Cultivar Variations of Australian‐Grown Danshen (Salvia miltiorrhiza): Bioactive Markers and Root Yields

Danshen (Salvia miltiorrhiza Bunge, Lamiaceae) is a commonly used and highly valued Chinese medicinal herb grown widely in China. In the present work, we studied cultivar variations of Australian‐grown Danshen in order to select optimal cultivars for local herbal production. Root yields of seven cultivars, V1–V7, were monitored in a one‐year field trial, and bioactive markers, including cryptotanshinone, tanshinone I, tanshinone IIA, and salvianolic acid B, were quantitatively determined using a validated RP‐HPLC method. Significant variations were found in root yields, root production efficiencies, and contents of the bioactive marker compounds. Linear correlations were observed among the contents of three tanshinones but not among those of tanshinones and salvianolic acid B. Among the cultivars, V6 was the best cultivar for production of tanshinones, and V4 and V5 were best for production of salvianolic acid B. The findings indicate that it is possible to achieve optimal root yields, and high contents of tanshinones and salvianolic acid B by selecting specific Danshen cultivars.     

Plant density-dependent variations in bioactive markers and root yield in Australian-grown Salvia miltiorrhiza Bunge

The plant density‐dependent variations in the root yield and content, and the yield of biomarkers in Australian grown Salvia miltiorrhiza Bunge , a commonly used Chinese medicinal herb for the treatment of cardiovascular diseases, were investigated in a field trial involving six different plant densities. The key biomarker compounds cryptotanshinone, tanshinone I, tanshinone IIA, and salvianolic acid B were quantified by a validated RP‐HPLC method, and the root yields were determined per plant pair or unit area. There were significant variations (p<0.05) in the root yields and contents and the yields of the biomarkers between the different plant densities. Positive linear correlations were observed between the contents of the three tanshinones, whereas negative linear correlations were revealed between the contents of the tanshinones and salvianolic acid B. The highest root yield per plant pair was achieved when the plants were grown at 45×30 cm or 45×40 cm, whereas the highest root production par unit area was obtained for a plant density of 30×30 cm. The highest contents of the three tanshinones and the most abundant production of these tanshinones per unit area were achieved when the plants were grown at 30×30 cm. However, the highest content of salvianolic acid B was found for a density of 45×40 cm, while its highest yield per unit area was obtained for densities of 30×40 cm or 45×30 cm. The findings suggest that the plant density distinctly affects the root yield and content and the yield of tanshinones and salvianolic acid B in Australian grown S. miltiorrhiza, which may be used as a guide for developing optimal agricultural procedures for cultivating this herb.     

Enhanced antioxidant bioactivity of Salvia miltiorrhiza (Danshen) products prepared using nanotechnology.

The traditional Chinese medicine, Salvia miltiorrhiza (Danshen), promotes blood circulation and relieves blood stasis, also demonstrating good antioxidant activity. In the present study, therefore, the antioxidant activities of medicinal plant materials prepared using nanotechnology or traditional grinding methods were compared using three biological assays. It was found that the nanotechnology preparation had stronger antioxidant bioactivities. Complementary quantitative analysis of four active constituents, salvianolic acid B, cryptotanshinone, tanshinone I and tanshinone IIA, by HPLC revealed only marked differences for salvianolic acid B. The results indicate that the polar active constituent in the nanotechnology samples was released faster compared to the traditionally powdered samples.     

生长调节物质和可溶性糖含量对丹参中丹酚酸类物质积累的影响

采用盆栽实验研究了生长调节物质水杨酸（SA）、茉莉酸甲酯（MeJA）、赤霉素（GA3）对不同生长时期丹参植株中非结构糖含量、碳/氮比及根中丹酚酸类物质积累的影响;并进一步测定培养基中不同浓度蔗糖、葡萄糖、果糖对丹参毛状根中丹酚酸类物质积累的影响,对盆栽实验的结论进行了验证。结果显示,SA处理的丹参幼苗及花后期植株中蔗糖含量有增加趋势,而MeJA处理的丹参幼苗及花后期植株及GA3处理的丹参花后期植株中蔗糖积累均有降低趋势;且SA、MeJA和GA3处理对花后期植株地上和地下部分碳/氮比的影响不同。然而,SA和MeJA处理的丹参幼苗及花后期植株地上部分和根中还原糖含量、GA3处理的花后期植株根中还原糖含量均显著增加;同时,SA和MeJA处理的丹参幼苗根中迷迭香酸含量,以及SA、MeJA、GA3处理的花后期植株根中迷迭香酸含量和丹酚酸类总量显著增加。毛状根培养结果进一步证明,葡萄糖促进毛状根中迷迭香酸的产生,增加丹酚酸类总量,毛状根中迷迭香酸、丹酚酸B的积累及丹酚酸类总量与培养基中蔗糖浓度不相关。可见,丹参（植株）根中丹酚酸类物质的产生和积累受SA、MeJA和GA3的诱导,其与碳/氮比及植株中蔗糖含量没有相关性,推测植株中葡萄糖含量的增加促进根中丹酚酸类物质的积累。     

丹参四倍体优良品系有效成分的含量测定及药理学试验

运用HPLC法测定丹参（Sabuia miltiorrhiza Bunge）同源四倍体优良品系61－2－22及安徽、江苏和陕西产丹参不同部位、不同生长年限的有效成分（3种丹参酮和2种水溶性酚酸）含量。丹参品系61－2－22的有效成分含量明显高于其他产地丹参的含量;根中有效成分含量明显高于茎和叶的含量;丹参酮和丹参素的含量呈明显相关。初步药理实验结果表明,在相同剂量下,优良品系61－2－22具有更好的药理活性。     

白花丹参与丹参活性成分积累比较研究

研究了山东产丹参（Salvia miltiorrhiza Bge．）、白花丹参（Salvia miltiorrhiza Bge．f．alba）不同生育阶段脂溶性与水溶性成分含量的动态变化,对活性成分积累过程进行数学模拟。结果表明：白花丹参脂溶性成分积累有两个高峰期,分别在7月底和10月底,丹参脂溶性成分的积累高峰在7月底;白花丹参和丹参水溶性成分的积累高峰期均出现在6—7月份。两年生白花丹参活性成分高于丹参;两种丹参的迷迭香酸含量均以地上部为高,其他活性成分均以地下部为高;白花丹参与丹参根中丹酚酸B的含量积累方程分别为y=-8．105X＋81．047和y=-13．777X＋117．917,即随着植株的不断发育,丹参根部丹酚酸B的含量呈下降趋势。该研究结果可为丹参栽培及其质量控制提供理论依据。     

Isolation and Analysis of Extractives from White Cinquefoil (<Emphasis Type="Italic">Potentilla alba</Emphasis> L.) Grown under Different Conditions

Potentilla alba L. is used as a medicinal plant that contains a variety of biologically active substances. Natural resources are not meeting the needs of the pharmaceutical industry. We obtained the first biomass of P. alba plant-regenerants using hydroponics technique with clonal micropropagation. We have conducted a comprehensive study of the chemical composition of a vegetable raw materials P. alba, obtained in the Biotechnology Department (plant-regenerants) in comparison with raw material obtained from plants grown under field conditions (intact plants). We have established the quantitative content of the structural components in the samples: cellulose in roots and rhizomes of intact plants, 15.4%, in the roots of plant-regenerants, 4.3%, in the leaves of plant-regenerants, 2.5%; lignin in roots and rhizomes of intact plants, 40.7%, in the roots of plant-regenerants, 37.0%, and in the leaves of plant-regenerants, 36.8%. We determined the content of extractives in P. alba samples, recovered by sequential treatment with hexane, 96% and 40% ethanol solution, water and 1% aqueous sodium hydroxide solution. We identified the total content of extractives in the roots and rhizomes of intact plants at 15.3%, in the roots of plants-regenerants, 11.2%, and in the leaves of plant-regenerants, 5.1%. We characterized the chemical composition of extractives by UV-spectroscopy. Phenolic substances (λ = 279.5–280.0 nm) were dominant in the composition of the extracts. We have established the authenticity of the samples by the TLC chromatography based on the presence of flavonoids and tannins. We showed the identity of the phytochemical composition of vegetable raw materials produced by micropropagation and grown hydroponically for two months.     

Quantification of Anti‐Addictive Alkaloids Ibogaine and Voacangine in In Vivo‐ and In Vitro‐Grown Plants of Two Mexican Tabernaemontana Species

Tabernaemontana alba and Tabernaemontana arborea are Apocynaceae species used in Mexican traditional medicine for which little phytochemical information exists. In this study, preliminary gas chromatography/mass spectrometry analyses of different organs obtained from wild plants of both species identified a total of 10 monoterpenoid indole alkaloids (MIAs) and one simple indole alkaloid, nine of which were reported for the first time in these species. Furthermore, callus cultures were established from T. alba leaf explants and regeneration of whole plants was accomplished via somatic embryogenesis. The anti‐addictive MIAs ibogaine and voacangine were then quantified by gas chromatography with flame ionization detection in wild plants of both species, as well as greenhouse‐grown plants, in vitro‐grown plantlets and embryogenic callus of T. alba. Ibogaine and voacangine were present in most samples taken from the whole plants of both species, with stem and root barks showing the highest concentrations. No alkaloids were detected in callus samples. It was concluded that T. alba and T. arborea are potentially viable sources of ibogaine and voacangine, and that these MIAs can be produced through somatic embryogenesis and whole plant regeneration of T. alba. Approaches to increase MIA yields in whole plants and to achieve alkaloid production directly in cell cultures are discussed.     

丹参四个栽培类型的脂溶性成分的聚类分析

为给丹参优质新品种选育研究提供一定的理论依据,本文采用HPLC法测定江苏栽培的四个丹参类型的丹参酮IIA、隐丹参酮的含量,采用紫外分光光度法测定其总丹参酮的含量,以DPS软件对其脂溶性成分的HPLC的8个峰的比例、隐丹参酮与丹参酮IIA的比值、丹参酮IIA、隐丹参酮和总丹参酮的含量进行聚类分析。结果表明四个类型丹参的脂溶性成分类别基本一致,其中小叶型丹参的隐丹参酮与丹参酮IIA的比值、隐丹参酮的含量与其它三个类型相差较大,聚类结果显示小叶型丹参独自成为一个类群,说明小叶型丹参为一个较为特殊的类型,也是一个优质的丹参材料。     

Cu2+胁迫对丹参生长及有效成分积累的影响

研究了土壤Cu²⁺胁迫对丹参（Salvia miltiorrhiza Bunge）的生长和有效成分积累的影响。结果表明,与对照相比,Cu²⁺胁迫下丹参的生长受到抑制,土壤和丹参体内Cu²⁺残留量、膜脂过氧化程度增加,而叶绿素含量降低。此外,与对照相比,Cu²⁺胁迫下丹参地上部分中水溶性酚酸类成分咖啡酸、丹参素和原儿茶酸含量增加,迷迭香酸、原儿茶醛和丹酚酸B含量显著降低,而根系中这6种酚酸类成分和4种脂溶性丹参酮类成分二氢丹参酮、隐丹参酮、丹参酮Ⅰ、丹参酮ⅡA的含量均降低。表明铜胁迫抑制了丹参的生长,影响了有效成分的积累。     

不同立地类型和土壤类型对"裕丹参"生长和有效成分含量的影响

采用大田种植和盆栽方法,分别研究了不同的立地类型(平地、阳坡、阴坡和坡地)和土壤类型(砂土、壤土和黏土)对产自河南省方城县的"裕丹参"(Salvia miltiorrhiza Bunge)生长和有效成分含量的影响效应.结果表明:在不同立地条件下,"裕丹参"的各项生长和根系产量指标以及根系中丹参酮ⅡA和丹酚酸B含量均有明显差异.随种植时间的延长,平地、阳坡和坡地种植的"裕丹参"单株地上部干质量均逐渐降低,而阴坡种植的单株地上部干质量先升高后逐渐降低;但在不同立地类型下,单株根系干质量和丹酚酸B含量均随种植时间的延长逐渐升高,而丹参酮ⅡA含量则先降低后升高.采收期,平地种植的"裕丹参"的单株地上部干质量、单株根系干质量和鲜质量以及根系鲜产量均最高,平地和坡地种植的"裕丹参"根直径较大,阳坡种植的"裕丹参"根长最长,阳坡和坡地种植的"裕丹参"单株根数、根系中丹参酮ⅡA和丹酚酸B含量也较高,而阴坡种植的"裕丹参"的各项生长和根系产量指标以及丹参酮ⅡA和丹酚酸B含量均最低.在不同类型土壤中,"裕丹参"的根系性状和根系中丹参酮ⅡA和丹酚酸B含量总体上差异显著,其中,用砂土种植的"裕丹参"的根直径、根长、单株根系鲜质量和干质量以及根系中丹参酮ⅡA和丹酚酸B含量均最高,但单株根数最少;用黏土种植的"裕丹参"根系生长总体最差,但单株根数最多;用壤土种植的"裕丹参"根系中丹参酮ⅡA和丹酚酸B含量则最低.综合研究结果显示:"裕丹参"适宜种植于砂土的坡地或阳坡.     

Roles of reactive oxygen species in methyl jasmonate and nitric oxide-induced tanshinone production in Salvia miltiorrhiza hairy roots

Salvia miltiorrhiza is one of the most popular traditional Chinese medicinal plants for treatment of coronary heart disease. Tanshinones are the main biological active compounds in S. miltiorrhiza. In this study, effects of exogenous methyl jasmonate (MJ) and nitric oxide (NO) on tanshinone production in S. miltiorrhiza hairy roots were investigated and the roles of reactive oxygen species (ROS) in MJ and NO-induced tanshinone production were elucidated further. The results showed that contents of four tanshinone compounds were significantly increased by 100 μM MJ when compared to the control. Application of 100 μM sodium nitroprusside (SNP), a donor of NO, also resulted in a significant increase of tanshinone production. Expression of two key genes encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) and 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) was up-regulated by MJ and SNP. Generations of O₂ ⁻ and H₂O₂ were triggered by MJ, but not by SNP. The increase of tanshinone production and up-regulation of HMGR and DXR expression induced by MJ were significantly inhibited by ROS scavengers, superoxide dismutase (SOD) and catalase (CAT). However, neither SOD nor CAT was able to suppress the SNP-induced increase of tanshinone production and expression of HMGR and DXR gene. In conclusion, tanshinone production was significantly stimulated by MJ and SNP. Of four tanshinone compounds, cryptotanshinone accumulation was most affected by MJ elicitation, while cryptotanshinone and tanshinone IIA accumulation was more affected by SNP elicitation. ROS mediated MJ-induced tanshinone production, but SNP-induced tanshinone production was ROS independent.     

Accumulation pattern of endogenous cytokinins and phenolics in different organs of 1‐year‐old cytokinin pre‐incubated plants: implications for conservation

A better understanding of phytohormone physiology can provide an essential basis to coherently achieve a conservation drive/strategy for valuable plant species. We evaluated the distribution pattern of cytokinins (CKs) and phenolic compounds in different organs of 1‐year‐old greenhouse‐grown Tulbaghia simmleri pre‐treated (during micropropagation) with three aromatic CKs (benzyladenine = BA, meta‐topolin = mT, meta‐topolin riboside = mTR). The test species is highly valuable due to its medicinal and ornamental uses. Based on UHPLC‐MS/MS quantification, mT and mTR pre‐treated plants had the highest total CK, mostly resulting from the isoprenoid CK‐type, which occurred at highest concentrations in the roots. Although occurring in much lower concentrations when compared to isoprenoid CKs, aromatic CKs were several‐fold more abundant in the root of mT pre‐treated plants than with other treatments. Possibly related to the enhanced aromatic CKs, free bases and ribonucleotides, plants pre‐treated with mT generally displayed better morphology than the other treatments. A total of 12 bioactive phenolic compounds, including four hydroxybenzoic acids, five hydroxycinnamic acids and three flavonoids at varying concentrations, were quantified in T. simmleri. The occurrence, distribution and levels of these phenolic compounds were strongly influenced by the CK pre‐treatments, thereby confirming the importance of CKs in phenolic biosynthesis pathways.     

丹参遗传转化体系建立及应用研究进展

丹参(Salvia miltiorrhiza Bunge)是我国一种传统的大宗中药材,主要活性成分丹参酮和丹酚酸被广泛用于治疗心脑血管等疾病。建立高效、稳定的遗传转化体系,对丹参进行定向改良,为丹参酮和丹酚酸类成分工业化生产及丹参药材产量与质量提高提供了可能。本文结合国内外的研究,对丹参遗传转化方法进行了系统总结,从外植体、培养基、菌液浓度等影响丹参遗传转化因素方面进行综述。对近5年丹参遗传转化研究的关键酶基因和转录因子进行归纳,并对未来的研究重点进行了展望。     

栽培与野生化血丹不同部位中两种化学成分含量分析

为探讨并分析栽培与野生化血丹植株中不同部位中两种化学成分的含量差异,该研究采用超声法提取、高效液相色谱法(HPLC)测定栽培与野生化血丹根、茎、叶、花、混合样等部位中桃叶珊瑚苷和梓醇的含量,并进行比较。结果表明:(1)桃叶珊瑚苷在栽培与野生化血丹植株内均有分布,含量均以根中最高,其在栽培与野生化血丹植株内的含量表现分别为根>叶>混合样>茎>花、根>混合样>茎>花>叶,栽培化血丹不同部位中桃叶珊瑚苷的含量均高于野生化血丹。(2)梓醇在栽培化血丹的茎中未检出,在栽培与野生化血丹其他部位均有分布,含量均以叶中最高,其在栽培与野生化血丹植株内的含量分别表现为叶>花>混合样>根、叶>混合样>茎>花>根,野生化血丹不同部位中梓醇的含量均高于栽培化血丹。(3)桃叶珊瑚苷和梓醇在栽培和野生化血丹植株不同部位中的含量均存在显著差异(P<0.05),栽培与野生同一部位间总体上无显著差异,为该濒危药用植物资源药用部位选择和合理开发利用提供实验参考。     

Micro‐particle‐induced X‐ray emission mapping of elemental distribution in roots of a Mediterranean‐type sclerophyll,Agathosma betulina (Berg.) Pillans,colonized by Cryptococcus laurentii

The role of rhizosphere yeasts as plant nutrient‐scavenging microsymbionts in resource‐limited Mediterranean‐type heathlands is unknown. This study, therefore, focused on quantitative elemental distribution within the roots of a medicinal sclerophyll, Agathosma betulina (Berg.) Pillans, grown under nutrient‐poor conditions, and colonized by Cryptococcus laurentii. Micro‐particle‐induced X‐ray emission (PIXE) was used to assess quantitative elemental distribution within the roots of A. betulina inoculated with viable C. laurentii, as well as within roots of control plants that received autoclaved yeast. To aid in the interpretation of heterogeneous elemental distribution patterns, apoplastic barriers (Casparian bands) in root tissues were located using fluorescence microscopy. In addition, root cross‐sections were examined for endophytic C. laurentii using light and transmission electron microscopy (TEM). The average concentrations of P, Fe and Mn were significantly (P < 0.05) higher in roots of yeast‐inoculated plants, compared to control plants. Casparian bands were observed in the exodermal cells of both treatments, and the presence of these bands was correlated with elemental enrichment in the epi/exodermal‐outer cortical tissues. Light and TEM micrographs revealed that the yeast was not a root endophyte. This is the first report describing the role of a soil yeast as a plant nutrient‐scavenging microsymbiont.     

Effects of electric cell fusion treatment among leaf protoplasts of Populus alba and alnus firma on growth, leaf morphology, and rapd pattern of eleven acclimatized plants

Summary This study reports the characterization of 11 plants regenerated from electrically fused protoplasts between Populus alba and Alnus firma. Growth characteristics of five regenerated plants (AP-1-AP-5) in terms of shoot height and leaf color showed small differences compared with those of P. alba grown, in pots, and showed no difference in shoot height and diameter compared with those grown in nursery field. There was also no difference in the RAPD pattern between the plants regenerated from interfamilial protoplast fusion and P. alba. In contrast, the lately regenerated plants (AP-6-AP-11) grown in pots showed a marked difference in leaf morphology and RAPD pattern. There was a variation in the ratio of longitudinal to transverse length of leaves among the 11 plants from interfamilial fusions compared with that of protoclones and intraspecific fused protoplasts of P. alba.     

PEG and ABA Trigger the Burst of Reactive Oxygen Species to Increase Tanshinone Production in Salvia miltiorrhiza Hairy Roots

Salvia miltiorrhiza is one of the most popular traditional Chinese medicinal plants because of its excellent performance in treating coronary heart disease. Tanshinones, a group of active compounds in S. miltiorrhiza, are derived from two biosynthetic pathways: the mevalonate (MVA) pathway in the cytosol and the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway in the plastids. Water stress is well known to stimulate the accumulation of secondary metabolites in plants. Reactive oxygen species (ROS) serve as important secondary messengers in water stress-induced signal transduction pathways. In this study, the effects of polyethylene glycol (PEG) and abscisic acid (ABA) on tanshinone production in S. miltiorrhiza hairy roots were investigated and the roles of ROS in PEG- and ABA-induced tanshinone production were further elucidated. The results showed that contents and yields of four tanshinones in S. miltiorrhiza hairy roots were significantly enhanced by 2 % PEG and 200?μM ABA. Simultaneously, the mRNA levels and activities of two key enzymes (3-hydroxy-3-methylglutaryl coenzyme A reductase and 1-deoxy-D-xylulose 5-phosphate synthase) involved in tanshinone biosynthesis were upregulated. Both PEG and ABA were able to trigger the burst of H₂O₂ and O₂ ^?. The PEG- and ABA-induced increases of tanshinone production, gene expression, and enzyme activity were all dramatically suppressed by two ROS scavengers, catalase and superoxide dismutase. In addition, ROS treatments resulted in a significant increase in tanshinone production. These results demonstrated that the MVA and MEP pathways were activated by PEG and ABA to stimulate tanshinone biosynthesis, and the increase of tanshinone production was probably via ROS signaling.     

Growth and Arbuscular Mycorrhizal Fungal Colonization of Two Prairie Grasses Grown in Soil from Restorations of Three Ages

I compared growth and arbuscular mycorrhizal fungal (AMF) colonization of two prairie grasses (Wild rye [Elymus canadensis] and Little bluestem [Schizachyrium scoparium]), an early‐ and a late‐dominating species in prairie restorations, respectively, grown in soil from restored prairies of differing age, soil characteristics, and site history. There were no consistent patterns between restoration age and soil inorganic nutrients or organic matter. The oldest restoration site had higher soil mycorrhizal inoculum potential (MIP) than 2‐ and 12‐year‐old restorations. However, MIP did not translate into actual colonization for two species grown in soils from the three restorations, nor did MIP relate to phosphorus availability. There were significant differences in root mass and colonization among Wild rye plants but not among Little bluestem plants grown in soils from the three restorations. Wild rye grown in 2‐year‐old restoration soil had significantly higher AMF colonization than when it was grown in soils from the 12‐ and 17‐year‐old restorations. Wild rye grown in 2‐year‐old restoration soil also had higher colonization than Little bluestem grown in 2‐ and 12‐year‐old restoration soils. Little bluestem had no significant correlations between shoot biomass, root biomass or colonization, and concentrations of soil P, total N, or N:P. However, for Wild rye, total soil N was positively correlated with root mass and negatively correlated with colonization, suggesting that in this species, mycorrhizae may affect N availability. Collectively, these results suggest that soil properties unrelated to restoration age were important in determining differences in growth and AMF colonization of two species of prairie grasses.