北青龙衣抗肿瘤谱效关系研究初探

目的:对北青龙衣抗肿瘤谱效关系进行初步研究。方法:采用乙醇冷浸提取,大孔树脂进行初步分离,所得到的各组分用MTT法进行体外细胞毒试验,测定各组分对人胃癌细胞BGC803的抑制活性,并用薄层色谱进行初步谱效关系研究。结果:大孔树脂经30%乙醇洗脱的组分显示较强的细胞毒活性,薄层色谱显示该组分为极性大和极性小的成分的复杂组合。结论:大孔树脂经30%乙醇洗脱的组分在北青龙衣抗肿瘤活性中起重要作用。     

金丝带缩酚酸类化学成分研究

对采自青海省的金丝带[Lethariella zahlbruckneri(Dr.)Krog.]的乙酸乙酯部分进行研究,采用硅胶柱色谱、半制备高效液相和重结晶技术进行分离纯化,通过1 H-NMR、13 C-NMR技术对化合物结构进行鉴定,结果共分离鉴定了5个化合物,包括:1个缩酚酸类:雪茶素(5);4个缩酚酸和缩酚酸环醚裂解产物:赤星衣酸甲酯(1),β-苔黑酚酸甲酯(2),苔色酸甲酯(3)和苔色酸乙酯(4)。其中化合物1、3、4、5均为首次从该植物中分得。     

RP-HPLC法测定北柴胡地上部分山柰酚的含量

目的:建立RP-HPLC法测定北柴胡地上部分山柰酚含量的色谱方法。方法:采用RP-HPLC,C18柱(150 mm×4.6 mm,5μm),流动相甲醇-(0.5%磷酸)水(体积比为48∶52),检测波长360 nm,柱温34℃。结果:山柰酚质量浓度在0~0.048 g/L内与峰面积呈良好的线性关系,r=0.999 5(n=6);平均回收率为100.7%,RSD=1.8%(n=6)。结论:测定方法准确,可靠,为北柴胡地上部分的质量评价提供了可靠的依据。     

地衣内生菌Elaphocordyceps sp.的代谢产物

【目的】通过对猫耳衣内生菌Elaphocordyceps sp.代谢产物的研究,以期获得结构新颖、抗菌活性强的化合物。【方法】采用PCR扩增和测定nrDNA的ITS(Internal transcribed spacer)序列,在GenBank数据库中比对,并采用构建系统发育树的方法,确定该菌株的分类地位。采用硅胶柱色谱、凝胶柱色谱和高效液相色谱方法进行成分分离。根据理化性质和波谱数据进行结构鉴定。采用倍比稀释法进行抑菌活性测定。【结果】鉴定出供试菌株为地衣内生菌Elaphocordyceps sp.。从Elaphocordyceps sp.的发酵液中分离得到3个化合物,结构鉴定为麦角甾醇、4,8-Dihydroxy-1-tetralone和De-O-methyldiaporthin。化合物1–3表现弱的抑制白色念珠菌活性。化合物3具有非常强的植物毒活性(叶片敏感度:4 nmol)。化合物3在地衣内生菌Elaphocordyceps sp.发酵液中的得率为0.75 mg/L(3.2?103 nmol),远远高于其对叶片敏感度4 nmol。【结论】由猫耳衣内生菌Elaphocordyceps sp.产生的De-O-methyldiaporthin可作为一种微生物源除草剂。     

治糜灵凝胶剂的薄层色谱鉴别研究

目的:将治糜灵栓剂(《中国药典》2010版一部)改为治糜灵凝胶剂,建立治糜灵凝胶剂的薄层色谱鉴别方法,为制定其质量控制标准制定依据。方法:参照《中国药典》2010版一部治糜灵栓剂项下黄柏、苦参、儿茶、冰片的薄层色谱鉴别方法,对处方中的主要药味进行定性鉴别。黄柏鉴别中以甲苯-乙酸乙酯-异丙醇-水(6:3:1.5:1.5)为展开剂;苦参鉴别中以甲苯-乙酸乙酯-甲醇(8:3:0.5)为展开剂;儿茶鉴别中以正丁醇-醋酸-水(3:2:1)为展开剂;冰片鉴别中以环己烷-三氯甲烷-乙酸乙酯(9:1:2)为展开剂。结果:薄层色谱上具有黄柏,苦参,儿茶,冰片的鉴别特征,且阴性对照无干扰。结论:色谱斑点清晰,专属性强;该方法操作简便,稳定性、重现性均很好。可作为质量标准的控制依据。证明治糜灵凝胶剂研究的方法可行。     

人参归脾丸薄层鉴别

目的:控制人参归脾丸的质量。方法:采用薄层色谱法(TLC)对人参归脾丸的主要成分人参、白术和甘草进行定性鉴别。结果:在TLC色谱中可检出人参、白术和甘草。结论:本方法准确可靠,可以用于人参归脾丸的质量控制。     

瑶药冷骨风质量控制方法研究

瑶药冷骨风来源于睡莲科植物萍蓬草(Nuphar pumilum)的干燥根茎,为瑶族人习用药材,具有止咳补虚、除蒸止汗、祛瘀调经、止血的作用,目前历版《中国药典》和地方标准并未有收载其质量控制方法。该研究收集了10批冷骨风药材,采用生药鉴别方法对冷骨风药材性状进行显微鉴别,用TLC(薄层色谱)法对药材进行定性鉴别,用高效液相色谱法对没食子酸含量进行测定。结果表明:(1)色谱柱为Welch Ultimate XB-C_(18)柱(4.6 mm×250 mm,5μm),流动相为甲醇-0.05%磷酸溶液(5∶95),流速为1.0 mL·min~(-1),检测波长为272 nm,柱温为室温。(2)建立了冷骨风药材性状、显微、TLC鉴别方法。(3)含量测定中没食子酸在0.072 1~0.360 5μg范围时,进样量与峰面积呈良好的线性关系,平均回收率为103.66%,RSD为2.03%(n=6)。(4)10批样品没食子酸含量为0.82%~1.23%,平均含量为0.94%。因此,建立的瑶药冷骨风质量控制方法具有较强的科学性,可用于冷骨风药材的质量控制。     

GC-MS/SIM法同时测定臭冷杉挥发油中柠檬烯和醋酸龙脑酯含量

目的:建立同时测定臭冷杉挥发油中柠檬烯和醋酸龙脑酯含量的方法。方法:采用气相色谱-质谱联用(GC-MS)技术,选择离子模式(SIM)检测,色谱柱为HP-5MS石英毛细管柱(30 m×0.25 mm×0.25μm),初始温度40℃,4℃·min-1升至260℃。载气为氦气。选择m/z为68和95的特征离子分别对柠檬烯和醋酸龙脑酯进行定量。结果:柠檬烯和醋酸龙脑酯在400~4 000μg·m L-1和800~8 000μg·m L-1浓度范围内与峰面积呈良好的线性关系,其相关系数为0.999 8和0.999 6;平均加样回收率为98.5%和98.7%;RSD值为1.3%和1.5%(n=6)。结论:该方法简便、快速,结果准确,可用于臭冷杉挥发油的质量控制。     

牵牛子(黑丑)挥发油成分研究

目的:研究牵牛子的化学成分.方法:利用有机溶剂-水蒸气蒸馏法提取牵牛子(Semen pharbitidis)挥发油,采用GC -MS进行测定,并应用色谱峰面积归—化法计算各成分的相对百分含量.结果:鉴别出35个化学成分,其化学成分为庚醛( 14.368％);反,反-2.4 -葵二烯醛(6.887％);2-戊基呋喃(5.327％);2-羟基-4-甲氧基苯甲醛(5.312％);萜品烯-4-醇(3.772％);苯乙醛(3.458％);十六烷酸乙酯(2.063％)等.结论:该文首次采用气相色谱-质谱联用法对牵牛子中的挥发性成分进行.     

纸莎草的化学成分研究

采用正相硅胶柱、Sephadex LH-20、高效液相色谱、MCI等色谱方法从纸莎草全株中分离得到19个化合物,采用1D、2D NMR、质谱等技术鉴定化合物的结构,分别鉴定为:α-香附酮(1)、香附酸(2)、广藿香烯酮(3)、4,5,6,7,8,8a-六氢-3,4,8,8-四甲基-1H-3a,7-亚甲基甘菊环-4-醇甲...     

Setting tools for the early assessment of the quality of thawed Pacific oyster (Crassostrea gigas) D-larvae

Parameters used to assess the survival of larvae after cryopreservation generally misestimate the damages that prevent larval development. The objectives of the present study were to 1) define the reliability of the survival rate, assessed at 2 and 7 days post fertilization, to estimate Pacific oyster larval quality after thawing, and 2) select complementary tools allowing an early and reliable estimation of their quality. Oyster larvae were reared for 25 h after fertilization at 19 °C and cryopreserved at early D-stage. Then, thawed larvae were incubated in 2-L beakers. At 2 days post fertilization, the survival rate of thawed Pacific oyster larvae was lower than that of fresh larvae for only one experiment (Experiment 3) among the four identical experiments carried out in this work (Experiments 1-4). By contrast, the survival of thawed larvae, as assessed 7 days after fertilization, was lower than that of fresh larvae for the four experiments. These results confirm that the quality of thawed larvae is lower than that of fresh larvae and that the survival rate, estimated 2 days post fertilization, is not adapted to a reliable estimation of the subsequent development ability of thawed larvae. Then, complementary parameters were tested at 2 days: the movement characteristics (Experiments 1 and 2) and the morphologic features (Experiments 3 and 4) of thawed larvae. Compared to values observed on fresh larvae, the percentage of thawed motile larvae was different for only one experiment (Experiment 2) of the two. Compared to control, a reduced Average Path Velocity (VAP) of larvae (determined at the D-larval stage using a CASA-Computer Assisted Sperm Analysis-system) was observed after thawing for both experiments (Experiments 1 and 2), suggesting the ability of larval movement velocity to assess the decrease of the quality of thawed oyster larvae. Using an ASMA (Automated Sperm Morphology Analysis) device, a lower area of thawed larvae was observed, compared to control and for the two experiments (Experiments 3 and 4). By contrast, the Crofton perimeter of thawed larvae was lower than that of control larvae for only one experiment (Experiment 3) and no significant difference of circularity between fresh and thawed larvae was recorded for Experiments 3 and 4. In conclusion, changes in the movement velocity (assessed by CASA) and in the area (measured by ASMA) of D-larvae allow an early and reliable estimation of the quality of thawed Pacific oyster larvae.     

In vitro polyploidization of mature ostrich fern sporophytes through rejuvenation

An in vitro method is described for producing ostrich fern (Matteuccia struthiopteris (L.) Todaro) polyploids from mature sporophytes as a possible means of plant improvement in this economically important fern species. The procedure is based on rejuvenating adult sporophytes (2n) to enable the aposporous production of diploid (2n) gametophytes, and then mating the gametophytes to produce tetraploid (4n) sporophytes. The adult sporophytes were rejuvenated by culturing excised shoot tips for a minimum of three months in a liquid medium (Murashige and Skoog salts) under conditions of extreme carbohydrate deprivation (0.01% sucrose). Apospory was induced by culturing leaves excised from the rejuvenated shoots for two months on a semi-solid medium lacking sucrose, resulting in the production of diploid gametophytes. The gametophytes were transferred to fresh medium and grown to sexual maturity for one or two months, then floated on the surface of a liquid medium containing 0.01% sucrose for up to two months to promote opening of the sex organs. Subsequent self-fertilization resulted in the successful production of tetraploid sporophytes in 11 of the 14 diploid clones in which polyploidization was attempted. Tetraploids (4n=156) were confirmed by cytological examination. This method permits polyploidization of mature, fully characterized plants.     

The effect of rejuvenation of aged erythrocytes on biochemical parameters in the perfused hind limb muscle preparation

(1) A systematic investigation was carried out into the use of time-expired erythrocytes in an isolated perfused skeletal muscle preparation. Comparisons were made between erythrocytes subjected to a process of 'rejuvenation' (Rennie and Holloszy (1977), Biochem. J. 168, 161-170) and untreated erythrocytes (controls). (2) The use of rejuvenated erythrocytes had no significant effect on concentrations of muscle ATP, phosphocreatine and lactate, nor fractional rates of muscle protein synthesis. However, muscle water concentrations were reduced when compared to controls. (3) There was an influx of K+ from the plasma into rejuvenated erythrocytes. This was accompanied by a substantial loss (17%) of intramuscular K+. There was also loss of K+ from control preparations but this amounted to approx. 1% of muscle content. (4) Erythrocyte fragility was greater in the control perfusate (6%, haemolysis) when compared to the medium with rejuvenated cells (1%, haemolysis). As a consequence of either erythrocyte storage, rejuvenation or haemolysis, plasma concentrations of phosphate, magnesium, calcium and potassium were significantly different from starting values, by as much as 300% in both groups, and varied throughout the study. (5) It is concluded that the use of rejuvenated erythrocytes does not confer any advantage in unexercised perfused skeletal muscle preparations. However, both types of erythrocyte induce changes in perfusate composition relative to starting or in vivo profiles.     

γ射线紫外线镰刀菌产生玉米赤霉烯酮毒素的影响

锭刀菌(Fusarium)经γ射线、紫外线辐射诱变后,接种于大米培养基中.经10℃低温产毒培养.采用薄层层析方法定性定量分析培养物中的镀刀菌毒素—玉米赤霉烯酮(zearalenone简称ZEA).结果表明:不同菌株对诱变剂的反应不同.产毒菌株NF5232、NF5946在较大剂量γ射线处理后,ZEA产量显著增加,但其产生的色素量下降.或者用紫外线处理,仍然产生ZEA.不产毒菌株NF6127、NF6123、NP6138和HD—002,经γ射线或紫外线处理后仍不产生ZEA.由此可知镰刀菌产生ZEA毒素的能力是相对稳定的,其产毒机制是由菌株本身的遗传特性决定的,一定剂量的γ射线可使低产毒菌株变为高产毒菌株.     

莲子草假隔链格孢SF 193菌株的复壮对其产孢量和致病性的影响

【背景】莲子草假隔链格孢是空心莲子草的重要生防菌,但长时间保存或继代培养会导致该菌株产孢量和致病性显著下降。【方法】通过对初始菌株回接、分离、纯化和鉴定相继获得复壮一代和二代菌株,分别在3、6、9d后对初始菌株、复壮一代和二代菌株进行产孢培养,比较其产孢量;同时比较分析了这3代菌株的液体发酵原液和1:10稀释液对空心莲子草的防除效果。【结果】获得了10株复壮一代和10株复壮二代菌株,20株菌对空心莲子草均有致病作用,且产生的分生孢子形态特征与初始菌株一致。与初始菌株相比,复壮菌株的产孢量显著提高且达到最大产孢量的时间显著短于初始菌株。其中,复壮二代菌株的孢子量是初始菌株的4.8倍,其次为复壮一代菌株,产孢量是初始菌株的4.1倍;复壮菌株达到最大产孢量的时间为3d,初始菌株为6d。复壮菌株一代和二代的原液对空心莲子草的致病性分别比初始菌株提高了4.65%和9.82%,1:10稀释液的致病性分别提高了25.79%和16.55%。【结论与意义】对长期保存或继代培养的空心莲子草生防菌复壮可显著提高其产孢量和致病性,对维持生防菌遗传性状的稳定性和提高对空心莲子草的防效具有重要意义。     

A protocol toward multiplication of the medicinal tree,Eucommia ulmoides oliver

Summary A protocol based on shoot cultures of 1-mo.-old seedlings was developed for rapid asexual multiplication of eucommia, the source of an antihypertensive medicinal. The explant is an excised shoot tip, 3–5 mm tall. MS basal medium supplemented with 1 mg/liter BA is employed to establish primary cultures and subsequently multiply shoots. Shoots are subculturable on the same medium and can be increased at a rate of 7.5 new shoots per 2-shoot sector every 3 wk. Rooting is achieved in a Gelrite medium with the MS salts reduced to 1/3 strength and the BA replaced by 0.1 mg/liter NAA. The method is not directly applicable to mature trees. Applicability will require explants from rejuvenated sources, possibly attainable by the method of repeated grafting of shoot apices onto juvenile rootstocks, repeated subculturing of shoots, or culturing shoot apical meristems.     

Comparative evaluation of fresh, fixed, and cryopreserved solid tumor cells for reliable flow cytometry of DNA and tumor associated antigen.

Five different protocols for the short-term preservation of cells used for multiparameter flow cytometric assay of tumour associated antigens (TAA) and DNA were assessed in cell suspensions prepared by mechanical disaggregation of 15 gynecological tumors. The protocols at 4 degrees C were 1) storage in buffer, 2) storage in 50% methanol, and 3) storage in buffer after formalin fixation. Tissues were also cryopreserved as cell suspensions and tissue blocks. When the TAA expression and DNA histograms of the preserved cells were compared with those in fresh cell suspensions, cryopreservation was found to be the best method: TAA expression was well preserved and there was a good correlation between TAA expression and the quality of the DNA histograms, respectively, in fresh and cryopreserved cells (RS: 0.82-0.91, P less than 0.001 for all TAAs). The cell suspensions preserved at 4 degrees C all showed a significant increase in background fluorescence (P less than 0.05) and a reduction in the TAA specific fluorescence (P less than 0.011). Methanol fixation was better than buffered formalin for the proteins studied, though both gave significantly worse results than cryopreservation. The quality of these cell suspensions and the correlation with TAA measurements in fresh cell suspensions deteriorated progressively with time, particularly if they were stored more than a week.     

Inactivation of a human norovirus surrogate by high-pressure processing: effectiveness, mechanism, and potential application in the fresh produce industry

Fresh produce is often a high-risk food for norovirus contamination because it can become contaminated at both preharvest and postharvest stages and it undergoes minimal or no processing. Currently, there is no effective method to eliminate the viruses from fresh produce. This study systematically investigated the effectiveness of high-pressure processing (HPP) on inactivating murine norovirus (MNV-1), a surrogate for human norovirus, in aqueous medium and fresh produce. We demonstrated that MNV-1 was effectively inactivated by HPP. More than a 5-log-PFU/g reduction was achieved in all tested fresh produce when it was pressurized at 400 MPa for 2 min at 4°C. We found that pressure, pH, temperature, and food matrix affected the virus survival in foods. MNV-1 was more effectively inactivated at 4°C than at 20°C in both medium and fresh produce. MNV-1 was also more sensitive to HPP at neutral pH than at acidic pH. We further demonstrated that disruption of viral capsid structure, but not degradation of viral genomic RNA, is the primary mechanism of virus inactivation by HPP. However, HPP does not degrade viral capsid protein, and the pressurized capsid protein was still antigenic. Overall, HPP had a variable effect on the sensorial quality of fresh produce, depending on the pressure level and type of product. Taken together, HPP effectively inactivated a human norovirus surrogate in fresh produce with a minimal impact on food quality and thus can provide a novel intervention for processing fruits intended for frozen storage and related products such as purees, sauces, and juices.     

Changes in fluidity of erythrocyte membranes after storage of erythrocytes and regeneration of cellular ATP level

The membrane fluidity of freshly collected human erythrocytes, of erythrocytes stored for 3–4 weeks and of stored erythrocytes rejuvenated with glucose and inosine was investigated by measuring polarization of fluorescence emission of 1,6-diphenyl-1,3,5-hexatriene and $\text{N-}\text{phenyl}\text{-1-}\text{naphthylamine}$. The fluidity of membranes prepared from stored erythrocytes was higher than that of fresh erythrocytes. After rejuvenation of erythrocytes with glucose and with or without inosine the membrane fluidity decreased. These changes were probably due to variations of ATP levels in the erythrocytes.