益生菌发酵中药的研究现状及产品开发

中药发酵技术历史悠久,是中药炮制的一种重要方法。随着全民健康意识的提高,人们将益生菌广泛应用于药食两用中药,利用益生菌发酵中药成为科学研究的热点,通过中药发酵技术开发出的一系列健康产品颇受消费者的青睐,拥有广阔的市场前景。中药经益生菌发酵后具有提高药效、更易人体吸收、降低毒副作用等优势,该文从单一菌种发酵中药和多菌种混合发酵中药两方面对益生菌发酵中药的研究现状进行阐述,并对益生菌发酵药食两用中药开发的相关产品进行综述,为该领域进一步深入研究探索及相关开发提供参考。

Apoptotic Effects of Hypocrellin A on HeLa Cells

Hypocrellin A（ HA）, a photosensitive perylenequinone compound of Hypocrella bambusae, inhibited the proliferation of several tumor cell lines. Human cervical cancer cells, HeLa ceils, were used as a model to elucidate the molecular mechanisms of HA-induced tumor cell death. The results show that HA can induce the oligonucleosomal fragmentation of DNA in HeLa cells and also can increase the expression of apoptosis inducer Bax mRNA and that it decreases the expression of apoptosis suppressor, Bcl-2 mRNA, in mitochondria. It can be concluded from the data that HA-induced apoptosis is related to the balance between Bcl-2 and Bax gene expressions.

Isolation,Purification and Bioactivities of Polysaccharides from Irpex lacteus

Irpex lacteus has been widely used for treating chronic glomerulonephritis as a traditional Chinese medicine.Seven water-soluble polysaccharide fractions（ILN I,ILN II,ILN III,ILA I,ILA II,ILB I and ILB II） were isolated and purified from Irpex lacteus by hot-water extraction,deproteinization,decolorization,dicthy laminoethyl（DEAE）-cellulose ion exchange and sephadex G100 chromatographies,respectively.The average molecular weights and monosaccharide composition of these polysaccharide fractions greatly differed from each other.The antitumor and antinephritis activities of the seven polysaccharide fractions were evaluated.It was found that ILN III displayed significant inhibition effects on both humar hepatocellular liver carcinoma（HepG2） and hentietta lacks（HeLa） tumor cells with IC 50 values of 60.95 and 99.95 μg/mL,respectively.ILA I exhibited significant inhibition effects on murine mesangial cells（HBZT-1） with an IC 50 value of 185.06 μg/mL.The inhibition effects of other polysaccharide fractions on these three cells were significantly different.These results suggest that the polysaccharide fractions isolated from Irpex lacteus have potential antitumor and antinephritis activities.

Extraction and Anti-diabetic Activity on Alloxan-induced Diabetic Mice of Polysaccharides from Huidouba

The polysaccharide extracted from Huidouba（PEH） exihibited a significant anti-diabetic activity. The polysaccharide contained glucose, mannose and galactose in a content ratio of 1.00：0.34：0.32. After administration with 50-- 200 mg/kg body weight of PEH to type II diabetic mice induced by alloxan for four weeks, the blood glucose level of mice was decreased significantly. Moreover, the SOD activity increased at the same time（p〈0.01）. PEH showed a significant anti-diabetic and anti-oxidation activity. In addition, PEH affected the BUN and ALT levels（p〈0.05）, which are important characteristics of diabetes mellitus. Through this work, PEH should be a potential anti-diabetic drug in the future.

Application of Near Infrared Diffuse Reflectance Spectroscopy with Radial Basis Function Neural Network to Determination of Rifampincin Isoniazid and Pyrazinamide Tablets

Partial least squares（PLS）,back-propagation neural network（BPNN）and radial basis function neural network（RBFNN）were respectively used for estalishing quantative analysis models with near infrared（NIR）diffuse reflectance spectra for determining the contents of rifampincin（RMP）,isoniazid（INH）and pyrazinamide（PZA）in rifampicin isoniazid and pyrazinamide tablets.Savitzky-Golay smoothing,first derivative,second derivative,fast Fourier transform（FFT）and standard normal variate（SNV）transformation methods were applied to pretreating raw NIR diffuse reflectance spectra.The raw and pretreated spectra were divided into several regions,depending on the average spectrum and RSD spectrum.Principal component analysis（PCA）method was used for analyzing the raw and pretreated spectra in different regions in order to reduce the dimensions of input data.The optimum spectral regions and the models＇ parameters were chosen by comparing the root mean square error of cross-validation（RMSECV）values which were obtained by leave-one-out cross-validation method.The RMSECV values of the RBFNN models for determining the contents of RMP,INH and PZA were 0.00288,0.00226 and 0.00341,respectively.Using these models for predicting the contents of INH,RMP and PZA in prediction set,the RMSEP values were 0.00266,0.00227 and 0.00411,respectively.These results are better than those obtained from PLS models and BPNN models.With additional advantages of fast calculation speed and less dependence on the initial conditions,RBFNN is a suitable tool to model complex systems.

Purification and Properties of Superoxide Dismutase(SOD) in Allium Sativum

Superoxide dismutases(SODs) were purified to homogeneity from Allium Sativum by means of ammonium sulfate precipitation and column chromatography with DEAE-cellulose(DE52) and Sephadex G-75. Based on sodium dodecyl sulfate\|polyacrylamide gel electrophoresis(SDS-AGE), Allium Sativum is predicted to contain four SODs. The molecular weights of the native SODs are 41 3 kD, 37 0 kD, 35 2 kD and 31 0 kD, which consist of subunits of 20 7 kD, 18 4 kD, 17 7 kD and 15 4 kD respectively. Because of their specific sensitivity to hydrogen peroxide, cyanogens potassium and chloroform\|alcohol, the SODs in Allium Sativum appear to be Cu, Zn-SOD isoenzymes. The isoelectric analysis indicates that three of the four isoenzymes are acidic proteins with isoelectric points at pH 3 5, 3 7 and 4 0, respectively, and the fourth one is a basic protein with isoeletric point at pH 8 5.

Purification and in vitro Activity of an Antimicrobial Peptide from Skin of Rana Temporaria Chensinensis,David

In this study, an antimicrobial component（RTCI） was purified from the skin of Rana temporaria chensinensis, David. Antimicrobial activities of RTCI against clinical multi-drug resistant bacterial strains, including Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureaus, Klebsiella oxytoca, Klebsiella pneumoniae, Enterococcus fae-calis, and Proteus mirabilis, were measured in vitro by means of minimal inhibitory concentration and time-kill studies. The results indicate that RTCI could inhibit the growth of these bacteria at a proper concentration and suggest that RT-CI shows a better antimicrobial activity to Gram-negative bacterial strains than to Gram-positive bacterial strains.

Synthesis of Codon-optimized Human Interleukin-18 Gene by Combination of Chemical and Enzymatic Method

According to the amino acid sequence and codon preference of E. coli, the human interleukin-18（IL-18） gene was optimized to avoid the rare codons. The total length of the synthesized gene is 571 bp; 18 oligonucleotides, DNA fragments were designed and synthesized by the phosphoramidite four-step chemical method. The whole DNA sequence was synthesized by a one-step total gene synthesis method, and then inserted in pUC18 vector. Five positive clones identified by blue-white colony screening were sent to Shanghai Sangon Biological Engineering Technology and Service Co., Ltd. for sequencing. The sequencing result shows that one clone contained the complete correct gene in all the five positive clones.

Protein Adsorption onto Nanosized Hydroxyapatite Particles for Controlled Drug Release

Nanosized hydroxyapatite（nsHAp） was synthesized to examine its possibility as a controlled release cartier of protein. To achieve effective protein release from nanosized hydroxyapatite, the study of the adsorptive properties of protein on nsHAp and different influence parameters such as pH, calcium, and phosphate concentrations during the adsorption process is necessary. Ovalbumin（OVA） was selected as the model of growth factors. The results show that the amount of OVA adsorbed onto nsHAp in acetic buffer（pH = 3.6） is more than that in acetic buffer（pH =5.6） because of the electric interaction. The amount of OVA adsorption in phosphate buffer solution（PBS） is smaller than that in acetic buffer because of surface complexation and surface hydroxylation. The presence of Ca^2＋ dramatically increases the adsorbed amount of OVA in acetic buffer on maintaining the same pH. Meanwhile, the release kinetics of OVA adsorbed onto nsHAp（nsHAp-OVA） was also examined. The amount of released OVA in PBS（pH = 5.6） was significantly smaller than that released in solution of pH = 7.0. All the results suggest that nanosized hydroxyapatite particles could be successfully used as controlled released carrier of protein.

Purification and Characterization of β-Glucosidase from a Newly Isolated Strain Tolypocladium cylindrosporum Syzx4

A higher β-glucosidase-producing strain was isolated and identified as Tolypocladium cylindrosporum syzx4 based on its morphology and internal transcribed spacer（ITS） rDNA gene sequence.The present study is to ferment,purify and characterize a β-glucosidase from T.cylindrosporum gams.The enzyme was purified to homogeneity by sulfate precipitataion,diethylaminoethyl cellulose anion exchange chromatography and Sephadex G-100 gel filtration with a 9.47-fold increase in specific activity and a recovery of 12.27%.The molecular weight（Mw） of the β-glucosidase was estimated to be 58600 by SDS-PAGE,which is much lower than that of the enzyme from other fungi.The β-glucosidase shows high activity over a wide range of temperature from 35℃ to 70℃ and the optimum pH is approximately 2.4.Then the kinetic parameters Km（0.85 mmol/L） and vmax[85.23 mmol/（L·s）] of the β-glucosidase were studied,respectively with high affinity p-nitrophenyl-β-D-glucopyranoside（p-NPG） as the substrate,inhibition constant Ki（39.5 mmol/L） with the tolerance of glucose.Although β-glucosidases have been purified and characterized from several other sources,T.cylindrosporum β-glucosidase with the unique optimal pH and higher tolerance of glucose distinguished from others makes the β-glucosidase a potential application in simultaneous saccharification and fermentation（SSF）.

Forced Degradation of Flavonol Glycosides Extraced from Ginkgo biloba

The degradation of flavonol glycosides extracted from Ginkgo biloba was performed under different conditions and the degraded products were determined by reversed-phase high performance liquid chromatography （RP-HPLC） method.Four stress conditions including acid（0.1 mol/L HCl）,base（0.1 mol/L NaOH）,temperature （70 ℃） and oxidation（0.03% H2O2,volume fraction） were used for the forced degradation studies.The pH stabilities of the flavonol glycosides were determined in phosphate buffers of varying pH values from 4.5 to 7.4.The degradation rate constants and half-life of three Ginkgo flavonol aglycones（quercetin,kaempferol and isorhamnetin） which represent Ginkgo flavonol glycosides were calculated in forced degradation and pH-stability studies of them.The resuits indicate that the three substances were more stable when incubated under acid condition and showed pH-dependent stability.The degradation was observed to follow first-order kinetics in all degradation studies.The stability results could provide important bases on development,preparation and storage of products of Ginkgo biloba extract and should be significantly considered during the further formulation development.