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italic>Anoectochilus roxburghii liquid (spray, a hospital preparation of Wu Mengchao Hepatobiliary Hospital of Fujian Medical University) has shown a good clinical treatment effect during the COVID-19 pandemic, but its material basis and mechanism of action are still unclear. In this study, network pharmacology and molecular docking methods were used to predict the molecular mechanism of A. roxburghii liquid against COVID-19, and pharmacodynamic experiments in vitro were conducted to study the interaction between the current targets with clear preventive and therapeutic effects and the key components of A. roxburghii liquid. UPLC-MS and database were used to compare and analyze the active ingredients in the liquid, and 17 potential active ingredients with good drug-like properties were screened by in vivo pharmacokinetics process in SwissADME database. SwissTargetPrediction and GeneCards were searched to find 93 common targets. Cytoscape 3.8.2 software was used to construct the "component-target" network map, and the Metascape platform was used for gene function annotation and pathway enrichment analysis. It was found that the extract could regulate the positive response to external stimuli, inflammatory response, cytokine production and other biological processes by binding the active ingredients such as isorhamnetin, kaempferol, luteolin, quercetin and apigenin to the common targets (NOS3, MPO, MMP3, etc.), and play an anti-COVID-19 role. In the angiotensin-converting enzyme 2 (ACE2) activity inhibition assay, it was found that the stock solution of A. roxburghii liquid (for spray), and the supernatant after removing polysaccharides (mainly containing flavonoids) could to some extent inhibit the activity of ACE2. Crucially, in the experiment of 2019-nCOV-S pseudovirus infecting HEK-293T-ACE2 cells, we found that A. roxburghii liquid may exert anti-COVID-19 effects by blocking the binding of SARS-CoV-S protein to ACE2.
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Based on ultra high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS), a rapid and simultaneous quantitative method for the measurement of seven components (kinsenoside; rutin; kaempferol-3-O-rutinoside; quercimeritrin; narcissin; isorhamnetin-3-O-glucoside; quercetin) of A. roxburghii was established. The separation was performed over 8.0 minutes on a Waters Acquity UPLC BEH Shield RP18 (2.1 mm × 100 mm; 1.7 μm) with a mobile phase consisting of acetonitrile (A) and 0.1% formic acid water solution (B) with gradient elution at a flow rate of 0.2 mL·min-1; the column temperature was 30 ℃ and the injection volume was 2 μL. Electrospray spray ionization source (ESI source) was used for mass spectrometry, and positive and negative ion modes were detected at the same time. The results showed good linearity (R2 ≥ 0.998 0), with good precision, repeatability and stability, and the average recovery was 97.71%-103.33%. Through cluster heat map and redundancy analysis, we found that kinsenoside was mainly distributed in stems, followed by leaves, and the lowest content in roots. The content of kinsenoside increased significantly in the stems of plants 6 months, but less change was evident in the roots and leaves. Flavonoids and flavonoid glycosides were mainly distributed in leaves. The UHPLC-MS/MS method established in this paper can be used for the quality control of A. roxburghii and provides a reference for establishing a more comprehensive quality detection method for this medicinal.
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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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italic>Anoectochilus roxburghii (Wall.) Lindl. is a medicinal species belonging to the Orchidaceae, whose whole plant can be used as a medicinal herb, known as "JinXianLian". It has antidiabetic, liver-protecting, anti-inflammatory, etc. A. roxburghii has long been used as food and medicine in Guangdong and Fujian provinces. With the wide recognition of the concept of "medicine and food homology" and the surge of market demand, wild A. roxburghii has been far from meeting the supply. It is important to establish an artificial propagation system. Resource characteristics are the key basis for optimizing germplasm and propagation systems. Therefore, this paper summarizes the germplasm resource characteristics and propagation technologies of A. roxburghii in China to provide a reference for sustainable development and subsequent mechanistic research.
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BACKGROUND: There is a large amount of industrial wastewater produced by the mushroom industry during the canning processing each year, which could provide abundant carbon, nitrogen and inorganic salts for microbial growth. The aim of this study was to optimize the culture conditions for Bacillus licheniformis cultured in the Agaricus bisporus industrial wastewater to produce the agricultural microbial fertilizer. RESULTS: In this work, the maximal biomass of B. licheniformis could be obtained under the following culture conditions: 33.7°C, pH 7.0, 221 rpm shaking speed, 0.5% wastewater, 2 (v:v, %) inoculum dose, loading liquid of 60 mL/250 mL and a culture time of 24 h, and the average experimental value obtained was 1.35 ± 0.04 × 109 Obj/mL, which was within the 95% confidence interval of the predicted model (1.291.38 × 109 Obj/mL), and met the national microbial fertilizers' standard in China. Furthermore, the field experiment results showed that the fermentation broth of B. licheniformis could significantly improve the yield of Anoectochilus roxburghii. CONCLUSIONS: Agaricus bisporus industrial wastewater can be used to produce agricultural microbial fertilizer.
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Orchidaceae/physiology , Fertilizers/microbiology , Bacillus licheniformis/physiology , Agaricus , Fermentation , Wastewater , Flow Cytometry , Hydrogen-Ion Concentration , Industrial WasteABSTRACT
Objective To optimize the process of ultrasonic extraction of polysaccharide in Anoectochilus roxburghii and to investigate the method of protein removal. Methods The extraction rate of polysaccharide was used as the detection index. On the basis of single factor investigation, Box-Behnken experimental design and response surface method were used to optimize the three factors of material-liquid ratio, ultrasonic time and ultrasonic extraction temperature. The five deproteinization methods including Sevage reagent method, TCA method, salt method (NaOH-CaCl2 and NaOH-NaCl) and hydrochloric acid method were investigated with the retention rate of polysaccharide and protein removal rate. Results The optimal extraction conditions of polysaccharide from Anoectochilus roxburghii were as follows: liquid-to-solid ratio was 10∶1, extraction temperature was 48 ℃ and extraction time was 36 min with extraction 2 times, ultrasonic power was 300 W, the extraction rate was 13.13%. NaOH-CaCl2 deproteinized methods∶ the loss rate of polysaccharide was 18.74%, and the removal rate of protein was 95.62%. Conclusion Ultrasonic extraction is easy to operate, and the optimized extraction method can achieve a high extraction rate. NaOH-CaCl2 deproteinization methods can get high protein removal rate and polysaccharide retention rate. This method is suitable for the research and development of the active components of the polysaccharides from Anoectochilus roxburghii.
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@#Transcriptome sequencing was performed for the first time on Anoectochilus roxburghii(AR)in different harvesting periods using RNA-seq high-throughput sequencing technique, and the results were verified and analyzed by Q-PCR and HPLC. A total of 51, 370 genes were obtained by transcriptome sequencing and annotated to the database of Nr, GO, Swiss-Prot, KEGG and KOG. The species that were sequenced according to the homology sequence were the same as AR monocotyledon plants. Through comparison of AR transcriptome in different periods, it was found that the differences were mainly in flavonoid biosynthesis-related genes. The expression levels of flavonoid biosynthesis-related genes(trans-cinnamate 4-monooxygenase, caffeoyl-CoA O-methyltransferase, chalcone synthase, flavonol synthase, shikimate O-hydroxycinnamoyltransferase and flavonoid 3′, 5′-hydroxylase)were verified by Q-PCR, and the results were consistent with those of transcriptome sequencing. The contents of 6 flavonoids(rutin, isoquercitrin, narcissin, quercetin, kaempferol and isorhamnrtin)were determined by HPLC. The results showed that the expression of flavonoid synthetic gene in AR increased with the growth time, and the variation trend of flavonoid compound content and gene expression were basically consistent. Combined with transcriptome data, the biosynthetic pathway of flavonoid content in AR was plotted. This study provides important genetic resources for the key genes of flavonoid synthesis in AR and the biosynthesis of flavonoids, as well as the basis for the development of its medicinal value.
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Objective The aim of this study is to explore the hypoglycemic effect of active components of Anoectochilus roxburghii on zebrafish models. Methods Anoectochilus roxburghii components were extracted and separated into three groups: the alcohol extraction group, macromolecular polysaccharide group (≥ 5 ×103) and small molecular polysaccharide group (＜5×103). Zebrafish embryos were exposed to 2% glucose solution (2% Glu) at 24 h to imitate acute hyperglycemia phenotype, and then treated with the three Anoectochilus roxburghii components. Based on this high-glucose model, the zebrafish embryos at 72 h were collected to detect the whole tissue glucose value. Furthermore, semi-quantitative PCR and whole mount in situ hybridization were performed to detect the expression of mRNA levels of glycometabolism-related genes. Results An acute diabetic zebrafish model was induced by high glucose stress. In this model, some key factors during glycometabolism such as insulin, pck-1 and pdx-1 were significantly affected, while the alcohol extracts of Anoectochilus roxburghii obviously reversed these abnormalities induced by high glucose stress, even to normal levels. Conclusions The alcohol extracts of Anoectochilus roxburghii has obvious hypoglycemic effect on diabetic zebrafish model. Our result suggest that Anoectochilus roxburghii has a potential application in the hypoglycemic drug screening.
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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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Anoectochilus roxburghii is a traditional Chinese medicine and natural health products. In the modern cultivation system, A. roxburghii is micropropagated in tissue culture, and the plants are transferred to soil cultivation for months. However, it remains unclear about the necessity of soil cultivation for the accumulation of health beneficial compounds. In this paper, we performed nontargeted metabolomic analysis using GC-TOF-MS and UPLC-Q-TOF-MS, on A. roxburghii plants at tissue culture stage or after 3 months of soil cultivation. The results showed that the primary metabolites such as alcohols and organic acids are abundant in the tissue culture plants. In contrast, polysaccharide, nucleoside, esters and secondary metabolites such as flavonoids, terpenoids were significantly accumulated in cultivated seedlings. Flavonoids and polysaccharides are considered as the principle effective components in A. roxburghii. Soil cultivation period is therefore essential for the accumulation of these metabolites.
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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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The current status of the domestic manufacturing and sales markets of Anoectochilus roxburghii were investigated and analyzed in the study. Some problems in the A. roxburghii industry were revealed and a variety of sustainable development countermeasures were also proposed. The main problems of A. roxburghii industry are the lack of protection for wild resources, the lag in the speed of variety breeding, the insufficient research on the quality systems, the low level of industry and product innovation capability, as well as the relatively low market cognition and brand competence. Therefore, strengthening the protection for breeding resources, establishing a dynamic monitoring system, promoting the variety breeding, constructing a propagation system for improved varieties, enhancing the quality of medicinal herbs, accelerating the adjustment of product structure, upgrading the industry technology, strengthening brand competence and expanding the market, will be the effective methods to realize the sustainable development of A. roxburghii industry.
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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 study the hypoglycemic effect and mechanism of n￣butyl alcohol extraction from Anoectochilus roxburghii. Methods The total of 12 normal rats were used as normal control group, and 48 diabetic model rats were divided into model control group, and high dose, middle dose and low dose treatment groups with 12 rats in each group,three treatment groups received Anoectochilus roxburghii at dose 600,400,200 mg.kg-1 , respectively, normal control group and model control group were administered with equal volume of 0.9% sodium chlorade solution once a day.After 21 days of drug administration, the weights of rats, the levels of blood glucose and insulin were examined. Results Compared with normal control group, the body weight in model control group decreased significantly(P<0.01), while blood glucose significantly increased (P<0.01).Compared with model control group, the body weight in Anoectochilus roxburghii groups exhibited no obvious difference,while blood glucose decreased significantly (P<0.01 or P<0.05).The level of insulin in high dose group increased remarkably (P<0.05). Conclusion The n￣butyl alcohol extraction from Anoectochilus roxburghii shows markedly hypoglycemic effect,specifically for which at high dose increases the level of insulin significantly,its mechanism may involve with promoting β cells regeneration.
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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 establish a method for the determination of rutin in Anoectochili roxburghii. Methods:The detection was carried out by HPLC. A Lanbo-Kromasil C18 column(250 mm × 4. 6 mm,5 μm) was used with methanol-0. 2% phosphoric acid solu-tion (40∶60) as the mobile phase;the detection wavelength was 257nm, the flow rate was 1. 0 ml·min-1 and the column temperature was 30℃. Results:Rutin showed a good linear relationship within the range of 0. 021-0. 433 μg(r=0. 999 6). The average recovery was 96. 65%(RSD=1. 67%, n=9). Conclusion:The method is simple, rapid, specific and reproducible.