From traditional resource to global commodities: a comparison of Rhodiola species using NMR spectroscopy - metabolomics and HPTLC

The fast developing international trade of products based on traditional knowledge and their value chains has become an important aspect of the ethnopharmacological debate. The structure and diversity of value chains and their impact on the phytochemical composition of herbal medicinal products has been overlooked in the debate about quality problems in transnational trade. Different government policies and regulations governing trade in herbal medicinal products impact on such value chains. Medicinal Rhodiola species, including Rhodiola rosea L. and Rhodiola crenulata (Hook.f. & Thomson) H.Ohba, have been used widely in Europe and Asia as traditional herbal medicines with numerous claims for their therapeutic effects. Faced with resource depletion and environment destruction, R. rosea and R. crenulata are becoming endangered, making them more economically valuable to collectors and middlemen, and also increasing the risk of adulteration and low quality. We compare the phytochemical differences among Rhodiola raw materials available on the market to provide a practical method for Rhodiola authentication and the detection of potential adulterant compounds. Samples were collected from Europe and Asia and nuclear magnetic resonance spectroscopy coupled with multivariate analysis software and high performance thin layer chromatography techniques were used to analyse the samples. A method was developed to quantify the amount of adulterant species contained within mixtures. We compared the phytochemical composition of collected Rhodiola samples to authenticated samples. Rosavin and rosarin were mainly present in R. rosea whereas crenulatin was only present in R. crenulata. 30% of the Rhodiola samples purchased from the Chinese market were adulterated by other Rhodiola spp. Moreover, 7 % of the raw-material samples were not labelled satifactorily. The utilisation of both 1H-NMR and HPTLC methods provided an integrated analysis of the phytochemical differences and novel identification method for R. rosea and R. crenulata. Using 1H-NMR spectroscopy it was possible to quantify the presence of R. crenulata in admixtures with R. rosea. This quantitative technique could be used in the future to assess a variety of herbal drugs and products. This project also highlights the need to further study the links between producers and consumers in national and trans-national trade.

基于ITS 序列和matK 基因序列的中国药用石斛及其混伪品的分子鉴定

为了从分子水平对中国药用石斛及其混伪品进行鉴定，本文选取了核rDNA ITS 序列和叶绿体DNA 的matK 基因序列进行研究。采用改良的CTAB 法提取石斛的基因组DNA，PCR 产物直接测序法对17 种（共32 份）药用石斛的核糖体内转录间隔区ITS 全序列进行测定，克隆测序法对12 种（共22 份）药用石斛的叶绿体的matK 基因序列进行测定，运用BioEd it，MEGA4.0 等生物软件分析了石斛属植物的rDNA ITS 序列及叶绿体的matK 基因序列的特征，比较了石斛属间、种间、种内不同居群（品种）间的序列碱基差异及遗传距离，应用邻接法构建分子系统树。主要研究结果如下： （1）建立了17 种（共32 份）药用石斛rDNA ITS 区碱基全序列数据库，其中，ITS1 的长度为228~234 bp，GC 含量为45.7%~53.0%，变异位点167 个，占总位点67.34%，信息位点106 个，占总位点42.74%，ITS2 长度为241~247 bp，GC含量为44.8%~55.7%，变异位点165 个，占总位点66.27%，信息位点115 个，占总位点46.18%。 （2）建立了12 种（共22 份）药用石斛的叶绿体matK 基因全序列数据库，叶绿体matK 基因长1410 bp，变异位点51 个，信息位点11 个。除了存在碱基替换的遗传变异外，还存在碱基的插入和缺失。 （3）通过ITS 序列比较分析了各材料间的遗传距离和碱基差异，属间的遗传距离为0.295，石斛种间的平均遗传距离为0.142，碱基相差2~156 个，种内各居群间的平均遗传距离为0.002，碱基相差1~2 个。属间的遗传距离大于种间的遗传距离，种间的遗传距离大于种内不同居群（品种）间的遗传距离。 （4）根据分析石斛叶绿体的matK 基因序列得到，外类群（密花石豆兰）与石斛属间最小遗传距离为0.027，石斛种间的平均遗传距离为0.008，种间最大的遗传距离0.014， 最小的遗传距离为0.003，碱基相差8~20 个。种内不同居群（品种）遗传距离为0.001，相差1~5 个碱基。 （5）利用17 种石斛的全序列数据库及遗传分析软件，通过对待检种rDNA I TS区进行序列测定，成功地对10 个待检种进行了鉴定，并且在原植物开花后得到了验证。 （6）运用12 种石斛的matK 基因全序列数据库及遗传分析软件，成功地对4个待检种进行了鉴定，同样在原植物开花后得到了验证。 （7）本文利用石斛的核糖体内转录间隔区ITS 序列和叶绿体的matK 基因序列数据库分别构建了NJ 树，外类群与石斛属间石斛种间以及种内不同居群（品种）间均能在NJ 树中明显分化开来，二者构建的分子系统树一致，为石斛的分子鉴定提供了依据。 In order to identify Chinese Herba Dendrobii and its adulterant species on molecular level, we studied the sequences of rDNA ITS and chloroplast matK gene. Genomic DNA of Dendrobium was extracted using the modified cetyltrimethyl ammonium bromide (CTAB) method. The PCR products of the rDNA ITS sequences of Dendrobium (32 materia ls) were purified and then sequenced. The PCR products of chloroplast matK gene of Dendrobium (22 materia ls) were purified, cloned and then sequenced. The characteristic of the sequences and the genetic dista nce were compared between Bulbophyllum odoratissimum and Dendrobium, Dendrobium interspecies, and different populations. Phylogenetic trees were constructed using the NJ method by the biology softwares including BioEd it, MEGA4.0 etc. The ma in results as follows: (1) It was built up that the database of rDNA ITS sequences of 17 species of Herba Dendrobii (32 materia ls). The ITS1 was 228~234 bp, the GC content accounting for 45.7%~53.0%. Its variable sites were 167, accounting for 67.34%. The Parsim-Informative positions were 106, accounting for 42.74%. The ITS2 was 241~247 bp, the GC accounting for 44.8%~55.7%. The variable sites were 165, accounting for 66.27%. The Parsim-Informative positions were 115, accounting for 46.18%. (2) The database of the chloroplast matK gene sequences was built up, which contained 12 species of Herba Dendrobii (22 materia ls). The matK gene sequences were about 1410bp in length. There were 51 variable sites and 11 Parsim-Informative sites. And there were nucleotides insertions and deletions in some species , in addition to the nucleotides substitutions. (3) The rDNA ITS sequences were compared and analyzed by the biology softwares. The genetic dista nce between Bulbophyllum odoratissimum and Dendrobium was 0.295. The avera ge genetic dista nce was 0.142 between Dendrobium species, and there were 2~156 variable nucleotides. The avera ge genetic dista nce between different populations was 0.002, and there were 2~156 variable nucleotides. The genetic dista nce between Bulbophyllum odoratissimum and Dendrobium was greater tha n that of Denrobium interspecies. Meanwhile, the genetic dista nce between Denrobium species was also greater tha n that of different populations (variaties). (4) The characteristics of the chloroplast matK gene sequences were obtained after analyzing by the biology softwares. The minima l genetic dista nce was 0.027 between Bulbophyllum odoratissimum and Dendrobium . The ma xima l genetic dista nce was 0.014 between Dendrobium species, and there were 20 variable nucleotides. The minima l genetic dista nce between populations was 0.003, and there were 8 variable nucleotides.The genetic dista nce between populations was 0.001, and there were 1~5 variable nucleotides. (5) The molecular Phylogeny tree was constructed on the database of rDNA ITS the sequences of 17 species of Herba Dendrobii using the biology softwares. Then we authenticated 10 materia ls on molecular level. What’s more, they had been proved when these pla nts flowered. (6) The molecular Phylogeny tree was built up on the database of chloroplast matK gene sequences of 12 species of Herba Dendrobii with the biology softwares.Then 4 materia ls were authenticated on molecular level. Moreover, they had also been proved when these pla nts were in flower. (7) The Phylogenetic trees were separately constructed on the sequences of rDNA ITS and chloroplast matK gene B. odoratissimum and Dendrobium all could be distinguished on the Phylogenetic trees. Meanwhile, the Phylogenetic trees based on two groups of sequences were coincident. rDNA ITS and matK gene sequence could be used as molecular markers for authentication of Herba Dendrobii.

A molecular marker that is specific to medicinal rhubarb based on chloroplast trnL/trnF sequences

"Da-Huang" (Radix et Rhizoma Rhei, medicinal rhubarb), a famous and important Traditional Chinese Medicine, has often been confused with the adulterant species in the same genus, Rheum. Through sequencing the trnL (UAA)/trnF (GAA) regions of chloroplast DNA of thirteen species of Rheum (three medicinal rhubarb species and ten adulterant ones), a molecular marker of the medicinal species was found. A pair of PCR primers based on the sequences, was thus designed, which amplified a highly specific DNA fragment in medicinal rhubarb exclusively, and absent in the adulterants at all under an optimized PCR condition.

Endangerment and Likeability of Wildlife Species: How Important are they for Payments Proposed for Conservation?

This paper examines empirically the relative influence of the degree of endangerment of wildlife species and their stated likeability on individuals' allocation of funds for their conservation. To do this, it utilises data obtained from the IUCN Red List, and likeability and fund allocation data obtained from two serial surveys of a sample of the Australian public who were requested to assess 24 Australian wildlife species from three animal classes: mammals, birds and reptiles. Between the first and second survey, respondents were provided with extra information about the focal species. This information resulted in the dominance of endangerment as the major influence on the allocation of funding of respondents for the conservation of the focal wildlife species. Our results throw doubts on the proposition in the literature that the likeability of species is the dominant influence on willingness to pay for conservation of wildlife species. Furthermore, because the public's allocation of fund for conserving wildlife species seems to be more sensitive to information about the conservation status of species than to factors influencing their likeability, greater attention to providing accurate information about the former than the latter seems justified. Keywords: Conservation of wildlife species; Contingent valuation; Endangerment of species; Likeability of species; Willingness to pay

Common And Endangered Species: How Does Society Allocate Support For Their Conservation

Examines how society allocates support for species’ conservation when numbers involved are large and resources are limited. Rational behaviour suggests that species in urgent need of conservation will receive more support than those species that are common. However, we demonstrate that in the absence of balanced knowledge common species will receive support more than they would otherwise receive despite society placing high existence values on all species. Twenty four species, both common and endangered and some with a restricted distribution, are examined. We demonstrate that balanced information is vital in order to direct more support for species that are endangered than those that are not. Implications for conservation stemming from the findings are discussed.

Electronic states of trans-polyacetylene, poly(p-phenylene vinylene) and sp-hybridised carbon species in amorphous hydrogenated carbon probed by resonant Raman scattering

Inclusions of sp-hybridised, trans-polyacetylene [trans-(CH)x] and poly(p-phenylene vinylene) (PPV) chains are revealed using resonant Raman scattering (RRS) investigation of amorphous hydrogenated carbon (a-C:H) films in the near IR – UV range. The RRS spectra of trans-(CH)x core Ag modes and the PPV CC-H phenylene mode are found to transform and disperse as the laser excitation energy ћωL is increased from near IR through visible to UV, whereas sp-bonded inclusions only become evident in UV. This is attributed to ћωL probing of trans-(CH)x chain inhomogeneity and the distribution of chains with varying conjugation length; for PPV to the resonant probing of phelynene ring disorder; and for sp segments, to ћωL probing of a local band gap of end-terminated polyynes. The IR spectra analysis confirmed the presence of sp, trans-(CH)x and PPV inclusions. The obtained RRS results for a-C:H denote differentiation between the core Ag trans-(CH)x modes and the PPV phenylene mode. Furthermore, it was found that at various laser excitation energies the changes in Raman spectra features for trans-(CH)x segments included in an amorphous carbon matrix are the same as in bulk trans-polyacetylene. The latter finding can be used to facilitate identification of trans-(CH)x in the spectra of complex carbonaceous materials.

A three species model to simulate application of hyperbaric oxygen therapy to chronic wounds

Chronic wounds are a significant socioeconomic problem for governments worldwide. Approximately 15% of people who suffer from diabetes will experience a lower-limb ulcer at some stage of their lives, and 24% of these wounds will ultimately result in amputation of the lower limb. Hyperbaric Oxygen Therapy (HBOT) has been shown to aid the healing of chronic wounds; however, the causal reasons for the improved healing remain unclear and hence current HBOT protocols remain empirical. Here we develop a three-species mathematical model of wound healing that is used to simulate the application of hyperbaric oxygen therapy in the treatment of wounds. Based on our modelling, we predict that intermittent HBOT will assist chronic wound healing while normobaric oxygen is ineffective in treating such wounds. Furthermore, treatment should continue until healing is complete, and HBOT will not stimulate healing under all circumstances, leading us to conclude that finding the right protocol for an individual patient is crucial if HBOT is to be effective. We provide constraints that depend on the model parameters for the range of HBOT protocols that will stimulate healing. More specifically, we predict that patients with a poor arterial supply of oxygen, high consumption of oxygen by the wound tissue, chronically hypoxic wounds, and/or a dysfunctional endothelial cell response to oxygen are at risk of nonresponsiveness to HBOT. The work of this paper can, in some way, highlight which patients are most likely to respond well to HBOT (for example, those with a good arterial supply), and thus has the potential to assist in improving both the success rate and hence the costeffectiveness of this therapy.