Molecular authentication of Panax ginseng species by RAPD analysis and PCR-RFLP

In order to develop convenient and reproducible methods for the identification of ginseng drugs at a DNA level, randomly amplified polymorphic DNA (RAPD) and PCR-restriction fragment length polymorphism (PCR-RFLP) analyses were applied within Panax species. To authenticate Panax ginseng among ginseng populations, RAPD analysis was carried out using a 20 mer-random primer. The similarity coefficients among the DNA of ginseng plants analyzed were low, ranging from 0.197 to 0.491. In addition, by using PCR-RFLP analysis, very different fingerprints were obtained within Korean ginseng plants. These results suggest that these methods are able to authenticate the concerned Panax species. Broader application of this approach to authenticate other morphologically similar medicinal materials is rationalized.     

Identification of Panax species in the herbal medicine preparations using gradient PCR method

In order to identify the existence of Panax species in herbal medicine preparations, the Ginseng specific marker primer was selected and created based on the sequence of Korean ginseng DNA fragment, 359 bp. The gradient PCR was performed on 40 types of the herbal medicines including the 7 types of Araliaceae that are in the same family with the Panax ginseng using the created Ginseng maker primer. As result, Panax notoginseng (Chinese), Panax japonicus (Japanese) and Panax quinquefolius (American), along with Panax ginseng (Korean) were the only ones amplified. However, in the case of Atractylodes lancea, one of the herbal medicines not categorized as Panax species, the DNA was prominently amplified by the Ginseng marker primer. The sequence of the amplified DNA of Atractylodes lancea was identified, resulting in enabling the differentiation from the Panax species by the Restriction Fragment Length Polymorphisms (RFLP) method. In addition, the results of the gradient PCR performed on the herbal medicine preparations that consists of Panax ginseng showed that 290 bp size of the original DNA fragments of Panax ginseng was amplified on the herbal medicine preparations containing Panax ginseng. Therefore, these results suggest a possibility of creating a new testing method for identifying specific herb medicines using the gradient PCR, a molecular biological method not only on Panax ginseng, but also on other herbal medicines and herbal medicine preparations.     

Authentication of Panax notoginseng by 5S-rRNA spacer domain and random amplified polymorphic DNA (RAPD) analysis

The great majority of Panax species are well-known herbal medicines in the Orient, and many of them share a close resemblance in appearance and chemical composition. Among these Panax species, the root of P. notoginseng (Sanqi) is a unique herb that has distinct clinical usage. Here, the 5S-rRNA spacer domains were isolated from P. notoginseng, P. japonicus var. major, P. stipuleanatus, P. quinquefolius, P. ginseng, P. zingiberensis, and P. wangianus, and four common adulterants of P. notoginseng including Curcuma wenyujin, Curcuma longa, Bletilla striata and Gynura segetum. The spacer domains were sequenced and compared, which showed over 75 % DNA identity among all Panax species, but not for the adulterants. In addition, random amplification of polymorphic DNA (RAPD) analysis was used to distinguish different members of Panax genus as well as the morphological variants of P. notoginseng. These molecular methods could be used in the authentic identification of P. notoginseng from other Panax species.     

A random amplified polymorphic DNA (RAPD) primer to assist the identification of a selected strain, aizu K-111 of Panax ginseng and the sequence amplified

Panax ginseng is widely used as a Chinese medicine, but it takes a long time to reach harvest and to establish its qualified strains. In the course of searching high quality Panax ginseng, we found a useful random amplified polymorphic DNA (RAPD) primer, which showed a 725 base pair band for a selected elite strain Aizu K-111 (now called Kaishusan) including its cultured tissues, while the other strains did not necessarily show this band. We sequenced the DNA fragment amplified and designed primers to improve electrophoretic profiles, based on the sequence.     

Using RAPD in Neisseria gonorrhoeae genotyping and transmission detection

The aim of this paper is to develop an applicable Random Amplified Polymorphic DNAs (RAPD) method for genotyping Neisseria gonorrhoeae strain, and discuss the possibility of using the RAPD method to trace N. gonorrhoeae strain transmission route. Four different pretreatment methods were used on the N. gonorrhoeae genomic DNA component, and the best adaptive extract method was selected for RAPD. Different RAPD primers sequence was used for amplification and their differentiating capabilities for N. gonorrhoeae strains were compared. Applicable RAPD primer was selected for N. gonorrhoeae genotyping and then applied into transmission detection. The results show that the so called cetyl-trimethylammonium bromide (CTAB) method for extracting genomic DNA could give integrated genomic DNA and give out relatively better RAPD fingerprint maps, subsequently, using selected RAPD primer could give out a group of amplification polymerase chain reaction bands. The fingerprint maps from different N. gonorrhoeae strains were distinctive. Some main segments were common to all the N. gonorrhoeae strains tested. Some segments were different among the N. gonorrhoeae strains. According to the fingerprint maps and similarity index of different N. gonorrhoeae isolates, isolates from a pair of sex-partners were very similar. Based on these findings, the best extracting method and suitable RAPD primer were chosen. The RAPD fingerprint maps could type N. gonorrhoeae effectively and could be used as an additional approach in molecular epidemiology for tracing infection sources.     

中药材分子鉴别新方法:锚定引物扩增多态性DNA的研究

为了寻找稳定性好、可操作性强的分子鉴定新方法，在充分吸取RAPD优势的基础上，对其引物和退火温度进行了改进。本文以人参、西洋参为例进行了方法的探索和各种验证，并推广应用到天花粉以及白芷类药材的鉴别。结果显示引物Pg-q36F得到人参、西洋参及其9种伪品的多态性条带。对于人参、西洋参的鉴别结果与文献鉴别方法结果一致，并且具有更高的稳定性。引物TkS1-64F得到了天花粉及其11种伪品的多态性条带，引物AfS1-100F得到白芷及其3种伪品的多态性条带，均能准确鉴别各种药材。实验结果证明本方法具有简单易行、稳定性和重复性好、提供的信息量大等优点，是一种极具前途的中药材分子鉴定新方法，被命名为锚定引物扩增多态性DNA(anchored primer amplification polymorphism DNA，APAPD)。     

野生人参RAPD指纹的研究

目的：分析山参遗传多样性及其遗传特性。方法：用随机扩增多态DNA(RAPD)标记方法对7个来源地不同的山参和1个园参样品进行遗传多样性检测和遗传分析。结果和结论：用14个10-mer寡聚核苷酸引物共检测111个位点,其中多态位点76个,占67.6%,远大于园参内的遗传变异,因此山参在人参育种上有很大利用价值。聚类分析表明,山参之间及其与园参之间的遗传变异,没有超出与近缘种西洋参之间的遗传差异;遗传因素在人参形态变异上的作用小于环境因素,这一结果为“山参”的培育提供了理论依据。     

Utilization of RAPD markers to assess genetic diversity of wild populations of North American ginseng (Panax quinquefolium)

The Catskill Mountains of New York State are an important source of wild-collected American ginseng (Panax quinquefolium) and, increasingly, of woods-cultivated ginseng. The objective of this study was to assess genetic diversity among 9 different wild ginseng populations in and adjacent to the Catskill Mountain region of New York State and to compare these to wild populations from other states including Kentucky, Tennessee, North Carolina, Pennsylvania, and Virginia, and one cultivated population from Wisconsin. Randomly amplified polymorphic DNA (RAPD) markers were used to estimate the genetic distance among samples from the 15 populations. Pooled DNA from 10 plants of each of 8 New York populations was initially screened with 64 random primers; subsequently, the 15 primers that exhibited the greatest number of reproducible polymorphic markers were selected for further experimentation. Gel electrophoresis with the selected 15 primers produced 124 highly reproducible polymorphic bands. The ratio of discordant bands to total bands scored was used to estimate the genetic distance within and among populations. Multidimensional scaling (MDS) of the relation matrix showed distinctly separate clusters between New York and non-New York populations, indicating separation between these two groupings. The MDS analysis was confirmed using pooled chi-square tests for fragment homogeneity. This study shows that RAPD markers can be used as population-specific markers for Panax quinquefolium, and may eventually be utilized as markers for ginsenoside assessment.     

AFLP法构建人参、西洋参基因组DNA指纹图谱

目的　采用扩增片段长度多态性DNA(AFLP)分子遗传标志技术,分析人参、西洋参基因组DNA多态性。方法　人参、西洋参干燥根基因组DNA,经EcoRI/MseI酶切并与其相应的人工接头连接后,使用选择性引物进行PCR扩增。结果　经变性聚丙烯酰胺凝胶电泳检测,成功构建出多态性丰富和重复性好的人参、西洋参DNA指纹图谱。结论　AFLP法有望成为一种独立的切实可行的手段,将在人参、西洋参等药用植物的鉴定、生物进化、系统发育研究及指导道地性药材的科学栽培等方面发挥重要作用。     

Molecular authentication of Panax ginseng and ginseng products using robust SNP markers in ribosomal external transcribed spacer region

Panax ginseng and Panax quinquefolius are the most widely used Panax species, but they are known to have different properties and medicinal values. The aim of this study is to develop a robust and accurate DNA marker for identifying P. ginseng and the origins of ginseng products. Two single nucleotide polymorphism (SNP) sites specific to P. ginseng were exploited from nuclear ribosomal external transcribed spacer (ETS) region. Based on the SNP sites, two specific primers were designed for P. ginseng and P. quinquefolius respectively. P. ginseng can be easily discriminated from P. quinquefolius by amplifying the two specific alleles using multiplex allele-specific PCR. Favorable results can also be obtained from commercial ginseng products. The established method is highly sensitive and can detect 1% of intentional adulteration of P. quinquefolius into P. ginseng down to the 0.1ng level of total DNA. Therefore this study provides a reliable and simple DNA method for authentication of the origins and purities of ginseng products.     

人参和其他中草药的遗传学鉴定(英文)

The main objective of this paper is to review the chemical and genetic methods used in authentication of ginseng, especially the recent advances in microsatellite genotyping and its application to the authentication of other traditional Chinese medicines (TCM). The standardization and modernization of TCM hinge on the authentication of their botanical identities. Analysis of well-characterized marker compounds is now the most popular method for identifying the herbal materials and quality control of TCM, eg, ginsenoside profiling for authentication of Panax species. However, in many herbal species the chemical composition of the plant changes with the external environment and processing conditions, which lowers the reliability of these authentication methods. In the light of the advances in molecular biotechnology in the past few decades, genetic tools are now considered to provide more standardized and reliable methods for authentication of herbal materials at the DNA level. These genetic tools include     

Application of sequence characterized amplified region (SCAR) analysis to authenticate Panax species and their adulterants.

A 420-bp RAPD fragment from Panax quinquefolius was converted to a sequence characterized amplified region (SCAR) marker. The main difference between the SCAR of P. quinquefolius and its homolog in P. ginseng is the presence of a 25 bp insertion in the latter. Primers derived from this sequence were successfully used to authenticate six Panax species and two common adulterants.     

Development of species specific AFLP-derived SCAR marker for authentication of Panax japonicus C. A. MEYER

Panax japonicus is an important medicinal plant. The aim of this study was to develop species-specific molecular markers for P. japonicus. Amplified fragment length polymorphism (AFLP) was compared among P. japonicus, P. ginseng and P. quinquefolius. A clear species-specific AFLP marker for P. japonicus was generated. After isolation and sequencing of the AFLP fragment, a DNA sequence (293 bp) was obtained and named JG14. Oligonucleotide primer (23 mer) was designed for amplifying 191 bp of the sequence of JG14. PCR analysis revealed a clear amplified band for P. japonicus but not in 3 other Panax species (P. ginseng, P. quinquefolius and P. notoginseng). This sequence characterized amplified regions (SCAR) marker will be used for rapid authentication of P. japonicus among other related Panax species. This is the first report of species-specific SCAR marker development in P. japonicus.     

Stimulation of nitric oxide synthesis by the aqueous extract of Panax ginseng root in RAW 264.7 cells.

1. In this study, we investigated the effect of Panax ginseng root aqueous extracts upon inducible nitric oxide synthesis in RAW 264.7 cells. Panax ginseng root extract has been used in the Asian world for centuries as a traditional herb to enhance physical strength and resistance and is becoming more and more popular in Europe and North America. 2. Incubation of murine macrophages (RAW 264.7 cells) with increasing amounts of aqueous extracts of Panax ginseng (0.05 - 0.8 microg microl(-1)) showed a dose dependent stimulation of inducible nitric oxide synthesis. 3. Polysaccharides isolated from Panax ginseng showed strong stimulation of inducible nitric oxide synthesis, whereas a triterpene-enriched fraction from an aqueous extract of Panax ginseng did not show any stimulation. 4. Inducible nitric oxide synthase protein expression was enhanced in a dose dependent manner as revealed by immunoblotting when cells were incubated with increasing amounts of Panax ginseng extract. This was associated with an incline in inducible nitric oxide synthase mRNA-levels as determined by semiquantitative polymerase chain reaction and electromobility shift assay studies indicated enhanced nuclear factor-kappaB DNA binding activity. 5. As nitric oxide plays an important role in immune function, Panax ginseng treatment could modulate several aspects of host defense mechanisms due to stimulation of the inducible nitric oxide synthase.     

Phytochemical and analytical studies of Panax notoginseng (Burk.) F.H. Chen

Panax notoginseng (Burk.) F.H. Chen is distributed throughout the southwest of China, Burma and Nepal. The root of this plant, called notoginseng or sanchi, has a long history of use as a remedy in Oriental traditional medicine. Modern studies have found that extracts and compounds from notoginseng exert various physiological effects. The active constituents are mainly recognized as saponins. In this review, we summarized the discovery and analysis of chemical constituents in notoginseng. Fifty-six saponins from notoginseng were isolated and elucidated. All of them are dammarane saponins, 35 of which can be classified as belonging to the protopanaxadiols group, and 21 as belonging to the protopanaxatriols group. Evidence from phytochemical studies on notoginseng demonstrated that no oleanane-type saponin, which exists in Asian ginseng (Panax ginseng) and American ginseng (Panax quinquefolius), was found. Other types of compounds such as non-protein amino acids, polyacetylenes, phytosterols, flavonoids, and polysaccharides, many of which have pharmacological activities, were also isolated from notoginseng. Analytical studies on notoginseng were carried out based on botanical and phytochemical advances. In the qualitative studies, identification of the herbal materials and extracts was the main objective. The utilization of high-performance liquid chromatography (HPLC) fingerprint and molecular biological methods made the identification accurate and efficient. Spectral, chromatographic and immunoassay methods were used for the quantitative analysis. HPLC methods are the main authority regarding the determination of saponins and other types of constituents. The chromatographic conditions and detectors employed in the HPLC are discussed.     

不同产地白及遗传多态性的RAPD分析

目的 研究国内不同产地白及的遗传多样性和亲缘关系。方法 利用随机扩增多态DNA （random amplified polymorphic DNA,RAPD）引物,RAPD分子标记技术分析国内50个不同产地的白及。结果 RAPD技术扩增产物经琼脂凝胶电泳检测,从50条引物中筛选出10条（S8,S9,S14,S19,S23,S25,S28,S29,S30,S31）条带清晰的引物,10条引物共扩增出88条DNA条带,其中多态性的DNA条带数目为81条,占总数的92.05%。每个引物能扩增出5~11条DNA条带,平均可扩增出8.8条;扩增最少的引物为S19,扩增出5条DNA条带;扩增最多的引物为S29,扩增出11条DNA条带。而每个引物能扩增的多态性DNA条带数为3~11条,平均可扩增出8.6条。结论 不同产地的白及具有较丰富的遗传多样性,RAPD可有效应用于白及的遗传多样性研究。     

Synergistic immunostimulating activity of pidotimod and red ginseng acidic polysaccharide against cyclophosphamide-induced immunosuppression

We investigated the synergistic effect of combined treatment with red ginseng acidic polysaccharide (RGAP) from Panax ginseng C.A. Meyer and pidotimod in cyclophosphamide-treated mice. The combination of pidotimod and RGAP restored concanavalin A-induced splenic T cell proliferation and LPS-stimulated B cell proliferation significantly. The production of nitric oxide from peritoneal macrophages was increased by the combinations. NK cell activity was increased by RGAP alone or in combination with pidotimod. A synergistic increase in the level of serum IL-12 and interferongamm was observed when the combination of the two was used. RGAP alone or in combination with pidotimod modulated the level of serum C-reactive protein to a near-normal level. These results indicate that combinations of pidotimod and RGAP are synergistic and suggest that combination therapy using pidotimod and RGAP for improving immune activity may provide an additional benefit over the use of the two drugs by themselves.     

Protective role of Panax ginseng extract standardized with ginsenoside Rg3 against acrylamide-induced neurotoxicity in rats

Acrylamide (ACR) is an industrial neurotoxic chemical that has been recently found in carbohydrate-rich foods cooked at high temperatures. ACR was designated as a probable human carcinogen by IARC (1994) and USEPA (1988). Panax ginseng extract has efficacies such as anticancer, antihypertension, antidiabetes and antinociception. The objective of the current study is to evaluate the protective effects of Panax ginseng extract against ACR-induced toxicity in rats. Sixty adult Sprague Dawley female rats were divided into six groups included a control group, a group treated orally with ACR (50 mg kg(-1) body weight; b.w.) for 11 days, a group treated orally with Panax ginseng extract (20 mg kg(-1) b.w.) for 11 days and groups treated orally with Panax ginseng for 11 days before, during or after 11 days of ACR treatment. The results indicated that treatment with ACR alone resulted in a significant increase in lipid peroxidation level and LDH activity in brain homogenate as well as in serum CK activity, whereas it caused a significant decrease in SOD activity and a small but statistically insignificant decrease in Na(+)K(+)-ATPase activity in brain homogenate. Serum serotonin, corticosterone, T3, T4, TSH, estradiol, progesterone and plasma adrenaline were significantly decreased in ACR-treated rats. Treatment with Panax ginseng before, during or after ACR treatment reduced or partially antagonized the effects induced by ACR towards the normal values of controls. It could be concluded that Panax ginseng extract exhibited a protective action against ACR toxicity and it is worth noting that treatment with Panax ginseng extract before or at the same time as ACR treatment was more effective than when administered after ACR treatment.     

Species identification from Ginseng drugs by multiplex amplification refractory mutation system (MARMS)

The multiplex amplification refractory mutation system (MARMS) was applied to the identification of 5 Panax species ( P. ginseng, P. japonicus, P. quinquefolius, P. notoginseng and P. vietnamensis). A set of specific primers, including 2-pair primers on chloroplast trnK gene and nuclear 18S rRNA gene regions, respectively, was designed and synthesized for each species on the basis of species-specific sequences of the 2 genes. By using 5 sets of specific primers, in turn, PCR amplifications were performed with total DNA extracted from 5 Panax species as template under appropriate condition, and each resulting product was detected by agarose gel electrophoresis. The results showed that two expected fragments, one from trnK gene and another from 18S rRNA gene regions, were observed simultaneously only when the set of species-specific primers encountered template DNA of the corresponding species. This assay could give more reliable results for identification of not only 5 Panax species but also corresponding Ginseng drugs by simultaneous detection of 4-site nucleotide differences on 2 completely different genes.     

用火焰原子吸收光谱法测定人参与人参果中的无机元素

目的：测定人参和人参果中9种无机元素的含量。方法：将人参和人参果粉碎后置于锥形瓶中,分别在电热板上经硝酸-高氯酸（4∶1）消解体系小火加热消解后,采用空气-乙炔火焰原子吸收光谱法测定。结果：人参和人参果样品中Zn、Cu、Mn、Fe、Ca、Mg、Cd、K、Na 9种无机元素含量测定结果的相对标准偏差为0.53%-3.4%,加样回收率分别为90.59%-110.00%和90.00%-114.29%。结论：该方法简单、快速,可用于人参和人参果中9种无机元素含量的测定,为其临床疗效研究提供了实验依据。