This study was aimed to establish a HPLC method for the content determination of liposoluble components, such as dihydrotanshione I, croptotanshinone, tanshinone I and tanshinone IIA, as well as the content determination of water-soluble components, such as danshensu, protocatechuic aldehyde, rosmarinc acid, salvianolic acid B, and salvianolic acid A in Danshen capsule simultaneously. For liposoluble components, the determination was performed on a Welch ultimate XB-C18 column (250 mm × 4.6 mm, 5 μm) by gradient elution using acetonitrile-water as the mobile phase. The flow rate was 1 mL·min-1. And the detection wavelength was set at 270 nm. For water-soluble components, the determination was performed on a Thermo Syncronis C18 column (250 mm × 4.6 mm, 5μm) by gradient elution using methanol-acetonitrile-0.02%phosphoric acid as the mobile phase. The flow rate was 1 mL·min-1. And the detection wavelength was set at 286 nm. The results showed that there were good linear relationships between components of peak areas and the ranges were 0.472-9.44 μg (r = 0.999 8) for danshensu, 0.352-7.04 μg (r = 0.999 9) for protocatechuic aldehyde, 0.244-4.88 μg (r = 1.000 0) for rosmarinc acid, 2.268-45.36 μg (r = 0.999 9) for salvianolic acid B, 0.168-3.36 μg (r = 0.999 9) for salvianolic acid A, 0.088-1.76 g(r=0.999 9) for dihydrotanshione I, 0.18-3.6μg (r=0.999 9) for croptotanshinone, 0.208-4.16μg (r=0.999 9) for tanshinone I, and 0.17-3.4μg (r=0.999 9) for tanshinone IIA. Average recoveries of the method were between 97.48%and 98.59%. It was concluded that the analysis was stable and reproducible, which can be used as a method for the analysis of Danshen capsule.

Objective To observe the effects of butylphthalide on the expression of autophagy-related protein and mRNA in l-meth-yl-4-phenyl-pyridiniumion (MPP+)-induced SH-SY5Y cells, and to explore the protective effect and possible mechanism of butylphthalide to the cell model of Parkinson's disease. Methods The SH-SY5Y cells were divided into control group (A), MPP+group (B), rapamycin pre-treated+MPP+group (C) and Butylphthalide pretreated+MPP+group (D). The relative viability of SH-SY5Y cells induced by MPP+was measured with MTT assay, the morphology of SH-SY5Y cells was observed. The expression of microtubule associated protein 1 light chain 3 (LC3)-II/I and Beclin 1 protein was detected by Western blotting. And the expression of LC3-II/I and Beclin 1 mRNA were assayed by re-al-time quantitative reverse transcription-PCR (RT-PCR). Results The viability rates of cells were significantly lower in group B than in group A (t=20.270, P<0.001), and were significantly higher in groups C and D than in group B (t>8.770, P<0.001), however, there was no significantly difference between groups C and D (t=2.270, P=0.064). The expression of LC3-II/I and Beclin 1 was higher in group B than in group A (t>6.647, P<0.01), and was higher in groups C and D than in group B (t>3.630, P<0.01), however, there was no significantly differ-ence between groups C and D (t<2.238, P≥0.05). Conclusion Butylphthalide could prevent the injury of SH-SY5Y cells induced by MPP+, which may affect Parkinson's disease by inducing autophagy.

Objective To establish a new quality control standard for danshen capsules. Methods The qualitative identification of danshen capsules was characterized by ultraviolet fluorescence and thin-layer chromatography( TLC ). The contents of tanshinoneⅡA,cryptotanshinone,salvianolic acid B,danshensu and protocatechuic aldehyde in danshen capsules were determined by high performance liquid chromatography(HPLC)on a C18 column. The flow rate was 1 mL·min-1,and the column temperature was 35 ℃. Results The HPLC linear ranges of tanshinone ⅡA,cryptotanshinone,salvianolic acid B, danshensu and protocatechuic aldehyde were 2. 046-40. 92 μg · mL-1 ,1. 482 25 -59. 29 μg · mL-1 ,1. 502 55 -60. 102 μg·mL-1 ,11. 49-459. 582 μg·mL-1 ,and 0. 617 4-24. 696 μg·mL-1 ,respectively,and r values were 0. 999 9. The average recoveries were 99. 66%(RSD of 0. 91%)for tanshinoneⅡA,99. 26%(RSD of 0. 88%)for cryptotanshinone,99. 09%(RSD of 0. 76%)for salvianolic acid B,100. 51%(RSD of 0. 62%)for danshensu,and 100. 62%(RSD of 0. 82%)for protocatechuic aldehyde,respectively. The contents of the tanshinoneⅡA,cryptotanshinone,salvianolic acid B,danshensu showed a certain high level in the 3 batches of danshen capsule samples,but protocatechuic aldehyde was low by comparison. Conclusion The HPLC method is proven to be sensitive,accurate,repeatable,and can be used for quality control of the danshen capsules.

This study was aimed to establish the HPLC fingerprint of liposoluble components inDanshen capsule for quality evaluation. HPLC was run on a Welch ultimate XB-C18 column (250 mm × 4.6 mm, 5 μm) with acetonitrile-water as mobile phase in gradient elution at a flow rate of 1 mL·min-1. The column temperature was 25℃. The detection wavelength was 270 nm. The injection volume was 10μL. The results showed that 7 and 11 common peaks were selected as the HPLC fingerprint of liposoluble components inDanshen capsule from two manufactures, respectively. The similarities of 20 batches of samples were above 0.9. It was concluded that the analysis was stable and reproducible, which can be used as a basis for evaluating the quality control of liposoluble components in Danshen capsule.

Objective To analyze the protective effects ofbutylphthalide(NBP) on apoptosis factors (p-JNK,Fas and FasL) of death receptor pathway in JNK pathway of cell model of Parkinson's disease (PD).Methods SH-SY5Y cell apoptosis model induced by MPP + was established in vitro.The cells were divided into four groups:normal control group,SHSYSY cells were treated with complete medium without drug intervention;MPP+ group,1 mmol·L-1 MPP+ was added into the cells;NBP+ MPP+ group,the cells were pretreated with 10 mol·L-1 NBP for 3 h and added with 1 mmol·L-1 MPP+;SP600125 + MPP+ group,the cells were cultured with 10 mol·L-1 JNK inhibitor SP600125 pretreatment for 3 h and 1 mmol·L-1 MPP+ was added.The proliferative potentiality of SH-SY5Y cells induced by MPP+ was measured by MTT.The apoptotic rate was analyzed by Annexin-V/PI (FCM).The morphology of SH-SY5Y cells was observed by inverted phase contrast microscope.The expression of apoptotic protein p-JNK,Fas,FasL was detected by Western blotting.Results The cell proliferative potentiality in the MPP+ group (49.30 ± 2.07)％ was significantly lower than that of the normal control group (100.00 ±0.00)％ (P ＜ 0.05).The cell proliferative potentiality in NBP + MPP + group and SP600125 + MPP + group were (71.90 ±2.10) ％ and (76.40 ± 2.80) ％,which was significantly higher than that of the MPP + group (P ＜ 0.05).Apoptosis rate in the MPP + group (32.27 ± 2.26) ％ was significantly higher than that of the normal control group (10.63 ± 2.07) ％ (P ＜ 0.05).The apoptosis rate in the NBP + MPP + group and SP600125 + MPP+ group were (21.13 ± 3.63) ％ and (19.15 ± 2.63) ％,and the apoptosis rate was significantly lower than that in the MPP+ group(P ＜0.05).The protein expression levels of p-JNK,Fas and FasL were significandy lower in NBP + MPP+ group and SP600125 + MPP+ group than that in the MPP+ group (P ＜0.05).Conclusion Butylphthalide can protect the injury of SH-SY5Y cells induced by MPP+.The mechanism of butylphthalide inhibiting apoptosis may be achieved through regulating p-JNK,Fas and FasL protein expression of death receptor pathway in JNK pathway and inhibiting the cell apoptosis.

Objective To establish a HPLC method for simultaneous determination of seven active components in Fufang Danshen tablets and Fufang Danshen dripping pills. Methods These seven compounds were analyzed simultaneously with a Zorbax C18 column by gradient elution using acetonitrile-0. 1% phosphoric acid solution as mobile phase, the flow rate was 1 mL·min-1 and the detection wavelength was set at 203, 270 and 281 nm, respectively. Results All the seven components showed good linear relation between peak area and concentration of the test, and the average recoveries were between 95. 1%-100. 4%. Tanshinone ⅡA was not detected in samples of dropping pills. Conclusion The HPLC method to determine the components including tanshinone ⅡA, salvianolic acid B, propanoid acid, protocatechuic aldehyde, notoginsenoside R1 , ginsenoside Rg1 and ginsenoside Rb1 of the two different Danshen preparations has been established, and it has the advantages of simplicity, high precision, good repeatability, and can be used for the quality control of two kinds of Fufang Danshen preparations. The content of tanshinone Ⅱ A in Fufang Danshen tablet was distinctly higher than that of dropping pills.

Objective To explore the effects of butylphthalide on the apoptosis of SH-SY5Y cells induced by the 1-methyl-4-phenylpyridinium iodide (MPP+). Methods SH-SY5Y cells were cultured with butylphthalide in the dosage of 1 µmol/L, 10 µmol/L and 20 µmol/L for 2 hours, and with MPP+ for 24 hours. The viability of cells was measured with trypan blue. The mitochondrial membrane potential was detected with Rhodamine 123. The content of P53 protein was detected with ELISA. The expression of P53 and cytochrome C (CytC) were detected with Western blotting. Results The viability of SH-SY5Y cells increased with the dosage of butylphthalide (P<0.05), while the mitochondrial membrane potential and the expression of P53 and CytC decreased (P<0.05). Conclusion Butylphthalide can prevents the SH-SY5Y cells from the injury induced by MPP+, which may associate with the inhibition of apoptosis through the expression of P53 and CytC.

It is well known that puerarin possesses protective activity on neurodegenerative diseases. However, the exact path way involved in the protective effect of puerarin on MPP+ -induced cell death is unclear. In this study, we focused on mitochondria im pairment in the apoptotic process of MPP+ -elicited SH-SY5Y cells and detected the protection of puerarin. As evidenced by Trypan blue assay, the cell viability was significantly decreased by 1 mmol x L(-1) MPP+, but reversed by different concentrations puerarin pre treatment. Flow cytometer analysis revealed that MPP+ -induced SH-SY5Y cells apoptosis and arrested the cells in G2/M phase, where as puerarin pretreatment concentration dependently reversed the apoptosis ratio. In addition to the apoptosis ratio, 50.0 micromol x L(-1) puerarin pretreatment even altered the MPP+ -induced G2/M phase arrest. JC-1 assay suggested that MPP+ significantly opened MMP of the SH-SYSY cells; pretreatment with puerarin attenuated the deterioration of the MMP. Both ELISA and Western blotting showed that puerarin prevented the release of cytochrome c from the mitochondrial interior to the cystol elicited by MPP+. DNA ladder showed that typical DNA ladder was present in the MPP+ -induced SH-SY5Y cells. Additionally, MPP+ enhanced caspase-9 and caspase-3 ac tivity, respectively, while not caspase-8. However,the enhancement was concentration dependently blocked by puerarin pretreatment. Taken together, puerarin can modulate mitochondrial membrane potential and inhibit the cytochrome c releasing-caspase cascade to pre vent MPP+ -induced cell injury.
1-Methyl-4-phenylpyridinium ; pharmacology ; Apoptosis ; drug effects ; Blotting, Western ; Caspase 3 ; metabolism ; Caspase 8 ; metabolism ; Caspase 9 ; metabolism ; Cell Cycle ; drug effects ; Cell Differentiation ; drug effects ; Cell Line, Tumor ; Enzyme-Linked Immunosorbent Assay ; Flow Cytometry ; Humans ; Isoflavones ; pharmacology ; Membrane Potential, Mitochondrial ; drug effects ; Mitochondria ; drug effects

OBJECTIVE: To observe the effect of puerarin on energy metabolism of the central nervous in acrylamide-exposed rats. METHODS: Specific pathogen free adult male SD rats were randomly divided into control group,model group,and puerarin low-,medium-,and high-dose groups,with 10 rats in each group. Intraperitoneal injection of acrylamide was given to rats in model group,and puerarin low-,medium-,and high-dose groups( 30 mg/kg body weight). Rats in control group were intraperitoneally injected with equal volume of 0. 9% sodium chloride solution. Rats in the puerarin low-,medium-,and high-dose groups were given puerarin of 40,80 and 160 mg/kg body weight,respectively after one hour of acrylamide exposure,three times a week for continuous 4 weeks. In the 4th week,the rats were sacrificed,the brain and spinal cord were isolated,and the ratio of adenosine diphosphate( ADP)/adenosine triphosphate( ATP),ATP activity and mitochondrial membrane potential( MMP) in brain and spinal cord tissue mitochondria were detected. RESULTS: The ADP/ATP ratio increased in mitochondria of brain and spinal cord tissue in model group and the three puerarin treatment groups( P < 0. 05),meanwhile the ATP synthase activity and MMP decreased compared with control group( P < 0. 05). The ADP/ATP ratio decreased in mitochondria of brain and spinal cord tissue( P < 0. 05),while MMP increased in mitochondria of brain tissue in the three puerarin treatment groups compared with model group( P < 0. 05). The ATP synthase activity increased in mitochondria of brain and spinal cord tissue of puerarin high-dose group( P < 0. 05),and MMP increased in mitochondria of spinal cord tissue of puerarin medium-does group and puerarin high-does group when compared with the model group( P < 0. 05). The ADP/ATP ratio in mitochondria of rat brain and spinal cord decreased with the increase of puerarin doses( P < 0. 05). The MMP of rat brain and spinal cord increased with the increase of puerarin dose( P < 0. 05). CONCLUSION: Puerarin enhances the energy metabolism function of the central nervous in rats by regulating mitochondrial ATP activity. It has certain protective effect on the mitochondrial membrane of central nervous system in rats exposed to acrylamide.