藏药蔓菁抗氧化活性多糖的提取及纯化工艺优选

目的: 优化藏药蔓菁中抗氧化活性多糖的提取、纯化工艺。 方法: 以粗多糖得率、总多糖质量分数和抗氧化活性的综合评分为指标,采用正交试验考察浸提次数、料液比及浸提时间对蔓菁多糖提取工艺的影响;以色素去除率和多糖损失率为综合评价指标,通过正交试验考察脱色温度、吸附时间、活性炭用量对脱色工艺的影响。 结果: 蔓菁多糖最佳提取工艺为料液比1:30,于90 ℃水浴浸提3次,每次2 h;最佳脱色工艺为加3%活性炭于60 ℃吸附40 min。蔓菁多糖纯度达4.66%,其清除DPPH自由基的IC₅₀=6.71 g·L^-1。 结论: 优选的提取、纯化工艺稳定可靠,所得多糖的含量高、杂质少,适合于蔓菁多糖的工业化生产。     

假单胞杆菌、灵芝等几种菌的发酵液对猪苓菌生长的影响

 目的：证明在猪苓菌发酵过程中加入假单胞杆菌、灵芝、金针菇等几种菌的发酵液对猪苓多糖产生是否具有促进作用。方法：采用平板培养法、发酵培养法。平板培养法直接测定生长量；发酵培养所得的发酵液采用3，5-二硝基水杨酸法测定多糖含量。结果：在猪苓菌发酵培养过程中加入假单胞杆菌、灵芝、金针菇等几种菌的发酵液可使猪苓菌球数量增多、重量大、多糖含量高。以假单胞杆菌发酵液的处理效果最佳；在平板培养时，培养25d菌落直径达72．25mm，在发酵培养过程中，在最佳产糖期发酵液中粗多糖含量达7．800mg·ml^－1，而对照分别为48．63mm和5．200mg·ml^－1。结论：在猪苓菌丝体培养过程中加入假单胞杆菌、灵芝、金针菇等几种菌的发酵液均对其菌丝的生长及发酵过程中多糖的产生具有促进作用。     

单因素考察活性炭脱除麦冬多糖色素

目的:优化麦冬粗多糖除色素的工艺。方法:以活性炭为吸附剂,选择多糖溶液质量浓度、活性炭用量、脱色时间、脱色温度等4方面进行单因素考察。结果:脱除麦冬多糖色素的最佳条件为多糖溶液质量浓度为5 g.L-1,加入2%的活性炭,在50℃温度下脱色30 min。结论:该脱色方法为麦冬多糖进一步纯化提供了基础。     

猴头菇多糖提取液脱色工艺研究

目的优化猴头菇多糖脱色工艺。方法以活性炭为吸附剂,选择多糖溶液浓度、活性炭用量、脱色时间、脱色温度和pH 5个方面因素进行单因素实验。结果猴头菇多糖活性炭脱色的最佳条件为多糖浓度为0.4 g/L,pH调至4,加入3%的活性炭颗粒,在70℃下脱色80 min。宜用活性炭颗粒脱色。结论该脱色方法简便,为猴头菇多糖进一步纯化提供了基础。     

白屈菜多糖果胶酶提取及脱色工艺的优化

目的优化白屈菜多糖的果胶酶提取及脱色工艺。方法以多糖得率和多糖含有量为指标,采用加权评分法(权重各占50%),以正交法优化果胶酶对白屈菜多糖的酶解提取效果;以脱色率和多糖保留率为指标,采用加权评分法(权重各占50%),利用正交试验分别确定活性炭和双氧水的最佳脱色工艺。结果白屈菜多糖最佳酶提条件为酶解温度40℃,酶用量5%,酶解pH 3.0,酶解时间2 h;活性炭最佳脱色工艺为白屈菜多糖溶液加入0.5%的活性炭,调pH至3.0,50℃条件下水浴搅拌40 min,多糖保留率为88.56%,多糖含有量为68.03%;双氧水最佳脱色工艺为白屈菜多糖溶液加入10%双氧水,调pH至10.0,50℃条件下水浴反应1 h,多糖保留率为79.17%,多糖含有量为71.34%。结论该方法稳定,重复性好,可用于提取白屈菜多糖果胶酶。     

玉米须多糖脱色工艺条件的研究

目的探讨玉米须多糖脱色的工艺条件。方法以活性炭和过氧化氢为脱色试剂,对玉米须多糖进行脱色试剂用量、脱色时间和加热温度等因素的考察,在单因素基础上进行正交试验。结果活性炭和过氧化氢对玉米须多糖脱色的最佳条件是：活性炭用量为2％,脱色时间为80min,加热温度为60℃,脱色率是14．76％,损失率是13．01％;过氧化氢的样液比为1：0．15,脱色时间2h,加热温度50℃,pH值为6,脱色率是90．7％,损失率是18．80％。结论过氧化氢对玉米须多糖脱色的效果优于活性炭。     

马齿苋多糖的活性炭脱色工艺优选

目的:优选马齿苋多糖的活性炭脱色工艺.方法:在单因素试验基础上,根据Box-Behnken中心组合设计原理,选取活性炭用量、脱色时间和脱色温度三因素三水平进行响应面分析,建立马齿苋多糖溶液脱色率的二次多项数学模型,分析各因素显著性,确定马齿苋多糖溶液的最佳脱色工艺.结果:最佳工艺条件为活性炭用量30 g·L-1,脱色时间50 min,脱色温度40℃,脱色率68.05％.结论:优选的脱色工艺简单、稳定、可预测性好.     

大孔吸附树脂对厚朴多糖的脱色工艺研究

目的从6种大孔吸附树脂中筛选出对厚朴多糖脱色效果最佳的树脂,并研究其对多糖脱色的最佳工艺。方法以脱色率、脱蛋白率和多糖保留率为指标,采用静态吸附法筛选出对厚朴多糖具有最佳脱色效果的树脂。以脱色率和多糖保留率为指标,采用正交试验法考察该树脂对厚朴多糖的最佳脱色条件。结果 ADS-7型大孔树脂对厚朴多糖的脱色效果最佳,该树脂最佳脱色条件为:多糖浓度为0.2%,脱色温度为45℃,脱色时间为2 h。结论在此条件下,厚朴多糖的脱色率可达到最大,同时也可以去除大量的蛋白。     

六味地黄生物制剂总多糖的精制工艺优选

目的:优化六味地黄生物制剂总多糖的精制工艺,为该制剂的开发提供参考。方法:以粗多糖质量为指标,采用正交试验考察醇沉浓度、醇沉时间、药液相对密度对总多糖醇沉工艺的影响。在单因素试验基础上,以蛋白质清除率和总多糖保存率的综合评分为指标,采用正交试验优化三氯乙酸(TCA)-正丁醇法脱蛋白工艺。以色素清除率和总多糖保存率的综合评分为指标,利用正交试验优化活性炭除色素工艺。结果:最佳醇沉工艺为醇沉浓度85%,醇沉时间6 h,药液相对密度1.05;粗多糖质量8.92 mg。最佳脱蛋白工艺为粗多糖溶液与TCA-正丁醇体积比1∶2,TCA-正丁醇(1∶15),振荡时间20 min,静置时间0.5 h;蛋白质清除率80.23%,总多糖保存率85.45%。最佳除色素工艺为脱色温度40℃,活性炭加入量6%,脱色时间40 min;色素清除率74.84%,总多糖保存率78.69%。结论:优选的精制工艺稳定可行,适用于六味地黄生物制剂总多糖保健产品的开发。     

白芍多糖脱色工艺研究

目的优化白芍多糖脱色的最佳工艺。方法分级醇沉技术得到不同组分的白芍多糖,研究粉末活性炭、颗粒活性炭、过氧化氢、次氯酸钠对其脱色效果的影响,单因素和正交试验优化脱色工艺,并对脱色前后多糖进行UV、IR分析。结果白芍多糖色素主要集中在PRPS90组分。该组分白芍多糖最优脱色工艺为温度为65℃,过氧化氢体积分数为20%,调节p H值为9,脱色3.5 h。经UV、IR分析,脱色前后结构未发生明显改变。结论该工艺稳定可行,易于操作,适合工业化生产。     

猪苓药材质量影响因素及质量评价的研究进展

通过对近年来相关文献进行整理,介绍了目前对猪苓化学成分的相关研究,包括对猪苓多糖、甾体类成分及其他成分的研究,系统地介绍了影响猪苓药材质量的有关因素,如品种、产地、栽培、采收与加工,同时比较、分析了评价猪苓质量的提取、鉴别和含量测定方法等.作为一种常用中药材,猪苓药材的质量影响着其在临床使用的疗效,通过对质量影响因素以及质量评价方法的整理,为保证猪苓药材质量提供合理的依据.     

猪苓与蜜环菌化学成分研究的相关分析进展

共生的猪苓Polyporus umbellatus与蜜环菌Armillaria mellea均为药食兼用真菌,具有降血糖、调节免疫、抑制肿瘤等多种生物活性。猪苓菌核经菌丝体发育而来,其生长过程与共生蜜环菌有关;受其侵染,猪苓菌丝体可形成菌核。该文通过分析猪苓菌丝体、菌核和蜜环菌的化学成分,发现三者均含有甾体和含氮杂环等化合物,且猪苓菌核与蜜环菌中还含有三萜类次生代谢产物。猪苓菌核及其菌丝体的甾体种类存在显著差异,但部分成分存在一定的相关性。此外,猪苓菌核还特有长链脂肪酸、酰胺和苯酚等多种化合物,推测这些可能是因蜜环菌入侵而形成的多种次生代谢产物;而蜜环菌自身主要产生倍半萜、二萜等物质。猪苓与蜜环菌的化合物含量、种类与其共生繁殖密切相关,目前尚需对二者的共生机制进行深入研究,为提高二者产量及质量提供科学依据。     

猪苓多糖硫酸酯的制备及活性测定

目的:制备猪苓多糖硫酸酯,同时测定其活性.方法:采用醇沉分级、大孔吸附树脂、聚酰胺树脂和脱蛋白法从中药猪苓中提取分离得到猪苓多糖,硫磺酸-吡啶法进行硫酸酯化,经Sephadex G-75、透析得到硫酸化猪苓多糖,并体外观察硫酸酯化多糖对羟基自由基清除作用.结果:猪苓多糖硫酸酯化前后均有清除Fenton反应由Fe2+-H2O2体系产生的羟自由基,硫酸酯化猪苓多糖对羟自由基的清除能力增强,并呈明显量效关系.结论:制备猪苓多糖硫酸酯的方法准确可靠,为猪苓多糖的进一步开发利用提供实验依据.     

药用真菌猪苓菌核渗出液理化性质的研究

探讨不同发育阶段人工培养的猪苓菌核渗出液的理化性质。对药用真菌猪苓进行培养,系统地观察不同发育阶段的猪苓菌核及其渗出液的形态特征;对不同发育阶段猪苓菌核渗出液的pH进行测定;采用硫酸-苯酚法测定猪苓菌核渗出液的多糖含量;并利用BCA蛋白含量测定方法测定猪苓菌核渗出液的蛋白含量,利用紫外吸收法测定猪苓菌核渗出液中过氧化氢酶（CAT）的含量。在猪苓菌核发育过程中,菌核渗出液pH出现先升高后降低的趋势;渗出液的多糖含量逐渐下降;渗出液的蛋白含量及CAT含量出现逐渐升高的趋势,说明猪苓菌核渗出液形成过程中伴随着高水平的氧化应激反应。添加维生素C以后,部分地消除了活性氧,因此CAT活力下降。猪苓菌核渗出液的pH在猪苓菌核形成过程中一直维持酸性状态,间接反应出酸性环境有利于猪苓菌核的形成;菌核渗出液具有逐渐产生且最终被菌核重新吸收的特征。     

Bioactivity-directed isolation, identification of diuretic compounds from Polyporus umbellatus

Aim of the study

Polyporus umbellatus is a fungus used as a diuretic medicine. The objective of this study was to isolate and elucidate the diuretic constituents of n-hexane, ethyl acetate, n-butanol and water extracts of Polyporus umbellatus and to evaluate their diuretic activity.

Materials and methods

The n-hexane, ethyl acetate, n-butanol and water extracts of Polyporus umbellatus were tested by diuretic experiment of normal rats in metabolic cage. The n-hexane extract and n-butanol extract were prepared separately by the bioassay-guided approach. Three isolated compounds doses (5, 10 and 20 mg/kg BW) were orally administered to normal rats. Water excretion rate, pH and content of Na⁺, K⁺ and Cl⁻ were measured in the urine of saline-loaded rats.

Results

n-Hexane extract (P < 0.05), n-butanol extract (P < 0.05) and three isolated compounds (ergosta-4,6,8(14),22-tetraen-3-one, ergosterol and d-mannitol) displayed diuretic activity.

Conclusions

The ergosta-4,6,8(14),22-tetraen-3-one was the strongest diuretic constituent in the three compounds. Ergosterol and d-mannitol were found to be also responsible for duiretic effects in Polyporus umbellatus for the first time. Data show that 20 mg/kg dose of the ergosterol for urine out put became significantly higher than in the control rats, but the ratio of Na⁺/K⁺ almost unaltered in the three doses. The highest dose of the d-mannitol was significant and increased the cumulative urine output. Regarding the electrolyte excretion, data show that the doses 10 and 20 mg/kg produce significant increase for excretion of Na⁺ and Cl⁻. The present results provide a quantitative basis explaining application of Polyporus umbellatus as a diuretic medicine. The result proved that its diuretic effects were also due to the contribution of multi-components in clinical application.     

Diuretic activity and kidney medulla AQP1, AQP2, AQP3, V2R expression of the aqueous extract of sclerotia of Polyporus umbellatus FRIES in normal rats

Aim of the study

Zhuling, sclerotia of Polyporus umbellatus FRIES, a Traditional Chinese Medicine, has long been used as a diuretic. The aim of the present study was to evaluate the diuretic effect on the urinary electrolyte concentration (Na⁺, K⁺, and Cl⁻) and regulation of the relative mRNA expression of aquaporin-1 (AQP1), aquaporin-2 (AQP2), aquaporin-3 (AQP3) and vasopressin V₂ receptor (V₂R) post-oral administration of sclerotia of Polyporus umbellata aqueous extract in normal rats.

Materials and methods

Aqueous extract of sclerotia of Polyporus umbellatus (50 mg/kg, 250 mg/kg, 500 mg/kg) or the reference drug, furosemide (10 mg/kg) were administrated orally to male SD rats and their urine output was quantified and collected 24 h and 8 days after the treatment. The kidney medulla AQP1, AQP2, AQP3 and V₂R mRNA relative expressions were measured with RT-PCR.

Results

After single dose of the exact of sclerotia of Polyporus umbellata, urine output was found to be significantly increased, which began at 4 h, and at 24 h after the treatment, the sclerotia of Polyporus umbellatus extract and furosemide treatment produced the similar total volume of urine excreted. The extract increases urinary levels of Na⁺, K⁺, and Cl⁻, to about the same extent, while furosemide increased urinary levels of Na⁺ and Cl⁻. After the 8-day doses, all two substances induced significant diuresis, natriuresis and chloriuresis. These two substances do not regulate the AQP1 and AQP3 mRNA level in normal rat kidney medulla. The AQP2 mRNA level of sclerotia of Polyporus umbellata extract was down-regulated significantly, the V₂R mRNA level of sclerotia of Polyporus umbellata extract 50 mg/kg dose group and 250 mg/kg dose group were down-regulated significantly too. Interestingly, the low-dose group had higher effect on regulation of AQP2 and V₂R mRNA level.

Conclusion

Aqueous extract of sclerotia of Polyporus umbellatus has conspicuous diuretic effect confirming its ethnopharmacological use. From the pattern of excretion of water, sodium, potassium, chlorine, AQP2 and V2R mRNA level, it may be logically concluded that it has effect from down-regulating AQP2, and down-regulate AQP2 by down-regulating V₂R.     

猪苓菌核2种热激蛋白基因的克隆及其表达分析

该研究采用RT-PCR技术从中国野生猪苓菌核Polyporus umbellatus中克隆得到2种热激蛋白基因,其中Pu Hsp90基因的开放阅读框2 091 bp,编码696个氨基酸,推导的蛋白质相对分子质量约78.9 k Da;Pu Hsp70基因的开放阅读框1 944 bp,编码647个氨基酸,推导的蛋白质相对分子质量约70.5 k Da;蛋白质结构预测及同源比对分析表明,这2个基因编码的核苷酸序分别具有Hsp90,Hsp70蛋白的保守结构域。进化树分析表明,Pu Hsp90与变色栓菌Hsp90聚为一类,Pu Hsp70与肝色牛排菌Hsp70聚为一类。qRT-PCR分析表明,蜜环菌侵染的情况下,这2个基因在猪苓菌核中均上调表达。这2个基因在蜜环菌侵染猪苓菌核的状态下有相同的表达模式,预示这2个基因其可能在抗生物胁迫中起重要作用。     

药用真菌猪苓菌核无机磷酸盐转运蛋白基因的分子克隆与特性分析

目的克隆猪苓Polyporus umbellatus菌核无机磷酸盐转运蛋白基因并进行生物信息学和表达模式分析。方法采用RT-PCR技术从猪苓菌核中克隆得到1个无机磷酸盐转运蛋白(inorganic phosphate transporter)基因,利用生物信息学软件预测蛋白的理化性质、结构域、信号肽和跨膜结构等分子特性;采用Clustal W2以及MEGA 6.0分别进行氨基酸多序列比对和进化关系分析;实时荧光定量PCR分析基因表达模式。结果猪苓无机磷酸盐转运蛋白基因命名为Pu Pi T(NCBI登录号:KU179154)。该基因开放读码框全长为1 590 bp,编码530个氨基酸。Pu Pi T蛋白相对分子质量为57 552,等电点6.82。氨基酸序列分析表明,Pu Pi T蛋白具有12个跨膜区。氨基酸序列多重比对及系统发育树结果显示Pu Pi T与Moniliophthora rorer亲缘关系最近,与Moniliophthora roreri、双色蜡蘑Laccaria bicolor、Heterobasidion irregulare有较高的同源性,荧光定量PCR结果表明,Pu Pi T在与蜜环菌共生的猪苓菌核及未与蜜环菌共生的菌核中都有表达,其中共生部分的表达量显著上调,约是未共生部位的12倍,说明其参与猪苓与蜜环菌共生过程。结论 Pu Pi T基因克隆和分子特征为进一步研究揭示其在猪苓菌核磷元素转运及与蜜环菌共生过程中的调控作用奠定基础。     

TLR4-mediated activation of macrophages by the polysaccharide fraction from Polyporus umbellatus(pers.) Fries

Aim of the Study

Zhu Ling (Polyporus umbellatus) is well-known to reduce the risk of a variety of diseases. In this study, we explored the molecular mechanism of its immunostimulatory potency in immune responses of macrophages, using polysaccharides prepared from Polyporus umbellatus (PPS).

Materials and methods

Splenocyte proliferation was analyzed with ³H-TdR incorporation method. Nitric oxide (NO) was measured by Griess method and cytokines of culture supernatants was detected by enzyme linked immunosorbent assay (ELISA). The fluoresceinamine-labeled PPS (Flu-PPS) and dextran (Flu-dextran) were prepared by the cyanogen bromide activation method. The cell-binding activity of Flu-PPS was analyzed with FACS and confocal microscopy. NF-κB activity was measured by ELISA assay.

Results

We found that PPS is able to strongly upregulate the functions of macrophages such as Nitric oxide (NO) production and cytokine expression. Compared with C3H/HeJ group, PPS significantly stimulated the proliferation of splenocytes and the production of TNF-α, IL-1β and NO of peritoneal macrophages from C3H/HeN mice. The function blocking antibodies to TLR-4, but not TLR-2 and CR3, markedly suppressed PPS-mediated TNF-α and IL-1β production. Flow cytometric and confocal laser-scanning microscopy analysis shown that fluorescence-labeled PPS (f-PPS) can bind specifically to the target cells, and the binding can blocked by unlabeled PPS and anti-TLR4, but not anti-TLR2 and CR3 monoclonal antibodies. Nuclear translocation and DNA binding activity of NF-κB was significantly induced by PPS.

Conclusions

Therefore, our data suggest that PPS may exert its immunostimulating potency via TLR-4 activation of signaling pathway.