Comprehensive evaluation of the quality of Amomum tsao -ko based on entropy weight TOPSIS method / 中国药房

OBJECTIVE To evaluate the quality of Amomum tsao -ko from different origins and harvesting periods comprehensively. METHODS The contents of total volatile oil in A. tsao -ko were determined by volatile oil measurement method A stated in 2020 edition of Chinese Pharmacopoeia （part Ⅳ）；the contents of total flavonoids and total polyphenols in A. tsao -ko were determined by aluminum nitrate-sodium nitrite colorimetry and folin-ciocalteu method. The contents of α-pinene，β-pinene， 1，8-cineole，α-terpineol，geraniol and trans-nerolidol in the volatile oil of A. tsao -ko were determined by gas chromatography ；the contents of protocatechuate and vanillic acid in A. tsao -ko were determined by ulta high performance liquid chromatography. The above 11 indicators were selected ，and entropy weight TOPSIS method was used to comprehensively evaluate the quality of 16 batches of A. tsao -ko. RESULTS The contents of total volatile oil ，total flavonoids ，total polyphenols ，α-pinene，β-pinene， 1，8-cineole，α-terpineol，geraniol，trans-nerolidol，protocatechuate and vanillic acid in 16 batches of A. tsao -ko were 15.833 3- 28.000 0 μL/g，29.100 5-78.199 6 mg/g，6.789 8-35.797 7 mg/g，0.088 7-0.401 3 mg/g，0.106 3-0.408 0 mg/g，3.709 6-8.533 1 mg/g，0.259 8-0.599 6 mg/g，0.314 8-1.324 1 mg/g，0.272 3-0.576 4 mg/g，9.301 2-19.818 5 μg/g，8.180 9-27.666 3 μg/g， respectively. Entropy weight TOPSIS results showed that the top three of relative closeness rankings were A. tsao -ko produced by Yunnan Baoshan in July ，Yunnan Honghe in October ，Yunnan Wenshan in September ；the last three of relative closeness rankings were A. tsao -ko produced by Yunnan Dehong in September ，Yunnan Dehong in November ，Yunnan Dehong in December. CONCLUSIONS A. tsao -ko produced by Yunnan Baoshan in July ，Yunnan Honghe in October and Yunnan Wenshan in September present better quality.

Chemical constituents and biological activities of essential oils of Amomum genus (Zingiberaceae)

Amomum Roxb. includes the aromatic and medicinal plants native to tropical and subtropical Asia belonging to the family Zingiberaceae. Members of Amomum genus have been used for a long time in traditional medicine for the treatment of throat trouble, congestion of lungs, inflammation of eyelids, and digestive disorders, etc. Amomum essential oils have been studied for their chemical profiles in which limonene, allo-aromadendrene, 1,8-cineole, camphor, farnesyl acetate, α-pinene, β-pinene, caryophyllene, camphene, D-camphor, santolina triene, methyl chavicol, bornyl acetate, β-elemene, δ-3-carene, etc. were the major compounds. Furthermore, the oils extracted from Amomum plants have been reported to possess antimicrobial, antioxidant, insecticidal, larvicidal, cytotoxic, anti-scabies, and anti-inflammatory activities. This review focuses on the chemical constituents and biological activities of the essential oils isolated from the different plant parts of Amomum plants. The objective of the present review is to highlight therapeutic potentials and provide evidence for future medicinal applications of these species of genus Amomum.

Selection of q RT-PCR reference genes for Amomum tsaoko seeds during dormancy release / 中国中药杂志

Freshly collected seeds of Amomum tsaoko demonstrate obvious dormancy. Therefore, the selection of stable reference genes during seed dormancy release is very important for the subsequent functional research of related genes. In this study, ten commonly used reference genes(GAPDH, 40S, actin, tubulin, EIF4A-9, EIF2α, UBC, UBCE2, 60S, and UBQ) were selected as candidates for quantitative Real-time polymerase chain reaction(qRT-PCR) of the embryo samples of A. tsaoko at different dormancy release stages. Three kinds of software(BestKeeper, geNorm, and Normfinder) and the Delta CT method were used to evaluate the expression stability of the candidate reference genes, and the RefFinder online tool was employed to integrate the results and generate a comprehensive ranking. The results showed that the expression levels of the ten candidate reference genes differed greatly in different embryo samples. GAPDH and UBC had high expression levels, as manifested by the small Ct values. GeNorm identified 40S and UBCE2 as the most stable genes. NormFinder ranked EIF2α as the most stable gene and UBC as the least stable gene. UBCE2 was found to be the most stable gene and actin the least stable one by BestKeeper. Delta CT analysis suggested that the expression of 40S was most stable. UBCE2 was recommended as the most stably expressed gene by RefFinder. Thus, UBCE2 is the ideal reference gene for qRT-PCR analysis of A. tsaoko seeds at different dormancy release stages. The results may lay a foundation for analyzing the expression of related genes during seed dormancy release of A. tsaoko.

Optimization of the Extraction Technology of Phenolic Acid from Amomum tsaoko / 中国药房

OBJECTIVE：To opt imize the extraction technology of phenolic acid from Amomum tsaoko . METHODS ：The extraction technology of phenolic acid from A. tsaoko was optimized by using Box-Behnken design-response surface methodology with ethanol volume fraction ，liquid-solid ratio and extraction time as factors ，using the total contents of protocatechuic acid and vanillic acid as response value. The optimizd extraction technology was vlidated. RESULTS ：The optimal extraction technology was as follows ：ethanol volume fraction 65％，liquid-solid ratio 4∶1（mL/g），extraction time 2.5 h. After 3 times of validation tests ， average total content of protocatechuic acid and vanillic acid were 12.32 mg/g（RSD＝0.26 ％，n＝3），average relative error of which with predicted value （12.63 mg/g）was 2.45％. CONCLUSIONS ：The optimal technology is stable and feasible .

Antimicrobial activity of the essential oils from the leaves and stems of Amomum rubidum Lamxay & N. S. Lý / Actividad antimicrobiana de los aceites esenciales de las hojas y tallos de Amomum rubidum Lamxay & N. S. Lý

This paper described the chemical compositions and antimicrobial activity of the essential oils from the leaves and stem of Amomum rubidumLamxay & N. S. Lý, collected from Bidoup Nui Ba National Park, Lam Dong, Vietnam. The essential oils were obtained by hydrodisitllation method while antimicrobial activity was evaluetd by microdilution broth susceptibility assay. The main constituents of the leaf essential oil were identified as 1,8-cineole (37.7%), δ-3-carene (19.5%) and limonene (16.3%) while δ-3-carene (21.9%), limonene (17.8%) and ß-phellandrene (14.6%) dominated in the stem essentialoil. The leaf and stem essential oils displayed stronger inhibition of Pseudomonas aeruginosa with MIC of 25 µg/mLand 50 µg/mL respectively. The stem essential oil was active against Candida albicans (MIC, 50 µg/mL) while both essential oils inhibited the growth of Fusarium oxysporum (MIC 50 µg/mL). This is the first report on chemical constituents and antimicrobial activity of the essential oils of A. rubidum.

Este artículo describe la composición química y la actividad antimicrobiana de aceites esenciales de las hojas y el tallo de Amomum rubidum Lamxay & N. S. Lý recolectados del Parque Nacional Bidoup Nui Ba, Lam Dong, Vietnam. Los aceites esenciales se obtuvieron mediante el método de hidrodisitilación, mientras que la actividad antimicrobiana se evaluó mediante un ensayo de susceptibilidad de caldo de microdilución. Los principales componentes del aceite esencial de la hoja se identificaron como 1,8-cineol (37,7%), δ-3-careno (19,5%) y limoneno (16,3%), mientras que δ-3-careno (21,9%), limoneno (17,8 %) y ß-felandreno (14,6%) dominaron en el aceite esencial del tallo. Los aceites esenciales de hoja y tallo mostraron una inhibición más fuerte de Pseudomonas aeruginosa con un MIC de 25 µg/mL y 50 µg/mL, respectivamente. El aceite esencial del tallo fue activo contra Candida albicans (MIC, 50 µg/mL) mientras que ambos aceites esenciales inhibieron el crecimiento de Fusarium oxysporum (MIC 50 µg/mL). Este es el primer informe sobre los componentes químicos y la actividad antimicrobiana de los aceites esenciales de A. rubidum.

Identification of Microscopic Characteristics of Amomum villosum from Different Habitats and Its Adulterants / 中国药房

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.

Complete chloroplast genome sequence of Amomum villosum and comparative analysis with other Zingiberaceae plants / 中草药·英文版

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.

Study on in vitro Antioxidant Activities of Different Polar Parts of Ethanol Extract from Amomum tsao-ko / 中国药房

OBJECTIVE：To evaluate in vitro antioxidant activities of 4 different polar parts of ethanol extract from Amomum tsao-ko，and to lay a foundation for the research and development of antioxidant chemical components in the plant. METHODS ： The dried fruits of A. tsao-ko were crushed ，then were hearted and reflux extracted with 95％ ethanol. The extraction fluid was concentrated by rotary evaporation and evaporated in water bath to obtain the ethanol extract. The extract was dispersed in water ， and then extracted with petroleum ether ，ethyl acetate and n-butanol organic solvents one by one. Each solvent extract was combined and the lower water phase were collected. Finally ，the petroleum ether part ，ethyl acetate part ，n-butanol part and water part were obtained，after rotary evaporation concentration and water bath evaporation. Through in vitro antioxidant activity tests ，using 2， 6-di-tert-butyl-4-methylphenol（BHT）as positive control ，DPPH radical ，superoxide anion radical scavenging ability and Fe 3+ reducing ability of different polar parts of ethanol extract from A. tsao-ko were investigated. RESULTS ：The scavenging rates of 4 polar parts of ethanol extract from A. tsao-ko on DPPH radical were all over 80％；the order of scavenging ability was ethyl acetate part＞BHT＞n-butanol part ＞petroleum ether part ＞water part. Those of the 4 polar parts to superoxide anion radical were between about 30％-40％ mostly；the order of scavenging ability was n-butanol part ＞petroleum ether part ＞water part ＞ethyl acetate part ＞ BHT；but those were weaker than their scavenging ability to DPPH r adical. The polar parts of ethanol extract also had a certain reduction ability to Fe 3+；the order of the reduction ability was n-butanol part ＞BHT＞ethyl acetate part ＞petroleum ether part ＞ water part on the whole ，but that of water part rose to the stron- gest when its concentration was 4.0 μg/mL. CONCLUSIONS： The different polar parts of ethanol extract from A. tsao-kom have certain in vitro antioxidant capacity ，but the order of antioxidant activity of different polar parts was not the same in different antioxidant activity tests ；ethyl acetate part has the 163.com strongest scave nging effect on DPPH radical ，n-butanol part has the strongest scavenging ability on superoxide anion radical and reducing ability on Fe 3+.

Study on the Optimization of Purification Technology of Total Flavonoids from Amomum tsao-ko by Macroporous Adsorption Resin / 中国药房

OBJECTIVE：To establish a method for the content determination of to tal flavonoids from Amomum tsao-ko ，and to optimize the purification technology by macroporous resin. METHODS ：The content of total flavonoids was measured by HPLC. The determination was performed on Eclipse Plus C 18 column with mobile phase consisted of acetonitrile- 1％ acetic acid solution （15∶85，V/V）at the flow rate of 0.8 mL/min. The column temperature was 40 ℃，and the detection wavelength was set at 256 nm. The sample size was 10 μL. Taking the adsorption and desorption performance as indexes，6 kinds of macroporous resins were screened out by static adsorption and desorption tests ；adsorption and desorption time were investigated by static adsorption and desorption kinetics tests. Using the content of total flavonoids （calculated by rutin ）as index ，with sample concentration ，sample pH，ethanol volume fraction and elution amount as factors ，based on single factor test ，orthogonal design was used to optimize the purification technology of total flavonoids from A. tsao-ko ，and validation test was performed. RESULTS ：The linear range of rutin were 0.028-0.281 mg/mL（r＝0.999 9）. The limit of quantification was 437.5 ng/mL and the limit of detection was 109.4 ng/mL. RSDs of precision ，stability and reproducibility tests were all lower than 2％；the recoveries were 96.24％-99.75％（RSD＜2％，n＝ 6）. The comprehensive capacity of adsorption and desorption of HPD 450 macroporous resin was the most suitable ，and the best static adsorption and desorption time both were 12 h. The optimal purification technology was 1.854 4 mg/mL ； ethanol elution was 8 times of the column volume. Vertificationtests show that after optimized ，the content of total flavonoids from A. tsao-ko increased from 22.556 7 mg/g to 57.728 2 mg/g. The purity of was 2.56 and stable for the content determination. Optimal purification technology is stable and feasible ，which is suitable for purifieation of total flavonoids from A. tsao-ko .

Forming Process of Gynandrium-like in Amomum villosum / 中国实验方剂学杂志

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.

Phosphate-solubilizing function evaluation and taxonomic identification of endophytic fungi separated from roots of Amomum villosum / 中草药

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.

Effect of Amomum subulatum seeds against cypermethrin induced haematological changes in wistar albino rats

Background: Cypermethrin is a well know agricultural pesticide used in the developing countries. It is associated with significant toxic potential on human health. Hence the present study was aimed to evaluate the protective role of Amomum subulatum against cypermethrin induced haematalogical changes in Wistar albino rats.Methods: The albino rats were divided into five different groups of six rats each. Group I considered as normal control, group II cypermethrin control (25mg/kg body weight p.o.), group III only test drug and group IV and V administered with cypermethrin 25mg/kg body weight along test drug 1.08 and 2.16mg/kg body weight for 28 consecutive days. At the end of 28th day blood was withdrawn and total haematalogical parameters were estimated.Results: In the cypermethrin control there was significant reduction in the WBC, Platelet, MCHC and considerable reduction in the haemoglobulin concentration in comparison to normal control. The test drug administered at both dose levels was significantly reversed the cypermethrin induced changes in haematalogical parameters.Conclusions: Authors can conclude that the Amomum subulatum has potency to reverse the cypermethrin induced haematalogical changes.

Callus Induction from Amomum villosum / 中国实验方剂学杂志

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.

Cloning and activity analysis of AvTPS1 promoter of terpene synthase from Amomum villousm / 中草药

Objective To clone the unknown sequence of terpene synthase (TPS) AvTPS1 promoter from Amomum villousm and analyze its activity. Methods In this research, AvTPS1 DNA sequence was amplified and cloned from genomic DNA (gDNA) of A. villousm leaves. Furthermore, the promoter of AvTPS1 was cloned by FPNI-PCR and the sequence was analyzed. The recombinant vector pCAM-AvTPS1p with AvTPS1 promoter for the expression of GUS gene was constructed. The activity of AvTPS1 promoter was verified by transient expression of Agrobacterium-mediated infiltration using the leaves of Nicotiana benthamiana. Results The gene sequence of AvTPS1 was 2 444 bp including seven exons and six introns. The 568 bp AvTPS1 promoter was successfully cloned using FPNI-PCR. Furthermore the sequence had ten kinds of cis-elements including conserved elements TATA-box, CAAT-box, MYC2 related element G-box and other elements. Finally, the GUS staining showed the tobacco leaves infiltrated by the pCAM-AvTPS1p were blue. Conclusion The AvTPS1 promoter can drive the transcription of GUS gene and then it was verified to have the promoter activity. These results give foundation for future research on the function of AvTPS1 involved in the terpenoid biosynthesis and its relationship with the transcription factors.

Nitric Oxide Inhibitory Constituents from the Fruits of Amomum tsao-ko

Bioactivity-guided fractionation of MeOH extract of the dried fruits of Amomum tsao-ko led to isolation of nine compounds (1 – 9). Their structures were elucidated by spectroscopic methods including extensive 1D and 2D-NMR, as alpinetin (1), naringenin-5-O-methyl ether (2), naringenin (3), hesperetin (4), 2′,4′,6′-trihydroxy-4-methoxy chalcone (5), tsaokoin (6), boesenbergin B (7), 4-hydroxyboesenbergin B (8), and tsaokoarylone (9). Of these, compound 8 was isolated from a natural source for the first time, which was previously reported as a synthetic product. The isolated compounds (1 – 9) were tested for their inhibitory effects on LPS-induced nitric oxide production in RAW 264.7 macrophages. Among them, three chalcone derivatives (compounds 5, 7, and 8) and a diarylheptanoid (compound 9) exhibited significant inhibitory activity on the NO production with IC₅₀ values ranging from 10.9 to 22.5 µM.

Study on ecological stereoscopic cultivation mode of Amomum villosum-Dimocarpus longan / 中国中药杂志

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.

Cloning and prokaryotic expression of transcription factor AvMYC4b in Amomum villosum / 中草药

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.

Chemical constituents from fruits of Amomum paratsao-ko / 中草药

Objective: To investigate the chemical constituents of Amomum paratsao-ko. Methods: The chemical constituents were separated and purified consecutively by silica gel, Sephadex LH-20 column chromatography, recrystallization as well as preparative HPLC. Their structures were determined by physicochemical properties and spectral analyses. Results: Fourteen compounds were isolated and identified as 2(E)-2-decene-1,10-di-yl-acetate (1), 2(E)-2-decene-1,10-diol (2), β-sitosterol (3), 3,5-dihydroxy- 7,4’-dimethoxyflavone (4), rhamnocitrin (5), kaempferol (6), rhamnetin (7), kaempferol-3,7,4’-O-trimethylether (8), ombuin (9), quercetin (10), kumatakenin (11), octyl ferulate (12), (E)-decyl-3-(4-hydroxy-3-methoxyphenyl) propenoate (13), and trans-p-hydroxycinnamic acid (14). Conclusion: Compound 1 is a new compound named paratsaokoster, and compounds 2-14 are isolated from this plant for the first time.

High-performance thin-layer chromatographic fingerprint analysis of Amomum villousm and its related species / 中国药学杂志

OBJECTIVE: To study the high-performance thin layer chromatographic (HPTLC) fingerprint of volatile oil constituents from Amomum villousm and its related species so as to set up the identification protocol of the medicinal plant and provide scientific information for its quality control. METHODS: TLC was used to analyze comparatively 10 batches of Amomum villosum Lour.samples, 10 batches of Amomum villousm crude drugs collected from different producing areas and stored for different time, 10 batches of the fruits of counterfeit species and 10 kinds of related species in the Zingiberaceae family. The samples were separated on silica gel G precoated plates with a mixture of cyclohexane-chloroform-ethyl acetate (13:2:2) as developing solvent system. The relative humidity was 67%. The spots were visualized with 5% vanillin sulfuric acid solution, then were analyzed by utilizing CHROMAP 1.5 solution software. RESULTS: The fingerprint of volatile oil of Amomum villosum, with 9 specific bands examined under natural light, was set up. The quality of Amomum villosum stored for different time or collected from different areas was distinctly variable. Obvious difference existed in the chemical composition of the volatile oils between Amomum villosum and its counterfeit and other related species. CONCLUSION: The HPTLC fingerprint analysis method can be used for rapid identification and quality control ofAmomum villosum.

Comparison of HPLC fingerprints of Amomum villosum Lour., amomurn villosum Lour. var. xanthioides T.L. Wu et Senjen and amomum longiligulare T.L. Wu / 中国药学杂志

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.