Characterization of co-amorphous complex with piperine and triterpenoids from Ligustri Lucidi Fructus and dissolution evaluation in vitro / 中草药

Objective To prepare a co-amorphous complex of piperine (PIP) and triterpenoids from Ligustri Lucidi Fructus (TLLF) to improve the dissolution of TLLF. Methods TLLF-PIP co-amorphous complex was prepared using solvent evaporation method. The microscopic structure of co-amorphous complex (CAC) was analyzed using differential scanning calorimetry (DSC), scanning electronmicroscopy (SEM), and powder X-ray diffraction (PXRD), the optimal ratio of the two components was investigated through the thermal stability experiment. Ultraviolet-visible spectrophotometry (UV-Vis) and high performance liquid chromatography (HPLC) were applied to determine the amount of TLLF, PIP and the individual components, ursolic acid (UA) and oleanolic acid (OA), respectively. The interaction of the optimal CAC was analyzed by Fourier transform infrared spectroscopy (FTIR), and its solubility and dissolution behavior in vitro were also studied to evaluate the formation of the preparation. Results PXRD analysis indicated that the TLLF-PIP-CAC with a mass ratio of 1:1 exhibited a stable amorphous state, which particles presented in near-spherical shape. FTIR results indicated that there are some hydrogen interactions between the moleculars of TLLF and PIP. The solubility and dissolution determination exhibited a pairwise released behavior with improved triterpenoids and decreased piperine dissolution. Conclusion The prepared TLLF-PIP-CAC can significantly improve the dissolution and amorphous stability of TLLF, which may provide a reference for the compatibility of insoluble compound in traditional Chinese medicine.

Application of Cre-loxP(*) system in constructing the markerless double-gene-deletion strain in Streptococcus mutans / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To construct double gene deletions at the htrA and clpP loci on the chromosome of Streptococcus mutans (Sm) and to remove the antibiotic resistance markers with the Cre-loxP(*) site-specific recombination system.</p><p><b>METHODS</b>The htrA gene was cloned into the pGEM-T-Easy TA cloning vector and then inactivated via the insertion of a kanamycin resistance cassette (lox71-Km-lox66), yielding pGEM-T-ΔhtrA/Km for deleting the htrA gene. Using the same method, the pGEM-T-ΔclpP/Sp was constructed for deleting the clpP gene. Following the transformation of pGEM-T-ΔhtrA/Km in Sm, the homologous recombination event was selected. One such mutant was transformed with a cre expression plasmid (pCrePA). The kanamycin resistance gene was then excised. The pCrePA was then easily eliminated at nonpermissive temperatures, resulting in a mutant strain (MSΔhtrA) carrying a deletion at the htrA loci without a selectable marker. This mutant was verified by PCR and DNA sequencing. Then, the clpP and spectinomycin resistance gene were deleted from MSΔhtrA, yielding markerless mutant strain lacking clpP and htrA.</p><p><b>RESULTS</b>The deletion of htrA, clpP and antibiotic resistance markers were confirmed by PCR analysis and DNA sequencing.</p><p><b>CONCLUSIONS</b>A mutant of Sm was constructed successfully which contained a deletion of the htrA and clpP gene without selectable marker. The Cre-loxP(*) system can be applied to Sm, which provides experimental evidence for generating markerless multiple gene deletion mutants.</p>

Function of luxS gene in sulfurmetabolism of Streptococcus mutans / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To investigate the function of luxS in sulfurmetabolism of Streptococcus mutans (Sm).</p><p><b>METHODS</b>The growth with absorbency (A) of the standards and mutant strains was measured and analyzed in the sulfur-limited defined medium at different periods. The laser scanning confocal microscopy (LSCM) was used to observe and compare the biofilm thickness of the two kinds of strains at different culture conditions.</p><p><b>RESULTS</b>The significant increases in the thickness of mutant strain biofilm and its growth were observed after the addition of cysteine, but did not reach the standards strain levels (P < 0.05). The growth and the biofilm thickness of the mutant strains were (1.301 ± 0.009) and (45.009 ± 0.429) µm. When methionine and S-adenosylhomocysteine of certain concentrations were respectively added, the biofilm thickness and the growth of mutant strain were raised but did not reach the level of the standards strain at 24 h (P < 0.05), but at 48 h they did. When the methionine was added in the mutant strains for 24 h, the biofilm thickness and the growth of mutant strain were (0.448 ± 0.028) and (37.068 ± 2.392) µm, as for the adding of S-adenosylhomocysteine were (0.460 ± 0.005) and (27.343 ± 1.107) µm. When adding the supernatant fluid of standard strains, the biofilm thickness and the growth levels of mutant strain were much higher than those of the standards strain. The biofilm thickness and growth of both kinds of strains decreased after the addition of S-adenosylmethionine.</p><p><b>CONCLUSIONS</b>luxS gene plays not only a role in quorum sensing but also a role in sulfurmetabolism.</p>

Detection of quorum-sensing pathway and construction of luxS gene allelic exchange plasmid of Streptococcus mutans / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To detect the AI-2 quorum-sensing pathway and construct the luxS g-ene allelic exchange plasmid of Streptococcus mutans.</p><p><b>METHODS</b>To detect AI-2 pathway in Streptococcus mutans, the Vibrio harveyi BB170 was used as reporter strain. The PCR fragments of the upstream and downstream regions of luxS and the Erythromycin resistance gene were amplified with the primers respectively, and these fragments were ligated into pUC19 vector with double endonuclease reaction sequentially, the ligated DNAs were transformed into Escherichia coli DH5alpha, then the reconstructed plasmids were isolated and identified by restricted endonuclease digestions.</p><p><b>RESULTS</b>Streptococcus mutans Ingbritt C could induce luminescence of BB170, suggesting the presence of AI-2 quorum sensing pathway in Streptococcus mutans, and such stimulatory activity was maximal at the mid-log growth phase. The recombinant plasmid pUCluxKO was digested by PstI-BamHI, and the digest product were 1000 bp and 5000 bp. When the pUCluxKO was digested by BamHI-KpnI, the digest product were 1500 bp and 4500 bp. While it was digested by KpnI-EcoRI, the digest product were 1000 bp and 5000 bp. All PCR product was in a single belt respectively.</p><p><b>CONCLUSIONS</b>The recombinant plasmid was cloned effectively and can be used in the construction of S.mutans luxS mutant.</p>

Enriching of total flavonoids from Herba Leonuri with polyamide and macroporous resin / 中国中药杂志

<p><b>OBJECTIVE</b>To study the enriching method of total flavonoid from Herba Leonuri with polyamide and macroporous resin.</p><p><b>METHOD</b>Seven enriching and purifying methods were compared with the yield and purity as indexes. The method of enriching with polyamide and macroporous resin was confirmed and the process of purifying was determined by orthogonal design.</p><p><b>RESULT</b>D101 resin is packed by wet method, the ratio of diameter to height is 1:7. After mixed with the extract liquids, the weight of wet resin increased to 3 times of the dry resin. Evaporated the wet resin to dryness, mixed well with a little of 95% ethanol and dry polyamide powder, evaporated them to dryness again. Elute with deionized water until the effluent being colourless, then loaded it on the macroporous adsorptive resin, elute with 50% ethanol, the volume of effluents was collected to 7 times of the column volume. The purity of total flavonoids reached to 23%, while the diversion rate from raw Herba Leonuri was to 69%.</p><p><b>CONCLUSION</b>The process is simple and convenient, and the regeneration of resin is easy, which has a good application foreground.</p>