OBJECTIVETo examine the in vitro dissolution of forsythin in Forsythia suspensa powder of different particle diameter, in order to give guidance to the grinding process.

METHODHPLC was used to determine the in vitro dissolution quantity and dissolution velocity of forsythin coarse powder, fine powder and ultramicroscopic powder.

RESULTThe dissolution curves of Forsythia suspensa coarse powder, fine powder and ultramicroscopic powder were basically inconformity to Weibull distribution. Specifically, T50 was 11.8, 10.5 and 6.8 min, respectively, and Q45 was 78.22%, 81.91% and 90.76%, respectively.

CONCLUSIONThe superfine milling process can significantly increase the dissolution quantity and dissolution velocity of forsythin.

Bridged Bicyclo Compounds, Heterocyclic ; chemistry ; Chromatography, High Pressure Liquid ; Forsythia ; chemistry ; Furans ; chemistry ; Particle Size ; Powders

Humans ; Bridged Bicyclo Compounds, Heterocyclic/therapeutic use* ; Hydrazines/therapeutic use* ; Leukemia, Myeloid, Acute/drug therapy*

Airway Extubation ; adverse effects ; Bridged Bicyclo Compounds, Heterocyclic ; therapeutic use ; Endoscopy ; Humans ; Nasal Surgical Procedures ; Tetrahydronaphthalenes ; therapeutic use ; Trachea

OBJECTIVETo explore the inhibitory effect of norcantharidin (NCTD) on the expression of DNA replication initiation protein Cdc6 in cancer cells.

METHODSMTT assay was performed to detect the inhibitory effect on different cancer cell lines, including HeLa, HepG2, Jurkat and Ramos cells. The effect of NCTD on Cdc6 protein level was detected by Western blotting, and BrdU incorporation assay was used to evaluate the DNA replication of the cells.

RESULTSNCTD significantly inhibited the proliferation of the cells and caused degradation of Cdc6 protein to result in the inhibition of the DNA replication of the cells shown by BrdU incorporation assay.

CONCLUSIONNCTD can induce the degradation of Cdc6 in cancer cells to produce an anti-cancer effect.

Bridged Bicyclo Compounds, Heterocyclic ; pharmacology ; Cell Cycle Proteins ; metabolism ; Cell Line, Tumor ; DNA Replication ; drug effects ; Humans ; Nuclear Proteins ; metabolism

In order to find the endogenous potential biomarkers of in vitro hepatic injury caused by NCTD-Na and elucidate the mechanism of hepatic injury of NCTD-Na,ultra-high performance liquid chromatography coupled quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF-MS/MS) was used for lipidomics detection.Multivariate statistical analysis was used to study the endogenous lipid metabolic changes of human normal liver cells LO2 injury after the treatment with sodium norcantharidate(NCTD-Na).The results showed that the half maximal inhibitory concentration(IC50) of NCTD-Na was 0.034 mmol·L-1.A total of 280 differential metabolites were found between the control group and the low-dose group,with VIP > 2.0 and P<0.05.At the same time,a total of 273 differential metabolites were found between the control group and the high-dose group,with VIP > 2.0 and P<0.05.Cell metabolite profiles showed clear separation among control group,the low-dose group and the high-dose group,and 111 differential metabolites were found,with VIP > 2.0,P<0.05,RSD<30% and in a dose-dependent manner.It was found that most of the above differential metabolites were lipid metabolites after the analysis of simple preparnation methods and database search.A total of 32 potential biomarkers were identified,including 3 phosphatidylcholine(PC),5 lysophosphatidylcholine(Lyso PC),3 ceramide(Cer),1 sphingomyelin(SM),1 phosphatidylethanolamine(PE),10 lysophosphatidylethanolamine(LysoPE),4 diacylglycerol(DG),1 Phosphatidic acid(PA),1 lysophosphatidic acid(Lyso PA),1 phosphatidyl glycerol(PG),1 fatty acid hydroxy fatty acid(FAHFA) and 1 phosphatidylserine(PS).The changes of PCs,Cers,SM,PE and DGs were closely related liver protection,DNA methylation and self-repair in hepatocytes,apoptosis,methylation and detoxification of carcinogens,as well as lipid peroxides production process.Also,they had impact on the proliferation of hepatocytes,differentiation and gene transcription disorders.Cells stimulated by NCTD-Na could promote the production of PA as well as the synthesis and catabolism of FAHFA in a variety of ways.The levels of Lyso PCs,LysoPEs and Lyso PA were correlated with PCs,PE and PA;PE and PS might have valgus during apoptosis,triggering phagocytosis.
Bridged Bicyclo Compounds, Heterocyclic ; pharmacology ; Cells, Cultured ; Hepatocytes ; drug effects ; metabolism ; Humans ; Lipid Metabolism ; Lipids ; analysis ; Tandem Mass Spectrometry

OBJECTIVETo explore the effect and mechanism of norcantharidin (NCTD) on hematopoiesis function in leucopenia model rat induced by cyclophosphamide (CTX).

METHODSLeucopenia model was replicated in SD rat with cyclophosphamide(CTX) and model animal was treated with NCTD. Peripheral blood and bone marrow tissue samples were collected from the rats in each experimental group. Peripheral white blood cells (WBC) were counted and analyzed by automatic blood cell analyzer. Histopathologic changes of the biopsied bone marrow tissues were observed by histopathological techniques. The cell cycle and apoptosis rate of bone marrow cells were detected by flow cytometry. Immunohistochemical method was applied to observe the expression of apoptosis-related proteins BCL-2 and BAX in bone marrow.

RESULTSAfter NCTD treatment in model rats, the WBC count of peripheral blood obviously increased, the cell structure of bone tissue significantly recovered, NCTD could promote the cell proliferation and cycle changes of bone marrow cells, inhibit the bone marrow cell apoptosis and necrosis induced with CTX, up-regulate the expression of apoptosis-related protein BCL-2 and downregulated the BAX.

CONCLUSIONNCTD can stimulate the bone marrow hematopoiesis and promote recovery of peripheral white blood cell level in the leukopenia model induced by CTX, and its mechanism may be related with NCTD regulating bone marrow cell cycle and with NCTD inhibiting cell apoptosis.

Animals ; Apoptosis ; Bone Marrow ; Bone Marrow Cells ; Bridged Bicyclo Compounds, Heterocyclic ; Cell Cycle ; Cell Proliferation ; Cyclophosphamide ; Disease Models, Animal ; Flow Cytometry ; Hematologic Diseases ; Hematopoiesis ; Rats ; Rats, Sprague-Dawley

OBJECTIVEMonoclonal antibody (mAb) conjugated with certain toxin to generate immunotoxin bears an important and promising effect as a new therapy for patients with hematopoietic malignancies. However, most toxic moieties conjugated with antibody proteins reported in the literature were toxic proteins which presented immunogenicity to patients capable of inducing anti-toxin antibody. Norcantharidin (NCTD) is a small molecule toxin. It does not have the immunogenicity to human body so that it bears a promising potential for development of new targeting drug. In this study, a new clone of self-made anti-CD19 mAb named ZCH-4-2E8 conjugated with NCTD was used to investigate its targeting efficacy against CD19+ lymphoid malignant Nalm-6 cells in vitro to provide the experimental data for the further development of this new targeting agent.

METHODSA monoclonal antibody named 2E8 was prepared from mouse ascites and purified by gel chromatography. The purity of the antibody protein was checked by SDS-PAGE assay. Immunotoxin 2E8-NCTD was successfully generated through conjugating CD19 mAb protein and Norcantharidin by the activated ester method. The binding activity of the immunoconjugate (2E8-NCTD) against CD19 antigens on cell surface and the expression levels of CD19 antigens on Nalm-6 and K562 cells were examined by flow cytometry. Comparisons of the inhibitory effects among PBS, purified 2E8 antibody, norcantharidin and immunotoxin 2E8-NCTD groups on cell growth of either Nalm-6 cells or K562 cells were made.

RESULTSThe purity of the purified 2E8 antibody was higher than 99.00% demonstrated by SDS-PAGE assay. 2E8 antibody in the supernatant reacted with 99.34% of Nalm-6 cells, while only 0.98% of K562 cells reacted with this antibody. The newly generated immunotoxin (2E8-NCTD) had a positive rate of 99.90% on Nalm-6 cells with little reduction of binding activity. From the in vitro study, both 2E8-NCTD and norcantharidin were shown to have significant inhibitory effects on the growth of CD19+ Nalm-6 cells (P < 0.001), while the purified 2E8 antibody did not show any significant influences on the growth of Nalm-6 cells. Since no significant inhibitory effects were identified among immunotoxin 2E8-NCTD, 2E8 antibody and control groups on CD19(-) K562 cells, a significant targeting effect of the 2E8-NCTD against Nalm-6 cells was confirmed.

CONCLUSIONSThe immunotoxin 2E8-NCTD was successfully synthesized by activated ester method with an excellent targeting killing effect on CD19+ Nalm-6 leukemia cells in vitro, which provides some experimental data for the further development of this new targeting agent.

Animals ; Antibodies, Monoclonal ; biosynthesis ; Antigens, CD19 ; immunology ; Bridged Bicyclo Compounds, Heterocyclic ; chemical synthesis ; immunology ; Electrophoresis, Polyacrylamide Gel ; Humans ; Hybridomas ; Immunotoxins ; immunology ; K562 Cells ; Mice ; Mice, Inbred BALB C

The skin of Bufo bufo gargarizans, originated from Bufo bufo gargarizans Cantor (Bufonidae), is widely used in traditional Chinese medicine for the treatment of hepatoma, lung cancer and etc. The preparation of the aqueous components has significant therapeutic effect against the digestive tract cancer. The water-soluble chemical constituents in the skin of Bufo bufo gargarizans were then investigated to make clear the active compounds. Six compounds were isolated and purified by recrystallization and column chromatography on silica gel and ODS, their structures were elucidated as 4-amido-3-hydroxymethyl-cyclooctylamidezotetra-alpha-furanone (I), bufogargarizanine C (II), bufothionine (III), dehydrobufotenine hydrobromide (IV), suberic acid (V) and succinic acid (VI) on the basis of physicochemical properties and spectral data (UV, IR, 1H NMR, 13C NMR and MS). Of the above compounds, compounds I and II are new compounds and named bufogargarizanine B and C, respectively.
Animals ; Bridged Bicyclo Compounds, Heterocyclic ; chemistry ; isolation & purification ; Bufo bufo ; Caprylates ; chemistry ; isolation & purification ; Dicarboxylic Acids ; chemistry ; isolation & purification ; Medicine, Chinese Traditional ; Molecular Conformation ; Molecular Structure ; Skin ; chemistry

Animals ; Bridged Bicyclo Compounds, Heterocyclic ; pharmacology ; Heterografts ; Male ; Mice ; Mice, Inbred Strains ; Mice, Nude ; Multiple Myeloma ; metabolism ; NF-kappa B ; metabolism ; Signal Transduction ; drug effects

The establishment of high specificity and sensitivity method of small molecule monoclonal antibody-based immunoassay has a great importance in the study of small molecule compounds in Chinese medicine, wherein synthesis of small molecule artificial antigen is a critical step in the preparation of small molecule antibodies. Oxidation method using sodium iodide was used to synthesize immunogenic antigen (FRn-BSA) and coating antigen (FRn-OVA) of forsythin. UV spectroscopy and thin layer chromatography showed that forsythin was successfully conjugated with BSA and OVA. After immuned FRn-BSA, the mice could specifically produce anti-forsythin antibodies with titer up to 1:8 000, and the linear range was from 1 mg x L(-1) to 100 mg x L(-1). In this paper, the artificial antigen of forsythin was successfully synthesized, which can be applied for preparation of monoclonal antibodies and establishment of appropriate immune method.
Animals ; Antibodies ; immunology ; Antigens ; chemistry ; immunology ; Bridged Bicyclo Compounds, Heterocyclic ; chemistry ; immunology ; Drugs, Chinese Herbal ; chemistry ; Furans ; chemistry ; immunology ; Male ; Mice ; Mice, Inbred BALB C