Preparation of trans-Crocetin with High Solubility, Stability, and Oral Bioavailability by Incorporation into Three Types of Cyclodextrins.

Crocetin (CRT), an active compound isolated from saffron, exhibits several pharmacological activities, including anti-tumor and immune-regulatory activities, and is effective against myocardial ischemia and coronary heart disease; however, its low stability and solubility limit its clinical application. Therefore, we investigated CRT inclusion complexes (ICs) with three cyclodextrins-α-CD, HP-ß-CD, and γ-CD-suitable for oral administration prepared using an ultrasonic method. Fourier transform infrared spectroscopy and powder X-ray diffraction indicated that the crystalline state of CRT in ICs disappeared, and intermolecular interactions were observed between CRT and CDs. 1H nuclear magnetic resonance and phase solubility studies confirmed CRT encapsulation in the CD cavity and the formation of ICs. In addition, we observed the morphology of ICs using scanning electron microscopy. All ICs showed a high drug encapsulation efficiency (approximately 90%) with 6500-10,000 times better solubilities than those of the pure drug. CRT showed rapid dissolution, whereas pure CRT was water-insoluble. The formation of ICs significantly improved the storage stability of CRT under heat, light, and moisture conditions. Further, the peak time of CRT in rats significantly decreased, and the relative bioavailability increased by approximately 3-4 times. In addition, the oral bioavailability of CRT IC was evaluated. Notably, the absorption rate and degree of the drug in rats were improved. This study illustrated the potential applications of CRT/CD ICs in the food, healthcare, and pharmaceutical industries, owing to their favorable dissolution, solubility, stability, and oral bioavailability.

Enhancement of Dissolving Capacity and Reducing Gastric Mucosa Irritation by Complex Formation of Resibufogenin with ß-Cyclodextrin or 2-Hydroxypropyl-ß-cyclodextrin.

Resibufogenin (RBG) is a natural medicinal ingredient with promising cardiac protection and antitumor activity. However, poor solubility and severe gastric mucosa irritation restrict its application in the pharmaceutical field. In this study, the inclusion complex of RBG with ß-cyclodextrin (ß-CD) and 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CD) was prepared using the co-evaporation method, and the molar ratio of RBG to CD was determined to be approximately 1:2 by continuous variation plot for both CDs. The formation of inclusion complexes between RBG and each CD (RBG/ß-CD and RBG/HP-ß-CD) was evaluated by phase solubility study, Fourier transform infrared spectroscopy, and thin-layer chromatography. Powder X-ray diffraction and differential scanning calorimetry confirmed drug amorphization and encapsulation in the molecular cage for both CDs. Moreover, the inclusion complexes' morphologies were observed using scanning electron microscopy. The dissolution rate of the inclusion complexes was markedly improved compared to that of RBG, and the complexes retained their antitumor activity, as shown in the in vitro cytotoxicity assay on a human lung adenocarcinoma cancer (A549) cell line. Moreover, less gastric mucosal irritation was observed for the inclusion complex. Thus, the inclusion complex should be considered a promising strategy for the delivery of poorly water-soluble anticancer agents, such as RBG.

Simultaneous quantitative analysis of 11 flavonoid derivatives with a single marker in persimmon leaf extraction and evaluation of their myocardium protection activity.

An improved, reliable and comprehensive method for assessing the quality of the ethyl acetate extract from persimmon leaves (EAPL) and its commercial preparation, Naoxinqing (Brain and Heart Clear capsules), has been developed and validated. Based on HPLC-DAD-ESI-Q-TOF-MS analysis, myricetin-3-O-ß-D-galactoside (1), myricetin-3-O-glucoside (2), quercetin-3-O-ß-D-galactoside (3), quercetin-3-O-ß-D-glucoside (4), quercetin-3-O-(2″-O-galloyl-ß-D-galactoside) (5), quercetin-3-O-(2″-O-galloyl-ß-D-glucoside) (6), kaempferol-3-O-ß-D-galactoside (7), kaempferol-3-O-ß-D-glucoside (8), kaempferol-3-O-(2″-O-galloyl-ß-D-galactoside) (9), kaempferol-3-O-(2″-O-galloyl-ß-D-glucoside) (10), quercetin (11) and kaempferol (12) were identified from 15 batch samples. A HPLC fingerprint analytical method was established. All compounds, with the exception of compound 2, were simultaneously quantified by the single standard to determine multi-components (SSDMC) method, using kaempferol-3-O-ß-D-glucoside as the internal standard. The rate of analysis was found to be faster with the SSDMC method than with current acid hydrolysis method (Pharmacopoeia of the People's Republic of China 2015 edition) and the results were more intuitive and reliable. Three-dimensional principal component analysis revealed that there were similar characteristics in persimmon leaf from same district. Analysis of the myocardial cell protection activity of 11 monomeric compounds showed that compounds 12, 11 and 10 were the main active ingredients that produce pharmacologic functions in EAPL. Among these compounds, the bioactive constituent of myricetin-3-O-ß-D-galactoside was determined for the first time in Diospyros khaki. Thus, we have established an effective assessment method that can be applied to the comprehensive quality evaluation of EAPL extract and Naoxinqing capsule.

A new indole alkaloid from the traditional Chinese medicine Chansu.

A new indole alkaloid N'-formylserotonin (1), along with five known indole alkaloids N'-methylserotonin (2), 5-hydroxy-1H-indole-3-carbaldehyde (3), N-acetylserotonin (4), 6-hydroxy-1-oxo-3,4-dihydro-ß-carboline (5), and bufoserotoin C (6), were isolated from the water extract of traditional Chinese medicine Chansu. Their structures were elucidated on the basis of spectral analyses. The cytotoxicities of 1-6 against human lung adenocarcinoma epithelial cells A549 were tested using the MTT method. Compound 6 exhibited stronger cytotoxic effect than 5-FU, and 1-5 showed no cytotoxic effects. Bufoserotonin C is one of the cytotoxic components in water-soluble extract of Chansu.

A New Indole Alkaloid from the Toad Venom of Bufo bufo gargarizans.

A new indole alkaloid named bufobutarginine (1), along with three known bufotenines, namely, serotonin (2), bufotenidine (3), and bufotenine (4), were isolated from the water extract of toad venom. Their structures were elucidated by spectral methods. This is the first time that arginine has been found to be involved in the biosynthesis of bufotenines in parotid of toad. The cytotoxic activities of these compounds have been assayed against A375 and A549 cell lines by the MTT method; however, they showed no cytotoxic activities.

Improvement of the Antitumor Efficacy of Intratumoral Administration of Cucurbitacin Poly(Lactic-co-Glycolic Acid) Microspheres Incorporated in In Situ-Forming Sucrose Acetate Isobutyrate Depots.

Localized drug delivery strategies for cancer therapy have been introduced for decades as a means of increasing drug concentration at tumor target site and minimizing systemic toxicities. In this paper, a combination of microspheres (MSs) and sucrose acetate isobutyrate (SAIB) in situ-forming implants (ISFIs) was evaluated for improving antitumor efficacy via intratumoral injection. Monodispersed cucurbitacin (Cuc)-loaded Poly (lactic-co-glycolic acid) (PLGA) MSs with mean diameter of about 5 µm were fabricated by Shirasu porous Glass (SPG) membrane emulsification technique, and their properties were investigated. The in vitro drug release pattern, antimelanoma efficiency, and drug distribution in tumor of three different intratumoral injection systems, that is, MSs, SAIB ISFIs, and combination of MSs and SAIB ISFIs (SAIB-MSs), was investigated. The Cuc-loaded MSs prepared by PLGA (LA/GA = 50:50, inherent viscosity = 0.87 dL/g), has an appropriate release pattern with lower initial burst and delayed drug release. SAIB-MSs have a much slower drug release rate than that of MSs or SAIB ISFIs. SAIB-MSs showed the best antitumor efficacy in melanoma-bearing mice model, and the results of drug distribution in tumor revealed that the incorporation MSs in SAIB solution obviously extended the residence of drug in tumor. The low Cuc concentration in tumor periphery region after intratumoral administration of SAIB-MSs demonstrated poor drug penetration of this system. For further improving the antitumor efficacy of intratumoral chemotherapy, elegant designing to carriers with both extended residency and wide drug distribution in tumor is needed.

[Cloning, prokaryotic expression and function of the Bufo bufo gargarizans tryptophan hydroxylase I gene].

Tryptophan hydroxylase I（TPH1） catalases the 5-hydroxylation reaction of L-Trp, which is the rate-limiting step in the synthesis of serotonin. Serotonin, a major component of Bufonis venenum, is involved in numerous physiological functions as an important neurotransmitter. In this study, BbgTPH1 cDNA was cloned from the parotid gland of Bufo bufo gargarizans. Genetic engineering techniques were used to construct a recombinant prokaryotic fusion expression plasmid pMAL-BbgTPH1, and the induced conditions to express the recombinant BbgTPH1 in E. coli TB1 cells were optimized. The full length of BbgTPH1 is 1 984 bp (GenBank accession No. JQ768313) with a 1 443 bp open reading frame (ORF) encoding a 480 amino acid residues. The deduced protein molecular weight is 55.2 k Da and its theoretical isoelectric point is 5.58. The sequence includes conserved domain and special signal sequence of the aromatic amino acid hydroxylase (AAAH) superfamily. Homologous alignment showed that BbgTPH1 shared a high homology with other species. Phylogenetic tree showed the closest relative to BbgTPH1 was Xenopus tropical-TPH1. The best induction conditions of recombinant BbgTPH1 were 0.5 mmol·L(-1) IPTG at 20 ℃ for 8 h. The function of BbgTPH1 was identified by in vitro enzymatic reaction and the recombinant BbgTPH1 was able to produce 5-hydroxytryptophan by catalyzing tryptophan. This study represents the first time of cloning and identification of the function of TPH1 in Bufo genus. The results of this study will be an important foundation for future studies of biosynthesis of bufotenines in the parotid gland of B. bufo gargarizans.

[Pharmacokinetics of Tongmai granule in acute myocardialischemic model beagles].

Tongmai granule (TM) is composed of Puerariae Lobatae Radix (Gegen), Salviae Miltiorrhizae Radix et Rhizoma(Danshen) and Chuanxiong Rhizoma(Chuanxiong). It has been used to treat ischemic cardio-cerebrovascular diseases for decades. For the purpose of elucidating its pharmacodynamic material foundation, the absorption and pharmacokinetic property of TM were investigated in acute myocardial ischemic model beagles. All serum samples were extracted before analysis with ethyl acetate after being acidified by hydrochloric acid. Under negative ESI detection mode, the chromatographic separation was carried out with monolithic C18 column for gradient elution. A simultaneous quantitative analysis was made on 15 polyphenols, including 8 from Gegen, 5 from Danshen and 2 from Chuanxiong, in 8.5 min. The validation result demonstrated the specificity, accuracy and precision of the method in line with the bioanalysis requirements. After TM solution was administrated to acute myocardial ischemic model beagles through duodenum injection, serum samples were collected after 6 h. The quantitative detection proved the prompt absorption of TM, all of the components were detectable in the blood samples 5 min later, and reached peak respectively at 0.18-3.83 h after administration. The components presented large variabilities. The most components were exposed in serum with puerarin and salvianic acid A, followed by 3'-methoxypuerarin, mirificin, and 3'-hydroxypuerarin. The study proves puerarin and salvianic acid A are dominating active components of TM in acute myocardial ischemic model beagles.