Identification of bulb from Fritillaria cirrhosa by PCR with specific primers

Bulb of Fritillaria cirrhosa is an important traditional Chinese herbal medicine. According to the Chinese Pharmacopoeia (1995), it is commonly used as an antitussive and expectorant. Many young bulbs from species of Fritillaria are similar to those of F. cirrhosa, but they are different in price and quality. Therefore, there are many young bulbs from species of Fritillaria that could fake those of F. cirrhosa on the commercial market. The coding region of 5S-rRNA is highly conserved in higher eukaryotes. The 5S-rRNA spacer region sequences of F. thunbergii, F. pallidiflora, F. ussuriensi, F. delavayi, F.cirrhosa, F. anhuiensis, F. puqiensis were cloned by PCR with a pair of primers located within the conserved coding region. Based on sequences analyses of the 5S-rRNA spacer region from the 7 species, a specific sequence was found in F. cirrhosa. A pair of specific primers was designed for differentiating the bulbs of F. cirrhosa from each other by PCR.This result indicated that the method is rapid, more accurate and applicable in identification of the bulbs of F. cirrhosa at the DNA level.     

伊犁贝母的生药学鉴定

目的：研究伊犁贝母的生药学鉴别特征。方法：利用性状、显微、薄层色谱与紫外光谱鉴别法为该药材进行初步鉴定研究,并确立其鉴别特征。结果：确定了伊犁贝母的性状、显微、薄层色谱与紫外光谱鉴别特征。结论：本研究可为伊犁贝母的鉴别、开发利用与质量控制提供依据。     

Molecular authentication of Alisma orientale by PCR-RFLP and ARMS

As a widely used medicinal plant, Alisma orientale is always a possible target for fraudulent labeling. The internal transcribed spacer (ITS) regions of nuclear ribosomal DNA (nrDNA) of the six species of genus Alisma were sequenced, and two variant sites were found to be specific for A. orientale. Polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analysis and amplification refractory mutation system (ARMS) analysis were applied to the ITS region for the identification of A. orientale. A restriction site for PSTI useful for PCR-RFLP analysis was detected and a pair of diagnostic primers DFZX-JB02S and DFZX-JB02X were designed for ARMS.     

Molecular diversity of 5S-rRNA spacer domain in Fritillaria species revealed by PCR analysis.

Beimu (bulbs of Fritillaria) is an important traditional Chinese herbal medicine commonly used as an antitussive and expectorant. There are about 25 species and varieties of Fritillaria that carry the name Beimu on commercial markets. The price for each Beimu may differ by more than 100-fold. However, the identification of the origin of a particular species on the market is difficult. Here, a molecular method was used to identify various species of Fritillaria regardless of their geographical origin. The 5S-rRNA coding sequence is highly conserved in higher eukaryotes, but the spacer sequence of the 5S-rRNA gene is variable among different species. Total genomic DNA from fresh leaves and bulbs of Fritillaria cirrhosa, F. puqiensis, F. anhuiensis, and F. thunbergii was extracted. The 5S-rRNA spacer region of the extracted DNAs was amplified by PCR with a pair of primers located within the conserved coding region. The isolated cDNA clones (approximately 600 bp) covering the 5S-rRNA spacer domain were sequenced. By aligning the isolated nucleotide sequences of the four Fritillaria species, sequence diversity was found in the spacer region. Furthermore, a unique EcoR I site was used for the rapid identification of different species of Fritillaria. To our knowledge, this is the first report on the detection of 5S-rRNA spacer domain sequences of Fritillaria and their use to identify species.     

药用植物束花石斛、流苏石斛及其形态相似种的PCR-RFLP鉴别研究药用植物束花石斛、流苏石斛及其形态相似种的PCR-RFLP鉴别研究

目的建立简易的采用rDNA ITS区作为分子标记对中药石斛的基源植物进行分子鉴定的技术。方法 用聚合酶链反应—限制性片段长度多态性(PCR-RFLP)方法获得ITS片段的限制性图谱。束花石斛及其形态相似种的PCR扩增产物用Cla I和Apa LI酶切,流苏石斛及其形态相似种的PCR扩增产物用Sph I酶切。结果根据预测的PCR-RFLP图谱,可以鉴别药用植物束花石斛、流苏石斛及它们的形态相似种。用这种方法鉴定了市场上收集的25件商品束花石斛、流苏石斛新鲜药材的原植物。结论rDNA ITS的PCR-RFLP可用于鉴定石斛药材的原植物,具有简便、费用低的优点。     

伊贝碱甙B的分离和结构测定

本文报道自吉林栽培的伊贝母(Fritillaria pallidiflora Schrenk)中分得一种新的C-去甲-D-异甾体生物碱甙,命名为伊贝碱甙B(yibeinoside B)。根据红外、质谱、氢谱和碳谱确定了结构。     

Identification of medicinal Atractylodes based on ITS sequences of nrDNA

Dried rhizomes of five species of Atractylodes (A. japonica, A. macrocephala, A. lancea, A. chinensis, and A. koreana), Compositae, have been used as crude drugs mainly for the treatment of stomach disorders and for their diuretic properties in Chinese and Japanese traditional medicines. The identification of the botanical origins of these crude drugs is generally difficult from their morphological and chemical features only. In this study, for identification with more reliable, nuclear ribosomal DNA (nrDNA), internal transcribed spacer (ITS) regions of five species of medicinal Atractylodes were sequenced. As a result, specific ITS genotypes were recognized by each species. The four species (A. japonica, A. macrocephala, A. lancea, and A. chinensis) prescribed in Chinese and Japanese Pharmacopoeias as botanical origins of crude Atractylodes drugs could be distinguished by their ITS sequences because they had difference genotypes on the ITS sequences. However, the genotype of A. koreana was the same as that of A. chinensis. Additionally, hybrids between A. lancea and A. chinensis were also recognized as nucleotide additives on their ITS sequences. In this study, several morphological characteristics were researched by their genotype, too. As this result, the hybrids recognized from the genetic analysis had intermediate morphological characteristics between A. lancea and A. chinensis. It was also recognized that A. lancea and A. chinensis except for their hybrids were significant differences. It is therefore suggested that ITS sequences of nrDNA would be useful for the identification of the crude drugs derived from Atractylodes species and their interspecific hybridizations.     

Molecular authentication of the traditional Tibetan medicinal plant Swertia mussotii

Swertia mussotii is an important species in Tibetan folk medicine. However, it is quite expensive and frequently adulterated, so reliable methods for authentication of putative specimens and preparations of the species are needed to protect consumers and to support conservation measures. We show here that the chloroplast (cp) DNA RPL16 intron has limited utility for differentiating S. mussotii from closely related species, since the cpDNA RPL16 sequences are identical in S. mussotii and two other species of Swertia. However, the rDNA internal transcribed spacer (ITS) sequences differ significantly between S. mussotii and all of 13 tested potential adulterants. Thus, the ITS region provides a robust molecular marker for differentiating the medicinal S. mussotii from related adulterants. Therefore, a pair of allele-specific diagnostic primers based on the divergent ITS region was designed to distinguish S. mussotii from the other species. Authentication by allele-specific diagnostic PCR using these primers is convenient, effective and both simpler and less time-consuming than sequencing the ITS region.     

Characters of nrDNA ITS region sequences of fruits of Alpinia galanga and their adulterants.

The fruits of Alpinia galanga (L.) Sw. are used as a traditional Chinese medicine; but the dry fruits of A. conchigera, A. suishaensis, A. maclurei and A. polyantha are also used as the medicine in local areas. Because dry fruits of these related plants are similar to those of Alpinia galanga (L.) Sw. in odor, morphological characters and chemical components, and even anatomical characters, it is difficult to identify the medicine. Nuclear ribosomal DNA internal transcribed spacer (ITS) regions of the five taxa were directly sequenced using an automated sequencer. Sequence analysis showed that the ITS 1 ranges from 177 to 178 base pairs (bp), and the ITS 2 from 225 to 234 bp. The size of the 5.8S coding region is 164 bp for all species. Also, the pairwise sequence divergence is higher and some molecular markers were determined. According to these molecular markers, Alpinia galanga (L.) Sw. and the related species can easily be distinguished from each other. Therefore, evidence from nrDNA ITS sequence variation can identify the medicine at the DNA level.     

Novel polymorphism of internal transcribed spacers (ITS) and their utilization in phylogenetic analysis of Neanthes glandicincta (Annelida: Polychaeta: Nereididae)

Sequences of internal transcribed spacers (ITS1 and ITS2) are increasingly being used to infer phylogenetic relationships at or below species levels. Here we report a novel case of ITS polymorphism within Neanthes glandicincta (Annelida: Polychaeta: Nereididae). Two types of ITS sequence (Type I and Type II) were cloned and sequenced, which showed significant differences both in nucleotide composition and length. Variations of these two types sequences also differed from each other with Type I was highly divergent while Type II was highly conserved. Phylogenetic trees inferred from ITS1 and ITS2 sequences showed striking discrepancy in N. glandicincta. Non-concerted evolution of multi-gene is suggested to be responsible for the high degree of polymorphism in ITS regions. Due to the two divergent types of ITS presented within a single N. glandicincta individual, the utilization of ITS regions for delineation of population or closely related species cannot be substantiated. The finding of different types of ITS in a single individual also stresses the need for analyzing a large number of clones whenever ITS sequences obtained by PCR amplification and cloning are being used in phylogenetic reconstruction.     

伊贝碱甙A的分离和鉴定

伊贝母Fritillaria pallidiflora Schreb系百合科贝母属伊贝母的干燥鳞茎,是药用贝母之一,已列入中华人民共和国1985年版药典。主产于新疆,具有产量高、生物碱含量高和抗病虫害能力强等优点。中国农科院左家特产研究所于1985年在吉林省引种栽培伊贝母获得成功。为阐明吉林省栽培的伊贝母的有效成分,以便扩大栽培和应用,我们研究了伊贝母的生物碱成分,曾分离到西贝素、西贝素-3β-D-葡萄糖甙和贝母辛。在继续研究中,又     

以ITS序列和花粉粒形态区分益母草和细叶益母草

作者测定了两种形态十分近似的生药益母草与细叶益母草的核核糖体DNA内转录间隔区(ITS)序列,并用扫描电镜观察了两者的花粉粒形态,结合二者可准确区分益母草与细叶益母草。     

DNA芯片技术用于贝母的基因分型和种类鉴别

目的通过对贝母几个种遗传多态性的研究来开发在分子水平上用于鉴别贝母基因型及不同种类的DNA芯片技术。方法用PCR扩增法和DNA直接测序法确定核苷酸多态性,用DNA芯片进行基因检测。结果首先提取来自多种贝母根茎的基因组DNA,对26S rDNA基因D2与D3区的多态性片段进行扩增和测序,然后将不同种属多态性片段的特异性寡核苷探针点置于经多聚赖氨酸处理包被的芯片。用来自不同种贝母的荧光素标记的PCR产物与DNA芯片进行杂交,可在芯片特定位置检测到不同种贝母的荧光信号。结论本研究显示DNA芯片技术可为植物种属的验证与质量控制提供一种快速、高通量的检测工具。     

Differentiation of medicinal Codonopsis species from adulterants by polymerase chain reaction-restriction fragment length polymorphism

DNA sequence analysis of rDNA internal transcribed spacer (ITS) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) were exploited for their applications in differentiating medicinal species Codonopsis pilosula, C. tangshen, C. modesta, and C. nervosa var. macrantha, from two related adulterants Campanumoea javania and Platycodon grandiflorus. The data demonstrated that the rDNA ITSI and ITSII sequences of the four Codonopsis are highly homologous but not identical, and are significantly different from those of the two adulterants. The sequence difference allows effective and reliable differentiation of Codonopsis from the adulterants by PCR-RFLP.     

Molecular analysis of the genus Mitragyna existing in Thailand based on rDNA ITS sequences and its application to identify a narcotic species: Mitragyna speciosa

In Thailand, there are four Mitragyna species; M. speciosa, M. hirsuta, M. diversifolia, and M. rotundifolia. One, M. speciosa, is a narcotic plant and has medicinal importance for its opium-like effect. Since the use of M. speciosa has been forbidden in Thailand, the leaves of M. diversifolia or others are frequently used as substitutes but are not considered as effective. Therefore, accurate authentication of M. speciosa is essential for both medicinal and forensic purposes. The nucleotide sequences of internal transcribed spacers (ITS) and the 5.8S coding region of nuclear ribosomal DNA (rDNA) of the Mitragyna species were analyzed. The whole length of ITS1-5.8S-ITS2 region was 608 bp in M. speciosa, 607 bp in the other species. Nineteen sites of nucleotide substitutions and 3 sites of 1-bp indels were observed, and M. speciosa showed specific sequence differed from the others. Based on the ITS sequences, a distinctive site recognized by a restriction enzyme XmaI in M. speciosa was found and then PCR-restriction fragment length polymorphism (RFLP) analysis was established to differentiate M. speciosa from the others. By the method, a 409-bp PCR fragment of ITS1-5.8S (partial) rDNA region from M. speciosa was cleaved into two fragments of 119 bp and 290 bp while the other species remained undigested. This method provides an effective and accurate identification of M. speciosa.     

Discrimination of Fritillary according to geographical origin with Fourier transform infrared spectroscopy and two-dimensional correlation IR spectroscopy

Fritillaria is a traditional Chinese herbal medicine for eliminating phlegm and relieving a cough with a long history in China and some other Asian countries. The objective of this study is to develop a nondestructive and accurate method to discriminate Fritillaria of different geographical origins, which is a troublesome work by existing analytical methods. We conducted a systematic study on five kinds of Fritillaria by Fourier transform infrared spectroscopy, second derivative infrared spectroscopy, and two-dimensional (2D) correlation infrared spectroscopy under thermal perturbation. Because Fritillaria consist of a large amount of starch, the conventional IR spectra of different Fritillaria only have very limited spectral feature differences. Based on these differences, we can separate different Fritillaria to a limited extent, but this method was deemed not very practical. The second derivative IR spectra of Fritillaria could enhance spectrum resolution, amplify the differences between the IR spectra of different Fritillaria, and provide some dissimilarity in their starch content, when compared with the spectrum of pure starch. Finally, we applied thermal perturbation to Fritillaria and analyzed the resulting spectra by the 2D correlation method to distinguish different Fritillaria easily and clearly. The distinction of very similar Fritillaria was possible because the spectral resolution was greatly enhanced by the 2D correlation spectroscopy. In addition, with the dynamic information of molecular structure provided by 2D correlation IR spectra, we studied the differences in the stability of active components of Fritillaria. The differences embodied mainly on the intensity ratio of the auto-peak at 985 cm(-1) and other auto-peaks. The 2D correlation IR spectroscopy (2D IR) of Fritillaria can be a new and powerful method to discriminate Fritillaria.     

鼠尾草属药用植物及其近缘种的ITS序列分析

采用分子系统学方法分析鼠尾草属药用植物及其近缘种的遗传多样性，为准确进行基源鉴定、阐明本属内的种间关系及发现新的药用资源提供分子证据。本文从野外采集的27个鼠尾草属植物叶片样品中分离提取DNA，PCR扩增ITS区及5.8S rDNA完整序列并测序，采用Mega 3.1软件进行系统学分析。27个鼠尾草样品的ITS及5.8S rDNA区序列全长为612～617 bp，邻接法(neighbor-Joining)构建的系统发生树部分支持了形态学的属下划分，但对部分种的系统位置特别是三叶鼠尾草和黄花鼠尾草两个亚种的处理上与形态学划分存在明显的分歧。序列分析显示5.8S rDNA序列相当保守，而ITS区段则在亚属间差异明显，且原产我国的该属植物与欧美引进种明显具有不同起源。ITS系统树对于亚属和组的处理较为合理，但对组下的划分则表现出了信息量不足，需要其他相关证据的支持。ITS分析支持了丹参组内其他近缘种作为丹参类药材替代资源的合理性，同时也揭示了甘西鼠尾类高山丹参在遗传上与丹参类药材的显著不同。     

PCR-based sensitive detection of medicinal fungi Hericium species from ribosomal internal transcribed spacer (ITS) sequences

Based on phylogenetic analysis of rDNA internal transcribed spacer (ITS) sequences, a pair of specific primers were designed for differentiating the Chinese traditional medicine Hericium species from other mushrooms by PCR. PCR was performed, with total DNAs as a template at an annealing temperature of 52-57 degrees C. Positive amplification was obtained from H. erinaceus with all DNA templates from different resources, but not from other related species. The result indicated that H. erinaceus could be clearly distinguished from other fungi by detection PCR, and no incorrect discrimination was found under the same reaction conditions. The primers were also successfully employed to identify H. erinaceus with different tissue types.     

伊犁贝母中西贝素和西贝素苷的高效液相色谱-蒸发光散射检测法

目的建立伊犁贝母中无紫外吸收的活性异甾体生物碱西贝素和西贝素苷的HPLC测定方法。方法HPLC-蒸发光散射检测器法。结果西贝素苷和西贝素的线性范围分别为48.72-194.88μg·mL^-1和60.72-161.92μg·mL^-1;RSD日内0.7%和0.9%,日间1.0%和1.5%;低、中、高浓度回收率分别为95.8%,96.9%,96.0%和97.1%,96.7%,96.8%;最低检测限分别为1.53和2.43μg·mL^-1。结论本法快速、简便、灵敏和分离度好,适用于伊贝母中活性生物碱的测定。     

暗紫贝母产生物碱内生真菌的筛选及生物碱抑菌活性的测定

目的从暗紫贝母Fritillaria unibracteata Hsiao et K.C.Hsia内生真菌中筛选产川贝母类生物碱及产活性生物碱的菌株。方法利用生物碱沉淀试剂和薄层层析(TLC)技术检测发酵液中的生物碱,采用平板打孔法分别测定生物碱碱粗提液的抑菌活性,结合形态学特征和ITS序列鉴定筛得的真菌。结果筛得5株能产生物碱的内生真菌(A1、A2、A3、A11、A14),其总生物碱对金黄色葡萄球菌均有抑制作用。其中,A1、A3、A14为镰刀属Fusarium sp.真菌,A2、A11属于粘帚霉属Gliocladium sp.真菌。结论筛选出的内生真菌产生的生物碱与暗紫贝母的不尽相同,但其抑菌效果效果显著,值得进一步研究。