天门冬多糖的化学结构及体外抗氧化活性

目的　研究天门冬多糖ACP1的化学结构及体外抗氧化活性。方法　用沸水提取天门冬块根粉粗多糖,经DEAE-cellulose及Sephadex G-150柱色谱纯化得纯多糖ACP₁。经酸全水解,PC,GC,IR,高碘酸氧化、Smith降解、CrO3氧化、全甲基化,1H及¹³CNMR等研究化学结构。并利用NADH-PMS-NBT系统产生,Fe2+-Vit C系统产生*OH,H₂O₂诱导红细胞氧化溶血来研究ACP₁清除自由基、抗氧化活性。 结果　ACP₁的分子量为1.04×10⁵, 比旋光度为［α］¹⁵_D=+64°（c 0.64,H₂O）。ACP₁是由Gal及Glc组成,摩尔比为1.00∶0.87。ACP₁是由8个β-（1→4）Gal及6个α-（1→6）Glc连接构成主链,并在其中1个Gal残基的3-O位上接有1个α-Glc残基的支链。ACP₁体外可清除NADH-PMS-NBT系统产生的超氧阴离子自由基,降低Fe²⁺-Vit C引起的小鼠肝微粒体脂质过氧化产物丙二醛的含量,抑制H₂O₂诱导的大鼠红细胞氧化溶血。结论　ACP₁为一半乳萄聚糖,有清除自由基及抗脂质过氧化活性。     

麦冬多糖MDG-1的分离纯化和结构分析

目的分离纯化麦冬多糖抗心肌缺血的活性部位，得到相对分子质量均一的组分MDG-1，并进行结构分析。方法水提醇沉得麦冬粗多糖，超滤后经DEAE Sepharose FF柱和Sephadex G25柱色谱纯化后得MDG-1。经完全酸水解、高碘酸氧化、Smith降解、甲基化分析、¹H NMR及¹³C NMR等研究分析MDG-1的化学结构。结果和结论 MDG-1为β-D-果聚糖，易溶于水，数均分子量(M_n)为3 400，重均分子量(M_w)为4 800，峰尖分子量(M_p)为5 000，以2→1连接的呋喃型果糖为主，平均每2.8个主链残基上有一个Fruf(2→6)Fruf(2→分支)。该多糖还含有微量葡萄糖(果糖∶葡萄糖=35∶1)，可能连接在多糖的还原端。     

半枝莲粗多糖对S180荷瘤小鼠红细胞膜脂类成分的影响

目的 探讨半枝莲粗多糖对S₁₈₀荷瘤小鼠红细胞膜脂类含量的影响。方法 48只小鼠,♂,随机分为正常对照组、模型对照组、阳性对照(黄芪多糖)组、半枝莲粗多糖低、中、高剂量(50,100,200 mg·kg^-1)组,除正常对照组外其他组小鼠右前腋皮下接种肿瘤。采用紫外分光光度计检测红细胞膜胆固醇、磷脂含量,采用GC-MS检测红细胞膜磷脂脂肪酸的百分含量。结果 与模型对照组比较,半枝莲粗多糖显著提高S₁₈₀荷瘤小鼠红细胞膜磷脂含量、降低胆固醇含量及二者比值,棕榈一烯酸(C_16∶1)、油酸(C_18∶1)、亚油酸(C_18∶2)、不饱和脂肪酸/总脂肪酸的比例均有不同程度的升高。结论 半枝莲粗多糖可增加S₁₈₀荷瘤小鼠红细胞膜磷脂含量,降低胆固醇及其比值,并且增加不饱和脂肪酸在总脂肪酸中的比例,可能通过这些指标共同作用提高红细胞膜的流动性,从而增加红细胞的免疫功能,实现抗肿瘤作用。     

山药多糖RDPS-I的结构分析及抗肿瘤活性

目的研究山药多糖的化学结构和抗肿瘤活性。方法用水提取山药块茎粗多糖,经Sevag反复脱蛋白8次,透析后经DEAE-cellulose及Sephadex G-100柱色谱纯化得山药多糖RDPS-I。经完全酸水解、部分酸水解,用PC,GC,IR,高碘酸氧化,Smith降解,甲基化分析,¹HNMR及¹³CNMR等研究山药多糖的化学结构。并用小鼠移植性实体瘤研究了RDPS-I的体内抗肿瘤作用。结果RDPS-I的分子量为41 000,比旋光度为［α］²⁰_D=+188.4°(c 0.80,H₂O),特性粘度为［η］=16.48×10^-3(g·mL^-1)。RDPS-I是由葡萄糖、甘露糖和半乳糖以1∶0.4∶0.1的摩尔比组成,以α-D-(1→3)-Glcp为主链,在6-O位有α-D-(1→2)-Manp-β-D-1)-Galp支链的杂多糖。RDPS-I对移植性黑色素B16和Lewis肺癌有很强的抑制作用。结论RDPS-I是由葡萄糖、甘露糖和半乳糖构成的杂多糖,有很强的体内抗肿瘤活性。     

大蒜多糖体外抗柯萨奇病毒B3作用

目的　研究大蒜多糖体外抗柯萨奇病毒B₃(Coxsackievirus group Btype3, CVB₃)作用。方法　观察大蒜多糖A,B,C的细胞毒性、对CVB₃直接灭活作用、抗CVB₃吸附作用及对CVB₃生物合成抑制作用。结果　大蒜多糖A,B,C对Hep-2细胞的半数中毒浓度(TC₅₀)分别为1000, 800, 4000μg/mL;均无直接灭活CVB₃及抗CVB₃吸附作用;除A外,B和C均可剂量依赖性抑制CVB₃生物合成.。结论　大蒜多糖B和C在体外通过抑制CVB₃生物合成而发挥抗CVB₃的作用。     

木贼麻黄多糖的提取及体外抗氧化活性研究

目的 优化木贼麻黄多糖的提取工艺,初步研究木贼麻黄多糖的体外抗氧化活性。方法 采用热水浸提法提取木贼麻黄多糖,利用单因素试验结合响应面法对提取条件进行优化,并研究木贼麻黄多糖的体外抗氧化活性。结果 木贼麻黄多糖的最佳提取条件为：提取时间2.5 h,提取温度73℃,液料比25∶1,在此条件下多糖提取率为（5.57±0.072）%。木贼麻黄多糖对DPPH自由基和羟基自由基具有较强的清除能力,IC₅₀分别为703.9 μg·mL^-1和2 135.1 μg·mL^-1;对超氧阴离子自由基具有一定的清除能力,对Fe³⁺有一定的还原能力。结论 该提取工艺能较好地提取木贼麻黄多糖,所得木贼麻黄多糖具有一定的体外抗氧化能力,但活性低于同浓度的维生素C。     

赤芝多糖肽GL-PP-3A的分离纯化和结构研究

分离纯化赤芝多糖肽，得到具有活性部位GL-PP-3A，对其进行结构分析。水提醇沉透析得赤芝多糖肽，经分级沉淀，Bio-Gel P-10柱色谱纯化得GL-PP-3A。经HPGPC、糖基组成、甲基化分析、 ¹H NMR和 ¹³C NMR等方法研究分析GL-PP-3A的结构。GL-PP-3A是以葡萄糖为主的杂多糖，含Rha、Xyl、Man、Gal、Glc糖残基和17种氨基酸。其M_w为1.7×10⁴；M_n为1.1×10⁴；M_w/M_n为1.49。M_p为1.3×10⁴。该多糖主链由1,6-或1,3-连接的β-D-Glcp构成，二者的比例约为2∶1，在部分1,6-连接的主链葡萄糖的2或3位有分支，分支的非还原末端均主要为β-D-Glcp,少量为鼠李糖，分支内部含有1～3个1,6-连接的β-D-Galp或1,3-连接的α-D-Manp。     

钝顶螺旋藻糖缀合物SPPA-1的理化性质及活性的研究

目的　研究螺旋藻多糖中的均一组分SPPA-1的分离方法、糖组成、分子量及生物活性。方法　采用丙酮分部沉淀,三氯乙酸去蛋白,再经SephadexG-75和CM-SephadexC-50柱色谱得到SPPA-1,用气相色谱法测定单糖组成,GPC法测定分子量,IR和NMR法确定糖苷键类型。结果　经高压液相色谱和毛细管电泳鉴定为均一性组分。其总糖含量为91.70%,N含量为0.96%,分子量为69.00×10⁴Da。糖组成分析表明其由单一的葡萄糖组成,红外光谱、核磁共振¹HNMR谱分析表明,其单糖类型为α-吡喃型。ESR研究表明SPPA-1具有清除O^-₂自由基活性。结论　SPPA-1为一具抗氧化活性的葡聚糖。     

6种药物对有机阴离子转运多肽OATP1B1及其基因多态性A388G、T521C转运作用的影响

目的 研究6种药物对有机阴离子转运多肽OATP1B1及其基因多态性A388G、T521C转运作用的影响。方法 体外培养稳定高表达OATP1B1和OATP1B1基因多态性A388G、T521C的人胚肾细胞（HEK293）株,高表达空白载体（Mock）的HEK293细胞为空白对照,实时荧光定量PCR（qRT-PCR）法检测各转运体细胞中mRNA表达;放射性标记化合物³Hestrone sulfate作为转运底物、利福平作为阳性抑制剂验证各高表达细胞的转运活性;测定30 μmol·L^-1的达比加群、辛伐他汀、替格瑞洛、卡培他滨、多西他赛、依那普利对各细胞³H-estrone sulfate摄入活性的抑制作用,并依据抑制试验结果,进一步测定辛伐他汀、替格瑞洛、多西他赛对转运体细胞的半数抑制浓度（IC₅₀）。结果 HEK293细胞内导入的各种转运体基因都呈现良好的复制表达;OATP1B1、OATP1B1/A388G、OATP1B1/T521C对底物³H-estrone sulfate（5 μmol·L^-1）的转运活性分别为Mock细胞的39、49和48倍,30 μmol·L^-1利福平添加后,可将细胞的转运活性抑制到50%以下;辛伐他汀、替格瑞洛、多西他赛对OATP1B1的抑制作用较强,30 μmol·L^-1给药的转运活性分别为对照组的（40.09±1.95）%、（33.82±0.61）%、（45.08±0.22）%;辛伐他汀对OATP1B1、OATP1B1/A388G、OATP1B1/T521C的IC₅₀分别为14.2、＞100、＞100 μmol·L^-1,替格瑞洛的IC₅₀分别为19.1、68.4、＞100 μmol·L^-1,多西他赛的IC₅₀分别为17.6、22.9、19.3 μmol·L^-1。结论 OATP1B1基因多态性在一定程度上改变了抑制剂对转运体活性的影响程度。     

抗癌抗生素C1027与单克隆抗体Fab片段偶联物的抗肝癌作用

抗人肝癌单克隆抗体(单抗)3A₅用木瓜蛋白酶消化得到Fab片段。单抗3A₅和Fab片段分别与抗癌抗生素C1027偶联,偶联物经克隆生成法测定对肝癌细胞有很强的杀伤作用,C1027,Fab-C1027和3;3A₅-C1027的IC₅₀分别为6.5×10^-16,8.6×10^-16和4.2×10^-14mol·L^-1;Fab-C1027偶联物对非靶细胞(KB)的IC₅₀值为1.4×10^-13mol·L^-1,与靶细胞(BEL-7402)的IC₅₀值相比,两者相差160倍。说明Fab-C1027的杀伤活性强于3A₅-C1027,并对靶细胞呈选择性杀伤作用。给皮下移植人肝癌的裸鼠iv剂量0.1 mg·kg^-1,结果C1027和Fab-C1027的抑瘤率分别为59和85%,说明Fab片段与C1027偶联物比游离C1027的疗效更高。     

UPLC-MS/MS法测定半枝莲中4个二萜烟酸酯的含量

目的:建立同时测定半枝莲中半枝莲碱A、半枝莲碱B、半枝莲萜C和半枝莲萜F含量的UPLC-MS/MS分析方法。方法:采用ACQUITY UPLC?BEH C₁₈色谱柱(100 mm×2.1 mm,1.7μm),以0.1%甲酸水溶液(A)-0.1%甲酸乙腈(B)为流动相,梯度洗脱(0~7 min,48%B→100%B;7~8 min,100%B;8~8.5 min,100%B→48%B;8.5~10 min,48%B),流速0.2 mL·min^-1,柱温30℃;质谱采用电喷雾离子源(ESI),多反应监测(MRM)正离子模式进行扫描。结果:半枝莲碱A、半枝莲碱B、半枝莲萜C、半枝莲萜F质量浓度分别在0.3344~13.38、0.2231~8.923、0.1624~6.496、0.1988~7.954μg·mL^-1的范围内线性关系良好(r皆大于0.999);平均回收率(n=9)分别为98.2%、98.6%、99.9%、93.3%,RSD分别为4.2%、6.2%、4.6%、6.3%;27批样品中上述4个成分含量范围分别为15.44~168.4、14.98~118.23、13.31~62.40、12.37~117.67μg·g^-1。结论:所建立的方法可用于半枝莲中主要二萜烟酸酯类成分的定量分析。     

半枝莲粗提物对小鼠肝脏药物代谢酶的调节作用

目的通过研究半枝莲水提物和乙醇提取物对昆明（KM）小鼠细胞色素P450酶（CYP）、谷胱甘肽S-转移酶（GST）活性的抑制作用,研究半枝莲与其他药物合用时可能发生的相互作用。方法按照随机对照原则,以半枝莲水提物和乙醇提取物1、2和5g·kg^-1·d^-1剂量为低、中和高剂量连续灌胃给药5d,以HPLC方法检测探针药及其代谢产物量来表示相应酶的活性;以分光光度法测定细胞溶质中GST酶活性。结果与对照组比较,中、高剂量半枝莲水提物组与乙醇提取物组CYP2C与CYP3A活性显著下降;高剂量水提物组GST活性显著下降;而各组CYP2D与CYP2E1活性未见改变。结论半枝莲水提物和乙醇提取物对KM小鼠的细胞色素P450酶和GST活性都有不同程度的抑制作用,呈现出剂量相关性。     

乳泰胶囊质量标准研究

目的研究乳泰胶囊的薄层色谱鉴别和含量测定方法。方法分别采用野黄芩苷、α-香附酮、野菊花为对照鉴别制剂中的半枝莲、香附、野菊花。采用高效液相色谱法测定陈皮、青皮中的有效成分橙皮苷的含量,色谱柱为AgilentTc-C18色谱柱(4.6mm×250mm,5μm),流动相为甲醇-0.025mol/L磷酸溶液(20:80),流速为1.0mL/min,检测波长为283nm,柱温为30℃。结果薄层色谱方法专属性强。橙皮苷线性范围为1.00～8.02μg(r=0.9996),平均回收率为99.06%,RSD为1.15%(n=9)。结论所建立的方法准确,能科学评价乳泰胶囊的质量。     

癌痛消颗粒质量标准研究

目的：建立癌痛消颗粒的质量标准。方法：采用薄层色谱法对处方中白花蛇舌草、半枝莲、黄芪、赤芍4味中药进行定性鉴别,采用HPLC法测定半枝莲中野黄芩苷的含量,以Kromasial C18柱（250 mm×4.6 mm,5μm）为色谱柱,流动相为甲醇-3﹪冰醋酸溶液（33∶67）,流速为1.0 ml·min^-1,检测波长为335 nm,柱温为25℃。结果：各供试品色谱中,在与各自对照品色谱相同的位置上显相同颜色的斑点,且阴性对照无干扰。HPLC法测定野黄芩苷在2.18-10.90μg·ml^-1范围内线性关系良好（r=0.999 9）,平均加样回收率为96.65%,RSD=1.45%（n=6）。结论：本方法简便、准确、重现性好,适用于癌痛消颗粒的质量控制。     

The interaction of aluminium with soluble protein kinase C from mouse brain

The interaction of aluminium ion species with soluble protein kinase C, Ca²⁺/phospholipid-dependent protein kinase, from mouse brain has been examined in vitro. The activity of protein kinase C was increased by addition of Ca²⁺ displaying an EC₅₀ value of 10.3±1.1 × 10^–6 M. The A1 species inhibited the activity with an IC₅₀ values of 8.6±0.5 × 10^–5 M and 2.7±0.3 × 10^–5 M in the presence of 0.5 mM Ca²⁺ and absence of Ca²⁺, respectively. Concerning the EC₅₀ for Ca²⁺ activation, this was increased by the A1 species in a dose-dependent manner. Moreover, the inhibition was of a non-competitive type with respect to H1 histone and of a mixed type with respect to ATP. It is likely that the inhibition was caused by 1) the blocking of Mg²⁺ binding to ATP, 2) the blocking of Ca²⁺ binding to protein kinase C. Our results suggested that protein kinase C was involved in neurotoxicity of A1.     

Purification and Characterisation of a Newly Isolated Stable Long-Life Tannase produced by F. subglutinans (Wollenweber and Reinking) Nelson et al.

A stable long-life tannase was synthesised by Fusarium subglutinans and the fermentation processing parameters were optimised. Maximum enzyme production (9.38 U/ml) was recorded after 96 h of incubation at 35°C, initial pH 5, in submerged culture (200 rpm) utilising 2% (w/v) tannic acid as a sole carbon source. The tannase produced was purified to electrophoretic homogeneity through two-step column chromatography and the purified form remained stable in a pH range of 6–8. Its midpoint of thermal inactivation (T _m) was recorded at 70°C after 60 min of exposure. Maximum tannase activity was enabled at pH 6 and 40°C. Ca²⁺, K⁺, Mg²⁺ and Mn⁺ showed a stimulatory effect while Ba²⁺, Co²⁺, Cu²⁺, Fe³⁺ and Zn⁺ showed a competitive inhibitory effect on enzyme activity. Values of K _m, V _max, K _cat and the molecular mass of the purified enzyme were 0.116 μM ml⁻¹ min⁻¹, 3.57 mM, 1.16 μM ml⁻¹ min⁻¹ and 150 kDa, respectively. The participation of the SH group and carbohydrates in the enzyme structure was also suggested by the results. The stability of the purified and partially purified enzyme at −15°C extended to 13 months.     

Quantitative analysis of mRNA expression levels of aldo-keto reductase and short-chain dehydrogenase/reductase isoforms in human livers

The aldo-keto reductase (AKR) and short-chain dehydrogenase/reductase (SDR) superfamilies are responsible for the reduction in compounds containing the aldehyde, ketone, and quinone groups. In humans, 12 AKR isoforms (AKR1A1, AKR1B1, AKR1B10, AKR1B15, AKR1C1, AKR1C2, AKR1C3, AKR1C4, AKR1D1, AKR1E2, AKR7A2, and AKR7A3) and 6 SDR isoforms (CBR1, CBR3, CBR4, HSD11B1, DHRS4, and DCXR) have been found to catalyze the reduction in xenobiotics, but their hepatic expression levels are unclear. The purpose of this study is to determine the absolute mRNA expression levels of these 18 isoforms in the human liver. In 22 human livers, all isoforms, except for AKR1B15, are expressed, and AKR1C2 (on average 1.6 × 10⁶ copy/μg total RNA), AKR1C3 (1.3 × 10⁶), AKR1C1 (1.3 × 10⁶), CBR1 (9.7 × 10⁵), and HSD11B1 (1.1 × 10⁶) are abundant, representing 67% of the total expression of reductases in the liver. The expression levels of AKR1C2, AKR1C3, AKR1C1, CBR1, and HSD11B1 are significantly correlated with each other, except between AKR1C2 and CBR1, suggesting that they might be regulated by common factor(s). In conclusion, this study comprehensively determined the absolute expression of mRNA expression of each AKR and SDR isoform in the human liver.     

Differential gene expression associated with inflammation and blood pressure regulation induced by concentrated ambient particle exposure

Epidemiological studies have indicated that exposure to particle matter (PM) increased the risk of respiratory and cardiovascular morbidity and mortality. It is suggested that PM smaller than 2.5 μm in aerodynamic diameter (PM_2.5) may contribute to these responses. However, the molecular mechanism is still unknown. To elucidate the changes in molecular level, we investigated the gene expression profile of concentrated ambient particles (CAPs)-exposed rats. Aged F344 rats were exposed with CAPs (594 μg/m³) or clean air 4?h per day for 3 days, and lung and heart tissues were then excised for DNA microarray analysis. Expression profiles related to inflammation and blood pressure regulation revealed differential expression of 7 genes in the lung and that of 3 genes in the heart ventricle. According to the complement activation-associated genes, complement factor B (Bf), complement component 2 and 4a (C4a), and C1 inhibitor genes were up-regulated in CAPs-exposed rat lung. Bf and C4a genes were also up-regulated in the heart. These suggest the treated animal ready for production of these proteins when activation of complement cascade is required. Pro-inflammatory cytokine, interleukin-1β, was also up-regulated in CAPs-exposed rat lung. Gene related with blood pressure regulation (angiotensin I converting enzyme) was also up-regulated in CAPs-exposed rat lung. Negative regulator of blood pressure (neuropeptide Y) was down-regulated in CAPs-exposed rat heart. These results indicate that CAPs may affect respiratory and cardiovascular organs by activation of inflammatory responses and disintegration of blood pressure regulation in early stage of CAPs exposure.     

In vitro intestinal and hepatic metabolism of Di(2-ethylhexyl) phthalate (DEHP) in human and rat

Species and organ differences in the intrinsic clearance and the enzymes involved in the metabolism of DEHP were examined in subcellular fractions of the intestine and liver as well as by recombinant cytochrome P450 (CYP) isoforms of human and rat. Estimated clearance (CL_int) of DEHP via esterase-mediated pathway in human intestine was 2.4-fold greater than that in human liver while its value in rat intestine was 1.7-fold less than that in rat liver. Ranks of CL_int for CYP-mediated oxidation/dealkylation of MEHP were human liver > rat liver > human intestine > rat intestine. Estimates of CL_int for the production of mono(2-ethyl-5-hydroxyhexyl) phthalate and mono(2-ethyl-5-oxohexyl) phthalate by human CYP2C9^*1 were 4.2- and 2.6-fold greater than those by rat CYP2C6, respectively. Total CL_int via hCYP2C9^*3-mediated oxidation was 1.9- and 2.6-fold less than those by hCYP2C9^*2 and 2C9^*1, respectively. Estimated CL_int for phthalic acid production by hCYP3A4 was 24.5 μL nmol CYP^?1 min^?1 while it was continuously produced by rCYP2C6 and 3A2 via passive mechanism. These species/organ differences in major metabolic pathway and CYP isoforms should be considered for appraisal of the potential adverse health effects of DEHP.