formic acid solution （gradient elution）. The detection wavelength was set at 278 nm， and the column temperature was 30 ℃. The flow rate was 1.0 mL/min. RESULTS： The linear range of forsythiaside A and forsythin were 0.61-6.1， 0.246-2.46 μg （r＝0.999 7， 0.999 9）， respectively； RSDs of precision， stability （within 20 h） and reproducibility tests were all lower than 2％ （n＝6）. Average recovery rates were 96.10％-99.37％ （RSD≤2.36％，n＝6） respectively. In F. suspensa， extraction rates of forsythiaside A and forsythin were 96.90％ and 66.67％. In F. suspensa compatible with PRZA， extraction rates of them were 101.61％ and 54.55％. In Menshi huwei formula， extraction rates of them were 98.39％ and 84.85％. CONCLUSIONS： After F. suspensa is compatible with PRZA， the extraction rates of forsythiaside A is increased while forsythin is decreased. After compatible with other medicinal material in Menshi huwei formula， extraction rates of both are increased slightly.
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Objective To establish a HPLC method for simultaneous determination of seven constituents in Pifubingxuedu tablet.Methods The determination was performed on Kromasil-C18 column (4.6 mm × 250 mm, 5 μm).The mobile phase was acetonitrile-0.5％ phosphoric acid aqueous solution with a flow rate of 0.8ml/min.The detector was PDA and detection wavelength was set at 245,325,403 nm, respectively.Results A method has been established for the determination of chlorogenic acid, hydroxy Safflower Pigment A, paeoniflorin, forsythiaside A, ferulic acid, berberine and glycyrrhizin acid in one run by HPLC.Their average contents and RSD in each Pifubingxuedu tablet were 0.299 5 mg/tablet (2.25％);0.700 0 mg/tablet(1.33％);0.429 2 mg/tablet (1.21％);0.039 1 mg/tablet (2.34％);0.014 8 mg/tablet(2.23％);0.209 0 mg/tablet (2.06％);0.272 7 mg/tablet (2.68％), respectively.Conclusion The established method is simple, convenient, accurate and reliable.It is suitable for the quality control of Pifubingxuedu tablet.
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OBJECTIVE:To establish a method for the simultaneous determination of 7 components in Tanreqing capsules.METHODS:HPLC method was adopted.The determination was performed on Ultimate XB C18 column with mobile phase consisted of acetonitrile-0.2％ formic acid (gradient elution) at a flow rate of 0.8 mL/min.The detection wavelength was set at 325 nm,and column temperature was 35 ℃.The sample size was 10 μL.RESULTS:The linear ranges of chlorogenic acid,isoforsythiaside A,forsythiaside A,isochlorogenic acid B,isochlorogenic acid A,isochlorogenic acid C and baicalin were 0.025-0.5 μg(r=0.999 6),0.025-0.5 μg (r=0.999 7),0.050-1.0 μg (r=0.999 9),0.025-0.5 μg((r=0.999 7),0.025-0.5 μg ((r=0.999 6),0.025-0.5 μg (r=0.999 6),0.750-1.5 μg(r=0.999 9),respectively.The limit of quantitation was no more than 1.5 ng,and the limit of detection was 0.5 ng.RSDs of precision,stability and reproducibility tests were all lower than 3.0％.The recoveries were 95.28％-106.30％ (RSD ranged 0.97％-2.14％,n=9).CONCLUSIONS:The method is simple,precise,stable and reproducible,and can be used for simultaneous determination of 7 components in Tanreqing capsules.
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Objective: To investigate the effect of deactivation of enzymes on chemical composition of Forsythiae Fructus using NMR based metabolomics approach. Methods: 1H-NMR-based metabolomics approach combined with multivariate statistical analysis was used to investigate the differential metabolites between raw Forsythiae Fructus (RF) and Forsythiae Fructus with deactivation of enzymes (DF). And degradation pathway of significant metabolites was inferred by biosynthetic pathway. Results: Twenty-four metabolites were identified in the 1H-NMR spectrum of Forsythiae Fructus, and principal component analysis (PCA) showed clear separation between RF and DF. The S-plot of orthogonal partial least square discriminate analysis (OPLS-DA) revealed that ten compounds contributed to the separation of RF and DF, and the levels of forsythiaside A, forsythiaside C, phillyrin, rutin, and surcose were higher in DF than those in RF, while the levels of rengyol, rengyoside, rengyolone, α-glucose, and β-glucose were higher in RF than those in DF. Moreover, the degradation of forsythiaside A into rengyol was proposed. Conclusion: This study reveals the chemical effect of deactivation of enzymes on Forsythiae Fructus in a holistic manner, which confirms rationality and necessity of deactivation of enzymes during the processing of Forsythiae Fructus. This study also serves as a basis for the bioactive compounds and quality standards research of Forsythiae Fructus.
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Objective: To establish an HPLC method for the fingerprint analysis of Shufeng Jiedu Capsule (SJC), so as to provide evidence for the quality control of it. Methods: HPLC method was applied with the chromatographic condition as follows: The chromatographic column was Unitary C18 column (200 mm×4.6 mm, 5 μm), acetonitrile-0.1% formic acid as the mobile phase with gradient elution, the flow rate was 1.0 mL/min, the detection wavelength was 250 nm, the column temperature was 30℃, and the injection volume was 10 μL. The HPLC fingerprint of SJC was analyzed with similarity analysis and principal component analysis (PCA) method. Results: The fingerprint chromatography included 22 mutual peaks, of which eight mutual peaks from Polygonum cuspidatum, six mutual peaks from Forsythia suspensa, three mutual peaks from Verbena officinalis, two mutual peaks from Glycyrrhiza uralensis, and one mutual peak from Thlaspi arvense. The similarity among 14 batches was more than 0.9.Based on the HPLC-MS2 data, 16 components were identified, which were forsythosid E, hastatoside, verbenalin, 5'-hydroxy-forsythiaside A, polydatin, phillyrin, forsythosid A, acteoside, iso-forsythosid A, aloe-emodin, emodin-8-O-grapeglycosides, parietic acid, monoammonium glycyrrhizinate, 3-hydroxyglabrol, emodin, and chrysophanol. Conclusion: It is the first time to establish the HPLC fingerprint of SJC. The method is simple, accurate, and reproducible, which could reflect the chemical composition information of SJC comprehensively and provide scientific evidence for the quality control.
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Objective:To establish the optimal extraction technology of Xiaoer Yinqiao granules by orthogonal test. Methods:The effects of water amount,extraction duration and extraction times were investigated by orthogonal design using the contents of forsythia-side A and chlorogenic acid as the indices. Results: The optimum extraction process was as follows: adding 8-fold amount of water, and extracting 1. 5 h for the first time, and then adding 6-fold amount of water, extracting 1 h for the second and third time, respective-ly. Conclusion:The extraction technology is simple, reasonable and reliable.