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Objective: Through network pharmacology, the network relationship between the active component of Sanqi Mixture, the target of hepatic ischemia- reperfusion injury(HIRI), and biological pathway was constructed to explore the key target and mechanism of effect of Sanqi Mixture on HIRI. Method: Through literature research at home and abroad, Traditional Chinese Medicine Systems Pharmacology (TCMSP) platform, Pharm Mapper, Swiss Target Prediction and other servers, oral availability (OB) and drug-likeness (DL) were selected as the limited conditions to collect the relevant targets for Sanqi Mixture for intervention in HIRI. The OMIM database was used to screen and collate HIRI related genes and protein targets. Excel table was used to merge and sort the intersection between disease and targets through Cytoscape3.7.2 software plug-ins Network Analyzer, with topological parameters (degree) ≥ 5 (average degrees of freedom 4.5) for the filter to find the core targets; And the intersection targets were imported to the server STRING, and with Confidence Score of 0.85 or higher for the filter conditions to build the core protein interactions (Hub-PPI) network. The intersection target was introduced into FunRich 3.0 software for biological process and biological pathway analysis, and Cytoscape3.7.2 was used to construct the network of "traditional Chinese medicine-active ingredient-HIRI target-biological pathway". Result: Sanqi mixture could reduce the expression of Aspartate aminotransferase (AST) and glutamate transaminase (ALT) in HIRI mice (P < 0.01). After screening, 45 active components of Sanqi Mixture were obtained, corresponding to 3 273 targets, and the main compounds included ursolic acid, oleanolic acid, brucine, quercetin, ginsenoside F2, paeoniflorin, etc. Among the 196 targets obtained by HIRI, 46 targets were intersected with components, including 11-β-hydroxysteroid dehydrogenase (HSD11B1), adenosine receptor A3 (ADORA3), cyclooxygenase 2 (PTGS2), adenosine receptor A1 (ADORA1), protein kinase C-ε (PKC), etc. With the STRING server setting the qualified condition of Confidence Score ≥ 0.85, the PPI network with high Confidence was obtained and clustered into three categories through cluster processing. Five biological processes including protein metabolism, signal transduction, negative regulation of enzyme activity, inflammatory response and transmembrane receptor protein tyrosine kinase signal pathway were analyzed by FunRich software (P < 0.05). 16 biological pathways including integrin-linked kinase signal, TNF receptor signaling pathway, P38 mitogen-activated protein kinase signaling pathway, and TRAIL signaling pathway (P < 0.01). Conclusion: It is preliminarily discussed that Sanqi Mixture intervenes HIRI through the interaction of multiple components and multiple targets, as well as the regulation of multiple biological pathways and biological processes. However, the key core targets and the specific regulation mechanism still need further experimental verification.
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OBJECTIVE：To prepare Brucine （shorted for “Bru”）bilayer polymer soluble microneedles ，and to investigate their in vitro transdermal permeation characteristics under different drug loading modes. METHODS ：Taking the degree of difficulty of microneedle film uncovering ，array integrity ，bubble amount ，needle shape ，tip hardness and backing toughness as the indexes ， tip and backing materials were screened. The swelling method and drying method of matrix were screened using the morphology of microneedles as index. The double-layer polymer soluble microneedle was prepared by two-step method ，then it was characterized and evaluated in the safety. The in vitro transdermal permeation characteristics of tip-loaded ，backing-loaded and full-loaded Bru bilayer polymer soluble microneedles were investigated by Franz diffusion cell. The in vitro skin penetration curve was drawn ，and the cumulative permeability amount （Q）and cumulative permeability rate were calculated. RESULTS ：The optimal preparation technology of bilayer polymer soluble microneedles included chondroitin sulfate （CS）and polyvinylpyrrolidone K 30（PVP K 30） （1∶1，m/m）as tip materials ，15％ polyvinyl alcohol （PVA）as backing material ，matrix swelling in the refrigerator at 4 ℃ for 1 h，and drying at room temperature for 24 h in dryer. Prepared microneedle array was complete and had good mechanical properties，and could successfully puncture aluminum foil and rat skin. After microneedle treatment ，the skin could return to its original state within 6 h. The results of in vitro transdermal test showed that microneedle drug delivery could greatly increase the cumulative transdermal permeability amount of GNYL Bru，and the tip material could dissolve and release the drug within 10 min； the tip-loaded microneedle was basically released within 8 h，Q8h was 102.185 μg/cm2 and the cumulative permeability rate reached 94.05％ ； the drug cumulativepermeability rate of backing-loaded and full-loaded microneedlesexceeded 50％ within 8 h and exceeded 90％ within 48 h；Q48h were 840.77 and 1 156.73 μg/cm2，showing sustained-release characteristics. CONCLUSIONS ：Bru bilayer polymer soluble microneedles with hard tip and tough backing material are successfully prepared to achieve effective transdermal delivery and sustained release through full-loaded mode.
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Objective: To study the bi-direction transport behavior of brucine and strychnine in the MDCK-MDR1 cell monolayer model. Methods: MTT method was employed to confirm the safe concentration of brucine and strychnine towards MDCK-MDR1 cells. The effects of transport time, drug concentration, and P-glycoprotein inhibitor verapamil on cumulative absorption concentration (Ccum) and apparent permeability coefficient (Papp) of brucine and strychnine in MDCK-MDR1 monolayer cells were studied. Results: The Papp value of brucine and strychnine was larger than 1 × 10-5 cm/s and the ratio of Papp(BL→AP) vs Papp(AP→BL) was less than 2. Brucine/ strychnine combined with verapamil decreased the ratio of Papp(BL→AP) vs Papp(AP→BL). Conclusion: The absorption of brucine and strychnine in MDCK-MDR1 cell monolayer model was well and the passive transference was its main intestinal absorption mechanism. The P-gp inhibitor verapamil has a significant inhibitory effect on brucine and strychnine absorption. Brucine and strychnine may be a substrate of P-glycoprotein.
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Objective: To study the pharmacokinetics and tissue distribution of procesed Strychni Semen in rats after multiple administration at clinical dose. Methods: The rats were given procesed Strychni Semen by intragastric administration for single and several times. The plasma concentrations of brucine and strychnine in plasma and tissues of rats were determined by UPLC-Q-Orbitrap HRMS method, and the organizational distribution differences were compared to explore whether there was accumulation in the body. Results: After single intragastric administration of procesed Strychni Semen, the main pharmacokinetic parameters of brucine and strychnine were as follow: t1/2 was 10.59 and 8.39 h, tmax was 0.77 and 0.64 h, t1/2Ka was 5.38 and 2.63 h, Cmax was 2.97 and 10.83 ng/L, AUC0-t was 87.36 and 172.24 ng∙h/L, CL/F was 40 637.08 and 38 370.26 L/(h∙kg); Brucine and strychnine in processed Strychni Semen reached a steady state after 3 d of administration in rats. After the last administration, the main pharmacokinetic parameters of brucine and strychnine in rats were as follow: t1/2 was 7.07 and 4.75 h, tmax was 0.48 and 0.46 h, t1/2Ka was 3.23 and 1.09 h, Cmax is 5.77 and 34.83 ng/L, AUC0-t was 32.80 and 107.86 ng∙h/L, and CL/F was 75 920.52 and 43 871.54 L/(h∙kg). Compared with the single administration, the content of brucine and strychnine in the liver and kidney tissues was significantly reduced after multiple administrations, and there was no significant change in other tissues. Conclusion: The pharmacokinetics of brucine and strychnine in healthy rats is consistent with one-compartment model. Both of them have the pharmacokinetic characteristics of fast absorption in rats. After 3 d of administration, the serum concentrations of brucine and strychnine reached steady state, and the blood concentration was increased continuously with the increase of administration times. The content of each tissue did not increase after single and multiple administrations. It is suggested that long-term administration of brucine and strychnine will not cause the superposition of the concentration of brucine and strychnine in the blood of rats, which may lead to the occurrence of poisoning.
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Objective: To prepare total alkaloids of Strychni Semen (TASS) - total glucosides of paeony (TGP) lipid-based cubic liquid crystalline nanoparticles (TASS-TGP LLCN) and investigate its percutaneous absorption behavior. Methods: TASS-TGP LLCN was prepared by precursor injection method, and the encapsulation efficiency (EE) was determined by ultrafiltration centrifugation. The prescription of TASS-TGP LLCN was optimized by uniform design with the encapsulation efficiency as the index, and the basic properties of the optimized TASS-TGP LLCN were evaluated. Meanwhile, Poloxamer 407 (F127) was used as matrix to prepare gel, Franz diffusion method was used to compare the in vitro percutaneous permeability of TASS-TGP LLCN gel with TASS-TGP ordinary gel. Results: The optimal formula of TASS-TGP LLCN was glycerol monooleate (GMO) 1.0 g, F127 0.25 g, dispersed phase 60 mL. The EE of brucine, strychnine and paeoniflorin were all more than 50%, the average particle size was about (245.3 ± 16.4) nm, the pH value was 6.62, and the cubic structure was uniform in size under transmission electron microscope. The cumulate osmotic quantities in 24 h, the permeation rate and the skin retention volume of TASS-TGP LLCN gel were all better than ordinary gel of TASS-TGP. Conclusion: TASS-TGP LLCN has dual effects of promoting permeability and skin reservoir, which has a potential development prospect.
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Objective@#To investigate the influence of Brucine on cell apoptosis of pancreatic cancer CFPAC-1 cells and the possible mechanism.@*Methods@#Brucine in different concentrations were used to treat CFPAC-1 cells. Cell proliferation was determined by MTT assay and cell apoptosis was determined by flow cytometer assay. Mitochondrial membrane potential was examined by JC-1 staining. The protein expression of Bax and Bcl-2 was measured by Western Blot.@*Results@#The growth inhibition rates of CFPAC-1 cells after being treated with 0 (control group), 0.4 and 0.8 mmol/L Brucine for 24, 48 and 72 h were 0, (30.23±0.55)%, (40.61±0.15)%, (46.98±1.27)% and(50.17±0.75)%, (61.23±0.91)%, (70.32±0.40)%, increasing with a concentration- and time-dependent increase, which was higher than that in control group; and the differences between either two groups at different time points were statistically significant (P<0.05). CFPAC-1 cell apoptosis rate after being treated with 0, 0.4 and 0.8 mmol/L Brucine for 48 h was (2.92±0.46)%, (4.64±1.31)% and (13.09±0.65)%, which increased gradually with the increased drug concentration. The apoptotic rate in 0.8 mmol/L treatment group was obviously higher than that in control group, and the difference was statistically significant (P<0.05). With the increase of the drug concentration, the red fluorescence gradually decreased, and the green fluorescence gradually increased, indicating that the mitochondrial membrane potential was severely damaged and thus decreased. The protein expression of Bcl-2 in CFPAC-1 cells were(0.92±0.12), (0.67±0.14)and(0.35±0.14)mmol/L, and the expression of Bax in CFPAC-1 cells were(0.56±0.12), (0.85±0.10)and(1.15±0.12)mmol/L. With the increase of brucine concentration, the expression of Bcl-2 was significantly reduced while the expression of Bax was significantly increased; and the difference was statistically significant (P<0.05).@*Conclusions@#Brucine can effectively induce the apoptosis of human pancreatic cancer CFPAC-1 cells through mitochondrial apoptotic pathway by up-regulating the expression of Bax and down-regulating the expression of Bcl-2.
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Aim: To establish an LC-MS/MS method to simultaneously determinate strychnine (STR), brucine (BRU), strychnine N-oxide(SNO) and brucine N-oxide(BNO) in rat tissue, and study their tissue distributions following single intragastric administration of total alkaloids from Semen Strychni at normal and high toxic dose. Methods: Ephedrine hydrochloride was used as the internal standard (IS). Analytes were extracted from tissue samples using liquid-liquid extraction with chloroform. Chromatographic separations were performed on a ZORBAX E-clipse XDB-C18 column(2.1 x 150 mm, 3. 5 μm) with the mobile phase of phase A(water, 10 mM ammonium acetate, adjusted to pH 4. 0 with formic acid) and B(methanol) at a flow rate of 0. 2 mL · min-1. The column temperature was maintained at 30 °C. Mass spectrometric detection was performed on an API4000+ triple quadrupole mass spectrometer equipped with an electrospray ionization (ESI) interface in the positive ion multiple reaction monitoring(MRM) mode. Results: Excellent linearity was observed in all analytes within their linear ranges. In-tra-and inter-day relative standard deviations (RSDs) were less than 14.90%, and accuracies were (85.94 ±1.43)% -(113. 77 ±5.19)%. STR, BRU and SNO distributed in all the tissues in different degree, among which content in kidney and liver was the richest. STR and BRU distributions were low in brain. Conclusions: The developed method is simple, sensitive, specific and reliable, which is suitable for simultaneous determination of STR, BRU, SNO and BNO in rat tissues.
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Objective To investigate the influence of Brucine on cell apoptosis of pancreatic cancer CFPAC-1 cells and the possible mechanism. Methods Brucine in different concentrations were used to treat CFPAC-1 cells. Cell proliferation was determined by MTT assay and cell apoptosis was determined by flow cytometer assay. Mitochondrial membrane potential was examined by JC-1 staining. The protein expression of Bax and Bcl-2 was measured by Western Blot. Results The growth inhibition rates of CFPAC-1 cells after being treated with 0 (control group), 0. 4 and 0. 8 mmol/L Brucine for 24, 48 and 72 h were 0,(30. 23 ± 0. 55)％,( 40. 61 ± 0. 15 )％, ( 46. 98 ± 1. 27 )％ and ( 50. 17 ± 0. 75 )％, ( 61. 23 ± 0. 91 )％, ( 70. 32 ± 0. 40)％, increasing with a concentration-and time-dependent increase, which was higher than that in control group; and the differences between either two groups at different time points were statistically significant (P<0. 05). CFPAC-1 cell apoptosis rate after being treated with 0, 0. 4 and 0. 8 mmol/L Brucine for 48 h was ( 2. 92 ± 0. 46 )％, ( 4. 64 ± 1. 31 )％ and ( 13. 09 ± 0. 65 )％, which increased gradually with the increased drug concentration. The apoptotic rate in 0. 8 mmol/L treatment group was obviously higher than that in control group, and the difference was statistically significant (P<0.05). With the increase of the drug concentration, the red fluorescence gradually decreased, and the green fluorescence gradually increased, indicating that the mitochondrial membrane potential was severely damaged and thus decreased. The protein expression of Bcl-2 in CFPAC-1 cells were(0.92 ±0.12),(0.67 ±0.14)and(0.35 ±0.14)mmol/L,and the expression of Bax in CFPAC-1 cells were(0. 56 ± 0. 12),(0. 85 ± 0. 10)and(1. 15 ± 0. 12)mmol/L. With the increase of brucine concentration, the expression of Bcl-2 was significantly reduced while the expression of Bax was significantly increased;and the difference was statistically significant (P<0. 05). Conclusions Brucine can effectively induce the apoptosis of human pancreatic cancer CFPAC-1 cells through mitochondrial apoptotic pathway by up-regulating the expression of Bax and down-regulating the expression of Bcl-2.
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Objective To optimize the prescription and preparation technology of brucine nanostructured lipid carriers (B-NLC). Methods The method of "the solvent emulsification ultrasound" was used to prepare B-NLC. The prescription and preparation was optimized using a single factor method combined with central composite design-response surface methodology (CCD-RSM). Results The resultant B-NLC was transparent liquid with light blue opalescence. The optimal conditions were that the dosage of drugs was 1.28 mg, the mass concentration of poloxamer 188 was 1.08%, and the ratio of solid lipid to liquid lipid was 1.45:1. The obtained NLC showed the average particle size of (136.89 ± 4.23) nm with a polydispersity index of 0.289 ± 0.005 and a zeta potential of (-34.46 ± 0.31) mV. The entrapment efficiency was calculated to be (68.98 ± 2.06)%, and the drug loading content was (1.90 ± 0.06)%. Conclusion B-NLC prepared by solvent emulsification ultrasound had a high entrapment efficiency and a narrow particle size distribution. The method was easy and simple and can be used to optimize the prescription and preparation of B-NLC, which provides a foundation for the further in vivo research of brucine.
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Objective To study the enzyme kinetics of brucine (BRU) and strychnine (STR), total alkaloids, and monomers active components of BRU and STR in extracts of Strychnos nux-vomica in rat liver microsomes, and investigate metabolic differences between BRU and STR monomer, total alkaloids, and BRU and STR in extracts in rat liver microsomes. Methods The contents of BRU and STR in the monomers, total alkaloids, and extracts in the metabolic system in vitro were measured by LC-MS/MS method, and the kinetic parameters of the enzyme were calculated. Results Compared with monomer group, Km and Vmax value of BRU and STR in total alkaloids and extracts of S. nux-vomica were obviously decreased; Compared with total alkaloids, Km and Vmax value of BRU and STR monomers in S. nux-vomica were obviously increased. The CLint of BRU among total alkaloids and monomer extracts groups had no significantly difference; And the CLint of STR in three groups was successively decreased. Conclusion Total alkaloids, BRU and STR in S. nux-vomica extracts, and BRU and STR monomer can be metabolized by the liver microsomes, and the pharmacokinetical parameters of BRU and STR in all groups have significant differences in metabolism.
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Objective To prepare a new kind of preparation formulation of the total alkaloids from seed of Strychni Semen (TASS), by which could improve their drug loading content and the transdermal function. Methods TASS-LLCN were prepared by hot solvent and high pressure homogeneous method. Using encapsulation efficiency (EE) measured by ultrafiltration as index, the prescription of TASS-LLCN was optimized by response surface methodology with central composite design, and the basic properties of the optimized TASS-LLCN was evaluated. The Franz diffuser method was used to compare the transdermal capacity of both LLCN gel and ordinary gel of TASS. Results The best prescription of TASS-LLCN was as follow: the content of glycerin monooleate (GMO) was 1 403.19 mg/mL, the ratio between GMO and F127 was 7.25:1, the drug loading content was 9.48% and the predicted encapsulation efficiency was 64.01%. The evaluation of basic properties of the optimized TASS-LLCN showed that the average grain diameter was about 186 nm, zeta potential was -33.1 mV, pH was 6.83, the stability was good. The transdermal experiment in vitro showed that the cumulate osmotic quantities in 24 h and the permeation rate of TASS-LLCN gel were both better than ordinary gel of TASS, which had obvious discrepancy (P < 0.05) and showed that LLCN could promote the absorption of active components; the skin retention volume of LLCN gel was still bigger than ordinary gel, which showed that LLCN gel of TASS could store in the skin and release the drug sustainedly. Conclusion TASS-LLCN could obviously enhance the drug loading content and the transdermal function. It is a potential new kind of preparation formulation which have the sustainable effect.
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Objective To prepare brucine solid lipid nanoparticles (SLN) and its lyophilized powder, and then hydrogel matrix sustained-release tablets (HMST) of brucine SLN (SLN-HMST) were prepared. The factors that may influence drug release in vitro and release mechanism were also investigated in present study. Methods Based on single factor test, orthogonal test was designed to gain the optimum prescription. Zero-order, First-order and Higuchi models were used for the model fitting of drug release. Ritger-Pappas models were employed to study release mechanism of brucine SLN-HMST. Results Brucine SLN-HMST was better agreed with First-order kinetics model. The equation was ln(1-Mt/M∞) = -0.212 1 t + 0.106 4 (r = 0.992 3). The cumulative release could achieve 91.48% in 12 h. The sustained release features were obviously. The drug release from the tablets was controlled by diffusion and degradation of the matrix. Conclusion The prepared brucine SLN-HMST can deliver drug continually for 12 h with good reproducibility.
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AIM To establish an HPLC method for the content determination of six constituents in Shiyifang Vinum (Notoginseng Radix et Rhizoma,Dipsaci Radix,Carthami Flos,etc.).METHODS The content determination of notoginsenoside R1,ginsenoside Rg1,ginsenoside Rb1 and asperosaponin Ⅵ was performed on a 30℃ thermostatic Inertsil(R) ODS-3 C18column (4.6 mm ×250 mm,5 μm),with the mobile phase comprising of acetonitrile-0.1％ phosphoric acid flowing at 1.0 mL/min in a gradient manner,and the detection wavelength was set at 203 nm.The content determination of brucine and strychnine was conducted on a 30 ℃ thermostatic Geminni(R) C18 110(A) column (4.6 mm × 250 mm,5 μm),with the mobile phase comprising of acetonitrile-mixed solution of 0.01 mol/L sodium heptanesulfonate and 0.02 mol/L potassium dihydrogen phosphate flowing at 1.0 mL/min in an isocratic elution manner,and the detection wavelength was set at 260 nm.RESULTS Six constituents showed good linear relationships within their own ranges (r ＞ 0.999 0),whose average recoveries were 98.52％-99.96％ with the RSDs of 2.0％-2.3％.CONCLUSION This simple,accurate and reproducible method can be used for the quality control of Shiyifang Vinum.
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Objective:To develop a determination method for strychnine and brucine in Shiyifang vinum by LC-MS/MS. Meth-ods:The samples were separated on an Agilent Zorbax SB-C18column(2.1 mm×100 mm,3.5 μm). The mobile phase was metha-nol-10 mmol·L-1ammonium formate solution (adjusting pH to 3.0 with methane acid)(25: 75), and the flow rate was 0.2 ml· min-1. The eletrospray ionization( ESI) with positive source of the mass spectrometer and multiple reaction monitor( MRM) mode were used to detect strychnine(m/z 395.2→243.8;m/z 395.2→212.8)and brucine(m/z 335.2→183.9;m/z 335.2→155.9). Re-sults:The linear range of strychnine was 0.242-7.733 μg·ml-1,and that of brucine was 0.302-9.661 μg·ml-1. The average re-covery was 100.1% (RSD=0.8%,n=6) and 99.4% (RSD=1.4%,n=6),respectively. Conclusion:The method is simple and accurate,which can be used to determine the contents of strychnine and brucine in Shiyifang vinum.
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OBJECTIVE:To establish a method for the determination of brucine concentration in plasma of rats,and to compare the pharmacokinetic differences between brucine and its nanostructure lipid carrier (NLC) in rats. METHODS:Sixteen male SD rats were randomly divided into brucine NLC solution group and brucine solution group(using normal saline as solvent, and containing brucine 1.28 mg/mL),with 8 rats in each group. They were given relevant solution 10 mg/kg via tail vein. Blood sample 0.5 mL was collected from fundus venous plexus capillary before medication and 15,20,30,40,45,60,90,120,150, 180,210,240,480 min after medication. HPLC method was adopted. The determination was performed on Dikma C18column with mobile phase consisted of methanol-water containing acetic acid and triethylamine(30∶70,V/V)at the flow rate of 1 mL/min. The detection wavelength was set at 265 nm,and column temperature was 30 ℃. Sample size was 10 μ L. Pharmacokinetic parameters of rats in 2 groups were calculated by using DAS 2.0 software,and the difference of them were compared by F test. RESULTS:The linear range of brucine plasma concentration were 1.03-66.00 μg/mL(R2＝0.999 6);the limit of quantitation was 1.03 μg/mL,and lowest detection limit was 0.515 μg/mL. RSDs of intra-day and inter-day were lower than 5%;method recoveries were 84.90%-100.88%, extraction recoveries were 80.60%-91.98%(all RSDs were lower than 10%). Average plasma concentration-time curve of single administration of brucine NLC solution and brucine solution were all in line with two-compartment model after medication via tail vein. The pharmacokinetic parameters included t1/2αwere(0.24±0.11)and(0.06± 0.03)h;t1/2 βwere (2.90 ± 0.22) and (0.57 ± 0.32)h;AUC0-twere (88.00 ± 6.98) and (28.50 ± 5.87)μg·h/mL;AUC0-∞were (109.96±7.99)and(45.06±6.66)μg·h/mL. Compared with brucine solution group,t1/2 α,t1/2 β,AUC0-tand AUC0- ∞of brucine NLC solution group were increased significantly;while CL, k10and k12were decreased significantly, with statistical significance (P＜0.05 or P＜0.01). There was no statistical significance in k21between 2 groups (P＞0.05). CONCLUSIONS: Established HPLC method is simple, specific,sensitive,precise and highly recoverable. It can be used for the determination of plasma concentration and phamacokinetic study of brucine in rats. After brucine NLC is prepared,the pharmacokinetic parameters of brucine change significantly;retention time of brucine is significantly prolonged and the clearance rate decreases significantly.
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OBJECTIVE:To improve the quality standard for Qiwei maqianzi pills.METHODS:TLC was used for the qualitative identification of Chebulae Fmctus and Aucklandiae Radix in the preparation.HPLC method was used for the content determination of hydroxy safflor yellow A,brucine and strychnine in preparation.The determination was performed on Phenomenex Prodigy C18 column with mobile phase consisted of methanol-acetonitrile-0.7％ phosphoric acid soulution(26 ∶ 2 ∶ 72,V/V/V,for hydroxy safflor yellow A),acetonitrile-0.01 mol/L sodium heptanesulfonate mixed with same quantity of 0.02 mol/L potassium dihydrogen phosphate (pH adjusted to 2.8 using 10％ phosphoric acid,21 ∶ 79,V/V,for brucine and strychnine) at the flow rate of 1.0 mL/min.The detection wavelengths were 403 nm (for hydroxy safflor yellow A) and 260 nm (for brucine and strychnine).The column temperature was 25 ℃C,and the injection volume was 10 μL.RESULTS:TLC spots of Chebulae Fructus and Aucklandiae Radix were clear and well-separated without interference from negative control.The linear range was 6.29-62.94 μg/mL for hydroxy safflor yellow A(r=0.999 3),1.83-18.30 μg/mL for brucine(r=0.999 4) and 2.11-21.11 μg/mL for strychnine (r=0.999 6).RSDs of precision,stability and reproducibility tests were lower than 2.0％.The recoveries were 101.66％-104.91％(RSD=1.14％,n=6),99.58％-104.55％ (RSD=1.75％,n=6) and 101.22％-104.04％ (RSD=0.99％,n=6),respectively.CONCLUSIONS:Improved standard can be better used for quality control of Qiwei maqianzi pills.
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Objective Brucine and strychnine monomer reference substance as extremely toxic substance had potential threat during transportation and utilization. In this study we investigated the homogeneity, stability and assignment accuracy of the mixture reference solution of strychnine and brucine, so as to provide reference for the quality control of extremely toxic chemical reference substances for Chinese medicine. Methods Following the assay in Chinese Pharmacopoeia volume I (2015), we prepared the mixture reference solution of brucine and strychnine, and investigated the solvents and the concentration of mixutre reference solution. The stability test lasted for 12 months. F-test was used for heterogeneity assay. Three researchers were involved for collaboration. Results Methanol and chloroform solution were selected as the solvents for the stability test. Results showed the difference was not statistically significant among various mixture solutions. The results of value assignment were 0.14 mg/mL for strychnine (sR = 0.5％)and 0.10 mg/mL for brucine (sR = 1.0％). The stability of mixture solution were better under the conditions of methanol solution at 4 ℃ or -20 ℃. Conclusion The results provide a possible way to develop the mixture solution in place of the monomer reference, and the mixture reference solution is expected for the quality control in the slices of Semen Strychni and its compound preparations.
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<p><b>OBJECTIVE</b>To examine the effect of brucine on the migration, invasion, adhesion and expressions of epithelial-to-mesenchymal transition (EMT) markers and matrix metalloproteinases (MMPs) in the highly metastatic breast cancer cell lines MDA-MB-231 and Hs578-T.</p><p><b>METHODS</b>MDA-MB-231 and Hs578-T cells were divided to 4 groups: the control group (0.1% DMSO), and 25, 50 and 100 μmol/L brucine groups. The cell viability was determined using a CellTiter-Glo® luminescent cell viability. The scratch wound healing assay and tanswell migration assay were used to determine the migration ability of these cells treated by different concentrations of brucine. The proliferation rate, invasive potential and adhesive ability were respectively performed by colony formation assay, transwell invasion assay and adhension assay. The protein and mRNA expressions of EMT biomarkers, MMP-2 and MMP-9 were investigated by real-time reverse transcription polymerase chain reaction and Western blot.</p><p><b>RESULTS</b>Compared with the control group, brucine had little effect on cell viability or proliferation (P>0.05), but led to a dose-dependent decrease on migration, invasion, adhension of MDA-MB-231 and Hs578-T cells (P<0.01). Furthermore, brucine increased the protein and mRNA levels of EMT markers such as E-cadherin and β-catenin in MDA-MB-231 and Hs578-T cells, and decreased the protein and mRNA levels of mesenychmal markers such as vimentin and fibronectin, as well as the expressions of MMP-2 and MMP-9 (all P<0.01).</p><p><b>CONCLUSION</b>Brucine inhibited triple negative breast cancer cells metastasis potentially through EMT reversion and MMP-2 and MMP-9 inhibition.</p>
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The inhibiting mechanisms of brucine to cancer cells included inducing the apoptosis of cancer cells, inhibiting the proliferation of cancer cells, preventing cell adhesion and transfer. This paper reviews the function of brucine and its anti-tumor preparations, and compares the efficacy and adverse reactions between brucine related preparations and traditional drugs to evaluate its anti-tumor effects. Furthermore, more exploration on the inhibiting mechanisms is necessary for the patients to obtain safe and effective drugs.
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AIM To investigate the equilibrium solubilities,oil-water partition coefficients and in vitro skin permeation features of brucine and strychnine in total alkaloids from Strychni Semen.METHODS Saturated dissolution method was applied to determining the equilibrium solubilities of two constituents in ethanol (10％,20％,30％,60％,90％,anhydrous ethanol),trichloromethane,n-octanol and surfactants (0.5％ tween,0.5％ sodium deoxycholate,0.5％ oleic acid).Shake-flask method was adopted in detecting their oil-water partition coefficients in PBS (pH 2.5,4.0,5.0,5.8,6.8,7.0,7.4,9.0).Modified Franz diffusion cell method was used for evaluating their in vitro skin permeation features in PBS,20％ ethanol and anhydrous ethanol.RESULTS Both brucine and strychnine showed the highest equilibrium solubilities in trichloromethane and the lowest equilibrium solubilities in surfactants.The equilibrium solubility of strychnine was higher than that of brucine in ethanol (＞ 20％) or PBS (pH ＜ 8.0),which reached the highest in 60％ ethanol and pH 2.5 PBS,respectively.The similar oil-water partition coefficients of two constituents,proportional to pH value,reached the highest at pH9.0.And they exhibited the highest accumulated transdermal absorptivities in anhydrous ethanol and pH 9.0 PBS,respectively.CONCLUSION Solvent type has obvious effects on the equilibrium solubilities,oil-water partition coefficients and in vitro skin permeation features of both brucine and strychnine.This study can provide a reference for the bioavailability improvement of transdermal drug delivery and development of related preparations.