Amomum villosum, serving as an important medicinal material, is complex in the genetic background of germplasm resources. Exploring the genetic diversity and genetic relationship of germplasm resources is conducive to clarifying the germplasm source and genetic background of A. villosum, so as to improve the efficiency of parent selection and variety breeding of A. villosum. Seventy-one pairs of SSR primers were used for PCR amplification of 84 A. villosum samples by polyacrylamide gel electrophoresis. Fifty-four pairs of SSR primers with high polymorphism were screened out for the analysis of genetic diversity. The results showed that 293 alleles were detected from 84 germplasm resources by 54 pairs of SSR primers, with an average of 5.32 alleles for each pair of primers, and a variation range of 3-8, and the primer AVL12 marked the highest number of alleles. The PIC value of each locus varied from 0.068 7 to 0.828 9, with an average of 0.529 9, and the highest was marked by AVL24. The genetic diversity of A. villosum was the highest in Yunnan, followed by Guangxi, and the lowest was found in Guangdong. The population structure analysis and cluster analysis showed that the samples were classified into two groups. In terms of origin, samples from Yunnan and Guangxi had a close genetic relationship, and there was no obvious differentiation of A, villosum resources from different origins. In this study, 54 pairs of SSR markers were used to analyze the genetic diversity and population structure of 84 germplasm resources, which can reflect the genetic relationship between A. villosum samples from different germplasm sources and different populations, thus providing a theoretical basis for the collection, research, and breeding of A. villosum resources.
Alleles ; Amomum/genetics* ; China ; Genetic Variation ; Microsatellite Repeats/genetics* ; Plant Breeding

Eight terpenoids were isolated from the fruits of Amomum villosum by silica gel, Sephadex LH-20, Rp-C_(18), MCI GEL CHP20 P column chromatography, preparative TLC, and HPLC. Their structures were identified by HR-ESI-MS, ~1H and ~(13)C-NMR, IR, UV, [α]_D, and ECD spectroscopic data as kravanhin A 3-O-β-D-glucopyranoside(1), kravanhin B(2), 6-eudesmene-1β,4β-diol(3), oplodiol(4), vicodiol(5),(1R,2S,4R,7S)-vicodiol 9-O-β-D-glucopyranoside(6),(1R,2S,4S,5R)-angelicoidenol 2-O-β-D-glucopyranoside(7), and(1S,2S,4R,6S)-bornane-2,6-diol 2-O-β-D-glucopyranoside(8). Compound 1 was a new compound, and compounds 2-5 were isolated from A. villosum for the first time. Their hypoglycemic activity was tested based on STC-1 cell model and two enzymatic models(GPa and PTP1 B). The results showed that compounds 1, 7, and 8 could stimulate GLP-1 with the secretion rates of 692.8%, 398.6%, and 483.3% at 25.0 μmol·L~(-1), and compound 6 showed inhibitory activity against GPa with an IC_(50) value of 78.6 μmol·L~(-1).
Fruit/chemistry* ; Terpenes/analysis* ; Amomum ; Hypoglycemic Agents/analysis* ; Chromatography, High Pressure Liquid

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.
Amomum ; Gene Expression Profiling ; Real-Time Polymerase Chain Reaction ; Reverse Transcriptase Polymerase Chain Reaction ; Seeds/genetics*

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.
Amomum ; Chalcone ; Ether ; Fruit ; Macrophages ; Nitric Oxide ; Zingiberaceae

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.
Agriculture ; methods ; Amomum ; growth & development ; Ecology ; Forests ; Soil ; Water

This paper is aimed to microscopic identification of traditional Chinese medicines (TCMs) using an in situ imaging method. In this study, two kinds of Zingiberaceae seeds, Amomi Rotundus Fructus and Alpiniae Katsumadai Semen, were investigated by synchrotron radiation in-line X-ray phase-contrast computed tomography (IXPCT) imaging method. The results showed that the microstructures of these Zingiberaceae seeds could be clearly obtained from the virtual slices information in different observing angles. It proves that IXPCT is an effective imaging method, which can provide the imaging information for the microscopic identification of the intact TCMs in situ and non-destructively.
Amomum ; cytology ; Imaging, Three-Dimensional ; methods ; Medicine, Chinese Traditional ; Seeds ; cytology ; Tomography, X-Ray Computed

OBJECTIVETo analyze the potential risks of fungal contaminants on Panax notoginseng and Amomum tsaoko.

METHODThe primary investigation was conducted in the P. notoginseng and A. tsaoko major production areas in Yunnan. Samples of P. notoginseng and A. tsaoko were collected from drugstores and markets in 3 cities of Yunnan. Dilution-plate method was applied for the isolation of fungi, the obtained species were identified according to morphological and molecular approaches.

RESULTPaecilomyces lilacinus and Penicillium citrinum were dominant on samples of Panax notoginseng. P. lilacinus and Aspergillus flavus were dominant on samples of Amomum tsaoko.

CONCLUSIONIn Yunnan province, the major fungal contaminants on P. notoginseng are P. lilacinus and P. citrinum and the major fungal contaminants on A. tsaoko are P. lilacinus and Aspergillus flavus. There exists a potential contamination risk of citrinin on P. notoginseng and aflatoxin on A. tsaoko.

Amomum ; microbiology ; China ; Drug Contamination ; prevention & control ; Drugs, Chinese Herbal ; Mitosporic Fungi ; isolation & purification ; physiology ; Panax notoginseng ; microbiology ; Risk

OBJECTIVETo compare inclusion effects and process conditions of two preparation methods-colloid mill and saturated solution-for beta-CD inclusion compound of four traditional Chinese medicine volatile oils and study the relationship between each process condition and volatile oil physical properties and the regularity of selective inclusion of volatile oil components.

METHODVolatile oils from Nardostachyos Radix et Rhizoma, Amomi Fructus, Zingiberis Rhizoma and Angelicaesinensis Radix were prepared using two methods in the orthogonal test. These inclusion compounds by optimized processes were assessed and compared by such methods as TLC, IR and scanning electron microscope. Inclusion oils were extracted by steam distillation, and the components found before and after inclusion were analyzed by GC-MS.

RESULTAnalysis showed that new inclusion compounds, but inclusion compounds prepared by the two processes had differences to some extent. The colloid mill method showed a better inclusion effect than the saturated solution method, indicating that their process conditions had relations with volatile oil physical properties. There were differences in the inclusion selectivity of components between each other.

CONCLUSIONThe colloid mill method for inclusion preparation is more suitable for industrial requirements. To prepare volatile oil inclusion compounds with heavy gravity and high refractive index, the colloid mill method needs longer time and more water, while the saturated solution method requires higher temperature and more beta-cyclodextrin. The inclusion complex prepared with the colloid mill method contains extended molecular weight chemical composition, but the kinds of components are reduced.

Amomum ; chemistry ; Angelica sinensis ; chemistry ; Gas Chromatography-Mass Spectrometry ; Medicine, Chinese Traditional ; methods ; Nardostachys ; chemistry ; Oils, Volatile ; chemistry ; isolation & purification ; Plants, Medicinal ; chemistry

OBJECTIVETo observe the effects of three traditional Chinese medicine (TCM) such as Amomi Fructus Rotundus, Perillae Folium and Angelicae Dahuricae Radix on lung-yang deficiency rats induced by compound factors.

METHODLung-yang deficiency rats were established with three-factor combination, such as smoking (exogenous evil effect on lung), swimming in common and ice water (cold body and exhaustoin) and drinking ice water (inhale cold). Meanwhile, rats were given water extracts of the three TCM by intragastric administration for 24 days everyday. Indexes such as general behavior, weight, rectal temperature, back temperature and grip strength were observed. Blood was collected to determine NO, IgG in blood serum. Lung and heart were dissected to measure organs index.

RESULTThe water extracts of Amomi Fructrs Rotundus, Perillae Folium and Angelicae Dahuricae Radix could markedly heighten weight, back temperature, grip strength, content of IgG in blood serum, reduce content of NO in blood serum, lung index and heart index. The water extracts of Amomi Fructrs Rotundus and Perillae Folium could heighten rectal temperature.

CONCLUSIONAmomi Fructrs Rotundus, Perillae Folium and Angelicae Dahuricae Radix were TCM with pungent-flavor, warm-nature and meridian tropism in lung, which could improve the symptoms of physique emaciation, aversion to cold of the back, weary and acratia and so on. It provides an important reference for the regularity of the properties theories about pungent-flavor, warm-nature and meridian tropism in lung.

Amomum ; chemistry ; Angelica ; chemistry ; Animals ; Body Temperature ; Disease Models, Animal ; Drugs, Chinese Herbal ; administration & dosage ; Enteral Nutrition ; Female ; Heart ; drug effects ; Immunoglobulin G ; blood ; Lung ; drug effects ; pathology ; Male ; Meridians ; Nitric Oxide ; blood ; Perilla ; chemistry ; Plant Extracts ; administration & dosage ; Rats ; Yang Deficiency ; drug therapy ; pathology

OBJECTIVEThe aim of this study was to optimize the testing methods for seed quality, and to provide a basis for establishing seed testing criterion and quality standard of Amomum villosum.

METHODReferring to the International Seed Testing Rules made by ISTA and Rules for agricultural seed testing, the seed quality of A. villosum from different collection areas was measured.

RESULTThe samples weight of A. villosum for purity analysis were at least 500 g and for test were at least 50 g. Verification of genuineness was assayed by seed appearance comparing and weight of per hundred seeds was determined, the moisture content test was carried out by high temperature drying method (3 hours). The seeds were stored in wet sand for 20 days and then dipping in the 100 mg x L(-1) GA3 for 30 days before germination, seeds on filter papers germinated at 30/20 degrees C. The first germination-counting time was the 15th day of the test and the final time was the 50th day. Seed viability was tested by TTC method.

CONCLUSIONThe seed testing methods for quality items of A. villosum, including sampling, purity analysis, verification of genuineness, weight, moisture content, percentage germination and seed viability of A. villosum had been initially established.

Amomum ; chemistry ; Germination ; Quality Control ; Seeds ; chemistry ; Water ; analysis