Multi-conformers caused by conformational change of A-ring in the C18- and C19-N-dealkyl diterpenoid alkaloids.

This paper describes a rare phenomenon of multi-conformers caused by conformational change of A-ring in the C18- and C19- N-dealkyl diterpenoid alkaloids. The possible reasons for the generation of multiple conformational isomers are complex, which could be affected by the substituents at C-1, C-3, C-13, C-14, and C-15, pH, solvents, the intramolecular hydrogen bond between 1α-OCH3/1α-OH and N-H groups, acid-base treatment, preparation methods, and work-up procedures.

Comparative Pharmacokinetic Investigation on Multiple Active Aminoalcohol-Diterpenoid Alkaloids after Single Oral Administrations of Monomers and Aqueous Extract of Fuzi (Aconiti Lateralis Radix Praeparata) by UFLC-MS/MS.

To further study the aminoalcohol-diterpenoid alkaloids (ADAs) in Fuzi (Aconiti Lateralis Radix Praeparata), a simple and sensitive UFLC-MS/MS method was established and validated for the determination of five ADAs, aconine, mesaconine, hypaconine, deoxyaconine and fuziline, in rat plasma to compare the pharmacokinetic characteristics of pure ADAs and Fuzi decoction. After precipitating protein with methanol, plasma samples were isolated at 0.5 mL/min flow rate on Waters Acquity UPLC BEH C18 column (100 mm × 2.1 mm, 1.7 µm). The mobile phase was composed of 0.1% formic acid-water and methanol with gradient elution. Mass spectrometric inspection was conducted on a 5500 UFLC-MS/MS system with an electrospray ionization source in patterns of positive ion and multiple reaction-monitoring (MRM). All calibration curves were proved to have acceptable linearity (r2 > 0.99) in linear ranges. Intra-day and inter-day precision and the accuracy met the requirements. The matrix effects of all analytes were between 85% and 115% of three concentration levels. This method has been under verification for comparative pharmacokinetic research after oral administration between aqueous extract of Fuzi and single pure ADAs. The results demonstrated that there are evident pharmacokinetic discrepancies between them, and administration in the extract form instead of pure form may contribute to higher absorption.

On the Famous Traditional Chinese Medicine "Fu Zi": Discovery, Research, and Development of Cardioactive Constituent Mesaconine.

This review summarizes the process of the discovery, research, and development of a cardioactive component, mesaconine, from the lateral roots of Aconitum carmichaelii ("Fu Zi"). To date, pre-clinical showed that mesaconine is a novel type of cardiotonic lead drug with relatively high potency, low toxicity, and a new mechanism.

Further Studies on Structure-Cardiac Activity Relationships of Diterpenoid Alkaloids.

The cardiac effect of thirty-eight diterpenoid alkaloids was evaluated on the isolated bullfrog heart model. Among them, twelve compounds exhibited appreciable cardiac activity, with compounds 3 and 35 being more active than the reference drug lanatoside. The structure-cardiac activity relationships of the diterpenoid alkaloids were summarized based on our present and previous studies [2]: i) 1α-OMe or 1α-OH, 8-OH, 14-OH, and NH (or NMe) are key structural features important for the cardiac effect of the aconitine-type C19-diterpenoid alkaloids without any esters. C18-diterpenoid alkaloids, lycoctonine-type C19-diterpenoid alkaloids, and the veatchine- and denudatine-type C20-diterpenoid alkaloids did not show any cardiac activity; ii) the presence of 3α-OH is beneficial to the cardiac activity; iii) the effect on the cardiac action of 6α-OMe, 13-OH, 15α-OH, and 16-demethoxy or a double bond between C-15 and C-16 depends on the substituent pattern on the nitrogen atom.

Simultaneous determination of thirteen aminoalcohol-diterpenoid alkaloids in the lateral roots of Aconitum carmichaeli by solid-phase extraction-liquid chromatography-tandem mass spectrometry.

Aminoalcohol-diterpenoid alkaloids have been reported as the cardioactive components in the lateral roots of Aconitum carmichaeli (Fuzi) according to recent studies. Determination of these effective components is of great significance for quality control purposes for Fuzi. Here we report, for the first, the development and validation of a new method to determine the 13 aminoalcohol-diterpenoid alkaloids in Fuzi by using a simple and accurate solid-phase extraction-liquid chromatography-tandem mass spectrometry. The chromatographic analysis was performed on an ODS column with methanol-0.1 % formic acid (80 : 20, v/v) as the mobile phase. The quantification was performed using MS/MS detection in the positive ion mode with multiple reaction monitoring. Linearity was observed within a range of concentrations of 20-2,000 ng/mL. For all the analytes, the r value was greater than 0.9990. The limit of detection and the limit of quantitation were less than 0.5 ng/mL and 2.0 ng/mL, respectively. The intraday and interday precisions were less than 5% and 10%, respectively. The accuracy was within the range of 90 to 105%. This method was successfully applied to determine the 13 aminoalcohol-diterpenoid alkaloids in Fuzi from different origins and with different processing methods.

[Identification of aminoalcohol-diterpenoid alkaloids in Aconiti Lateralis Radix Praeparata and study of their cardiac effects].

In order to affirm the cardioactive components in Fuzi, we identified a group of aminoalcohol- diterpenoid alkaloids in Fuzi using ultra high-performance liquid chromatography coupled with electrospray ionization mass spectrometer (UPLC-ESI-MS) method. Among a total of forty-one isolated ingredients, thirteen major aminoalcohol-diterpenoid alkaloids were identified by comparing their retention times and MS spectra with those of the reference substances. Moreover, Fuzi samples from different places of origin and with different processing methods were examined and their components displayed a pattern of high similarity, though the relative abundance varies probably due to their different processing methods. Furthermore, the cardiac effect of each identified alkaloid was individually evaluated using the isolated bullfrog heart perfusion experiment. Among the thirteen aminoalcohol diterpenoid alkaloids tested, six of them significantly enhanced the amplitude rates. Taken together, we affirm that the cardioactive components in Fuzi are aminoalcohol-diterpenoid alkaloids, shedding light on future studies of the mechanisms and development of these cardioactive compounds.

Structure-cardiac activity relationship of C19-diterpenoid alkaloids.

Thirty three C19-diterpenoid alkaloids, twenty-two prepared from known C19-diterpenoid alkaloids and eleven isolated from Aconitum and Delphinium spp. were evaluated for their cardiac activity in the isolated bullfrog heart assay. Among them, eleven compounds exhibited cardiac activity, with average rate of amplitude increase in the range of 16-118%. Compound 7, mesaconine (17), hypaconine (25), and beiwutinine (26) exhibited strong cardiac activities relative to the reference drug. The structure-activity relationship data acquired indicated that an alpha-hydroxyl group at C-15, a hydroxyl group at C-8, an alpha-methoxyl or hydroxyl group at C-1, and a secondary amine or N-methyl group in ring A are important structure features necessary for the cardiac activities of the aconitine-type C19-diterpenoid alkaloids without any ester groups. In addition, an alpha-hydroxyl group at C-3 is also helpful for the cardiac activity of these alkaloids.

Cardioactive C19-diterpenoid alkaloids from the lateral roots of Aconitum carmichaeli "Fu Zi".

Bioassay-guided fractionation of an n-BuOH extract of the lateral roots of Aconitum carmichaeli. led to the isolation of 5 cardioactive C(19)-diterpenoid alkaloids: N-deethylaconine (1), beiwutinine (2), hypaconine (3), mesaconine (4), and 15α-hydroxyneoline (5). N-Deethylaconine and beiwutinine are new aconitine-type C(19)-diterpenoid alkaloids. Hypaconine was isolated from this species for the first time. Among them, mesaconine, hypaconine, and beiwutinine showed the strongest cardiac actions on the isolated perfused bullfrog heart. Furthermore, mesaconine has protective effects, including improved inotropic effect and left ventricular diastolic function, on myocardial ischemia-reperfusion injury in rat at a dose of 10(-9) mol/L. However, mesaconine has almost no effect on heart rate.

Cardioprotective effect of water and ethanol extract of Salvia miltiorrhiza in an experimental model of myocardial infarction.

ETHNOPHARMACOLOGICAL RELEVANCE: Salvia miltiorrhiza has long been used in the traditional Chinese formulations for the treatment of heart ischemic diseases. AIM OF THE STUDY: We investigated the cardioprotective effect of purified Salvia miltiorrhiza extract (SME) in an experimental model of acute myocardial infarction. MATERIALS AND METHODS: Following induction of acute myocardial infarction in rats by adminstration of isoproterenol, hemodynamic and electrocardiographic parameters were monitored and recorded continuously, cardiac enzymes and parameters of oxidative stress were measured, and histopathological examination of heart tissue was performed. Experiments were performed in rats treated with SME or vehicle, as well as in those treated with Fufang Danshen Tablet (FDT) as a positive control which has previously been shown to prevent myocardial ischemia. RESULTS: Isoproterenol-treated rats showed reductions in left ventricular systolic pressure as well as in maximum and minimum rate of developed left ventricular pressure, together with an increase in left ventricular end-diastolic pressure. They also demonstrated ST-segment elevation, together with increases in serum levels of lactate dehydrogenase, glutamic oxalacetic transaminase, creatine kinase and malondialdehyde, as well as decreases in serum activities of glutathione peroxidase and superoxide dismutase. Oral administration of SME (29.76 or 59.52 mg/kg) blunted all of the hemodynamic and biochemical changes induced by isoproterenol, as did FDT (1210 mg/kg). The protective effect of SME on isoproterenol-induced myocardial damage was further confirmed by histopathological examination. CONCLUSIONS: Our results suggest that SME affords protection against isoproterenol-induced myocardial infarction.

[Identification of main related substances in potassium sodium dehydroandrographolide succinate].

To identify the structure of three related substances in potassium sodium dehydroandrographolide succinate (PSDS), an HPLC preparation method was used to separate the impurities. These main impurities were identified using LC-ESI/TOFMS, LC-ESI/MSn, NMR, UV and IR. One of the main impurities was a hydrolyzed and oxidized product of PSDS, which has not been reported previouely. The other two impurities were hydrolyzed products of PSDS after losing different succinic acids. The results indicate that PSDS can be easily hydrolyzed and oxidized. It should be stored at cool and dry places.

Pharmacokinetic study of salvianolic acid A in rat after intravenous administration of Danshen injection.

In order to research the pharmacokinetics of salvianolic acid A (SalA), a herbal ingredient isolated from Salvia miltiorrhiza Bunge, after intravenous administration to rats, a specific and accurate high-performance liquid chromatography (HPLC) was developed. The assay procedure involved simple liquid-liquid extraction of SalA and internal standard (IS, ethyl-p-hydroxybenzoate) from plasma into ethyl acetate. The organic layer was separated and evaporated under reduced pressure at 40 degrees C. The residue was reconstituted in the mobile phase and analyzed on an Inertsil C8 column, monitored at 285 nm. The mobile phase, which consisted of methanol-acetonitrile-water-formic acid (10:20:70:0.4, by vol), was used at a flow rate of 1.0 mL/min. The ratio of the peak area of the analyte to IS was applied to quantify the plasma samples. The standard curve for SalA was linear (r2 = 0.9999) in the concentration range of 0.75-150 microg/mL. The limit of quantitation (LOQ) of SalA was 0.75 microg/mL. The intra- and inter-day precisions (RSD) of the quality control (QC) samples were in the ranges of 2.17-3.29 and 1.24-5.28%, respectively. Accuracy in the measurement of QC samples ranged from 94.7 to 101.1%. This method was validated for specificity, accuracy and precision and was successfully applied to the pharmacokinetic study of SalA in rat plasma after intravenous administration of Danshen injection.

[Determination of alkaloids in Radix Aconiti Lateralis Preparata by RP-ion-pair HPLC].

AIM: To separate and quantitatively determine six alkaloids: aconitine, mesaconitine, hypaconitine, beiwutine, benzoylaconine and benzoylmesaconine in the Chinese traditional medicine Radix Aconiti Lateralis Preparata (Fuzi). METHODS: A RP-ion-pair HPLC method was established. An AichromBond-1 C18 column was used at a column-temperature of 35 degrees C. The mobile phase was CH3CN5 mmol x L(-1) NaH2PO4(50:50) containing 7 mmol x L(-1) SDS at a flow-rate of 1.0 mL x min(-1). The detector was set at UV 235 nm. RESULTS: These six alkaloids can be completely separated and determined quantitatively. CONCLUSION: This method is accurate and suitable for the determination of six alkaloids in Fuzi.

Solid lipid nanoparticles of mitoxantrone for local injection against breast cancer and its lymph node metastases.

In an attempt to improve the therapeutic effect of mitoxantrone (MTO) against breast cancer and its lymph node metastases, solid lipid nanoparticles (SLN) of MTO were prepared, characterized and evaluated on mice. Film dispersion-ultrasonication method was used to prepare MTO-SLN, optimized by central composite design. MTO-SLN were prepared with a mean size of 61 nm, drug content (DC) of 4.18+/-0.10% and encapsulation yield (EY) of 87.23+/-2.16%. MTO-SLN were lyophilized and their mean size became 79 nm without significant change in DC and EY. The in vitro release study revealed a profile of sustained release of MTO from MTO-SLN without burst effect: the cumulative release rate Q24 h = 25.86 +/- 0.82%, t50 = (5.25 +/- 1.10)d and t90 = (28.38 +/- 4.50)d. The drug concentration of MTO-SLN in local lymph nodes was much higher and the drug concentrations in other tissues lower than that of MTO solution (MTO-Soln). Human MCF-7 breast cancer in nude mice and animal model of P388 lymph node metastases in Kunming mice were applied to investigate the therapeutic effects. There was no observed toxicity to the main tissues after local injection of MTO-SLN, but, for MTO-Soln, medium to serious toxicity to liver and lung was produced. The percent inhibition of MTO-SLN against breast cancer was 81.81 +/- 14.03%, while that of MTO-Soln with a double dose was 82.86 +/- 11.13%. The tests for lymph node metastases showed that MTO-SLN gave a mean size of lymph node of 41.85 +/- 27.42 mm3, while that of the MTO-Soln was 119.32 +/- 57.30 mm3 and that of the placebo was 186.83 +/- 77.71 mm3. This study opens a new perspective of active delivery of antitumor drug against breast cancer and its lymph node metastases with inspiring therapeutic effect and little side effects.

[Isolation and structure identification of the main photolysis product in vitamin K1].

OBJECTIVE: To isolate and identify an unknown photolysis product in Vitamin K1. METHODS: A column chromatography method was used to prepare the impurity, and spectrometric analyses of ultraviolet spectrum, infrared spectrum, mass spectrum and nuclear magnetic resonance spectrum were carried out to identify its structure. RESULTS: Its structure is 2-methyl-3-(3-hydroxy-3, 7, 11, 15-tetramethyl-1-hexadecenyl)-1, 4-naphthoquinone. CONCLUSION: The structure of the photolysis product has been identified.

[Determination of progesterone and its main metabolite in rat plasma and uterus using HPLC].

AIM: To quantify progesterone (P) and one of its metabolites 20alpha-hydroxy-4-pregnen-3-one (20alpha-OHP) in rat plasma and uterus after im administration of progesterone. METHODS: Plasma and uterus samples were prepared by liquid-liquid extraction and separated through Shimadzu VP-ODS column (150 mm x 4.6 mm ID, 5 microm). The mobile phase consisted of acetonitrile and water (60: 40, adjusted to pH 4.0 with phosphoric acid). The detector was set at 240 nm. Norgestrel was used as the internal standard. RESULTS: Cmax of P in plasma was (508 +/- 62) microg x L(-1), Tmax was (3.2 +/- 0.4) h, T1/2 (ke) was (10 +/- 4) h and mean AUC0-48h was (5886 +/- 1573) microg x L(-1) x h. The maximum concentration of P in uterus was (1.7 +/- 1.1) microg x g(-1) and the peak time was (5.2 +/- 1.11) h. 20alpha-OHP showed a similar Tmax with P. CONCLUSION: The method is accurate and convenient. It can be used to determine P and its main metabolite 20alpha-OHP simultaneously for studying their preclinical pharmacokinetics.

[Determination of creatine, phosphocreatine and adenosinephosphates in experimental hydrocephalus tissue by reversed-phase high performance liquid chromatography].

OBJECTIVE: To investigate the state of impaired cerebral energy metabolism of the hydrocephalus tissue. METHODS: Adult male dogs were used for establishing the model of kaolin-induced hydrocephalus. A simple and rapid method for the simultaneous determination of creatine (Cr), phosphocreatine (Crp), and adenosinephosphates (AMP, ADP, ATP) in different stages of experimental hydrocephalus tissue by reversed-phase high performance liquid chromatography (RP-HPLC) has been established. The chromatographic conditions were as follows: Inertsil ODS-3 C18 column (4.6 mm x 250 mm i.d. 5 microns), the mobile phase being composed of KH2PO4 buffer (330 mmol/L)-acetonitrile-TBA (45 mmol/L) (94:5.5:0.5) (pH = 6.27) and detector at 210 nm. RESULTS: The calibration curve showed a good linearity in the mass concentration range of 5.69-3640.50 mumol/L (r = 0.9993) for Cr, 3.47-555.50 mumol/L (r = 0.9999) for Crp, 2.69-1723.00 mumol/L (r = 0.9993) for AMP, 2.66-1704.00 mumol/L (r = 0.9999) for ADP and 2.94-1883.50 mumol/L (r = 0.9999) for ATP. The recoveries ranged from 90.10% to 107.00% with relative standard deviations from 1.58% to 3.88%. The detection limits of this method were 3.55-5.84 mumol/L. By means of this method, the Cr, Crp, AMP, ADP and ATP in different stages of 16 dogs experimental hydrocephalus tissue were determined with satisfactory results. CONCLUSION: This method is rapid, precise, accurate and suitable for the determination of the high energy nucleotides in hydrocephalus tissues.

[Determination of danshensu in urine and its pharmacokinetics in human].

AIM: To determine Danshensu in urine and study its pharmacokinetics in human. METHODS: A solid phase extraction-HPLC method was used for determination of Danshensu in urine of human. HPLC separation is performed on a Shim-pack CLC-ODS column (150 mm x 6.0 mm ID, 5 microns) with a mobile phase composed of acetonitrile -0.01 mol.L-1 KH2PO4 (adjusted to pH 2.8 with phosphoric acid). The flow rate was 1.0 mL.min-1 and the UV detector was set at 280 nm. The linear range of Danshensu was 0.2-50 mg.L-1 (r = 0.9999), and its limit of detection was 1.5 ng. The mean recovery was 99.4% (RSD = 2.9%). RESULTS: The pharmacokinetics of Danshensu after p.o. administration of two kinds of pharmaceutical preparations containing Danshen (with 20 mg of Danshensu) were investigated in 6 healthy human volunteers by determining the Danshensu in urine samples. The elimination half lives (T1/2) of Danshensu after p.o. administration of compound granule preparation A and decoction of Danshen were (0.92 +/- 0.16) h and (0.94 +/- 0.21) h, respectively. Their excretions of Danshensu in urine were (6.2 +/- 2.8)% and (14 +/- 4)% of the dose in 8 hours, respectively. CONCLUSION: Under normal doses, Danshensu can be eliminated from kidney. There is no evident difference on elimination half lives of Danshensu after p.o. administration of the two doses, but the excretions of Danshensu by urine after p.o. administration of compound granule preparation A were lower than that of decoction of Danshen.