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OBJECTIVE：To identify Amomum villosum from different habitats and its adulterants. METHODS ：Through the identification methods of microscopic characteristics ，microscopic characteristics maps of 9 batches of A. villosum from genuine producing areas ，domestic commercially available A. villosum and its adulterants were obtained. The feature maps were extracted digitally and analyzed by SPSS 21.0 software. RESULTS ：Commercially available A. villosum was mainly from Guangdong ， Guangxi，Yunnan and Fujian ；the collected adulterants of A. villosum included A. villosum Lour. var. xanthioides T.L.Wu et Senjen ， A. aurantiacum H. T. Tsai et S. W. Zhao and other A. species from Yunnan Xishuangbanna ， Laos and Myanmar. Under the microscope，it was observed that microscopic characteristics of surface （such as exocarp color ，prickle，non-glandular hairs ， endocarp color ，endocarp oil chamber ） of A. villosum from different habitats and its adulterants were different. There was statistically significant difference in fruit width values and endocarp oil point diameter among all samples （P＜0.05）. CONCLUSIONS：The microscopic characteristics maps of A. villosum from different habitats and its adulterants by the microscopic characteristics identification methods will make up for the deficiency of traditional experience identification. The quantitative analysis of micro-property and the establishment of micro-property database of A. villosum can provide reference for the property identification and quality control of this medicinal material.
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Objective: To study the characteristics of endophytic fungi separated from the roots of Amomum villosum grown in Jinping County of Yunnan Province, including the culture, evaluation of phosphorus-solubilizing ability and taxonomic identification of target strains. Methods: Endophytic fungi in the roots of A. villosum were separated by culturing in the mediums of PDA and MEA, and purely cultured in PDA. The endophytic fungi with phosphorus-solubilizing ability were screened by solid and liquid mediums of Pikovaskaia’s (PVK) prepared with inorganic phosphorus source. Then, the phosphorus-solubilizing capacity and reasonable mechanism were analyzed by growth circle, biomass, effective phosphorus content, pH value, and phosphatase activity. Moreover, molecular identification of target strains with the capacity of phosphorus-solubilizing would be carried out by ribosome 18 S PCR amplification. Results: The results showed that 24 endophytic fungi were separated from the roots of A. villosum in total, 10 of which were dark septate endophytes (DSE). Eight strains could grow on PVK solid medium and produce phosporus-dissolved growth circle. The growth circle diameters of JP-20 and JP-23 were larger than others, and more than 9 cm, followed by JP-15 with the growth circle of 6.06 cm. Furthermore, it was shown that JP-23 had a strong ability of phosphate-solubilizing due to presenting a high biomass in the PVK liquid medium instead of the medium prepared by soluble phosphorus source. The content of effective phosphorus of JP-23 in PVK liquid medium was significantly increased with an obviously decreasing pH and a sharply rising of acid phosphatase (ACP) activity. Moreover, the strain JP-23 was preliminarily identified as Cladosporium sp. (GenBank: MK629004) by molecular identification. Conclusion: An assumption was concluded that strain JP-23 could decompose and use insoluble phosphorus sources by adjusting the pH value and secreting ACP in medium. Our findings would provide data support and theoretical basis for studying the phosphate absorption mechanism of plant-microbial symbiosis system and the ecological plantation of A. villosum.
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Objective: Amomum villosum (AV) is an herb whose dried fruit has been extensively used in modern medicine to treat digestive system diseases such as dysentery, vomiting and abdominal pain. This paper aims to supplement chloroplast (cp) genomic resources and to be used in phylogenetic studies and identification of AV related plants. Methods: High-throughput sequencing technology was used to determine the complete sequence of the AV cp genome, and the sequence was then compared with three related species. Results: The genome size of AV we obtained was 163,968 bp with an obvious tetrad structure. The AV cp genome was observed to contain 125 unique genes and 81 simple sequence repeat (SSRs) had been determined and the majority of which were adenine–thymine (AT)-rich. Comparative analysis of genome sequence of four ginger plants showed that the atpF, clpP and rpl32 genes are potential markers for identifying Amomum species. Phylogenetic analysis suggested that AV was closely related to A. kravanh and A. compactum. Conclusion: These results have brought useful genetic resources for further identification researches, DNA barcoding, resolving taxonomy and understanding the evolutionary mode of Zingiberaceae cp genome.
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Objective::To study the forming process of the gynandrium-like in Amomum villosum. Method::The flowerets were divided into 8 growth periods from 0.5 cm in length to the day after flowering. Fresh sample were anatomized, and paraffin sectioning was performed on the flowerets. The height of anther chamber, the pollen sac angles, the width of anther gap, the diameter of style, the filament-labellum angle (α), and the filament-anther angle (β) were determined. Result::The angle of the pollen sac had no obvious change before flowering, but decreased from 32° to 17° after flowering. The width of anther gap increased to 0.29 mm in the 5th growth period, while the diameter of style was 0.32 mm in the same period, the ratio of them was 92%. Compared with the day before flowering, the angle α decreased from 83° to 42° during flowering, and the angle β decreased from 186° to 147°. In the filament, the abaxial side had 1 to 5 layers of cells more than the adaxial side. In the style, it was found that the adaxial side had 1 to 6 layers of cells more than the abaxial side. Conclusion::The asymmetry of the cell structure at abaxial and adaxial sides of the filament and style is the basis of the movement. In the 5th growth period, the width of anther gap increased almost to the size of style, so the style was able to slide in. When blossoming, the pollen sacs quickly squeezed to the gap in middle, and the entrance for style to access was blocked. Therefore, the style had to remain in the gap of the pollen sacs. Meanwhile, angles α and β drastically decreased, resulting in the stamen sandwiched the pistil and bending together toward the labellum. The gynandrium-like structure was formed.
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Objective: To set up a callus induction system for Amomum villosum by tissue culture. Method: The rhizome buds of A. villosum and stem segments,root tip segments of sterile A. villosum plantles were used as explants and cultured in MS media with different concentrations of 6-BA,NAA and 2,4-D (the pH of each medi is about 5.8). A callus induction system was established to explore the effect of different explants and different medium on callus induction for A. villosum. Result:The findings showed that the rhizome buds and sterile plantlet stems and root tip segments of three different explants can be successfully induced into calli. The most suitable medium for callus induction from rhizome buds and sterile plantlet stems was MS with 6-BA (1.5 mg·L-1),2,4-D (1.0 mg·L-1) and NAA (0.5 mg·L-1) with the highest induction rates of 15% and 60% respectively. MS medium combined with 6-BA (2.0 mg·L-1),2,4-D (1.0 mg·L-1) and NAA (1.0 mg·L-1) was the most suitable proposal for inducing the callus from sterile root tip segments with the highest induction rate of 76%. Conclusion:Under certain culture conditions,rhizome buds,stem or root tip segments of sterile plantlet can be effectively induced into callus. The callus induction system of A. villosum is preliminarily established, and root tip segments of sterile plantlet are the optimal explant.
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Objective: The transcription factor AvMYC4b selected from transcriptome databases, which might be closely related to the terpene biosynthesis, was cloned from Amomum villosum for sequence analysis and prokaryotic expression. Methods: Based on the transcriptome data of A. villosum, specific primers were designed to obtain the AvMYC4b core sequence. In this study, the whole cDNA sequence of AvMYC4b was obtained by RACE method, then the GATEWAY TOPO cloning vector, and the express vector pDEST17 were constructed by ligation and LR method, respectively, and prokaryotic protein expression was performed. Induced by IPTG and arabinose, the recombinant protein AvMYC4b was successful expressed at the temperature of 16 ℃. Collected bacteria were processed through lysis, ultrasound and purification, and were used to determine the protein expression by SDS-PAGE. Results: AvMYC4b cDNA gene had 2 579 bp, including 165 bp 5'UTR, 1 995 bp ORF, and 389 bp 3'UTR, which encoded a deduce protein of 644 amino acid with a calculated molecular weight of 72 211. Bioinformatics analysis indicated that AvMYC4b had the conserved domain of transcription factor MYC family and predicted that AvMYC4b could be located in nucleus. The SDS-PAGE result showed that the AvMYC4b protein was expressed in Escherichia coil with a molecular mass of about 80 000, which was consistent with the predicted molecular weight. Conclusion: AvMYC4b gene of the bHLH family was cloned from A. villosum and had the whole ORF. The recombinant AvMYC4b protein also was successful expressed in Escherichia coil Rosetta (DE3). Therefore, this study could provide fundamental information for the function characterization of AvMYC4b in terpene biosynthesis pathway of A. villosum.
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In order to set up a technical standard for planting Amomum villosum in wood forest in the future, we analyzed the relationship between the ecological factors and the yield of A. villosum planted in five Dimocarpus imocarpus longan wood forests and five miscellaneous wood forests in Yangchun city, to find out the dominant factors that affect the yield of A. villosum. The results showed that agricultural measures of fertilization, artificial irrigation and removing the old plants were positively correlated with the yield of A. villosum, the pesticide spraying and soil pH value were negatively correlated with the yield of A. villosum. But the effects of ecological factors on the yield were not significantly. High yield regions are generally located in the ravine, two sides of mountain stream and other places where water is more adequate. The slope of cultivated field with high yield is generally less than 30°, lighting and ventilation are more appropriate; soil type is generally sandy or loam, shade density is generally about 50%, and pollinators are many in quantity and variety. And we found that there was a large difference in mineral nutrient contents of soils among ten plantations. Results indicate that the yield of A. villosum is determined by the combination of each ecological factor. Suitable light intensity, moisture, ventilation and reasonable fertilization are conductive to increase the yield of A. villosum, but the use of pesticides and soil alkalization hinder the increase of A. villosum production. Too high shade density and the abuse of pesticides may be the main reason for limiting the yield of A. villosum planted in D. longan wood forests. This study has obtained key techniques of the ecological stereoscopic cultivation mode of A. villosum-D. longan, which lays a theoretical foundation for the guidance of farmers in planting A. villosum in the D. Longan forest in the future.
Sujets)
Agriculture , Méthodes , Amomum , Écologie , Forêts , Sol , EauRésumé
"Amomum Villosum Lour is to keep to hot flavour and warm, oiliness, sharp, light with dry quality. It contains abundant amount of Bornyl acetate, Flavonoid, Polysaccharide, organic compound. In Mongolian medicine, Amomum Villosum Lour have many pharmacological effects such as to heal renal disease with cold quality, remove accumulated Qi in renal and heart, anti vomiting, improve appetite etc. Also in Chinese medicine mainly used to remove dampness to improve appetite, warm spleen to treat diarrhia, regulate Qi to prevent miscarriage etc. This research is to summarized the research of chemical major compound and research of pharmacological effect systematically on the basis of reviewing every researched aspects of Amomum Villosum Lour and it will provide theoretical evidence for further research of Amomum Villosum Lour."
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OBJECTIVE: To establish the HPLC fingerprints of Amomum villosum Lour., Amomum villosum Lour. var. xanthioides T. L. Wu et Senjen and Amomum longiligulare T. L. Wu and find their differences. METHODS: The samples were extracted with 75% ethanol aqueous and then analysis was carried out on an Agilent ZORBAX SB-Aq C18 column with the mobile phase consisting of methanol (A) and 0.05% formic acid solution (B). Gradient elution (0 min, 5% A; 5 min, 5% A→15% A; 10 min, 15% A→26% A; 20 min, 26% A→40% A; 45 min, 40% A→70% A; 58 min, 70% A→100% A, 63 min, 100% A) was carried out at the flow rate of 1.0 mL·min-1. The column temperature was maintained at 30℃, and the detection wavelength was set at 263 nm. The software "Similarity Evaluation System for Chromatographic Fingerprint of TCMs (Version 2012.0) " was employed to generate the mean chromatogras and carry out the similarity analysis of the samples. SPSS21.0 was employed to carry out the cluster analysis. RESULTS: The HPLC fingerprints of the three varieties were different according to fingerprinting and cluster analysis. Amomum villosum Lour. var. xanthioides T. L. Wu et Senjen was obvilously differernt fromAmomum villosum Lour. and Amomum longiligulare T. L. Wu. There were 25 common peaks in the former HPLC fingerprint and 29 common peaks in the latter. CONCLUSION: The HPLC fingerprints of three kinds of Amomum villasums were set up for the first time and they provide reference for the identification and quality control of Amomum villosum Lour., Amomum villosum Lour. var. xanthioides T. L. Wu et Senjen, and Amomum longiligulare T. L. Wu.
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In recent years, with the price rise of Amomum villosum, the quality of A. villosum in the market has been in disorder. To understand the quality status of A. villosum in the market and provide reference for the commercial size fifty-seven samples were collected from different producing areas or markets from August 2013. The samples were detected with evaluation on appearance quality, determination of the contents of bornyl acetate, determination of pesticide residues and heavy metals residues based on Chinese Pharmacopoeia 2015. The results showed that the pesticide residues and heavy metals residues met the requirments, all the samples from different producing areas were qualified except one sample from Fujian province. The qualified rate of native products and imports products samples from market were 43.75% and 14.29%, respectively, the qualified rate of the samples of Yunnan province from producing areas was higher than that from the market. There are two ports at the national level in Yunnan province, where the southern herbs from. A. villosumis one of import medicines from Southeast Asia, and lots of A. villosum samples import to China from Yunnan ports. Most of pharmacists believed that all of the samples from Yunnan province produced in Yunnan. The great majority of commercial species was A. villosum, but A. longiliglare was scarce. Through the survey, it isfound that the main factors affecting the quality of Amomi Fructus was source, lots of A. villosum samples have been replaced by the Amomi Fructus, so the source of imports Amomi Fructus was not clear, which was also more difficult to identify. The quality of A. villosum needs to protect, optimize germplasm, strict control of medicinal sources, specification for medicinal harvesting and processing technology.
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Objective To explore the seed protein extraction method for Amomum villosum Lour., so as to lay the foundation for the study of differential proteomics of Amomum villosum Lour. during seed dormancy and germination. Methods We used the four kinds of commonly-used protein extraction methods for the experiment, including Tris-HCl extraction method, trichloroacetic acid (TCA) /acetone precipitation method, direct lysis buffer method and improved Tris-saturated phenol method. The obtained proteins were quantified, and then were analyzed by sodium dodecyl sulphate- polyacrylamide gel electrophoresis (SDS-PAGE) . Results The protein yield was the highest when using direct lysis buffer method, which was mainly composed of small molecule protein. The protein yield was the lowest using Tris/HCl method, characterized by the diffused stripes in SDS-PAGE spectra. The protein yield was moderate using TCA/acetone method and improved Tris-saturated phenol method, and the obtained protein by the TCA/acetone method had less stripes in SDS-PAGE spectra than that by Tris-saturated phenol method, and was characterized by macromolecular protein. Conclusion The combination of direct lysis buffer extraction method and improved Tris-saturated phenol method may provide reference for seed protein extraction method of two-dimensional electrophoresis for Amomum villosum Lour..
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Objective: To explore the pharmacological activities of volatile oil in new hybrids of Amomum villosum (Spring No.1) and compare to the female parent "Longfruit No.2". Methods: The auricle swelling model of mice was induced by xylene, the diarrhea model of mice was induced by 10% Senna, the writhing body model of mice was induced by 0.6% acetic acid solution to observe the pharmacological actions of volatile oil in "Spring No.1". Results: The volatile oil in both "Spring No.1" and "Longfruit No.2" had significant antidiarrheal effect, their effect was equivalent to the positive drug (Montmorillonite Powder). There was no significant difference in anti-inflammatory effect compared with the control group, the analgesic effect was significant. Both of them had the bidirectional regulation on gastric emptying. High dose of them had the stimulative effect in the small intestinal propulsion, and low dose of them had no significant effect. Conclusion: There is no significant difference in pharmacological activity between the new hybrid species "Spring No.1" and its female parent. It could be comparable to the traditional cultivars of A. villosum. This study provides a scientific basis for expanding A. villosum medicinal resources.
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Objective To compare the contents of main compounds and the composition of volatile oil from the new hybrid of Amomum villosum Lour. Spring No.1-F4 with those in its female parent Longfruit No.2. Methods Steam distillation, gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) methods were used for analyzing the content differences of main compounds and compositions of volatile oil between Spring No.1-F4 and Longfruit No.2. Results The contents of volatile oil and bornyl acetate from Spring No.1-F4 and Longfruit No.2 met the standard required by Chinese Pharmacopoeia. The content of volatile oil from Spring No.1-F4 was higher than that of Longfruit No.2, and there were some differences between the two in the composition of volatile oil and their relative contents. Conclusion The effective constituents in Spring No.1-F4, a new hybrid species of Amomum villosum, has reached the standard required by Chinese Pharmacopoeia, which is expected for solving the problems of difficulty in pollination and for improving the yield and quality of Amomum villosum.
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Objective: To clone the full length cDNA encoding diketide CoA synthase (DCS) gene which plays an important role in curcumin biosynthesis pathway in Amomum villosum, and to provide the basis for the further studies on biosynthesis and gene regulation of curcumin. Methods: According to the annotation of root transcriptome of A. villosum, primers were designed and cDNA of DCS gene was cloned from A. villosum by PCR. Results: The complete coding sequence of DCS gene was 1 170 bp and it encoded a protein of 389 amino acids. The deduced DCS amino acid sequence exhibited 96% identity to the DCS of Curcumae Longae and 66%-69% identity to the chalcone synthase (CHS) of Narcissus tazetta. Phylogenetic analysis on the amino acid sequence of DCS with those of other plants showed that DCS was closely related to Curcumae Longae. Conclusion: The DCS gene is cloned from A. villosum for the first time. This research lays a foundation for studying the gene expression pattern and regulatory functions of DCS in curcumin biosynthesis.
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Objective: To study the identification between the Amomum villosum hybrid offspring and the female parent by useing the Fourier transform infrared (FTIR) spectroscopy. Methods: Using the FTIR to determine the infrared spectra of A.villosum hybrid offspring and female parent. Switching and processing the atlas with derivative spectrometry and Fourier self-deconvolution, to contrast for the characteristic differences in absorbance of them. Results: There were no significant differences between the A. villosum hybrid offspring and female parent by using FTIR. But 1 051.014 cm-1 was found to be the most obvious in the fourth-derivative spectrum, 771.563 4 and 1 612.432 5 cm -1 to be the most obvious in the Fourier self-deconvolution spectra. Conclusion: There are obvious differences between the A. villosum hybrid offspring and female parent in the derivative spectrometry and Fourier self-deconvolution spectroscopy atlas. Therefore, this method can be used to identify the A.villosum hybrid offspring and female parent simply, rapidly, and accurately.
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[Objective] To observe the influence of different kinds of biological pesticide on the fruiting rate and yield of Amomum villosum Lour. (AVL) . [Methods] Medicinal plant field experiment method was used to observe the influence of biological pesticide A (highly effective immuno-biologic bactericide, 75.76mg?m-2 for one time) and its 1000-fold, 800-fold and 500-fold diluent on the fruiting rate and yield of AVL. Meanwhile, the influence of biological pesticide B (edible oligosaccharide, 30.30mg?m-2 for one time) and biological pesticide C (deguelin emulsion, 1.89mg?2m-2 for one time) was also observed. [Results] The three biological pesticides, as well as the diluents of biological pesticide A increased the fruiting rate and yield of AVL, the influence of the moderate-concentration biological pesticide A being the greatest. [Conclusion] Proper application of biological pesticide is one of the important ways to raise the yield of AVL.
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[Objective] To compare and identify the different cultivated varieties of Amormun villosum for providing scientific evidence for the selection, breeding, standardization and industrialization. [Methods] Source appraisal, character identification and scanning electron microscope technique were applied to observe and compare the appearance of the original plant, the properties of the medicinal material and the outside characteristics of pollen of Amomum villosum in Yangchun, Guangdong province. [Results] The two cultivated varieties of Amomum villosum in this area, long-fruit and round-fruit types, are quite different in the appearance and the characteristics of pollen. [Conclusion] It is suggested that long-fruit Amomum villosum and round-fruit Amomum villosum should be the selected varieties for breeding.
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[Objective] To observe the effect of biological fertilizers on the yield and quality of Amomum villosum (AV). [Methods] Field experiment method was used to observe the effect of different kinds and different amount of biological fertilizers on AV. Biological fertilizers were: A (mainly composed of fermented chicken manure), B (mainly composed of fermented filter of sugarcane) and C (mixed with fertilizer A and biological fertilizer at certain proportion). The fertilizer amount was 150, 112 and 75 g****m-2 per time. Content of bornyl acetate in AV was detected by gas chromatography. [Results] The three kinds of biological fertilizers increased the yield of AV but had no obvious effect on its quality; high- and moderate-amount fertilizer increased the yield of AV, in particular fertilizer A (112 g?m-2 per time). The three kinds of fertilizers had no effect on the content of bornyl acetate in AV. [Conclusion] Reasonable application of fertilizers is one of the important ways to raise the yield of AV.
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Objective To optimize the soaking treatment method for increasing the germination of Amomum villosum Lour.(AVL) seeds.Methods Nine groups were set up: the control group(without soaking treatment),treatment group 1(soaking AVL seeds with naphthylacetic acid 15 mg/mL for 8 hours),treatment group 2(soaking with gibberellin 100 mg/L for 30 hours) and treatment groups 3~8(soaking with clean water for 8,20,30,40,45 and 50 hours respectively).The sowing amount was 100 grains in each group for each time,and the seeds were planted in the outdoor pot at a planting space of 4cm?4cm.Results Naphthylacetic acid had an obvious inhibition on the germination of AVL seeds and delayed the shooting.Gibberellin promoted the germination and shooting of the seeds.Soaking with clean water for 8 hours had no obvious effect on the seed germination,but soaking for 20~50 hours increased the seed germination to various degrees,in particular soaking for 30~50 hours.Conclusion Soaking with gibberellin or clean water can increase the germination of AVL seeds.This method is simple and practical and economic,and is worth of extensively applying in the sowing and breeding of AVL seeds.
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Objective An investigation of new cultivars of Amomum villosum Lour.(AVL) with high yield and good quality was carried out,thus to supply evidences for the identification of AVL cultivars in accordance with the morphological features of their flowers and fruits.Methods An investigation of AVL species from the genuine producing areas of Yangchun city of Guangdong province was performed.The morphological features of flowers and fruits of two cultivars(Changguo and Yuanguo) as well as one breeding type(Chunxuan type) were examined.Results Specific and significant features were screened out in different cultivars of AVL.Conclusion There exit specific features in flowers and fruits of different cultivars of AVL from Yangchun.