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OBJECTIVE@#Diterpenoids with a wide variety of biological activities from Anoectochilus roxburghii, a precious medicinal plant, are important active components. However, due to the lack of genetic information on the metabolic process of diterpenoids in A. roxburghii, the genes involved in the molecular regulation mechanism of diterpenoid metabolism are still unclear. This study revealed the complex metabolic genes for diterpenoids biosynthesis in different organs of A. roxburghii by combining analysis of transcriptomics and metabolomics.@*METHODS@#The differences in diterpenoid accumulation in roots, stems and leaves of A. roxburghii were analyzed by metabonomic analysis, and its metabolic gene information was obtained by transcriptome sequencing. Then, the molecular mechanism of differential diterpenoid accumulation in different organs of A. roxburghii was analyzed from the perspective of gene expression patterns.@*RESULTS@#A total of 296 terpenoid metabolites were identified in the five terpenoid metabolic pathways in A. roxburghii. There were 38, 34, and 18 diterpenoids with different contents between roots and leaves, between leaves and stems, and between roots and stems, respectively. Twenty-nine metabolic enzyme genes with 883 unigenes in the diterpenoid synthesis process were identified, and the DXS and FDPS in the terpenoid backbone biosynthesis stage and CPA, GA20ox, GA3ox, GA2ox, and MAS in the diterpenoid biosynthesis stage were predicted to be the key metabolic enzymes for the accumulation of diterpenoids. In addition, 14 key transcription factor coding genes were predicted to be involved in the regulation of the diterpenoid biosynthesis. The expression of genes such as GA2ox, MAS, CPA, GA20ox and GA3ox might be activated by some of the 14 transcription factors. The transcription factor NTF-Y and PRE6 were predicted to be the most important transcription factors.@*CONCLUSION@#This study determined 29 metabolic enzyme genes and predicted 14 transcription factors involved in the molecular regulation mechanism of diterpenoid metabolism in A. roxburghii, which provided a reference for the further study of the molecular regulation mechanism of the accumulation of diterpenoids in different organs of A. roxburghii.
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Objective To study the quality standard of the imitation ecological planting Anoectochilus roxburghii under forest covering. Methods The identification of medicinal properties, microscopic characteristics, thin-layer chromatography (TLC) and content determination of A. roxburghii cultivated under forest were carried out in this study, and the moisture content, ash and acid-insoluble ash were determined according to Chinese Pharmacopoeia of 2015 edition. Results A. roxburghii cultivated under forest exhibits specific properties in characteristics, microscopic features and TLC results. The average moisture content of A. roxburghii cultivated under forest from five planting bases was 8.69%, the average ash was 11.93%, and the average acid-insoluble ash was 3.27%. Content determination results of the average quality score of quercetin, isorhamnetin and kaempferide were at 0.021 0%,,0.024 7%, and 0.027 3%, respectively. Conclusion The above method is simple, specific and reproducible, which will provide the basis for the quality standard of the imitation ecological planting A. roxburghii under forest covering.
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Objective: To compare the contents of mineral elements and amino acids in different germplasms of Anoectochilus roxburghii. Methods: The contents of amino acids and mineral elements in 11 different germplasms of A. roxburghii were determined by automatic amino acid analyzer and atomic absorption spectrometer. Results: The range of essential amino acids in the different species of A. roxburghii was 2.81%-4.47%, and the range of total amino acids was 11.38%-17.06%. Aspartic acid, glutamic acid, and lysine contents were significantly higher than those of other amino acids. The content of potassium was the highest, and the order was: K > Ca > Mg > Fe > Mn > Zn > Cr > Cu > Pb > Cd. The principal components analysis showed that alanine, serine, glutamic acid, aspartic acid, threonine, isoleucine, alanine, phenylalanine, and leucine were characteristic amino acids in A. roxburghii. Fe, Zn, Mn, Ca, Cr, and Mg were characteristic elements of A. roxburghii. Conclusion: There are regional differences of A. roxburghii between amino acids and the content of mineral elements.
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Objective: To study the chemical constituents of Anoectochilus roxburghii. Methods: The chemical constituents of A. roxburghii were separated and purified by silica gel column chromatography, recrystallization, macroreticular resin, polyamide resin, ODS C18, and HPLC. Compound structures were identified by the spectral data and the physical and chemical properties. Results: From this plant, nineteen compounds were obtained, such as (22E, 24ξ)-24-isopropenyl-22-dehydrocholesterol (1), anoectosterol (2), stigmasterol (3), β-sitosterol (4), hircinol (5), moscatin (6), 5, 3'-dyhydroxy-3, 7, 4'-trimethoxyflavone (7), linoleic acid (8), 8, 9-epoxyheptadeca-1, 11, 14-triene (9), acetovanillone (10), parahydroxybenzaldehyde (11), parahydroxyacetophenone (12), 5-hydroxy-3', 4', 7-trimethoxyflavonol-3-O-β-D-rutinoside (13), isorhamnetin-3-O-robinobioside (14), β-adenosine (15), isorhamnetin-3-O-β-gentiobioside (16), loroglossin (17), corchoinoside C (18), and parahydroxybenzylalcohol (19). Conclusion: Compounds 1, 16, and 19 are isolated from Orchidaceae for the first time; Compounds 5, 6, 7, 10, and 12 are isolated from Anoectochilus Bl. for the first time; Compounds 9, 15, and 18 are isolated from this plant for the first time.
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Objective: To reveal the genetic relationship of germplasms of Anoectochilus roxburghii and develop an effective and valuable molecular marker. Methods: In this study the polymorphic random amplified polymorphic DNA (RAPD) and specific SCAR markers were developed based on 20 different germplasms from various places. Results: Twenty-eight 100 RAPD primers have significant polymorphism, generated 135 polymorphic bands among 20 germplasms. On the basis of RAPD results, 20 germplasms were clustered into six groups on genetic distance of 0.748. Conclusion: Clustering analysis shows that there are the significant genetic differences among germplasms derived from different regions. A total of five specific bands from RAPD results are transferred into sequence characterized amplified region (SCAR) markers. Amplified results of SCAR markers among different germplasms show that SCAR markers are significant specific to different germplsms. This study has laid a solid foundation for accelerating the breeding of A. roxburghii.
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Objective: To establish the inductively coupled plasma mass spectrometry (ICP-MS) method for content determination of five kinds of heavy metals (copper, arsenic, cadmium, mercury, and lead) in Anoectochilus roxburghii. Methods: After the treatment of microwave digestion, ICP-MS method was used in the determination. Results: The standard liquid working curves of Cu, As, Cd, Hg, and Pb had good linearity, with the correlation coefficients of more than 0.998. The average recovery of Cu was 104.3% with relative standard deviation (RSD) of 4.0%, the average recovery of arsenic was 102.2% with RSD of 4.3%, the average recovery Cd was 100.5% with RSD of 0.4%, the average recovery rate of Hg was 101.0% with RSD of 3.6%, and the average recovery of Pb was 101.1% with RSD of 1.1%. Conclusion: The method is stable, reliable, fast, and accurate, and has high sensitivity and good reproducibility. It can be used for the simultaneous determination of Cu, As, Cd, Hg, and Pb in A. roxburghii.
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Objective: To provide a new material for exploring the products of Anoectochilus roxburghii, and study the effect of explant lengths, BA concentration and light intensities on multiple shoot biomass and effective material accumulation of A. roxburghii; This paper also explored the feasibility of the bioreactor culture on the rapid proliferation of A. roxburghii. Methods: The optimum explant lengths for multiple shoot proliferation were selected, the effect of different BA concentration and light intensities on multiple shoot biomass and effective material accumulation were investigated. Additionally, the comparison was carried out between bioreactor culture and conventional solid culture for multiple shoot biomass and effective material accumulation of A. roxburghii. Results: Explants (5 mm) promoted multiple shoot proliferation, mutiple shoot biomass was significantly higher than explants (1 mm). MS medium supplemented with 2.5 mg/L BA was optimal for multiple shoot biomass accumulation but the maximum productivities of polysaccharide, flavonoid, and total phenolic were found at 2.0 mg/L BA. Multiple shoot biomass and effective material accumulation were more suitable under light condition than under dark condition. The biomass and production of polysaccharide, flavonoid, and total phenolic (678.3, 12.2, and 17.8 mg/L) of multiple shoot at 5 g/L of inoculation density were significantly better than in solid culture. Conclusion: Multiple shoot biomass and productivities of polysaccharide, flavonoid, and phenolic compound are enhanced when 5 mm explants are inoculated in MS medium supplemented with 2.0 mg/L BA and cultured under light condition. In addition, bioreactors are used for large scale propagation of multiple shoot in less time. The accumulation of biomass and bioactive compound during bioreactor culture is higher than in solid culture.
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Objective: To establish a method for determining the contents of quercetin, kaempferide, and isorhamnetin in Anoectochilus roxburghii and to provide scientific method for quality control of A. roxburghii and its related products. Methods: HPLC was used. Using Lanbo-Kromasil-C18 column, with methanol -0.2% phosphoric acid solution (50:50) as mobile phase; The detection wavelength was 360 nm, flow rate was 1.0 mL/min, and column temperature was 30 ℃. Results: Quercetin, kaempferide, and isorhamnetin showed a good linear relationship in the range of 11.0-165.6 (r = 1.0000), 11.4-170.4 (r = 0.9998), and 10.4-156.0 ng (r = 0.9995). The average recoveries of quercetin, kaempferide, and isorhamnetin were 97.69% (RSD = 1.1%, n = 9), 95.09% (RSD = 1.6%, n = 9), and 95.86% (RSD = 1.7%, n = 9). Conclusion: The method is simple, rapid, accurate, reproducible, and can be used as an ideal method for quality control.
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Objective: To research the pharmaceutical identification of Anoectochilus roxburghii (Wall.) Lindl., A. formosanus Hayata and A. chapaensis Gagnep. Methods: Using the methods of freehand section, microexamination, and digital photography to compare the plant morphology, tissue structures of root, stem and leaf transverse, and dried medicinal powder. Results: The plants of A.roxburghii and A. formosanus were smaller than that of A. chapaensis. The colors of their leaf veins were golden yellow, silver and light red, respectively, which should be the key morphologic identification. As for characteristics of histology, there were more xylem bundles and broad pulp in the root, and more external vascular bundles in the stem of A. chapaensis than the other two plants. The upper epidermal cells of A. formosanus were triangle shape differ to the oval shape in the other two plants. There were few red materials on the upper surface of A. roxburghii. Many red materials on the upper surface of A. formosanus were dispersed existence. The red materials on the upper surface of A. chapaensis showed to concentrate a column. The result showed their chemical compositions were large differences. Conclusion: The large differences on plant morphology and microstructure of the different sources plant were carried out. Our works will provide the basis for the establishment of quality standard of Anoectochilus.
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Objective: To investigate the genetic polymorphism of wild Anoectochilus roxburghii and its allied species by molecular marker and find out the genetic differences between them. Methods: Direct amplification of length polymorphism (DALP) was applied to evaluate the genetic variation of 18 populations of A. roxburghii and its allied species from different places. Results: Six pairs of primers were selected to detect 355 polymorphic loci in 18 populations, the percentage of polymorphic bands (PPB) was 100%, and the amplified average polymorphic locus by each pair of primers was 59.2. The percentage of PPB of 14 populations of A. roxburghii was 95.77%, its observed number of alleles (Na) was 1.2734, effective number of alleles (Ne) was 1.074, Nei's gene diversity (H) was 0.0562, Shannon's information index (I) was 0.0969, coefficient of gene differentiation (Gst) was 0.4230, and estimated gene flow (Nm) was 0.6819. Conclusion: The results indicate that A. roxburghii has larger genetic differentiation and lower gene flow among populations. Geographic isolation and wild resource loss may be the major causes of the limited gene flow.
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Objective: To study the germplasm resources of Anoectochilus roxburghii. and the genetic diversity of its spurious breed. Methods: By ISSR-PCR amplification, 24 samples were PCR amplified, and then electrophoresis detected. After marking the electrophoresis strips with "0" and "1" matrix, the genetic distances were calculated by NTSYSpc software, and the dendrogram was constructed by UPGMA. Results: A. roxburghii, A. formosanus, and A. sp. were identified. A. roxburghii with different geographical distribution and morphology had the close genetic relationship. Conclusion: The wild population of A. roxburghii resources have higher genetic diversity, witch could provide a great genetic library for varieties breeding.
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Object To obtain a clear view on the present status of this rare and precious medicinal herb for its further rational preservation and exploitation Methods A survey was carried out by literature retrieval, interview of veteran folk herbalists and on the spot field inspection Results A deeper understanding on the biological characteristics, ecological environment, growth and reproduction habits and natural distribution were obtained and reported Conclusion Based on the above results, measures for the preservation and exploitation of A roxburghii in the future were suggested