何首乌化学成分及其药理活性的研究进展（英文）

何首乌(Pleuropterus multiflorus)是一种珍贵的多年生中药,为蓼科(Polygonaceae)何首乌属植物,主要分布在四川、云南、贵州以及山西和甘肃南部,可用于治疗肝损伤、癌症、糖尿病、脱发、动脉粥样硬化以及神经退行性疾病。近年来,国际上有报道称摄入何首乌会引起肝损伤,因此,建立何首乌的安全监测和风险管理模型对何首乌的开发利用具有重要意义。对何首乌的化学成分、药理活性及其毒副作用进行了综述,为何首乌的临床应用、科学研究和生产质量控制提供参考。     

何首乌块根中砷、镉、汞和铅含量的检测及其富集特性

何首乌是著名传统常用中药,为蓼科(Polygonaceae)植物何首乌(Polygonum multiflorum Thunb.)的块根.生首乌具有解毒、消痈和润肠通便的作用,制首乌则有补肝肾、益精血、乌须发和强筋骨之功效[1].何首乌最早出现于唐代李翱所著的<何首乌传>,<本草图经>及<本草纲目>描述何首乌"叶叶相对",展学峰等认为二者所指为现今的萝摩科(Asclepiadaceae)植物白首乌(Cynanchum bungei Decne.)[2].何首乌野生资源分布于陕西南部、甘肃南部及华东、华中、华南和西南,日本也有分布[3]     

不同中药水提物对孤苓核生柄孔菌发酵产胞内三萜类物质和胞外多糖的影响

将11种中药水提物分别加入孤苓核生柄孔菌液体发酵中,以生物量、胞外多糖和三萜类物质为指标,从中选出最佳中药、最佳中药浓度以及最佳中药浓度组合。结果表明,对孤苓核生柄孔菌生长与发酵产物影响最大的中药是何首乌和菊花2种。菊花对三萜类物质的影响较大,何首乌则对孤苓核生柄孔菌生长和发酵胞外多糖影响较大。孤苓核生柄孔菌发酵生物量达到最大(23.97g/L)的条件为添加何首乌水提物浓度15g/L;产胞外多糖的最佳(4.15g/L)条件为添加何首乌水提物浓度5g/L;总三萜类物质产量达到最高值(397.99mg/L)的条件为组合中药水提物:何首乌和菊花水提物浓度均为25g/L。     

发酵法炮制何首乌的初步研究

为了筛选出可以专一性降解何首乌中蒽醌类成分而不破坏其有效成分二苯乙烯苷的菌种,降低何首乌的毒副作用,我们建立了一种炮制何首乌的新方法.采用HPLC法和UV分光光度计法,对不同菌株发酵的何首乌样品及其发酵前后的化学成分进行比较分析.结果显示:米根霉(Rhizojpus oryzae)、菌株YMS-010(Mucor.sp.)发酵样品的游离型总蒽醌、大黄素、大黄素甲醚的含量均有较大幅度的降低.菌株YMS-006(Aspergillus)、黑曲霉(Aapergillus nigers)、酿酒酵母(Saccharomyces cerevisiae)三株菌株发酵何首乌样品有未知成分产生,且含量较高.通过筛选发现米根霉、菌株YMS-006具有专一性降解蒽醌类成分而不破坏二苯乙烯苷的能力,获得了具有发酵炮制作用的菌株.     

何首乌种质资源核型分析及进化趋势研究

以32份何首乌种质为研究材料,采用常规压片法,对其进行核型分析及进化趋势研究。结果表明:(1)32份何首乌种质存在二倍体(2n=2x=22)和三倍体(2n=3x=33)两种倍型;核型不对称系数As.K%和AI值分别在51.85%～61.52%和0.37～1.40;核型有"1A"、"1B"和"2A"3种类型,属于进化程度较低的植物。(2)在32份何首乌种质中,53.13%的种质为三倍体,表明多倍化在何首乌种质的进化中起着重要作用,但染色体结构变异也是其进化的主要途径之一。(3)何首乌各种质染色体形态变化较大,进化程度参差不齐,可能与种质的生境有一定相关性。(4)何首乌最初的起源中心可能在高纬度地区,在向低纬度地区迁移过程中发生了染色体数目及结构的变化。     

土壤pH值对何首乌生理及其光合特性和有效成分含量的影响

该研究采用盆栽试验,探究不同土壤pH (4.5、5.5、6.5、7.5、8.3、9.5)对何首乌生长、生理特性及有效成分含量的影响,以明确何首乌种植的适宜土壤pH。结果表明：(1)何首乌在土壤pH 6.5~9.5时能正常生长,在pH 4.5和5.5时出现叶片枯萎脱落,少数不能存活;叶面积在pH 4.5～9.5范围内先增加后降低,并在pH 6.5和7.5达到高峰值,且显著高于其余处理。(2)在土壤pH 4.5~9.5范围内,何首乌叶片叶绿素含量、净光合速率、气孔导度和蒸腾速率均呈现先增加后降低的趋势,并在pH 6.5和7.5时达到峰值;叶片脯氨酸、可溶性糖和可溶性蛋白含量总体上随pH升高呈降低趋势;叶片丙二醛含量随pH升高先降低而后增加,其超氧化物歧化酶(SOD)、过氧化物酶(POD)和过氧化氢酶(CAT)活性均随pH升高先升高后降低。(3)在土壤pH 4.5~9.5范围内,何首乌块茎有效成分含量随pH升高而降低,但均达到《中华人民共和国药典》标准,其中二苯乙烯苷含量和结合蒽醌含量分别为1.89% ～ 5.41%和0.10% ～ 0.25%;土壤pH为4.5时,何首乌块茎中二苯乙烯苷和总蒽醌含量达到最高,分别为5.41%和0.38%。研究认为,何首乌对土壤酸碱有一定的耐受性,但在进行何首乌人工驯化和种植时要尽量避免强酸性环境,且宜选择pH在6.5 ～ 8.3的中性至弱碱性土壤。     

不同种源何首乌的ITS序列分析及其亲缘关系研究

采用PCR直接测序法,测定了10个种源何首乌的核糖体DNA ITS区序列,并结合GenBank中相关植物的ITS序列(以萹蓄Polygonu maviculare为外类群),应用遗传距离与系统树分析法对不同种源何首乌的亲缘关系进行了分析。结果表明:(1)ITS1序列长度为195bp,G C含量为69.23%~72.31%,ITS2序列长度为189bp,G C含量为77.25%~80.95%,序列间遗传分化距离为0.00175~0.04945;(2)10个种源何首乌构成2个分支,广西田阳种源自成一支,与其它种源亲缘关系较远;(3)结合形态、分布与化学成分,支持将田阳何首乌作为何首乌的一个变种——棱枝何首乌。     

楮实子对药物性肝损伤大鼠氧化应激因子的影响

为了探讨楮实子对对乙酰氨基酚(APAP)诱导的药物性肝损伤大鼠的保护作用以及对过氧化物酶体增殖物激活受体γ(PPAR-γ)、过氧化物酶体增殖物激活受体α(PPAR-α)、C-Ros癌基因1(ROS1)的调控作用。实验将50只SD大鼠随机分为正常组、模型组、水飞蓟宾组(44mg/kg)和楮实子高、低剂量组(4.2、1.05g生药/kg),每组10只。灌胃给予对乙酰氨基酚(1.2kg/kg)制备肝损伤模型,给药组造模的同时给予相应药物治疗,连续30天。实验结束,收集血清、肝组织标本进行指标检测。结果显示,楮实子各剂量均能降低药物性肝损伤大鼠血清中谷丙转氨酶(ALT)和谷草转氨酶(AST)活性,降低总胆红素(TBIL)和直接胆红素(TBIL)的含量,升高血清中谷胱甘肽(GSH)含量、超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-Px)活性,降低丙二醛(MDA)含量以及ROS1的表达,上调PPAR-αmRNA的表达,下调PPAR-γmRNA的表达。以上研究结果表明,楮实子能防治对乙酰氨基酚所致肝损伤,其作用机制可能是通过降低ROS1的表达、调节转录因子PPAR-α和PPAR-γ的基因表达,从而缓解氧化应激损伤来实现的。     

基于UPLC-Triple TOF-MS/MS技术分析同基源何首乌和首乌藤中差异化学成分

基于超高效液相-串联四极杆飞行时间高分辨质谱法(UPLC-Triple TOF-MS/MS)结合多元统计分析技术分析同基源何首乌和首乌藤中化学成分的差异。何首乌样品用甲醇在室温下超声提取后进样,通过二级串联质谱分析,对采集的图谱进行峰匹配、峰对齐、滤噪处理等进行特征峰提取;用主成分分析(PCA)和偏最小二乘法-判别分析(PLS-DA)进行数据处理;根据一级质谱精确质荷比和二级质谱碎片信息,结合软件数据库搜索及相关文献进行成分鉴定。结果显示,同基源何首乌和首乌藤样品间的化学组成得到明显区分;初步筛选并鉴定出42种同基源何首乌和首乌藤差异显著的化学成分,其中12种为共有差异化学成分,且呈现出不同的变化规律。该结果可为揭示同基源何首乌和首乌藤中次生代谢产物合成积累的差异性提供基础资料,亦为何首乌和首乌藤资源的综合开发利用提供了科学依据。     

何首乌生物技术研究进展

综述了何首乌的快速繁殖、愈伤组织的诱导和培养、毛状根的诱导和培养以及活性成分的产生,并展望了何首乌生物技术的前景。     

何首乌不同种质花粉形态及进化趋势研究

采用电镜扫描法,对48份何首乌种质的花粉形态进行了观察和分析。结果表明:何首乌花粉形状主要为椭球形,少量为长椭球形和近球形,部分种质花粉形状正处于椭球形向长椭球形进化的阶段;极面观主要为三裂圆形,部分种质的变异为半球状、四边形、凹陷状或平三角形;赤道面观主要为椭圆形,部分种质为三角形或四边形;萌发沟3条,狭长,几达两极,部分种质的1条或2条萌发沟中部或近极端处隆起,2条或3条萌发沟在极端处相交;表面纹饰为穴状,部分种质的穴中出现小孔。研究结果表明,何首乌物种正处于活跃进化阶段且因区域及生境的差异其进化速度不一致,低纬度地区的种质进化程度较高纬度地区的高,其最初的起源中心可能位于高纬度地区,在向低纬度地区迁移过程中其花粉形态发生了不同程度的变异。     

Molecular cloning of the lectin and a lectin-related protein from common Solomon's seal (Polygonatum multiflorum)

The most prominent protein ofPolygonatum multiflorum (common Solomon's seal) rhizomes has been identified as a mannose-binding lectin. Analysis of the purified lectin demonstrated that it is a tetramer of four identical subunits of 14 kDa. Molecular cloning further revealed that the lectin from this typical Liliaceae species belongs to the superfamily of monocot mannose-binding proteins. Screening of cDNA libraries constructed with RNA isolated from buds, leaves and flowers ofP. multiflorum also yielded cDNA clones encoding a protein, which contains two tandemly arranged domains with an obvious sequence homology to the mannose-binding lectins. Molecular modelling of thePolygonatum lectin and lectin-related protein indicated that the three-dimensional structure of both proteins strongly resembles that of the snowdrop lectin. In addition, this approach suggested that the presumed carbohydrate-binding sites of the lectin can accommodate a mannose residue whereas most of the carbohydratebinding sites of the lectin-related protein cannot.Abbreviations GNA Galanthus nivalis agglutinin - HCA hydrophobic cluster analysis - LECPMA cDNA clone encoding PMA - PM30 30 kDa protein isolated fromPolygonatum multiforum - PMA Polygonatum multiflorum agglutinin - PMLRP Polygonatum multiflorum lectin-related protein     

Molecular and cytogenetic characterization of repetitive DNA sequences from Lolium and Festuca: applications in the analysis of Festulolium hybrids

Summary A set of species-specific repetitive DNA sequences was isolated from Lolium multiflorum and Festuca arundinacea. The degree of their species specificity as well as possible homologies among them were determined by dot-blot hybridization analysis. In order to understand the genomic organization of representative Lolium and Festuca-specific repetitive DNA sequences, we performed Southern blot hybridization and in situ hybridization to metaphase chromosomes.Southern blot hybridization analysis of eight different repetitive DNA sequences of L. multiflorum and one of F. arundinacea indicated either tandem and clustered arrangements of partially dispersed localization in their respective genomes. Some of these sequences, e.g. LMB3, showed a similar genomic organization in F. arundinacea and F. pratensis, but a slightly different organization and degree of redundancy in L. multiflorum. Clones sequences varied in size between 100 bp and 1.2 kb. Estimated copy number in the corresponding haploid genomes varied between 300 and 2×10⁴. Sequence analysis of the highly species-specific sequences from plasmids pLMH2 and pLMB4 (L. multiflorum specific) and from pFAH1 (F. arundinacea specific) revealed some internal repeats without higher order. No homologies between the sequences or to other repetitive sequences were observed. In situ hybridization with these latter sequences to metaphase chromosomes from L. multiflorum, F. arundinacea and from symmetric sexual Festulolium hybrid revealed their relatively even distribution in the corresponding genomes. The in situ hybridization thus also allowed a clearcut simple identification of parental chromosomes in the Festulolium hybrid.The potential use of these species-specific clones as hybridization probes in quantitative dot-blot analysis of the genomic make-up of Festulolium (sexual and somatic) hybrids is also demonstrated.Abbreviations bp Base pair (s) - CMA chromomycin A₃ - DAPI 4,6-diamidino-2-phenylindole - IPTG isopropyl -D-thio-galactopyranoside - kb kilobase pair(s) - NBT nitroblue tetrazolium chloride - X-gal 5-bromo-4-chloro-3-inonyl -D-galactopyranoside     

Relationship between Contents of Chlorophyll (a+b) (SPAD values) and Nitrogen of Some Temperate Grasses

In a field experiment the chlorophyll (a+b) (SPAD readings) and nitrogen contents of three grass species (Festuca arundinacea Schreb., Lolium perenne L., and Lolium multiflorum Lam.) and three intergeneric hybrids of Festuca pratensis Huds. × Lolium multiflorum Lam. and Festuca arundinacea Schreb. × Lolium multiflorum Lam. were measured. Close relationships were found between SPAD readings and nitrogen leaf content (r ² = 0.873^** 0.491^** and 0.938^**) for the 1^st, 2^nd, and 3^rd cut, respectively. SPAD readings and N contents were closely correlated (r = 0.836^**) confirming that SPAD measurements could be used in grass selection and/or breeding for high N concentration in herbage. This revised version was published online in August 2006 with corrections to the Cover Date.     

Identification of parental and recombined chromosomes in hybrid derivatives of Lolium multiflorum × Festuca pratensis by genomic in situ hybridization

Genomic in situ hybridization (GISH) was used to identify Festuca chromatin in mitotic chromosomes of Lolium multiflorum (Lm) × Festuca pratensis (Fp) hybrids and hybrid derivatives. In two inverse autoallotriploids LmLmFp and LmFpFp, in situ hybridization was able to discriminate between the Lolium and Festuca chromosomes. In a third triploid hybrid produced by crossing an amphiploid of L. multiflorum × F. pratensis (2n=4x=28) with L. multiflorum (2n=2x=14), the technique identified chromosomes with interspecific recombination. Also, in an introgressed line of L. multiflorum which was homozygous for the recessive sid (senescence induced degradation) allele from F. pratensis, a pair of chromosome segments carrying the sid gene could be identified, indicating the suitability of GISH in showing the presence and location of introgressed genes. By screening backcross progeny for the presence of critical alien segments and the absence of other segments the reconstitution of the genome of the recipient species can be accelerated.     

The use of a triploid hybrid for introgression in Lolium species

Summary Triploid hybrids of Lolium multiflorum (4x) x L. perenne (2x) behaved cytologically as autotriploids but the segregation of isozyme variants did not always agree with the expected trisomic ratios. The overall effect of these deviations from expectation was a greater proportion than expected of diploid progeny from the cross L. multiflorum (2x) x triploid hybrid which did not include any of the L. perenne alleles at the three marker isozyme loci. The possible mechanisms for these aberrant segregation ratios are discussed together with the advantages of the crossing scheme to accelerate the recovery of the genotype of the recurrent parent in a backcrossing programme to transfer characters from one species to another.     

Experimental evidence for an alkali ecotype of <Emphasis Type="Italic">Lolium multiflorum</Emphasis>, an exotic invasive annual grass in the Central Valley,CA, USA

One of the characteristics of highly invaded ecosystems is that exotic species are often poor invaders of edaphically severe sites, which become refuges for native flora. To investigate the invasive potential of Lolium multiflorum (Per.) into alkali sites in California, an ex-situ reciprocal transfer experiment was carried out using seeds from populations of L. multiflorum taken from three sites differing in alkalinity (and inundation), including alkali sink soils (pH 8.5) and sink matrix soils (pH 7.4) located within meters of each other, and non-sink soils (pH 5.0) located several km away. Survivorship, plant height, leaf number and seed production were assessed. In addition, a native composite, Hemizonia pungens (Hick.), commonly found on alkali sinks was also sampled at the sink and sink matrix microsites. Lolium multiflorum plants grown from alkali sink and sink matrix seeds produced fewer leaves and seeds but were taller than plants grown from non-alkali seeds, the latter perhaps an adaptation to frequently inundated soils. Non-alkali genotypes fared poorly in sink soils for all traits, both in comparison to their growth on non-sink soils, and in comparison to the sink and sink edge genotypes. This suggests the existence of L. multiflorum ecotypes adapted to inundated alkali sinks, a genotypic difference that occurs on a broad spatial scale (kilometers), but not so obviously on the micro-site scale (meters) between sink and sink matrix populations. These data suggest that the absence of exotic invasives from alkali sites may be temporary if they are evolving tolerance for these severe sites, and this may threaten the future of the native alkali specialists that currently find refuge in these sites.     

Characterization of a cDNA encoding the protein moiety of a putative arabinogalactan protein from Lycopersicon esculentum

The nucleotide sequence of a cDNA prepared from poly(A)⁺ RNA from Lycopersicon esculentum fruit codes for a protein, M _r 20812, with features representative of the protein core of arabinogalactan proteins. The deduced amino acid sequence resembles that of peptides of arabinogalactan proteins isolated from carrot and rose and is most similar to the sequence of tryptic peptides from Lolium multiflorum (Gleeson et al., Biochem J 264 (1989) 857–862). The similar sequences include a number of Ala-Pro repeats, a feature considered distinctive of arabinogalactan proteins. The amino acid composition is similar to that of the peptide core of the Lolium multiflorum arabinogalactan protein; alanine, serine and proline account for 57% of the polypeptide. The mRNA corresponding to the cDNA sequence was detected in roots, leaves and fruit. The levels of mRNA are reduced in older leaves, in fruit that have commenced ripening and in leaves and fruit that have been wounded.     

Distribution of nutrients and phytotoxic metal ions in the soil and in two forest floor plant species of a beech (Fagus sylvatica L.) stand

In a beech (Fagus sylvatica L.) stand in north-west Germany vegetation of two transects (25m:1m and 20m:1m) was mapped and contents of macronutrients (Ca, Mg and K), micronutrients (Fe, Mn, Zn and Cu), and potentially phytotoxic metals (Pb, Cd, Ni and Al) were measured in different soil compartments and in roots, rhizomes, stems and leaves of two forest floor plant species (Mercurialis perennis L. and Polygonatum multiflorum L.). NH₄Cl extractable cation contents, pH and other soil variables were also determined.The highest macronutrient contents could be found in the leaves of M. perennis and P. multiflorum. Heavy metals and Al accumulated in the roots. Correlation analysis suggests a considerable translocation of Zn and Cd between below- and above-ground organs of both investigated forest floor plants. No significant correlation was found between the contents of the other elements in the below- and above-ground parts.Available data indicate a considerable uptake by the plants not only of nutrients, but also of heavy metals from the upper mineral soil. Amounts of heavy metals and Al solubilized in the presence of NH₄Cl increased with decreasing pH, whereas levels of soluble Ca and Mg were maximal at high pH-values of the extracts. It can be concluded that element uptake in the investigated plants is indirectly controlled by the pH of the upper mineral soil.     

Genetic segregation in relation to chromosome pairing in tetraploid hybrids between Lolium perenne and L. multiflorum

Summary Segregation at one of the loci controlling tiller-base pigmentation was studied to determine the mode of inheritance in tetraploid hybrids between Lolium perenne and L. multiflorum. The results could be explained by tetrasomic inheritance and thus did not support previous reports of a degree of preferential chromosome pairing in this material. However, double reduction and aneuploidy may to some extent have masked any tendency to disomic segregation brought about by preferential pairing. Moreover, there was significant heterogeneity between families in the segregation ratios which may indicate genetically controlled differences in pairing behaviour. The results are related to previous cytological and genetic studies.