蛹虫草固体发酵菌丝体多糖的提取纯化与含量测定

［摘要］目的建立蛹虫草固体发酵菌丝体多糖提取和含量测定方法,并探讨多糖的纯化工艺。方法采用正交实验确定最佳提取条件,利用苯酚 硫酸法测定菌丝体中多糖的含量,并用葡聚糖凝胶G 100对粗多糖按相对分子质量进行分段纯化。结果水提醇沉总糖产率为1.36%;多糖CP 1经葡聚糖凝胶柱层析得到5种组分;CP 1经硫酸水解后硅胶板层析得出主要单糖组分是葡萄糖。结论该提取测定方法操作简单,重复性好,结果准确可靠,适合蛹虫草固体发酵菌丝体多糖的提取、分离和纯化。     

蒙山九州虫草中黄酮类化合物提取、鉴定和含量分析

目的比较蒙山九州虫草不同部位黄酮类化合物的含量及不同提取方法对黄酮类化合物得率的影响,探讨蒙山九州虫草中粗黄酮的最佳提取工艺,并对提取所得黄酮类化合物进行定性和定量分析。方法分别采用乙醇回流法、微波法、超声波法提取蒙山九州虫草中黄酮类化合物,以分光光度法测定总黄酮的含量。结果蒙山九州虫草子座中黄酮含量最高,微波法提取得率最高,最佳提取工艺为:功率为140 W,提取时间为1.5 min,料液比为1∶25,pH 10。结论微波提法具有工艺简单、耗费小、产物纯、提取得率较高等特点,适于实验室条件下使用。     

九州虫草的核苷类成分及不同部位两种活性成分含量的分布九州虫草的核苷类成分及不同部位两种活性成分含量的分布

目的快速测定九州虫草中的部分核苷类成分及其主要活性成分虫草素与腺苷的含量，并考察九州虫草不同部位虫草素和腺苷含量的分布。方法用高效毛细管区带电泳法确定野生和人工九州虫草中的核苷类成分,测定虫草素和腺苷的含量，并比较它们在九州虫草不同部位含量的分布。结果蒙山九州虫草中含有至少8种核苷或碱基，其中抗肿瘤活性成分虫草素的含量远高于冬虫夏草和蛹虫草,且人工栽培九州虫草中虫草素的含量又显著高于野生九州虫草。腺苷主要存在于九州虫草子座中，而虫草素则不仅在九州虫草子座中含量甚高，人工栽培九州虫草虫体中虫草素含量也较高。结论天然和人工九州虫草及其不同部位的核苷和碱基成分存在一定差异，九州虫草中的虫草素含量高，具有良好的栽培、药用与开发前景。     

九州虫草的核苷类成分及不同部位两种活性成分含量的分布

目的快速测定九州虫草中的部分核苷类成分及其主要活性成分虫草素与腺苷的含量,并考察九州虫草不同部位虫草素和腺苷含量的分布.方法用高效毛细管区带电泳法确定野生和人工九州虫草中的核苷类成分,测定虫草素和腺苷的含量,并比较它们在九州虫草不同部位含量的分布.结果蒙山九州虫草中含有至少8种核苷或碱基,其中抗肿瘤活性成分虫草素的含量远高于冬虫夏草和蛹虫草,且人工栽培九州虫草中虫草素的含量又显著高于野生九州虫草.腺苷主要存在于九州虫草子座中,而虫草素则不仅在九州虫草子座中含量甚高,人工栽培九州虫草虫体中虫草素含量也较高.结论天然和人工九州虫草及其不同部位的核苷和碱基成分存在一定差异,九州虫草中的虫草素含量高,具有良好的栽培、药用与开发前景.     

锌、金属硫蛋白、超氧化物岐化酶对镉中毒拮抗作用的比较

本研究用CdCl_2制备小鼠镉中毒模型,使发生明显的脂质过氧化损伤,锌、金属硫蛋白和超氧化物岐化酶分别预处理后,可降低中毒鼠死亡率,肝、肾组织中丙二醛含量明显降低,金属硫蛋白含量明显升高,肝组织中锌/铜比例升高。提示锌、金属硫蛋白和超氧化物岐化酶能拮抗镉引起的致死作用和脂质过氧化作用。     

发酵虫草菌粉指纹图谱研究

目的通过对发酵虫草菌粉指纹图谱的深入研究,为发酵虫草菌粉类产品的质量提高提供思路。方法液质联用确定发酵虫草菌粉类产品(百令胶囊)指纹图谱中6个主色谱峰的化学成分;采用中药色谱指纹图谱相似度评价系统,比较天然虫草和不同厂家发酵虫草菌粉指纹图谱相似度及不同干燥方式对指纹图谱的影响。结果百令胶囊指纹图谱中的6个主色谱峰成分为尿苷5-单磷酸、鸟苷酸、5’-腺嘌呤核苷酸、尿苷、鸟苷、腺苷。发酵虫草菌粉的指纹图谱与天然虫草均存在差异,其中百令胶囊的指纹图谱与天然虫草最接近。干燥方式对指纹图谱影响较大,采用沸腾干燥方式对应的指纹图谱主色谱峰的面积比最接近天然虫草晒干方式。结论指纹图谱可准确反映发酵虫草菌粉质量及质量控制方式的有效性。     

重组人胰岛素样生长因子-Ⅰ工程菌高密度发酵工艺优化

目的优化表达重组人胰岛素样生长因子-Ⅰ(IGF-Ⅰ)工程菌的高密度发酵条件。方法考察培养基以及诱导时机、诱导剂量和诱导时间对蛋白表达的影响;用自控发酵罐进行分批补料培养实验,确定优化工艺条件。结果以2×YT+0.5%葡萄糖为发酵培养基,经0.8 mmol/L IPTG(异丙基-β-D-硫代半乳糖苷)诱导5 h,通过pH-stat反馈补料方式实现工程菌高密度发酵与目的蛋白高效表达,每1 L发酵液收获干菌体50.1 g,IGF-Ⅰ含量达5.25 g/L。结论建立了IGF-Ⅰ工程菌优化的高密度发酵工艺,为IGF-Ⅰ的下游纯化和工业化生产奠定了基础。     

真菌诱导子对北虫草深层培养的影响

目的研究4种真菌诱导子对北虫草液体培养菌丝体干重和虫草素产量的影响。方法在北虫草培养时,加入真菌诱导子,并与空白对照组比较。结果 0.02%的番茄灰霉对北虫草中菌丝体和虫草素的促进作用最明显,菌丝体干重为对照组的1.16倍,虫草素产量为对照组的5.02倍。0.02%番茄灰霉通过延长北虫草的对数生长期,提高了菌丝体干重和虫草素的产量。酵母菌对北虫草无明显作用,而其他两种真菌诱导子对菌丝体和虫草素的产量都有不同程度的抑制作用。结论 4种真菌诱导子对北虫草深层培养有不同程度的影响。     

家兔肝脏中锌-金属硫蛋白提取工艺的优化

用正交试验法对硫酸锌诱导的兔肝锌-金属硫蛋白(Zn-MT)的提取工艺进行优选，以原子吸收法测得锌提取量和差示紫外分光光度法测得巯基活性为评价指标，选用L9(3^4)正交表进行正交实验。考察影响提取Zr-MT的四个因素分别为：pH值8．6的Tris-HCl缓冲溶液浓度、温度、用量和-20℃预冷乙醇-氯仿(1：0．08)混合液与缓冲溶液体积比。所得最佳方案为：每克兔肝加3ml 80℃，pH值8．6的0．05mol／L Tris-HCl缓冲溶液，匀浆，再按每克兔肝加2ml乙醇氯仿(1：0．08)混合液，摇匀，提取。最佳方案所得提取液经凝胶色谱法初步分离得Zn-MT收率约为4．2mg／g肝。     

金属硫蛋白对铅中毒小鼠的保护作用

目的 :观察金属硫蛋白对已发生铅中毒的小鼠的保护作用。方法 :用Balc小鼠制成铅中毒模型 ,连续 7天给予不同浓度的金属硫蛋白解毒 ,测定 7天内的排泄物和血、肾、骨的铅、锌含量及脾脏重量 ,同时做对照试验。结果 :服用金属硫蛋白的小鼠排泄铅随其剂量的增加而明显增加 ,体内蓄积铅随其剂量的增加而明显减少 ,并且脾脏重量得到明显恢复 ,缺锌状态得到改善。结论 :金属硫蛋白有治疗铅中毒的疗效     

The toxicology and metabolism of chlorothalonil in fish. IV. Zinc coexposure and the significance of metallothionein in detoxication in Salmo gairdneri

Rainbow trout, Salmo gairdneri, were subjected to various regimes of exposure to zinc (0.36 mg/l) and ¹⁴C-chlorothalonil (¹⁴C-TCIN, 10 μg/l). Gel column chromatography of hepatic cytosol after exposure showed no binding of ¹⁴C-TCIN to metallothionein proteins with or without pre-exposure or with coexposure to zinc. ¹⁴C-TCIN did not induce the production of metallothionein with or without 96 h preexposure to 0.36 mg/l zinc, whereas induction did occur with zinc coexposure over 156 h. ¹⁴C-TCIN exposure did not affect cytosolic zinc levels, but exposure to zinc did reduce the level of cytosolic ¹⁴C-TCIN over the observed protein molecular weight range. It appears that metallothionein does not play a significant role in TCIN detoxication at this sublethal exposure level.     

Role of metallothionein on Ag accumulation in hepatic and renal cytosol after Ag injection to rats.

To examine the role of metallothionein on Ag accumulation in liver or kidney of rat after Ag injection, the relative Ag-binding capacity of Ag-induced metallothionein in hepatic or renal cytosol of rat after Ag injection was determined. The greater part of Ag increment in hepatic cytosol was attributable to a low molecular weight protein, while the main part of Ag increment in renal cytosol was ascribed to high molecular weight proteins. The low molecular weight, metal-binding protein was identified as metallothionein using ELISA. The maximal levels of hepatic and renal metallothionein mRNA induced by Ag occurred at 7 hr after Ag injection. There was a close relationship between Ag contents in the hepatic or renal cytosol and metallothionein after Ag injection. In dose-response and time-course studies, approximately 60-70% of the Ag increments in hepatic cytosol and approximately 30% of the Ag increments in renal cytosol were bound to metallothionein. These results suggest that the role of metallothionein on Ag accumulation in the liver after Ag injection is different from that of metallothionein on Ag accumulation in the kidney.     

Role of Metallothionein on Ag Accumulation in Hepatic and Renal Cytosol after Ag Injection to Rats

Abstract: To examine the role of metallothionein on Ag accumulation in liver or kidney of rat after Ag injection, the relative Ag-binding capacity of Ag-induced metallothionein in hepatic or renal cytosol of rat after Ag injection was determined. The greater part of Ag increment in hepatic cytosol was attributable to a low molecular weight protein, while the main part of Ag increment in renal cytosol was ascribed to high molecular weight proteins. The low molecular weight, metal-binding protein was identified as metallothionein using ELISA. The maximal levels of hepatic and renal metallothionein mRNA induced by Ag occurred at 7 hr after Ag injection. There was a close relationship between Ag contents in the hepatic or renal cytosol and metallothionein after Ag injection. In dose-response and time-course studies, approximately 60–70% of the Ag increments in hepatic cytosol and approximately 30% of the Ag increments in renal cytosol were bound to metallothionein. These results suggest that the role of metallothionein on Ag accumulation in the liver after Ag injection is different from that of metallothionein on Ag accumulation in the kidney.     

The effect of heavy metal-induced metallothionein on Zn, Cu and Cd accumulation in rat kidney.

To examine the role of metallothionein on heavy metal accumulation in kidneys of rats after Zn, Cd or Cu injection, the relative Zn, Cd or Cu-binding capacity of heavy metal-induced metallothionein in renal cytosol of rats after Zn, Cd or Cu injection was determined. The Zn or Cu increment in renal cytosol after Zn or Cu injection was attributable to low and high molecular weight proteins, while most of the Cd increment was attributable to a low molecular weight protein. The low molecular weight, metal-binding protein was identified as metallothionein using a competitive ELISA. There was a close relationship between heavy metal contents in the renal cytosol and metallothionein of all heavy metal-injected rats. In dose-response and time-course studies, approximately 45, 40 and 85% of the Zn, Cu and Cd increments in renal cytosol were bound to metallothionein after Zn, Cu and Cd injection, respectively. Therefore the order of relative binding capacities of Zn, Cu and Cd-induced metallothionein in kidney was determined to be Cd>Zn approximately Cu. These results suggest that the role of metallothionein in Zn or Cu accumulation in the kidney after Zn or Cu injection is different from that of metallothionein in Cd accumulation in the kidney after Cd injection.     

The Effect of Heavy Metal-Induced Metallothionein on Zn,Cu and Cd Accumulation in Rat Kidney

Abstract: To examine the role of metallothionein on heavy metal accumulation in kidneys of rats after Zn, Cd or Cu injection, the relative Zn, Cd or Cu-binding capacity of heavy metal-induced metallothionein in renal cytosol of rats after Zn, Cd or Cu injection was determined. The Zn or Cu increment in renal cytosol after Zn or Cu injection was attributable to low and high molecular weight proteins, while most of the Cd increment was attributable to a low molecular weight protein. The low molecular weight, metal-binding protein was identified as metallothionein using a competitive ELISA. There was a close relationship between heavy metal contents in the renal cytosol and metallothionein of all heavy metal-injected rats. In dose-response and time-course studies, approximately 45, 40 and 85% of the Zn, Cu and Cd increments in renal cytosol were bound to metallothionein after Zn, Cu and Cd injection, respectively. Therefore the order of relative binding capacities of Zn, Cu and Cd-induced metallothionein in kidney was determined to be Cd>Zn Cu. These results suggest that the role of metallothionein in Zn or Cu accumulation in the kidney after Zn or Cu injection is different from that of metallothionein in Cd accumulation in the kidney after Cd injection.     

Effect of cadmium ingestion on cadmium and zinc profile in male and female rat liver cytosol

A study of the changes in rat liver cytosol zinc and cadmium metalloprotein profiles obtained by G-75 Sephadex chromatography of male and female rats given oral cadmium chloride chronically at different low doses showed that the changes were dose related. Marked disturbances in the zinc-containing regions of the protein profile preceded any extensive formation of metallothionein, and the earliest signs of cadmium metalloprotein formation involved three equally prominent regions, a high molecular weight region (I), the metallothionein region (V) and a low molecular weight region (VI). The low molecular weight region (estimated at 3500 daltons) is of interest in that it is a newly recognized zinc-containing region even in the controls. Significant quantitative differences were found between males and females. Female livers consistently contained higher concentrations of cadmium and zinc. There also were important quantitative differences in the zinc and cadmium-containg cytosol fractions of the protein profiles of livers of males and females exposed to oral cadmium.     

Metallothionein expression during liver regeneration after partial hepatectomy in cadmium-pretreated rats

Metallothionein is a low molecular mass protein inducible mainly by heavy metals, having high affinity for binding cadmium, zinc and copper. In the present study we investigated the expression of metallothionein in regenerating liver, at different time intervals, in cadmium pretreated partially hepatectomized rats. Liver metallothionein is highly expressed during regeneration in-duced by partial hepatectomy in rats, providing zinc within the rapidly growing tissue. Cadmium pretreatment caused inhibition of the first peak of liver regeneration, while metallothionein expression was markedly more prominent in the liver residues of cadmium-pretreated rats. These results demonstrate that although metallothionein able to bind temporarily metal ions as zinc and cadmium has been highly expressed, the liver regenerative process was inhibited possibly due to the effects of cadmium on other pivotal events necessary to the DNA replication.     

Role of metallothionein in biliary metal excretion

We have studied the effect of acute induction of metallothionein on the biliary excretion of a bolus of metal in the rat. Female Sprague-Dawley rats were administered zinc chloride (6.5 mg/kg) or dexamethasone (2 mg/kg), ip in saline daily for 3 d; control animals received saline alone. Zinc causes a 17-fold induction in hepatic metallothionein levels, while dexamethasone caused a 5-fold induction. A bolus of metal chloride, either zinc, mercury, or cadmium, 1 mg/kg iv, was administered, and bile and plasma samples were collected and analyzed for metal content. At 3 h the rats were killed, livers excised, and both metallothionein and the metals associated with metallothionein estimated. Cadmium was excreted into the bile in inverse proportion to the hepatic metallothionein content, while metallothionein content did not appear to bear any relationship to biliary excretion of mercury or zinc. Metallothionein from rats pretreated with zinc contained twice as much zinc per molecule of metallothionein as that found in control rats. Cadmium, which exhibits a very high affinity for metallothionein, replaced this zinc found in association with metallothionein. Conversely, mercury only partially replaced the zinc associated with metallothionein, and a bolus of zinc was completely unable to bind to the already zinc-saturated metallothionein. Consequently, the bolus zinc was found associated with alternative cytosolic proteins, here termed the high-molecular-weight fraction. These findings support the hypothesis that the inhibitory effect of metallothionein on the biliary excretion of metals is dependent on the ability of the metal in question to replace any existing metals associated with metallothionein. Biliary metal excretion was directly proportional to the free metal content, regardless of the metal studied. Consequently, acute induction of metallothionein was inhibitory to biliary cadmium excretion, slightly inhibitory to biliary mercury excretion, and without effect on biliary zinc excretion.     

均匀设计法优选葛根总黄酮提取工艺

目的:筛选超声法提取葛根总黄酮的最佳工艺条件。方法:采用均匀设计法考察乙醇浓度、乙醇用量、提取时间、提取温度、超声提取次数等5个因素对葛根总黄酮提取量的影响;利用紫外-可见分光光度法测定葛根总黄酮的含量;以提取率和提取物中葛根总黄酮含量为指标优选工艺。结果:最佳提取工艺为乙醇浓度75%,乙醇用量20mL.g-1,提取时间40min,提取温度68℃,超声提取5次。结论:该提取工艺合理、可行,可为工业化生产提供理论依据。     

Induction of hepatic metallothionein in the rabbit fetus following maternal cadmium exposure

Timed-pregnant rabbits were administered a single, sc injection of cadmium chloride (0, 0.125, 0.25, or 0.50 mg cadmium/kg body wt) 48 hr prior to being killed at term. Although the doses chosen were below that shown to produce significant fetal lethality, the 0.50 mg/kg dose did produce a significant reduction in fetal body weight, liver weight, kidney weight, and crown-rump length while having no effect on placental weight. Fetal hepatic metallothionein levels were significantly increased at all three doses; however, a significant increase in metallothionein zinc content was seen only at the two higher doses. Metal distribution studies indicated that the increase in fetal metallothionein and the accompanying increase in cytosolic zinc coincided with an apparent redistribution of the metal from extrahepatic sites to the liver. The whole fetal content of zinc was not significantly altered by the maternal administration of cadmium.