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Objective To establish the HPLC fingerprints of Tangbikang Granules; To scientifically evaluate and effectively control the quality of Tangbikang Granules; To ensure its production stability. Methods HPLC was performed on the column of Germany Merck RP-18 endcapped (250 mm × 4.6 mm, 5 μm) with mobile phase of acetonitrile-0.1% formic acid water; column temperature was 40 ℃; flow rate was 1.0 mL/min; detection wavelength was 240 nm; volume injection was 20 μL. Fingerprint Similarity Evaluation Software (edition 2004A) of Chinese Pharmacopoeia Commission was used to evaluate the similarity of the 10 batches of Tangbikang Granules, and to analyze the correlations of 9 ingredients in Tangbikang Granules. Results Wogonoside was used as the reference peak, and the common mode for the HPLC fingerprints was set up. The similarities of the 10 batches of Tangbikang Granules were above 0.930, and altogether 25 common peaks in the chromatograms were found, of which 18 peaks were assigned to Chinese materia medica in Tangbikang Granules. Conclusion The method has good separability and is accurate and simple, which can provide references for the quality control of Tangbikang Granules.
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This study was aimed to improve the drug activity of three kinds of isoflavones from Chickpeas.Biochanin A,formononetin and genistein were used as raw materials.Acetone was used as solvent.Potassium carbonate was used as catalyst.The etherification reaction was with 1,3-dibromopropane,1-bromopropane and 3-bromopropene.The results showed that 9 isoflavone ramifications were synthesized.This method was simple,easy to control with high yield.It was concluded that the product structure was confirmed by 1H-NMR,13C-NMR and ESI-MS analysis.It laid a foundation for the structural study basis in the further research of its drug activity.
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This study was aimed to observe the effects of gymnema sylvestre total saponins (GA) on insulin resistance (IR) in adipose tissues of KKay mice,in order to discuss the action mechanism from the PI3K/AKT signal transduction pathway.A total of 27 KKay mice were randomly divided into the model group (DM),Pioglitazone group (BG) and GA group.Nine normal C57BL/6J mice were used as the normal control group (NC).Intragastric administration of drugs was given for eight weeks.The bodyweight and food intake of all mice were tested each week.After the experiment was completed,fasting plasma insulin (Fins) and fasting plasma glucose (FPG) were detected and the insulin sensitive index (ISI) was calculated.Expressions of PDK-1,AKT,P-AKT (Ser473),P-AKT (Thr308) in adipose tissues of epididymis in mice were detected.And expressions of PI3K-p85 mRNA,PTEN mRNA,APN mRNA were also measured.The results showed that compared with DM group,bodyweight of BG and GA groups were decreased (P＜0.05,P＜0.05);FPG and Fins level of BG and GA groups were decreased (P＜0.05,P＜0.05,P＜0.05,P＜0.05),ISI increased (P＜0.05,P＜0.05);APN mRNA increased in BG and GA group (P＜0.01,P＜0.01);PI3K-p85 mRNA increased in GA group (P＜0.05);P-AKT (Thr308) protein expression increased in BG and GA group (P＜0.05,P＜0.01);P-AKT (Ser473) protein expression increased in GA group (P＜0.05);the phosphorylation of AKT (Thr 308) was enhanced in BG and GA group (/9＜0.01,P＜ 0.01);the phosphorylation of AKT (Ser473) was enhanced in GA group (P＜0.01,P＜0.01).PDK-1 protein expression was decreased in BG and GA group (P＜0.05);PTEN mRNA decreased in BG and GA group (P＜0.05,P＜0.01).It was concluded that GA can ameliorate IR by sensitizing PI3κ/AKT signal pathway in adipose tissues of KKAy mice.
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This paper was aimed to study the effect of ethyl acetate extracts of Gentianella acuta on the gene and protein of insulin significant signal IRS-1 and Akt in insulin resistance (IR) HepG2 cells.The CCK-8 method was used to detect the HepG2 cell activity.HepG2 cells of human liver cancer were cultured with high concentration insulin (10-6 mol· L-1)for 36 hours to establish IR cell model.According to the results of CCK-8,the control group,model (IR) group,ethyl acetate extracts of Gentianella acuta IR + 50 μg· mL-1,IR + 500 μg· mL-1 group,and the metformin group were divided.Glucose consumption was measured with a glucose assay kit.The expressions of IRS-1 and Akt gene in IR HepG2 cells were detected by RT-PCR after 6-hour using of ethyl acetate extracts of Gentianella acuta.Western blot was used to detect the expression of IRS-1 and Akt protein after 6-hour using of ethyl acetate extracts of Gentianella acuta.The results showed that when the concentration of ethyl acetate extracts of Gentianella acuta was 500 μg· mL-1,the survival rate reached 95％.When the concentration was higher than 500 μg· mL-1,the survival rate decreased.Compared with the IR group,the IR + 50 μg· mL-1 group and the IR + 500 μg· mL-1 group promoted glucose consumption of IR HepG2 cells,but its effect was less than that of the metformin hydrochloride group.The expression of IRS-1 and Akt in IR HepG2 cells was significantly increased by using RT-PCR in the group of IR + 50 μg· mL-1 and IR + 500 μg·mL-1 compared with the IR group after 6-hour using of ethyl acetate extracts of Gentianella acuta.The expression of IRS-1 and Akt protein in the group of IR + 50 μg· mL-1 and IR + 500 μg· mL-1 was significantly higher than that in the IR group after 6-hour medication detected by western blot.It was concluded that the ethyl acetate extracts of Gentianella acuta can increase the expression of IRS-1,Akt gene,the expression of IRS-1 and Akt protein in HepG2 cells,which may be the mechanism of IR improvement.
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Through comprehensively characterizing components in blood after oral administration of Tang-Bi-Kang (TBK) granules by UPLC-ESI-MSn,this study was aimed to explain the pharmaceutical material basis of TBK initially.UPLC-LTQ-Orbitrap was used under both positive and negative ion modes of electrospray ionization.The blank serum and rat serum after oral administration of TBK were analyzed.Components in rat serum were identified and characterized based on ion fragment information,evenelectron law,nitrogen rule and so on.Reference data was used to establish the UPLC-ESI-MSn method.The results showed that after oral administration of TBK granules,15 components were detected in the serum,of which 13 components were taken as the prototype to blood and 2 metabolites.It was concluded that constituents of TBK granules in rat serum were generated from compatibility of all herbal medicines.In rat serum,most of the components had been absorbed by rat's metabolism;a few were absorbed as the prototype.This research provided references for pharmacodynamic material basis and metabolism of TBL granules in vivo.
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This paper was aimed to study the albiflorin activity of Tang-Bi-Kang (TBK) granules in protecting Schwann cells (SCs) of rat's sciatic nerve.The establishment of SCs oxidative stress model was the condition of 150 mmol×L-1 Dglucose with different concentrations.And the incubation time was 48 h.The experiment groups were the high-dose,middle-dose and low-dose (100 μM,20 μM,4 μM) albiflorin group,the model group,the vitamin C (100 μM) group,and the normal group.Flow cytometry was used to detect the content of ROS in SCs.Fluorescence microscope was used to observe the condition of ROS fluorescence in SCs.And CCK-8 was used to detect the cell activity.The results showed that by using CCK-8 to detect cell proliferation,after 48 h,there was a significant difference between the model group and the normal group (P＜0.01);the albiflorin group compared with the model group (P＜0.01).It indicated that albiflorin can promote the proliferation of SCs.Detecting the ROS fluorescence content,it showed that compared with the model group (Glu 150 mM),the 100 μM,20 μM,and 4 μM albiflorin group,it was P＜0.01 for each group.It showed that albiflorin could relieve the ROS in SCs and alleviate oxidative stress.It was concluded that albiflorin can increase the proliferation of SCs and improve the state of oxidative stress with the protection of SCs.
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Objective To explore the effects of Chinese herbal compound Tangbikang on the expressions of p38 MAPK of sciatic nerve and plasma TNF-α in diabetic rats. Methods Ten of the sixty male SD rats were selected randomly as normal group, and the rest were fed with high-fat diet and low-dosage STZ was used to induce type Ⅱdiabetic rat models. Model rats were randomly divided into model group, mecobalamine group and Tangbikang low-, medium-, and high-dosage groups, 10 rats in each group. Each medication group was intervened with relevant medicine. Rat unilaterals sciatic nerves were taken after 16 weeks. The content of TNF-α in plasma was determined by radioimmunoassay. Western blot method was used to detect the expressions of p38 and p-p38 MAPK protein of sciatic nerve. Results Compared with normal group, the expressions of p38 and p-p38 protein and content of TNF-αin model group significantly increase (P<0.05, P<0.01). Compared with the model group, the expressions of p-p38 protein and the content of TNF-α significantly decreased after medicine intervention in different doses Tangbikang groups and mecobalamin group (P<0.05, P<0.01). The expression of p38 protein in Tangbikang high-dose group significantly decreased (P<0.05), with statistical significance (P<0.05). Conclusion Tangbikang can reduce the expression of p38 and p-p38 MAPK protein of the rat sciatic nerve, and reduce the content of TNF-α protein in rat plasma, which may be one of the effective targets of neuroprotection and abirritation of diabetic peripheral neuropathy.
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This study was aimed to investigate the action mechanism ofHypoxis Hemerocallidea (African Potato, AP) on the AMPK signal pathway of skeletal muscles in diabetic rats. Among 40 male SD rats, 10 rats were used as the normal group, and the other 30 rats were fed with high-fat food for one month, and then injected with STZ for the model establishment. After the successful model establishment, rats were divided into the model group, pioglitazone hydrochloride group and the AP group. Intragastric administration was given for 5 weeks in each group. Then, the skeletal muscle tissues were embedded and sliced for immunohistochemistry test. The protein expression of p-AMPKα, p-AS16 and GLUT4 in skeletal muscles was detected by western blot. The 100 mmol·L-1 glucose was used in the establishment of C2C12 skeletal muscle cells insulin resistance model. AP drug-containing serum was used in the establishment of the treatment group. The control group was the normal cells. Glucose consumption, cell proliferation, SOD content, and MDA content were detected. And the protein expressions of p-AMPKα, p-AS160, GLUT4 were detected with the western blot and RT-PCR. The results showed that compared with the normal group, AP can up-regulate p-AMPKa protein express (P < 0.01), increase skeletal AS160 phosphorylation level (P < 0.01), and up-regulate the GLUT4 level (P < 0.01). Compared with the normal group, the high glucose caused the decrease of C2C12 skeletal muscle cell activity and the decrease of glucose consumption (P < 0.05), decrease of SOD, increase of MDA (P < 0.01), and the decrease of p-AMPKα, p-AS160, GLUT4 protein expression (P < 0.01). After 48 h intervention, the SOD of C2C12 skeletal muscle cells in the AP drug-containing serum group was significantly increased (P < 0.01), the MDA content was decreased (P < 0.05), the AMPKa and AS160 phosphorylation levels were increased (P < 0.01), the GLUT protein expression was increased (P < 0.01). It was concluded that the induced AMPKa and AS160 phosphorylation promoted GLUT 4 expression may be one of the action mechanism of insulin resistance of skeletal muscles in diabetes.
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This study was aimed to observe the effect ofGuava leaf total flavonoids on HIT-T15 pancreaticβcell insulin resistance. Effective part of FSL was prepared. The dosing time, concentration and high glucose concentration of FSL were confirmed by observing HIT-T15 pancreaticβ cell growth curve and the influences of HIT-T15 pancreaticβ cell proliferation by different concentrations of glucose and FSL. Afterwards, the influence of FSL on HIT-T15 pancreaticβ cell insulin secretion, the expression of insulin receptor mRNA and insulin receptor substrate (IRS) 1 protein were measured under the environment of high glucose. The results showed that 50 mmol·L-1 glucose can significantly inhibit the proliferation of HIT-T15 pancreaticβ cell (P < 0.01). The 50μg·mL-1 FSL can significantly promote the proliferation of HIT-T15 pancreaticβ cells (P < 0.01), the insulin secretion (P < 0.05), the expression of insulin receptor mRNA (P < 0.05), and the protein expression of IRS 1 (P <0.01). It was concluded thatGuava leaf total flavonoids can promote the insulin secretion of HIT-T15 pancreaticβ cells under the circumstance of high concentration of glucose which may be related to its effect of increasing expression of insulin receptor mRNA and IRS-1 protein.
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This study was aimed to establish a method for the content determination of three components in Tribulus terrestris L., in order to compare index components of different growing periods, production places and medical parts for the scientific explanation of the harvesting time and growing places. Contents of three effective components inTribulus terrestris L., which were quercetin-3-O-β-D-gentiobioside, isorhamnetin-3-O-β-D-gentiobioside and Terrestrinone A2, were determined by HPLC. The results showed that contents of three evaluation indexes all gradually increased until June and then started reducing. The contents in all evaluated indexes were in the order of the stem and leaf > fruit > root. And the content in the root was below the detection limit. The content indexes ofTribulus terrestris L. of the Inner Mongolia were much higher than that of other regions. It was followed byShandong,Hebei andShanxi province. It was concluded that the method was simple and accurate with good repeatability and stability, which was suitable for quality control ofTribulus terrestris L.
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Spectrophotometry and single factor test were used in the investigation of effects on extraction results of total flavonoids from the fruit ofVernonia esculenta Hemsl. from aspects of ethanol concentration, frequency of extraction, length of extraction time, and the solid-liquid ratio. The extraction conditions were optimized by orthogonal test. The results showed that the optimum extraction conditions were ethanol concentration of 70%, extracted for 4 times, 1 h for each time, and the solid-liquid ratio of 1:20. Under these extraction conditions, the content of total flavonoids in the fruit ofVernonia esculenta Hemsl. was 103.55 mg·g-1.
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This article was aimed to study the content determination methods ofTang-Nai-Kang (TNK) granules. The content of rosemary acid in TNK granules was determined by HPLC method, using C18 chromatographic column (150 mm×4.6 mm, 5μm), the mobile phase of 0.1% trifluoroacetic acid - methanol (60:40),λ of 330 nm. The theoretical plate number was more than 2 000. The standard curve method was used in the determination. Contents of ginsenosides Rg1, Re and Rb1 were determined by HPLC method, using C18 chromatographic column (150 mm× 4.6 mm, 5μm), the mobile phase of water-acetonitrile, gradient elution method,λ of 203 nm. The theoretical plate number was more than 2 000. The standard curve method was used in the determination. The results showed that rosmarinic acid was linear in the range of 0.300-0.500 mg·mL-1 with good linearity. The regression equation wasY = 1E + 07X -7 334,R2 = 0.999. Its sample recovery was 97.32% (RSD 1.25%). Ginsenoside Rg1 was linear in the range of 0.138-0.220 mg·mL-1 with good linearity. The regression equation wasY = 2E + 06X +34 864,R2 = 0. 999. Ginsenoside Re was linear in the range of 0.126-0.250 mg·mL-1 with good linearity. The regression equation wasY = 2E + 06X + 39 879,R2 = 0. 999. Ginsenoside Rb1 was linear in the range of 0.132-0.220 mg·mL-1 with good linearity. The regression equation wasY = 2E + 06X + 39 070,R2 =0. 999. Their sample recoveries were Rg1 97.97% (RSD 1.83%), Re 101.80% (RSD 1.83%), Rb1 98.35% (RSD 1.82%). It was concluded that the content determination method established for the quality control of TNK granules was simple, fast and reliable.
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Through the ethoxylation structure modification of three isoflavones which were genistein, biochanin A and formononetin in chickpea, the hypoglycemic activity and the synergistic hypoglycemic activity were studied. Ethoxylation structure modification was given on three kinds of isoflavones. The hypoglycemic activity of three kinds of isoflavones and their derivatives were studied. The synergistic hypoglycemic activity of the compounds was also studied. The insulin-resistance HepG2 cell was selected as the model of hypoglycemic activity screening. The results showed that four ethoxylation products were synthesized. And the hypoglycemic activity of genistein was better than biochanin A and formononetin with significant difference (P 0.05). Effects of the compound b and compound c were not as good as the compound a and compound d. There was statistical difference (P 0.05). It was concluded that the combinations of compounds played synergistic effects with better hypoglycemic effect. It provided a basis for developing hypoglycemic agents with the intellectual property rights.
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Guava leaf (GL) has been widely used as an herbal medicine for diabetes treatment in tropical and sub-tropical regions. GL has the function of drying dampness and fortifying the spleen, clearing heat and resolving toxins. Modern pharmacological study showed that GL contained flavonoid, phenolic acids, terpene, sesquiterpenoids and other chemical compounds, which regulated hyperglycemia. In recent years, the clinical and basic research on GL has received certain progress. This article reviewed effective components of GL, as well as research progress on clini-cal and basic research of type 2 diabetes.
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This article was aimed to study effects and mechanisms of Gymnema sylvestre on protein kinase B (PKB) and its phosphorylation in adipose tissues of KKAy mice which were mainly characterized by insulin resistance (IR). A total of 18 KKAy mice were randomly divided into the diabetes model (DM) group and Gymnema sylvestre (GS) group according to body weight levels. And 9 normal C57BL/6J mice were used as the normal control (NC) group. Intragastric administration of medication was given to mice for 8 weeks. At the end of the experiment, all animals were tested for fasting plasma glucose (FPG) and fasting insulin level (Fins) for evaluation of insulin sensitivity index (ISI). Expressions of phosphoinositide-dependent kinase-1 (PDK1), PKB, P-PKB (Ser473), P-PKB (Thr 308) in adi-pose tissues of epididymis were determined. The expression of phosphatase and tensin homolog (PTEN) mRNA was also determined. The results showed that compared with the DM group, the GS group showed lower FPG and Fins, higher ISI. The expression of P-PKB (Ser473) phosphorylation and P-PKB (Thr 308) were increased, and the PDK1 and PTEN mRNA were decreased. It was concluded that GS can improve insulin sensitivity of KKAy mice through activating PKB by up-regulate the expression of P- PKB (Ser473) and its phosphorylation ratio and P- PKB (Thr 308) in adipose tissues.
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In this paper, three higher content of flavonol glycosides from the stems and leaves of Tribulus terrestris L. were determined by reversed phase HPLC. The results showed that there was good liner relationship between the peak areas and the sample concentration at the ranges of 0.011 2~0.280 0 μg (r=0.999 6), 0.064 8~2.592 0 μg (r=0.999 8) and 0.018 4~0.460 0 μg (r=0.999 8) for isorhamnetin-3-O-β-D-gentiobioside-7-O-β-D-glucoside, quercetin-3-O-β-D-gentiobioside and isorhamnetin-3-O-β-D-gentiobioside respectively. The verage recovery rates (n=9) of three flavonol glycosides compounds were 100.15%(RSD=1.32%), 100.02% (RSD=1.14%), 99.77% (RSD=1.16%), respectively. This method is considered to be simple, accurate, stable, good precision and reproducibility, which is available for the quality control and evaluation of T. terrestris L.
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To develop a method for determining 3 Xanthone glycosides (1, 5-dihydroxy- 3-methoxyxanthone 8-O-β-D-glucopyranoside(F1), 1-hydroxy-3, 4-dimethoxyxanthon 7-O-β-D-glucopyranoside (F2), 1, 8-dihydroxy-3, 4-dimethoxyxanthone 5-O-β- D-glucopyranoside (F3) of Gentianella acuta by HPLC. The Thermo syncronis C18 (4.6 mmí250 mm, 5 μm) was used for simultaneous determination of 3 Xanthone glycosides in G. acuta. Isocratic elution with water and acetonitrile was 75.5:24.5. The flow rate was 1.0 mL·min-1 and the detection wavelength was set at 254 nm. The column temperature was 35℃. The linear concentration ranges of F1,F2,F3 were 14.06 ~ 281.28 μg·mL-1 (R2=0.999 7),0.56~11.16 μg·mL-1 (R2=0.999 8),0.46~9.20 μg·mL-1 (R2=0.999 9), respectively; The average recov-eries (n = 9) were 99.70% (RSD=1.06%),99.78% (RSD=1.21%),100.28% (RSD=1.15%), respectively. The method is simple, accurate and sensitive, and can be used for the quality control of G. acuta as a reference.
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Objective To observe the effects of genistein(GEN)on oleic acid(OA)induced lipid accumulationin in H4 ⅡE cells and to discuss the possible mechanism of GEN in the pointof AMPK.Methods H4ⅡE cells were cultured in vitro.The control group(NOR),OA treatment group(MOD group)and GEN treatment group were established according to the experimental requirements.The effects of GEN on the proliferation of H4 Ⅱ E cells were measured with MTT assay.The intraeellular TG mass was quantified spectrophotometrically using TG test kit.Cell protein was determined by DCTM Protein Assay kit.The intracellular TG concentration which was used to evaluate lipid accumulation was corrected using protein content as an internal standard.Western blotting was applied to determine the expression of AMPK and P-AMPK (Thr172).Accordingly the phosphorylation levels of AMPK was by means of P-AMPK(Thr172)/AMPK.Results OA treatment can induce lipid accumulation in H4 Ⅱ E cells while GEN treatment can decrease the intracellular TG concentration through up-regulate the phosphorylation levels of AMPK,though the effect was blocked by Compound C that is the inhibitor of AMPK.Conclusion GEN has anti-accumulation of lipid effect in H4ⅡE cell.The mechanism of GEN protective effect is partially due to AMPK.
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ObjectiveTo research the metabolism of components in the Radix of Paeonia lactiflora Pall. Methods(①) we established the HPLC fingerprint of water extract of Paeonia lactiflora Pall. and real-time monitored the chemical composition. (②) We established the HPLC fingerprint of rats' serum samples from hepatic portal vein, serum samples from aorta abdominalis and samples of intestinal absorption of Paeonia lactiflora Pall. (③) On this basis, using the established methods, I-IPLC fingerprint spectrum of serum samples,the sample of herb, the sample after intestinal metabolism, rats' serum samples from hepatic portal vein and rats'serum samples from aorta abdominalis were analyzed and compared in order to infer the metabolism of components in the Radix of Paeonia lactiflora Pall. Results24 compositions were detected, seven of which were metabolized by intestinal flora and could not be absorbed into blood; six of them could not be absorbed directedly into intestinal; eight new compounds were absorbed into blood after bowel metabolism while they were not detected in water extract in Paeonia lactiflora Pall. ConclusionWe could infer the metabolic processes of chemicals of Paeonia lactiflora Pall. in oral administration with this method.