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.

A rare CHD5 haplotype and its interactions with environmental factors predicting hepatocellular carcinoma risk.

BACKGROUND: CHD5 is a conventional tumour-suppressing gene in many tumours. The aim of this study was to determine whether CHD5 variants contribute to the risk of hepatocellular carcinoma (HCC). METHODS: Gene variants were identified using next-generation sequencing targeted on referenced mutations followed by TaqMan genotyping in two case-control studies. RESULTS: We discovered a rare variant (haplotype AG) in CHD5 (rs12564469-rs9434711) that was markedly associated with the risk of HCC in a Chinese population. A logistical regression model and permutation test confirmed the association. Indeed, the association quality increased in a gene dose-dependent manner as the number of samples increased. In the stratified analysis, this haplotype risk effect was statistically significant in a subgroup of alcohol drinkers. The false-positive report probability and multifactor dimensionality reduction further supported the finding. CONCLUSIONS: Our results suggest that the rare CHD5 gene haplotype and alcohol intake contribute to the risk of HCC. Our findings can be valuable to researchers of cancer precision medicine looking to improve diagnosis and treatment of HCC.

A new ocotillol-type ginsenoside from American ginseng berry.

A new ocotillol-type ginsenoside, named pseudoginsenoside F12 (1), was isolated from American ginseng berry, whose structure was elucidated as 6-O-[α-L-2,3-epoxy-rhamnopyranosyl-(1-2)-ß-D-glucopyranosyl]-dammar-20S,24R-epoxy-3ß, 6α,12ß,25-tetraol. In addition, the known alkaloids ß-carboline-1-carboxylic acid (2) and anoectochine (3) were isolated for the first time from the Araliaceae family. The new compound 1 was evaluated for cytotoxicity against MDA-MB-231 breast cancer cell line.

Identification of anti-inflammatory components in Panax ginseng of Sijunzi Decoction based on spectrum-effect relationship.

Objective: This study aimed to identify the main medicinal active components of Panax ginseng (P. ginseng) in the compatibility environment of clinical application. For this purpose, the anti-inflammatory ingredients of P. ginseng were investigated based on its therapeutic effect in Sijunzi Decoction (SJD) which is a widely used traditional Chinese formula. Methods: The fingerprints of 10 batches of SJD consisting of different sources of P. ginseng were established by UPLC technique to investigate the chemical components. At the same time, the anti-inflammatory effects of these components were evaluated by dextran sulfate sodium-induced ulcerative colitis mouse model. Grey relational analysis was applied to explore the correlation degree between fingerprints and anti-inflammatory effects in SJD. Lipopolysaccharide-stimulated RAW264.7 murine macrophages were established to evaluate the anti-inflammatory action of the screened effective substances of P. ginseng. Results: According to grey relational analysis, notoginsenoside R1, ginsenoside Rg2 and ginsenoside Rb3 of P. ginseng were the major anti-inflammatory contributions in SJD. They had been proven to be closely associated with the anti-inflammatory process of SJD and displayed a close effect compared with SJD by LPS-stimulated RAW264.7 murine macrophages. Conclusion: Our work provides a general strategy for exploring the pharmacological ingredients of P. ginseng in traditional Chinese formulas which is beneficial for establishing the quality standards of traditional herbs in traditional Chinese medicine prescription based on their clinical therapeutic effect.

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.

The Function, Role and Process of DDX58 in Heart Failure and Human Cancers.

Background: Heart failure (HF) is the most common outcome of cardiovascular disease, and an increasing number of patients with heart failure die from noncardiac causes, such as cancer. Epidemiological data suggest that ischemic cardiomyopathy-induced HF (ischemic HF) may be associated with an increased incidence of cancer. This study aimed to investigate the possible mechanisms of the association between ischemic HF and cancer, as well as potential therapeutic targets. Methods: Weighted gene co-expression network analysis was performed to analyze the correlations between phenotypes and gene modules using immune cells as phenotypes. Differential analysis was then performed to screen differentially expressed genes (DEGs) in ischemic HF and normal control samples. The macrophage-related Brown module was identified as the key module, and immune-related DEGs were obtained by taking the intersection of the Brown module, DEGs, and immune-related genes using a Venn diagram. DDX58 was identified as the key gene using a protein-protein interaction network and expression analyses and validated using immunohistochemistry. Kaplan-Meier survival analysis was performed to analyze the correlation between DDX58 expression and tumor prognosis. Spearman correlation analysis was performed to assess the correlation between DDX58 expression and immune cell infiltration. Results: DDX58 was identified as a key immune-related gene associated with ischemic HF and was highly expressed in most cancer types. The survival analysis revealed a significant negative correlation between high DDX58 expression and prognosis in multiple tumor types. Moreover, DDX58 expression was significantly associated with immune cell infiltration and immune checkpoint gene expression in many cancer types. Conclusion: DDX58 is a key immune-related gene in ischemic HF and may play a crucial role in the relationship between ischemic HF and cancer. Pan-cancer analysis suggests that DDX58 is a promising clinical prognostic marker for most cancers and may be a therapeutic target for cancer patients and ischemic HF patients at an increased risk of cancer.

[Effect of light intensity on growth and quality of Asarum heterotropoides var. mandshuricum].

To study the infection rate of leaf spot disease, the drying rate of root and volatile oil content of Asarum heterotropoides var. mandshuricum at the unwrapping stage, blooming stage, the initial fruit stage, fructescence and wither stage during the growth period under the different sunlight intensity of 100% (I), 50% (II), 28% (III), 12% (IV). The volatile oil content was measured according to the method of Chinese Pharmacopoeia and the oil composition was determined by GC-MS. The unwrapping stage, blooming stage and the early fruit stage postponed about 2 days with decrease of the sunlight intensity. The infection rate of leaf was 88.46%, 70.00%, 0.23%, 0.07% under light intensity of I, II, III and IV, respectively, the drying rate was 25.14%, 28.27%, 30.23%, 31.57% under light intensity of I, II, III and IV, respectively, and the volatile oil content was 18.1, 17.6, 16.3, 15.3 mL x kg(-1) under light intensity of I, II, III and IV, respectively. The composition of the oil determined by GC-MS was different between the groups, but the content did not changed significantly with the decrease of the light intensity.

Interference RNA (RNAi)-based silencing of endogenous thrombopoietin receptor (Mpl) in Dami cells resulted in decreased hNUDC-mediated megakaryocyte proliferation and differentiation.

Recently our laboratory reported evidence showing that hNUDC acts as an additional cytokine for thrombopoietin receptor (Mpl). Previously known as the human homolog of a fungal nuclear migration protein, hNUDC plays a critical role in megakaryocyte differentiation and maturation. Here we sought to further clarify the hNUDC-Mpl ligand-receptor relationship by utilizing interference RNA (RNAi) to knockdown Mpl expression in a megakaryocyte cell line. We created U6 promoter driven constructs to express short hairpin RNAs (shRNA) with affinity for different sites on Mpl mRNA. By including Mpl-EGFP fusion protein in these constructs, we were able to effectively screen the shRNA that was most efficient in inhibiting Mpl mRNA expression. This shRNA was subsequently transferred into a lentivirus vector and transduced into Dami cells, a cell line which constitutively expresses endogenous Mpl. This lentiviral vector was also designed to simultaneously express EGFP to monitor transfection efficiency. Our results show that lentivirus can be used to effectively deliver shRNAs into Dami cells and cause specific inhibition of Mpl protein expression after transduction. Furthermore, we show the functional effects of shRNA-mediated Mpl silencing by demonstrating reduced hNUDC stimulated megakaryocyte proliferation and differentiation. Thus, the use of a RNAi knockdown strategy has allowed us to pinpoint the connection of hNUDC with Mpl in the regulation of megakaryocyte maturation.

Loss of FGFR3 Delays Acute Myeloid Leukemogenesis by Programming Weakly Pathogenic CD117-Positive Leukemia Stem-Like Cells.

Chemotherapeutic patients with leukemia often relapse and produce drug resistance due to the existence of leukemia stem cells (LSCs). Fibroblast growth factor receptor 3 (FGFR3) signaling mediates the drug resistance of LSCs in chronic myeloid leukemia (CML). However, the function of FGFR3 in acute myeloid leukemia (AML) is less understood. Here, we identified that the loss of FGFR3 reprograms MLL-AF9 (MA)-driven murine AML cells into weakly pathogenic CD117-positive leukemia stem-like cells by activating the FGFR1-ERG signaling pathway. FGFR3 deletion significantly inhibits AML cells engraftment in vivo and extends the survival time of leukemic mice. FGFR3 deletion sharply decreased the expression of chemokines and the prolonged survival time in mice receiving FGFR3-deficient MA cells could be neutralized by overexpression of CCL3. Here we firstly found that FGFR3 had a novel regulatory mechanism for the stemness of LSCs in AML, and provided a promising anti-leukemia approach by interrupting FGFR3.

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.

[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.

Recombinant keratinocyte growth factor 1 in tobacco potentially promotes wound healing in diabetic rats.

Keratinocyte growth factor 1 (KGF1) is a growth factor that promotes epidermal cell proliferation, migration, differentiation, and wound repair. It is expressed at low levels in a form of inclusion body in E. coli. In order to increase its expression and activity, we produced tobacco plants expressing KGF1 via Agrobacterium-mediated transformation using a potato virus X (PVX)-based vector (pgR107). The vector contained the sequence encoding the KGF1 gene fused with a green florescence protein. The recombinant plasmid was introduced into leaf cells of Nicotiana benthamiana (a wild Australian tobacco) via Agrobacterium-mediated agroinfiltration. As determined by fluorescence and Western blot of leaf extracts, the KGF1 gene was correctly translated into the tobacco plants. The recombinant KGF1 was purified from plant tissues by heparin affinity chromatography, and cell proliferation in NIH/3T3 cells was stimulated by the purified KGF1. The purified KGF1 was also applied to the wounds of type-II diabetic rats. KGF1 had accumulated to levels as high as 530 µ g/g fresh weight in the leaves of agroinfected plants. We show that plant-derived KGF1 can promote the proliferation of NIH/3T3 cells and have significant effects on the type-II diabetic rat. The present findings indicated that KGF1 from tobacco maintains its biological activity, implying prospective industrial production in a plant bioreactor.

[Construction and transduction of hNUDC shRNA interference lentiviral vector].

AIM: To construct a lentiviral vector carrying human nuclear distribution protein C (hNUDC)-specific shRNA (sh-NUDC-F) and knock down hNUDC expression in Dami cells by infection of the lentivirus. METHODS: After labeling of green fluorescent protein (GFP), sh-NUDC-F was cloned into lentiviral vector pRRL-cPPT-CMV-X-PRE-SIN, and the high-quality plasmid was transfected into 293T cells to produce lentiviral particles by the calcium phosphate method. After high-speed centrifugation, lentiviral particles were collected and determined for its titer. The high-titer lentiviral particles were then transduced into Dami cells. By detecting the expression of GFP in lentiviral vector using a microscope, the transduction efficiency was readily figured out. And hNUDC protein level was detected by Western blotting. RESULTS: hNUDC was totally knocked down by the efficient transduction of Dami cells with the lentivirus carrying sh-NUDC-F. CONCLUSION: Lentiviral vector containing sh-NUDC-F can be successfully packaged in 293T cells and then efficiently transduced into Dami cells to silence hNUDC gene.

[The construction of NUDC shRNA interference vector with red fluorescence protein].

AIM: Used RFP gene to construct a RNA interference vector for convenience to obtain the good effective hairpins sequence. METHODS: NUDC and RFP genes were cloned into pDs vector separately, resulting in pDs-NUDC- RFP. as above, human U6 promoter and 9 hairpins sequence of NUDC were cloned into the pDs- NUDC-RFP vector separately.The RNA interfererence vectors target to 9 points of NUDC were constructed. Construct- ed recombinant vectors and then were identified by restrictive digestion and DNA sequencing.293T cells were transfected with the recombinant DNA samples by the liposome complex method, and then fluorescence photographs were taken. RESULTS: Enzyme digestion and DNA sequencing showed that the target gene and shRNA fragments were correctly inserted in pDs vector. fluorescence photographs showed that shNUDC-A is the best effective fragment. CONCLUSION: The NUDC gene targeted shRNA and its vector are successfully constructed.

High levels of expression of fibroblast growth factor 21 in transgenic tobacco (Nicotiana benthamiana).

Fibroblast growth factor-21 (FGF21) is a hepatic hormone that plays a critical role in metabolism, stimulating fatty acid oxidation in the liver and glucose uptake in adipose tissue. In this study, we produced tobacco plants expressing human recombinant FGF21 (hFGF21) via Agrobacterium-mediated transformation using a potato virus X (PVX)-based vector (pgR107). The vector contained the sequence encoding the human FGF21 gene fused with green florescence protein and a histidine tag. The recombinant plasmid was introduced into leaf cells of Nicotiana benthamiana (a wild Australian tobacco) via Agrobacterium-mediated agroinfiltration. As determined by fluorescence and Western blot of leaf extracts, the hFGF21 gene was correctly translated in tobacco plants. Seven days after agroinfection, the recombinant hFGF21 had accumulated to levels as high as 450 µg g(-1) fresh weight in leaves of agroinfected plants. The recombinant hFGF21 was purified from plant tissues by Ni-NTA affinity chromatography, and the purified hFGF21 stimulated glucose uptake in 3T3/L1 cells. This indicated that the recombinant hFGF21 expressed via the PVX viral vector in N. benthamiana was biologically active.

[Plant as bioreactor].

Plant can be used as bioreactor for heterogenous protein expression. We reviewed different expression systems of plant bioreactor as well as recent relevant developments. In addition, we discussed perspectives in combination with our own experience.