Metabolic Analysis of Medicinal Dendrobium officinale and Dendrobium huoshanense during Different Growth Years

Metabolomics technology has enabled an important method for the identification and quality control of Traditional Chinese Medical materials. In this study, we isolated metabolites from cultivated Dendrobium officinale and Dendrobium huoshanense stems of different growth years in the methanol/water phase and identified them using gas chromatography coupled with mass spectrometry (GC-MS). First, a metabolomics technology platform for Dendrobium was constructed. The metabolites in the Dendrobium methanol/water phase were mainly sugars and glycosides, amino acids, organic acids, alcohols. D. officinale and D. huoshanense and their growth years were distinguished by cluster analysis in combination with multivariate statistical analysis, including principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA). Eleven metabolites that contributed significantly to this differentiation were subjected to t-tests (P<0.05) to identify biomarkers that discriminate between D. officinale and D. huoshanense, including sucrose, glucose, galactose, succinate, fructose, hexadecanoate, oleanitrile, myo-inositol, and glycerol. Metabolic profiling of the chemical compositions of Dendrobium species revealed that the polysaccharide content of D. huoshanense was higher than that of D. officinale, indicating that the D. huoshanense was of higher quality. Based on the accumulation of Dendrobium metabolites, the optimal harvest time for Dendrobium was in the third year. This initial metabolic profiling platform for Dendrobium provides an important foundation for the further study of secondary metabolites (pharmaceutical active ingredients) and metabolic pathways.     

Genetic diversity assessment and ex situ conservation strategy of the endangered Dendrobium officinale (Orchidaceae) using new trinucleotide microsatellite markers

Dendrobium officinale (Orchidaceae) is an endangered plant species with important medicinal value. To evaluate the effectiveness of ex situ collection of D. officinale genetic diversity, we developed 15 polymorphic trinucleotide microsatellite loci of D. officinale to examine the genetic diversity and structure of three D. officinale germplasm collections comprising 120 individuals from its germplasm collection base and their respective wild populations consisting of 62 individuals from three provinces in China. The three germplasm collections showed reductions in gene diversity and average number of alleles per locus, but an increase in average number of rare alleles (frequency?≤?0.05) per locus in comparison to their wild populations. However, the differences in gene diversity between the germplasm collections and wild populations were not statistically significant. The analysis using STRUCTURE revealed evident differences in genetic composition between each germplasm collection and its wild population, probably because the D. officinale individuals with distinct genotypes in each wild population were unevenly selected for establishing its germplasm collection. For conservation management plans, we propose that D. officinale individuals with rare alleles need to be conserved with top priority, and those individuals with the most common alleles also should be concerned. The 15 new microsatellite loci may be used as a powerful tool for further evaluation and conservation of the genetic diversity of D. officinale germplasm resources.     

Genetic variation and conservation of the endangered Chinese endemic herb Dendrobium officinale based on SRAP analysis

Dendrobium officinale (Orchidaceae) is used for traditional medicine and is critically endangered in China. To investigate the genetic structure of this species and to offer some advice on conservation strategies, 84 individuals from nine wild populations of D. officinale were analyzed using the method of sequence-related amplified polymorphism. A high level of genetic diversity was detected (PPB = 88.07%, H _E = 0.2880) at the species level. However, the genetic diversity at the population level was lower (PPB = 51.68%, H _E = 0.1878) in comparison with other species with similar life history characteristics. Based on analysis of molecular variation, there was moderate variation between pairs of populations with Φ _ST values ranging from 0.1327 to 0.4151 and on average 27.05% of the genetic variation occurred among populations. Two main clusters were shown in UPGMA using TFPGA, which is consistent with the result of principal coordinate analysis using NTSYS. In situ conservation is the first advocated and and ex situ should be proposed at the same time to protect the endangered plant and to preserve germplasm resources.     

Specificity and preference of mycorrhizal associations in two species of the genus Dendrobium (Orchidaceae)

Dendrobium is a large genus of tropical epiphytic orchids. Some members of this genus are in danger of extinction across China. To investigate orchid mycorrhizal associations of the genus Dendrobium, plants from two Dendrobium species (Dendrobium officinale and Dendrobium fimbriatum) were collected from two habitats in Guangxi Province, China, and clone libraries were constructed to identify the mycorrhizal fungi of individual plants. A low and high degree of specificity was observed in D. officinale and D. fimbriatum, respectively. Phylogenetic analysis revealed that the majority of Dendrobium mycorrhizal fungi are members of the Tulasnellaceae, but, in some plants, members of the Ceratobasidiaceae and Pluteaceae were also found. In D. officinale, individual plants associated with more than three fungi simultaneously, and, in some cases, associations with five fungi at the same time. One fungus was shared by individual plants of D. officinale collected from the two habitats. In D. fimbriatum, only one fungal partner was found in each population, and this fungus differed between populations. The two species of Dendrobium sampled from the same habitat did not share any fungal taxa. These results provide valuable information for conservation of these orchid species.     

福建泰宁野生铁皮石斛种群的ISSR亲缘关系分析

为了解铁皮石斛(Dendrobium officinale)种质间的亲缘关系,利用ISSR技术对34份铁皮石斛种质资源进行亲缘关系和遗传多样性分析。结果表明,9条ISSR引物在34份种质中共扩增出78条带,多态位点百分率达100%。UPGMA聚类分析表明,种质的相似系数为0.61~0.92,在相似系数0.626处,福建省泰宁的野生铁皮石斛与栽培铁皮石斛分为两大类。泰宁野生铁皮石斛种群的Nei’s基因多样性(H)和遗传分化系数(Gst)分别为0.3111和0.4609,均高于栽培种群(0.3056和0.4204),表明泰宁野生铁皮石斛具有较丰富的多样性和较高的种群分化系数。AMOVA分析表明,铁皮石斛种群内变异指数为74%,种群间变异指数为26%,表明不同种群间可能存在基因交流。这些为不同地域的野生铁皮石斛资源的有效保护及利用提供理论依据及技术参考。     

Reflections on univariate and multivariate analysis of metabolomics data

Metabolomics experiments usually result in a large quantity of data. Univariate and multivariate analysis techniques are routinely used to extract relevant information from the data with the aim of providing biological knowledge on the problem studied. Despite the fact that statistical tools like the t test, analysis of variance, principal component analysis, and partial least squares discriminant analysis constitute the backbone of the statistical part of the vast majority of metabolomics papers, it seems that many basic but rather fundamental questions are still often asked, like: Why do the results of univariate and multivariate analyses differ? Why apply univariate methods if you have already applied a multivariate method? Why if I do not see something univariately I see something multivariately? In the present paper we address some aspects of univariate and multivariate analysis, with the scope of clarifying in simple terms the main differences between the two approaches. Applications of the t test, analysis of variance, principal component analysis and partial least squares discriminant analysis will be shown on both real and simulated metabolomics data examples to provide an overview on fundamental aspects of univariate and multivariate methods.     

转录组和代谢组分析瘤菌根菌对铁皮石斛的促生机制

为筛选出促进铁皮石斛(Dendrobium officinale)生长的差异表达基因和差异代谢物,对瘤菌根菌与无菌盆栽铁皮石斛苗共生后形成的侧根根系进行转录组、代谢组和双组学联合分析。结果表明,转录组分析共找到262条差异表达基因富集到了35条通路中,其中内质网蛋白质加工通路途径的差异基因最多,其次为氨基糖和核苷酸糖代谢通路。代谢组分析共检测出194个差异代谢物富集到33个KEGG通路中,其中代谢途径的差异代谢物最多有133个,其次为不同环境的微生物代谢途径的差异代谢物有70个。通过联合分析,有9个差异基因的差异表达导致丝氨酸、谷氨酸、D-甘露糖和激素等代谢物的积累量发生变化,这可能是瘤菌根菌促进铁皮石斛生长的重要原因。因此,推测瘤菌根菌促进铁皮石斛生长与氨基酸、糖、植物激素的积累及相关基因的表达变化有关。     

Fingerprint and relationship composition-antioxidant activity of the essential oil of Saccocalyx satureioides Coss. & Dur

Saccocalyx satureioides Coss. & Dur. is an endemic Algerian plant species growing in pre-desert area. The aerial parts are commonly used in the folk medicine. The fingerprint of the aerial part of S. satureioides essential oil (EO) collected periodically at different growing stages was established by GC and GC/MS analyses. Besides, the seasonal variations of the EO components were also investigated. In order to highlight the effect of seasonal variation/variability of the EO composition on the fingerprint pattern, chemometrics methods including principal component analysis (PCA) and heatmap associated with hierarchical clustering analysis (HCA) were applied. The antioxidant activity of the EO was measured using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP) assays. The relationship between GC fingerprint and antioxidant activity named spectrum-effect relationship was investigated by partial least squares (PLS) regression. The EO fingerprint was essentially marked by the three major oxygenated monoterpenes which were α-terpineol (34.48–42.43%), borneol (15.24–23.37%) and thymol (19.27–26.96%). Successfully, multivariate models were developed to relate the antioxidant activities responses to the EO chemical composition.     

Acaricidal and repellent effects of <Emphasis Type="Italic">Cnidium officinale</Emphasis>-derived material against <Emphasis Type="Italic">Dermanyssus gallinae</Emphasis> (Acari: Dermanyssidae)

The acaricidal activity of a methanolic extract and fractions from the rhizome of Cnidium officinale against Dermanyssus gallinae adults was investigated. The C. officinale methanolic extract exhibited 100% acaricidal activity after 48 h of treatment at a dose of 4000 ppm. The acaricidal constituents of the plant were sequentially partitioned with several solvents and then purified using silica gel column chromatography and high-performance liquid chromatography. Gas chromatography–mass spectrometry and nuclear magnetic resonance spectroscopy revealed (Z)-ligustilide as a constituent of C. officinale. Acaricidal activity was examined in three experimental tests (spray, fumigation and contact), with the spraying method being the most effective. The methanolic extract of C. officinale showed both contact and fumigant activities, though only fumigant activity was observed with (Z)-ligustilide. The fumigant effects of the methanolic extract and (Z)-ligustilide caused 86.5 and 62.6% mortality, respectively, of D. gallinae adults at 48 h. Among (Z)-ligustilide, acaricides (bifenthrin, cypermethrin and spinosad) and butylidenephthalide, bifenthrin displayed the highest acaricidal activity, and the activity of butylidenephthalide was 2.3-fold higher than that of (Z)-ligustilide. These results suggest that C. officinale-derived material can be used for the development of a control agent for D. gallinae.     

Inter-genomic relationships among three medicinal herbs: Cnidium officinale, Ligusticum chuanxiong and Angelica polymorpha

Cnidium officinale, Ligusticum chuanxiong and Angelica polymorpha have long been used as Chinese medicinal herbs to treat the same conditions. To elucidate the inter-specific relationships among these species, molecular and molecular cytogenetic analyses were conducted. Cytogenetically, C. officinale and A. polymorpha were found to be diploid with 2n=2x=22, but L. chuanxiong had a triploid chromosome number (2n=3x=33). FISH analysis revealed that each two pairs of the 45S- and 5S rRNA genes were detected on the different chromosomes of C. officinale, and that triplet of the rRNA genes were localized on the same loci of the homologue in L. chuanxiong. GISH conducted using a genomic DNA probe detected strong cross-hybridization of genomes between C. officinale and L. chuanxiong, while no distinct GISH signal between C. officinale and A. polymorpha was observed. ITS and NTS data also revealed a very high sequence homology (95–96%) between C. officinale and L. chuanxiong. The RFLP profile of the rDNA-ITS enabled us to confirm the genetic distance among species. An effective species-specific SCAR marker for the identification of the wild plant, A. polymorpha, was also developed. Taken together, the results of this study suggest two possibilities; (1) L. chuanxiong originated from C. officinale, or (2) L. Chuanxiong and C. officinale diverged from the same parental plant.     

硝酸镧和兰科菌根真菌对铁皮石斛生理特性的影响

利用盆栽的方式研究了不同硝酸镧水平(0、1.0、3.0、5.0和7.0mg·L-1)下接种兰科菌根真菌对铁皮石斛生物量、多糖和蛋白质合成的影响,并分析了叶绿素含量、丙二醛(MDA)含量以及超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和过氧化物酶(POD)活性的变化,以探讨硝酸镧和兰科菌根真菌对铁皮石斛生理特性的影响。结果表明:(1)添加适量的硝酸镧有利于菌根真菌侵染和菌根发育,提高铁皮石斛幼苗生物量。(2)在接种菌根真菌的同时添加5.0mg·L-1的硝酸镧,铁皮石斛的根重、茎叶重和总生物量均达到最大,分别是未添加硝酸镧以及未接种对照组的4.26倍、4.98倍和4.87倍,其菌根侵染率也高达92.8%;而且可显著提高叶片中叶绿素含量,并显著降低细胞内的丙二醛含量。(3)在适量(5.0mg·L-1)的硝酸镧水平下接种菌根真菌能促进铁皮石斛幼苗多糖和蛋白质的合成,并显著提高细胞内SOD、CAT和POD活性。研究认为,菌根真菌与适宜浓度硝酸镧(5.0 mg·L-1)联合使用能显著促进铁皮石斛菌根的形成,增强植株的生理活性和适应能力,提高其生物量和多糖等活性成分的积累,有效改善铁皮石斛的药用品质。     

兰科菌根真菌对铁皮石斛光合性能及pepc基因表达的影响

为了阐明兰科菌根真菌对铁皮石斛光合作用的影响及机制,采用盆栽方式研究了兰科菌根真菌对铁皮石斛幼苗生长的影响,并分析了叶片中叶绿素含量、光合参数、叶绿素荧光参数以及pepc基因表达的变化。结果表明：（1）兰科菌根真菌促进了铁皮石斛幼苗生长,接种兰科菌根真菌的铁皮石斛的株高、根重、茎叶重和总生物量分别是未接种对照组的1.21、1.54、1.71和1.68倍;而且可显著提高叶片中叶绿素含量、叶片净光合速率（P_n）、蒸腾速率（G_s）和气孔导度（T_r）。（2）接种兰科菌根真菌的铁皮石斛叶片潜在光化学效率（F_v/F₀）、最大光化学效率（F_v/F_m）、光化学猝灭系数（q_P）、非光化学猝灭系数（q_N）、实际光化学反应量子效率（Yield）和表观光合电子传递速率（ETR）均高于未接种对照组。（3）菌根真菌能促进pepc基因的表达,增强PEPC活性,提高铁皮石斛叶片的光合碳同化能力。研究表明,菌根的形成可以提高铁皮石斛叶片光合性能和pepc基因的表达水平,促进铁皮石斛幼苗的生长。     

Antioxidant activity in essential oils of Cnidium officinale makino and Ligusticum chuanxiong hort and their inhibitory effects on DNA damage and apoptosis induced by ultraviolet B in mammalian cell

Background: Owing to their high volatile aroma, the dried rhizomes of Cnidium officinale (C. officinale) and Ligusticum chuanxiong (L. chuanxiong) are used as herbal drugs to treat blood pressure depressant, a deficiency disease of antivitamin, inhibition of small intestine sympathetic nerve and as cosmetics for skin care. However, little has been known about the protective effect of their essential oils against ultraviolet B (UVB)-induced DNA damage. Methods: In this study, we report antioxidant activity of their essential oils using DPPH and ABTS scavenging assay. In addition, the composition of essential oils was measured by GC/MS. We also investigated whether these essential oils could inhibit UVB-induced DNA damage and apoptosis in the mammalian cell using intracellular DNA migration and expression level of phospho-H2A.X. Results: Twenty constituents in the essential oil were identified and they showed good antioxidant properties, in that IC₅₀ value in DPPH and ABTS showed 6.79 and 7.33 μg/ml and 1.58 and 1.58 μg/ml in C. officinale and L. chuanxiong. Their treatment inhibited the migration of damaged DNA induced by UV-B; furthermore, they decreased p21 expression and increased cyclin D1 expression as apoptosis-regulatory genes. Conclusions: These results suggest that essential oils in C. officinale and L. chuanxiong may exert inhibitory effects on DNA damage and apoptosis induced by UVB through their high free radical scavenging ability.     

Prediction of in situ root decomposition rates in an interspecific context from chemical and morphological traits

Background and Aims

We quantitatively relate in situ root decomposition rates of a wide range of trees and herbs used in agroforestry to root chemical and morphological traits in order to better describe carbon fluxes from roots to the soil carbon pool across a diverse group of plant species.

Methods

In situ root decomposition rates were measured over an entire year by an intact core method on ten tree and seven herb species typical of agroforestry systems and were quantified using decay constants (k values) from Olson''s single exponential model. Decay constants were related to root chemical (total carbon, nitrogen, soluble carbon, cellulose, hemicellulose, lignin) and morphological (specific root length, specific root length) traits. Traits were measured for both absorbing and non-absorbing roots.

Key Results

From 61 to 77 % of the variation in the different root traits and 63 % of that in root decomposition rates was interspecific. N was positively correlated, but total carbon and lignin were negatively correlated with k values. Initial root traits accounted for 75 % of the variation in interspecific decomposition rates using partial least squares regressions; partial slopes attributed to each trait were consistent with functional ecology expectations.

Conclusions

Easily measured initial root traits can be used to predict rates of root decomposition in soils in an interspecific context.     

Discrimination of the rare medicinal plant Dendrobium officinale based on naringenin, bibenzyl, and polysaccharides

The aim of this study was to establish a method for discriminating Dendrobium officinale from four of its close relatives Dendrobium chrysanthum, Dendrobium crystallinum, Dendrobium aphyllum and Dendrobium devonianum based on chemical composition analysis. We analyzed 62 samples of 24 Dendrobium species. High performance liquid chromatography analysis confirmed that the four low molecular weight compounds 4??,5,7-trihydroxyflavanone (naringenin), 3,4-dihydroxy-4??,5-dime-thoxybibenzyl (DDB-2), 3??,4-dihydroxy-3,5??-dimethoxybibenzyl (gigantol), and 4,4??-dihydroxy-3,3??,5-trimethoxybibenzy (moscatilin), were common in the genus. The phenol-sulfuric acid method was used to quantify polysaccharides, and the monosaccharide composition of the polysaccharides was determined by gas chromatography. Stepwise discriminant analysis was used to differentiate among the five closely related species based on the chemical composition analysis. This proved to be a simple and accurate approach for discriminating among these species. The results also showed that the polysaccharide content, the amounts of the four low molecular weight compounds, and the mannose to glucose ratio, were important factors for species discriminant. Therefore, we propose that a chemical analysis based on quantification of naringenin, bibenzyl, and polysaccharides is effective for identifying D. officinale.     

A chemometric approach to the quality control of Sutherlandia (cancer bush)

Sutherlandia frutescens (Fabaceae) commonly known as cancer-bush, is a well-known traditional phytomedicine in South Africa used to treat a range of ailments. There is limited information available on the phytochemistry and chemical variation within and between the S. frutescens and Sutherlandia microphylla species complex. This paper aims to elucidate the chemical variation of phytoconstituents (other than the non-protein amino acids) between the two species S. frutescens and S. microphylla and also between the wild and cultivated varieties of S. frutescens. An UPLC–MS analysis in tandem with chemometric analysis has been performed to assess the metabolite content of aerial plant parts obtained from different populations. Principal component analysis (PCA) was performed to observe groupings and trends in the data matrix. An orthogonal partial least square discriminant analysis (OPLS-DA) was performed which resulted in clear groups between the two taxa. Several flavonoid and triterpenoid glycoside derivatives contribute to the quantitative chemotypic variation within and between the species as observed. The identification of these compounds using advanced chromatographic techniques (UPLC–MS) and chemometric analysis leads to a better understanding of the phytochemical variation of Sutherlandia which can aid in quality control of raw material, phytomedicines and commercial herbal products.