Five novel and highly efficient endophytic fungi isolated from Huperzia serrata expressing huperzine A for the treatment of Alzheimer's disease.

Huperzine A (Hup A) is an important drug for treating Alzheimer's disease (AD) and mainly extracted from the Huperzia serrata (Thunb.) Trevis. (Lycopodiaceae) (HS). Nevertheless, the content of Hup A in HS is very low of 0.007% with growing circle of 8 to 10 years, and the chemical synthesis of Hup A still has some insurmountable limitations in the industrialized production. So, the available resources of Hup A for clinical treatment of AD are scarce. The purpose of this work was to construct a biosynthesis platform based on the endophytic fungi from HS. In this work, five endophytic fungi Mucor racemosus NSH-D, Mucor fragilis NSY-1, Fusarium verticillioides NSH-5, Fusarium oxysporum NSG-1, and Trichoderma harzianum NSW-V were firstly found and isolated from the Chinese folk medicine HS, which were identified according to their morphological characteristics and nuclear ribosomal DNA ITS sequences. The highest efficient fungus could effectively biosynthesize Hup A in a liquid culture of 319.8 ± 0.17 mg/L which were 112 times higher than that of other reported conventional endophytic fungi. Moreover, these fungi with higher hereditary stability could possess the initial expressing ability of Hup A after 40 generations, and the expressed Hup A from these biosynthesis systems has prior physicochemical properties, a better inhibition activity of acetylcholinesterase and a lower cytotoxicity compared with the listed active pharmaceutical ingredients (APIs) of Hup A. These results provide promising alternative resources for producing Hup A at an industrial scale by biosynthesis, and it may also shed light on millions of AD patients. KEY POINTS: • Five novel endophytic fungi with high stability could highly express prior Hup A Graphical abstract.

A novel embolic microspheres with micro nano binary progressive structure for transarterial chemoembolization applications.

In this work, a novel embolic microspheres with micro nano binary progressive structure (MN-Ms) were developed for transarterial chemoembolization (TCE) applications. The Bletilla striata polysaccharide (Bsp) polymer can inhibit neovascularization and having a dimensional porous network structure, which as the first level of micron structure (microspheres) and will play a role on tumor embolization and inhibition of ischemia-induced neovascularization. The nano flexible liposomes which were embedded by the Bsp polymer microspheres as the second level nano structure to deliver drug across biological membrane barriers. And the micro nano binary progressive structure of MN-Ms was easily formed by using an emulsion crosslinking method. The MN-Ms appeared as perfect round shape with desired swelling and suspensibility characteristics, this was very convenient for embolizing operation by TCE. Due to the binary progressive structure, the MN-Ms could effectively site-specific delivery drug to the targeted liver tissue by enhancing the permeability of Sodium dimethyl-cantharidate (SC) across vessel walls & tissue matrix and delaying drug release at the site of administration, this caused the administrated SC mostly accumulated in the liver, also a higher cytotoxicity to human hepatoma cells. This work indicate that the MN-Ms may be a promising embolic agent for TCE applications for advanced liver cancer.

Level of Coagulation Function Indexes and Its Clinical Significance in Patients with Lymphoplasmacytic Lymphoma / 中国实验血液学杂志

OBJECTIVE@#To analyze the level of coagulation function indexes in patients with lymphoplasmacytic lymphoma (LPL) and its clinical significance.@*METHODS@#The clinical data of 32 patients with initial LPL (LPL group) and physical examination data of 25 healthy persons (control group) who underwent physical examination in our hospital during the same period were collected. The differences of platelet (Plt), D-Dimer (D-D), fibrinogen (Fib), thrombin time (TT), prothrombin time (PT) and activated partial thrombin time (APTT) between the two groups were compared.@*RESULTS@#The Plt count in LPL group [ (137.06±40.14)×10/L] was significantly lower than that in control group [ (215.07±33.25)×10/L], D-D [ (1.01±0.16) mg/L, PT [ (13.01±1.37) s] and APTT [ (40.96±7.24) s] in LPL group were significantly higher than those in control group [ (0.37±0.09) mg/L, (11.96±0.87) s, (25.07±5.13) s] (P＜0.01); there was no significant difference in TT and Fib levels between the two groups (P＞0.05). There was no significant difference in Plt, D-D, Fib, AP, TT and APTT among LPL patients secreting different types of immunoglobulin (Ig) (P＞0.05). After treatment, the coagulation function of LPL patients returned to normal, and no death cases occurred due to hemorrhage or thrombosis.@*CONCLUSION@#LPL patients have hypercoagulable state blood and abnormal coagulation function, but which not closely relates to with the type of Ig secreted by patients.

Expression Level and Target Gene Prediction of miR-181b in Patients with Chronic Lymphocytic Leukemia / 中国实验血液学杂志

OBJECTIVE@#To analyze the diagnostic value of multiple reverse transcription-polymerase chain reaction (RT-PCR) for detecting different fusion genes in children with primary acute lymphoblastic leukemia (ALL).@*METHODS@#The clinical data of 80 children with ALL treated in the 2 affiliated hospital of Xi'an Medical College from September 2012 to September 2017 were collected and retrospectively analyzed. Immunophenotype, chromosome karyotype and fusion gene were analyzed.@*RESULTS@#Immunophenotyping showed that there were 2 cases of mixed expression of myeloid + B system, 2 cases with pre- B expression, 58 cases with former B expression, 11 cases with CD13 combined with pre- B expression, 4 cases with CD5 combined with pre- B expression, and 3 cases with CD2 combined with pre- B expression. The results of chromosome karyotype analysis showed that among 72 cases of karyotype analysts 5 cases could not be analyzed, 27 cases were determined to be normal karyotype, 11 cases with abnormal karyotype and 29 cases without mitotic phase. Six fusion genes were expressed in 30 cases (37.50%) of 80 ALL children, including MLL/AF9, CBF/MYH 11, BCR/ABL, TLS/ERG, MLL/ENL and TEL/AML1. Among the 3 cases with MLL/AF9 fusion gene expression [t(9;11)], 2 cases showed a poor response to early treatment, but achieved complete remission after intensive chemotherapy, and 1 case accepted bone marrow transplantation; in 1 case with CBF/MYH 11 fusion gene expression, treatment was abandoned by family members, and 4 cases with BCR/ABL fusion gene expression [t (9;22) (q34; q11)] were all showed poor response to early treatment, and achieved complete remission after intensive chemotherapy. All the fusion genes were positive during remission, including 2 cases of bone marrow transplantation; 1 case with TLS/ERG fusion gene expression [t (16;21)] displayed poor response to early treatment, and completely remitted after intensive chemotherapy; 2 cases with MLL/ENL fusion gene expression [t (11;19)] recurred during chemotherapy; 19 cases with TEL/AML1 fusion gene expression [t (12;21)] also achieved complete remission. 4 cases achieved a partial remission.@*CONCLUSION@#Genotyping can make up for the insufficiency of MICM typing, and multiplex RT-PCR can be used to rapidly detect the fusion genes caused by chromosomal aberration in children with ALL.

Preparation of matrine loaded microspheres based on Bletilla striata polysaccharide / 药学学报

Microspheres (MS) are an excellent transarterial chemoembolization carrier for cancer treatment. Then the Bletilla striata polysaccharide (BSP) that was isolated from the rattan of Bletilla striata was used as skeleton material, and the matrine (ME) loaded Bletilla striata polysaccharide microspheres (ME-BSPMS) were prepared by emulsify-chemical crosslinking method. ME-BSPMS was characterized for appearance shape, particle size, drug loading, swelling ratio, suspension property, drug entrapment condition and in vitro release characteristics. The results showed that the ME-BSPMS appeared as round spherical and smooth shape by SEM, with an average size of (85 ±7) μm. ME-BSPMS with a good suspension in physiological saline and the swelling ratio could reach upwards of (53 ±4.2)% in 20 minutes, also with a large amount of drug loading of (30.12 ±3.25)%. The results of DSC scanning indicate that good compatibility exists between the ME and BSP, and the ME could be embedded fully in the matrix of the ME-BSPMS. The accumulation drug release from ME-BSPMS was (25.38 ±1.57)% at 12 h, this suggests that the ME-BSPMS has a good sustained release effect. These results indicate that the ME-BSPMS may be a promising transarterial chemoembolization carrier for cancer treatment.

The effects of essential oils of traditional Chinese medicine on the pharmaceutical properties of flexible nano-liposomes loaded with palmatine chloride / 药学学报

Flexible liposomes are an excellent drug delivery nanocarrier, however, the leakage of drugs from liposomes has become common technical obstacle in the industry and also hindered its further application seriously. It is very urgent and necessary to avoid or reduce the leakage of drugs from liposomes. In this work, five kinds of essential oils such as Folium Artemisiae Argyi oil (FA), Folium Eucalypti oil (FE), Arabian Jasmine oil (AJ), Syzygium Aromaticum oil (SA) and Fructus Forsythiae oil (FF) were encapsulated in the lipid bilayer of palmatine chloride (PC) loaded flexible nano-liposomes (PFL), then the optimal essential oil and its dosage level were determined by the external leakage curve of PC. The female Japanese white rabbits were used to evaluate the vaginal irritancy potential of liposomes samples. The pharmaceutical properties such as encapsulation efficiency, particle size, zeta potential, deformability and structure of liposomes samples were evaluated. In order to investigate the permeability of liposomes samples to deliver PC across skin and mucous membrane in vitro, the side-by-side diffusion cells were used. The results showed that the leakage of hydrosoluble PC from PFL was reduced at different degrees by the essential oils in the lipid bilayer of PFL, however, the reduction in leakage degree was obviously higher for FA than thoses of FE, AJ, SA and FF (P < 0.05), and the highest reduction in leakage degree was obtained when the FA and lipid mass ratio was 1:6. The encapsulation efficiency, particle size, zeta potential and deformability of PFL were not significantly changed after FA was encapsulated in the lipid bilayer of the PFL (P > 0.05), so did the lamellar structure of PFL. In addition, the transdermal and transmucosal permeability of PC were also enhanced obviously by encapsulating FA in the lipid bilayer of PFL, and there was no vaginal/vulvar irritation observed in the rabbits. In summary, the drug leakage was reduced by encapsulating suitable essential oil (such as FA) in the lipid bilayer of flexible liposomes, and the vaginal mucosa permeability were improved for the drug. These results provide a novel technique in the improvement of flexible nano-liposomes for drug delivery.

Detection and identification of high producing huperzine A by an endophytic fungus from Huperzia serrata / 国际药学研究杂志

Objective To screen endophytic fungal strains isolated from wild Huperzia serrata which can highly produce huperzine A. Methods The strain producing huperzine A was identified through thin layer chromatography (TLC) and high- performance liquid chromatography(HPLC) with authentic huperzine A. The fungus was identified according to its morphological characteristics and nuclear ribosomal DNA ITS sequence. Results The strain was identified as Fusarium oxysporum NSG-1 according to its morphological characteristics and nuclear ribosomal DNA ITS sequence. The amount of huperzine A produced by the fermentation liquor of this endophytic fungus was quantified to be 1.11 mg/100 ml by HPLC, which was higher than that of previously reported endophytic fungi. Conclusion Endophytic fungus producing high huperzine A can be obtained from H. serrata. The production of huperzine A based on mutagenesis and transformation of the obtained strain may adapt to the industry production.

Identification of alkaloids and huperzine A-producing endophytic fungi isolated from wild Huperzia serrata / 国际药学研究杂志

Objective To screen endophytic fungal strains isolated from wild Huperzia serrata which can produce alkaloids and huperzine A (HupA). Methods Thirty-three endophytic fungal strains were obtained from H.serrata.Alkaloid production was assayed with alkaloid precipitation and acid dye colorimetry. Then, the crude extracts of alkaloids produced from fungal strains were analyzed by modulation of acetylcholinesterase(AChE) activity, TLC and HPLC. Identification of endophytic fungi producing HupA was based on morphology analysis. Results Seven alkaloids produced by endophytic fungal strains were determined based on alkaloid precipitation and acid dye colorimetry, among which strain V generated HupA in vitro. Morphology analysis showed that strain V belonged to Penicillium. Conclusion A high percentage of fungal strains isolated from H.serrata leaves has alkaloid producing potentials. It is the first report that Penicillium species produced HupA. The screening of endophytic fungi producing HupA based on alkaloid precipitation and acid dye colorimetry, AChE activity and structure identification are more efficient and directive.

Effect of Tangshenkang Granule containing serum on renal mesangial cells' proliferation and TGF-β1/Smad2/3 pathway in the high glucose condition / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To study the effect of Tangshenkang Granule (TG) containing serum on renal mesangial cells' (RMCs) proliferation and TGF-β1/Smad2/3 pathway in the high glucose condition.</p><p><b>METHODS</b>Twelve SD rats were randomly divided into four groups, i.e., the low dose TG group, the middle dose TG group, the high dose TG group, and the blank control group, 3 in each group. After 7-day gastrogavage via portal vein blood, rats were sacrificed and their serum samples were collected. RMCs were cultured in common rat serum and TG containing serum respectively. The proliferation of mesangial cells was determined by methly thiazolyl tetrazolium (MTT) assay to determine the optimal TG containing serum concentration. Expression levels of TGF-β1 mRNA and protein were determined by real time quantitative PCR and ELISA. Smad2/3 protein expression and phosphorylation were determined by Western blot and immunofluorescence.</p><p><b>RESULTS</b>TG containing serum at different doses could inhibit high glucose induced RMC cells' proliferation, TGF-β1 over-expression and Smad2/3 phosphorylation.</p><p><b>CONCLUSION</b>TG containing serum could inhibit high glucose induced RMC cells' proliferation, and its mechanism might be possibly associated with inhibiting TGF-β1/Smad2/3 signaling pathway.</p>

Effect of Chinese herbal compounds for blood activating stasis removing, qi benefiting Shen invigorating on high glucose stimulated proliferation of renal mesangial cells and expressions of fibronectin / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To study effects of Chinese Herbal Compounds (CHC) for blood activating stasis removing (BASR), qi benefiting Shen invigorating (QBSI) on high glucose stimulated proliferation of renal mesangial cells (RMCs) and expressions of fibronectin (FN).</p><p><b>METHODS</b>Rats' RMCs were dealt with high glucose and different concentrations of Chinese medicine for 24 and 48 h respectively. The proliferation of RMCs was detected with 4-A thiazolyl blue. mRNA expressions of FN was detected by real time quantitative PCR. The protein expression of FN was detected by ELISA.</p><p><b>RESULTS</b>Compared with the control group, the proliferation obviously increased (P < 0.05, P < 0.01) after 24 and 48 h of treatment in the high glucose group, mRNA and protein expressions of FN also increased (P < 0.01). There was no statistical difference in the proliferation of RMCs or expressions of FN at 24 h between each CHC group and the high glucose group (P > 0.05). Compared with the high glucose group, the proliferation of RMCs and expressions of FN at 24 h each obviously decreased in the CHC group (P < 0.05, P < 0.01).</p><p><b>CONCLUSIONS</b>High glucose could promote the proliferation of RMCs and induce expressions of FN. No obvious effect could be stimulated by CHC treatment for 24 h. The proliferation of RMCs, protein and mRNA expressions of FN could be reversed by CHC treatment for 48 h.</p>

Preparation of sinomenine hydrochloride loaded nano flexible liposomes and their characteristics / 中草药

Objective: To study the preparation of sinomenine hydrochloride (SH) loaded nano flexible liposomes and investigate the mechanism of the flexible liposomes for enhanced in vitro transdermal drug delivery. Methods: The SH loaded nano flexible liposomes were prepared by film dispersion method, and the effects of concentration of phosphatidylcholine (PSC), cholesterol (CH), and propyleneglycol (PG) on the entrapment efficiency of SH were also investigated. The SH content was determined by HPLC. The physical property was evaluated by the atomic force microscope (AFM), transmission electron microscope (TEM) and photon correlation spectrometer (PCS). The side-by-side diffusion cells were used to evaluate transdermal delivery of SH by nano flexible liposomes. At the end of the transdermal experiment, the treated skin was carefully observed by scanning electron microscopy (SEM). Results: The SH loaded nano flexible liposomes were prepared by film dispersion method with the PSC (3%), CH (0.02%), and PG (25%); The SH entrapment efficiency was (66±2.3)%. The prepared nano flexible liposomes had a closed spherical or elliptical shape showed by AFM images, the TEM images appeared as multi-lamellar vesicles. The calculated mean size was (170±26) nm, the zeta potential values of -(43±3.4) mV. The SH loaded nano flexible liposomes caused the structure of stratum corneum (SC) layer disturbed and disordered the intercorneocyte domain wider, this increased the skin permeability of drug. Conclusion: The SH loaded nano flexible liposomes are obviously resulted in a remarkable enhancement of the SH transdermal drug delivery, which could act as a new nanodimensional vehicle for transdermal delivery of SH.