Identification and Expression Analysis of R2R3-MYB Transcription Factors Associated with Flavonoid Biosynthesis in Panax quinquefolius.

Panax quinquefolius L. is an important medicinal plant, and flavonoids are among its main secondary metabolites. The R2R3-MYB transcription factor plays an irreplaceable role in plant growth, development, and secondary metabolism. In our study, we identified 159 R2R3-MYBs and analyzed their physical and chemical properties in P. quinquefolius. The protein length of 159 PqMYBs varied from 107 to 1050 amino acids. The molecular weight ranged from 12.21 to 116.44 kDa. The isoelectric point was between 4.57 and 10.34. We constructed a phylogenetic tree of P. quinquefolius and Arabidopsis thaliana R2R3-MYB family members, and PqMYB members were divided into 33 subgroups. Transcriptome data analysis showed that the expression patterns of PqMYBs in root, leaf, and flower were significantly different. Following the MeJA treatment of seedlings, five candidate PqMYB genes demonstrated a response. A correlation analysis of PqMYBs and candidate flavonoid pathway genes showed that PqMYB2, PqMYB46, and PqMYB72 had correlation coefficients that were higher than 0.8 with PqCHS, PqANS4, and PqCCoAMT10, respectively. Furthermore, a transient expression assay confirmed that the three PqMYBs were localized in the nucleus. We speculated that these three PqMYBs were related to flavonoid biosynthesis in P. quinquefolius. These results provided a theoretical basis and a new perspective for further understanding the R2R3-MYB gene family and the biosynthesis mechanism of secondary metabolites in P. quinquefolius.

Genome-Wide Identification and Expression Analysis of Auxin Response Factor (ARF) Gene Family in Panax ginseng Indicates Its Possible Roles in Root Development.

Auxin-responsive factors (ARFs) are an important class of transcription factors and are an important component of auxin signaling. This study conducted a genome-wide analysis of the ARF gene family in ginseng and presented its findings. Fifty-three ARF genes specific to ginseng (PgARF) were discovered after studying the ginseng genome. The coding sequence (CDS) has a length of 1092-4098 base pairs and codes for a protein sequence of 363-1565 amino acids. Among them, PgARF32 has the least number of exons (2), and PgARF16 has the most exons (18). These genes were then distributed into six subgroups based on the results obtained from phylogenetic analysis. In each subgroup, the majority of the PgARF genes displayed comparable intron/exon structures. PgARF genes are unevenly distributed on 20 chromosomes. Most PgARFs have B3 DNA binding, Auxin_resp, and PB1 domains. The PgARF promoter region contains various functional domains such as plant hormones, light signals, and developmental functions. Segmental duplications contribute to the expansion of the ARF gene family in ginseng, and the genes have undergone purifying selection during evolution. Transcriptomic results showed that some PgARFs had different expression patterns in different parts of ginseng; most PgARFs were affected by exogenous hormones, and a few PgARFs responded to environmental stress. It is suggested that PgARF is involved in the development of ginseng by regulating hormone-mediated genes. PgARF14, PgARF42, and PgARF53 are all situated in the nucleus, and both PgARR14 and PgARF53 noticeably enhance the growth length of roots in Arabidopsis. Our findings offer a theoretical and practical foundation for exploring PgARFs' role in the growth of ginseng roots.

Discrimination for geographical origin of Panax quinquefolius L. using UPLC Q-Orbitrap MS-based metabolomics approach.

American ginseng, Panax quinquefolius L., is an important medicinal plant with multiple pharmacological effects and high nutritional value. American ginseng from different geographical origins varies in quality and price. However, there was no approach for discriminating American ginseng from different geographical origins to date. In this study, a metabolomic method based on the UPLC-Orbitrap fusion platform was established to comprehensively determine and analyze metabolites of American ginseng from America and Canada, Heilongjiang, Jilin, Liaoning, and Shandong provinces in China. A total of 382 metabolites were detected, including 230 saponins, 30 amino acids and derivatives, 27 organic acids and derivatives, 25 lipids, 17 carbohydrates and derivatives, 10 phenols, 8 nucleotides, and derivatives, as well as 35 other metabolites. Metabolite differences between North America and Asia producing areas were more obvious than within Asia. Twenty metabolites, contributed most to the differentiation of producing areas, were identified as potential markers with prediction accuracy higher than 91%. The results provide new insights into the metabolite composition of American ginseng from different origins, which will help discriminate origins and promote quality control of American ginseng.

Identification and analysis of BAHD superfamily related to malonyl ginsenoside biosynthesis in Panax ginseng.

Introduction: The BAHD (benzylalcohol O-acetyl transferase, anthocyanin O-hydroxycinnamoyl transferase, N-hydroxycinnamoyl anthranilate benzoyl transferase and deacetylvindoline 4-O-acetyltransferase), has various biological functions in plants, including catalyzing the biosynthesis of terpenes, phenolics and esters, participating in plant stress response, affecting cell stability, and regulating fruit quality. Methods: Bioinformatics methods, real-time fluorescence quantitative PCR technology, and ultra-high-performance liquid chromatography combined with an Orbitrap mass spectrometer were used to explore the relationship between the BAHD gene family and malonyl ginsenosides in Panax ginseng. Results: In this study, 103 BAHD genes were identified in P. ginseng, mainly distributed in three major clades. Most PgBAHDs contain cis-acting elements associated with abiotic stress response and plant hormone response. Among the 103 genes, 68 PgBAHDs are WGD (whole-genome duplication) genes. The significance of malonylation in biosynthesis has garnered considerable attention in the study of malonyltransferases. The phylogenetic tree results showed 34 PgBAHDs were clustered with genes that have malonyl characterization. Among them, seven PgBAHDs (PgBAHD4, 45, 65, 74, 90, 97, and 99) showed correlations > 0.9 with crucial enzyme genes involved in ginsenoside biosynthesis and > 0.8 with malonyl ginsenosides. These seven genes were considered potential candidates involved in the biosynthesis of malonyl ginsenosides. Discussion: These results help elucidate the structure, evolution, and functions of the P. ginseng BAHD gene family, and establish the foundation for further research on the mechanism of BAHD genes in ginsenoside biosynthesis.

Immunomodulatory Effect of Ginsenoside Rb2 Against Cyclophosphamide-Induced Immunosuppression in Mice.

Ginsenoside Rb2 (Rb2), a fundamental saponin produced and isolated from ginseng (Panax ginseng C.A. Meyer), has a wide range of biological actions. The objective of this investigation was to see if ginsenoside Rb2 has any immunomodulatory properties against cyclophosphamide (CTX)-induced immunosuppression. For the positive control group, levamisole hydrochloride (LD) was used. We discovered that intraperitoneal injection of Rb2 (5, 10, 20 mg/kg) could relieve CTX-induced immunosuppression by enhanced immune organ index, reduced the pathological characteristics of immunosuppression, promoted natural killer (NK) cells viability, improved cell-mediated immune response, boosted the IFN-γ (Interferon-gamma), TNF-α (Tumor necrosis factor-alpha), IL-2 (Interleukin-2), and IgG (Immunoglobulin G), as well as macrophage activity like carbon clearance and phagocytic index. Rb2 significantly elevated the mRNA expression of IL-4 (Interleukin-4), SYK (Tyrosine-protein kinase-SYK), IL-2, TNF-α, and IL-6 (Interleukin-6) in the spleen of CTX-injected animals. Molecular docking results showed that Rb2 had excellent binding properties with IL-4, SYK, IL-2, TNF, and IL-6, indicating the target protein might be strongly correlated with the immunomodulatory effect of Rb2. Taken together, ginsenoside Rb2 can improve the immune function that is declined in CTX-induced immunosuppressed mice, the efficacy maybe due to the regulation of related cytokine and mRNA expression.

Authentication of Hippophae rhamnoides ssp. sinensis and ssp. mongolica Based on Single Nucleotide Polymorphism at Ribosomal DNA and Their Vitamin Content Analysis.

Hippophae rhamnoides widely known as sea buckthorn berries (SB) are rich in vitamins and phytonutrients. The subspecies ssp. sinensis and ssp. mongolica are highly valued for their medicinal properties and vitamin contents, hence domesticated widely across Eurasia and Southeast Asia. Due to the frequent usage of these two subspecies, accurate identification is required to prevent economically motivated adulteration. In this study, we report the single nucleotide polymorphism (SNP) based molecular markers to easily distinguish these two subspecies at 45S nrDNA region. From the determined 45S rDNA region, we designed two primers (5' sinensis and 5' mongolica) and developed a multiplex PCR profile. The developed primers effectively distinguished the sea buckthorn subspecies in commercial products as well. Along with the development of subspecies specific primers, we have profiled vitamin contents from H. rhamnoides ssp. sinensis and ssp. mongolica and found ascorbic acid and riboflavin contents were high in both ssp. sinensis and spp. mongolica, yet the content of folic acid was high only in ssp. mongolica. Thus, we provide species specific primers and vitamin profile as an effective authentication of H. rhamnoides.

Dynamic changes of ginsenosides in Panax quinquefolium fruit at different development stages measured using UHPLC-Orbitrap MS.

RATIONALE: Some studies have shown that Panax quinquefolium fruit (PQF) could also be used as a potential medicinal resource. However, little is known about the composition of ginsenosides and their dynamic changes at different development stages of PQF. Therefore, this study is of great significance for the metabolomics and rational utilization of PQF. METHODS: The samples were analyzed using ultra-high-performance liquid chromatography combined with an Orbitrap mass spectrometer (UHPLC-Orbitrap MS), and the method of metabonomics was applied to profile the dynamic changes of ginsenosides in PQF at different development stages. RESULTS: A total of 109 ginsenosides were identified or tentatively characterized. Samples collected from different development stages were significantly discriminated according to ginsenoside contents. A total of 25 potential chemical markers enabling the differentiation were discovered. CONCLUSIONS: For the first time, the study developed an UHPLC-Orbitrap MS-based approach to detect ginsenoside in PQF at different development stages using a non-targeted mode. This comprehensive phytochemical profile study revealed the structural diversity and discrimination of ginsenosides in PQF at different development stages, which could provide the basis for the metabolomics and rational application of PQF.

Advances in Saponin Diversity of Panax ginseng.

Ginsenosides are the major bioactive constituents of Panax ginseng, which have pharmacological effects. Although there are several reviews in regards to ginsenosides, new ginsenosides have been detected continually in recent years. This review updates the ginsenoside list from P. ginseng to 170 by the end of 2019, and aims to highlight the diversity of ginsenosides in multiple dimensions, including chemical structure, tissue spatial distribution, time, and isomeride. Protopanaxadiol, protopanaxatriol and C17 side-chain varied (C17SCV) manners are the major types of ginsenosides, and the constitute of ginsenosides varied significantly among different parts. Only 16 ginsenosides commonly exist in all parts of a ginseng plant. Protopanaxadiol-type ginsenoside is dominant in root, rhizome, leaf, stem, and fruit, whereas malonyl- and C17SCV-type ginsenosides occupy a greater proportion in the flower and flower bud compared with other parts. In respects of isomeride, there are 69 molecular formulas corresponding to 170 ginsenosides, and the median of isomers is 2. This is the first review on diversity of ginsenosides, providing information for reasonable utilization of whole ginseng plant, and the perspective on studying the physiological functions of ginsenoside for the ginseng plant itself is also proposed.

Changes of 5-hydroxymethyl-2-furfural in fresh and processed ginsengs.

The study estimated changes of 5-hydroxymethyl-2-furfuraldehyde (5-HMF) in different ginseng products with different temperatures and time pretreatment. Heat treatment was performed at various temperatures for 1.50, 2.00, 2.50, and 3.00 hr, respectively. Ultrasonic extraction and reflux extraction were used to evaluate the extraction rate and different solvents (such as 80% methanol, dichloromethane, ethyl acetate, and an extraction with both dichloromethane and ethyl acetate solvents) using two extraction methods (liquid-liquid extraction and solid-phase extraction) to remove matrix interference. An ultraperformance liquid chromatography-mass spectrometer (UPLC-MS) method was used for quantitative and changing analysis of 5-HMF in different ginseng samples. The results indicated that the content of 5-HMF increased dramatically with heating temperature and time, and the 5-HMF in the ginseng samples ranged from 0.01 to 112.32 g/kg protein. The highest value was observed in the honey-added ginseng samples with the highest amount of addition and highest temperature treatment, and the lowest value was found in the fresh ginseng samples. These results implied that 5-HMF may be as an indicator to estimate the honey addition level and heat treatment degree during the processing of ginseng products, and the content of 5-HMF is a promising parameter to evaluate the quality of products (ginseng). The production and regulation of potentially harmful Maillard reaction products (PHMRPs)-5-HMF in ginseng manufacture will provide an important reference for safe ginseng processing.

[Isolation,screening and identification of endophytic fungi and detection of its antifungal effects against Alternaria panax].

To obtain biocontrol fungus for Alternaria panax,the antifungal effects of one strain of endophytic fungi isolated from leaves of healthy ginseng were screened and evaluated by using dual-culture method,and the taxonomic assignment of the screened strain was identified based on the morphological characters and ITS sequence analysis. The results of dual-culture showed that one of the endophytes marked as FS-01 had good antifungal effects and the inhibitory rates of FS-01 strain to A. panax was( 60. 21±0. 12) %.The hyphae junction of the both strains,A. panax dissolved,broke and winded,while the hyphae of FS-01 strain remained normal. The inhibitory rates of non-sterilized FS-01 strain fermentation liqud was( 13. 94±0. 21) %. Strain FS-01 identified as Chaetomium globosum.

[Rapid determination of pesticide multiresidues in panax ginseng by QuEChERS-gas chromatography-mass spectrometry].

In this study, the extraction and purification improvements of the QuEChERS method were coupled with gas chromatography-mass spectrometry (GC-MS) to determine 20 pesticides in panax ginseng. Dry ginseng powder was mixed with water, and the solution was then extracted with acetonitrile and purified with N-propylethylenediamine (PSA) and MgSO4 to remove co-extractives that might interfere with the results. The target compounds were detected after electron-impact ionization in selected ion monitoring (SIM) mode. Under optimum conditions, the method gave a good linear relationship for the determination of the pesticides within a certain concentration range, with correlation coefficients greater than 0.990. Moreover, the limits of detection (S/N=3) were 0.002-0.007 mg/kg, the limits of quantification (S/N=10) were 0.002-0.024 mg/kg, and the average recoveries for pesticides in panax ginseng were 70.41%-114.06%, with relative standard deviations of 0.76%-15.47%. In comparison with previous methods, the new procedure has the advantages of simple sample preparation and higher sensitivity.