Preparation, characterization and cytotoxicity assessment of a novel selenized polysaccharide from Morchella sextelata.

Selenylation modification has been widely developed to improve the biological effects of natural polysaccharides. In this study, a purified new polysaccharide (MSP-4) was isolated from Morchella Sextelata, and selenized into SeMSP-4 using the HNO3-Na2SeO3 method. The selenium (Se) content of SeMSP-4 was 101.81 ± 9.90 mg/kg, and the molecular weight of SeMSP-4 was 1.23 × 105 Da. The FT-IR, XRD and AFM results showed that MSP-4 was successfully combined with the Se element. The structure characters of SeMSP-4 were analyzed by methylation analysis combined with 1D and 2D NMR spectroscopy. And, the radical scavenging test revealed that SeMSP-4 exhibited higher antioxidant capacities in vitro than MSP-4. The cytotoxicity analysis indicated that SeMSP-4 could dose-dependently inhibit the proliferation of HepG2 and HeLa cells, but did not show a cytotoxic effect on normal cells (HEK293). Furthermore, SeMSP-4 stimulation significantly increased the macrophage viability and enhanced NO production in macrophage cells. This study suggested that SeMSP-4 could be utilized as a potential selenium source with antioxidant, antitumor, and immunostimulatory activities.

Multifunctional biocompatible Ni/Ni-P nanospheres for anti-tumor "neoadjuvant phototherapy" combining photothermal therapy and photodynamic therapy.

Gastric cancer, a gastrointestinal tumor with high morbidity and lethality, is often treated using strategies that are not as effective as they could be due to the locally advanced stage. Although pre-operative neoadjuvant chemotherapy can degrade the tumor stage to afford the possibility of surgery, it still possesses the problems of high systemic toxicity and low selectivity. In this work, we constructed an intelligent multi-functional nanoplatform (NNPIP NPs) with synergistic effects of photothermal therapy (PTT) and photodynamic therapy (PDT), which consisted of the nickel/nickel phosphide (Ni/Ni-P) nanosphere as the core, polyethyleneimine (PEI) as the shell, and the loaded indocyanine green (ICG). The mutual reinforcement of heat generated by the core and photosensitizer under 808 nm NIR laser irradiation is highly effective in the synergistic action of PTT. And co-delivery of ICG with nanoparticles into the cell enhances the PDT effect by reducing the consumption of singlet oxygen (1O2). Ultimately, this therapeutic strategy in vivo not only shrunk tumors but even eliminated tumors completely in a quarter of samples, which may be considered as a potential alternative to neoadjuvant chemotherapy and called "neoadjuvant phototherapy". In addition, as a nanoplatform based on transition metal nickel, NNPIP NPs could also be considered as a potential contrast agent for T1-weighted magnetic resonance imaging (MRI). Herein, we can diagnose and achieve pre-surgical downstaging of tumors and hope to improve R0 resection rates with lower toxicity and higher selectivity.

Synthesis, characterization, and cytotoxicity analysis of selenium nanoparticles stabilized by Morchella sextelata polysaccharide.

Natural bioactive molecules have been widely used as stabilizers in the functional improvement of selenium nanoparticles (SeNPs) in recent years. In this study, Morchella sextelata polysaccharide (MSP) was introduced as a novel stabilizer for the synthesis of SeNPs based on the redox system of sodium selenite and ascorbic acid. The size, morphology, stability, and anti-cancer cell activities were respectively analyzed by various methods. The results showed that the synthesized SeNPs with MSP were 72.07 ± 0.53 nm in size, red in color, spherical in shape, and amorphous in nature. MSP-SeNPs showed high scavenging activity against DPPH and ABTS radicals. And, these MSP-SeNPs exhibited a significant anti-proliferation effect on human liver (HepG2) and cervical cancer (Hela) cells in vitro, while no significant cytotoxicity against normal human kidney cells (HK-2) was observed. Moreover, the mitochondria-dependent apoptosis pathway triggered by MSP-SeNPs in HepG2 cell was identified. The expression levels of p53, Bax, cytochrome c, caspase-3 and caspase-9 were all up-regulated in HepG2 cells after MSP-SeNPs treatment, while Bcl-2 expression was down-regulated. These results suggest that MSP-SeNPs have strong potential as the food supplement for application in cancer chemoprevention.

Colorectal cancer therapy mediated by nanomedicines.

Colorectal cancer is the third most malignant gastrointestinal tumor. Although traditional chemotherapy and radiotherapy have been widely used for treating colorectal cancer, the treatment effect is still unsatisfactory, resulting in a high mortality rate and a low 5-year survival rate. In recent years, with the development of molecular biology of colorectal cancer, many promising therapeutic strategies based on nanomaterials have been developed for colorectal cancer. In this review, we focus on recent advances in colorectal cancer treatment-related nanomedicines. We first discuss the exploration of stimuli-responsive drug delivery systems (DDSs) for colorectal cancer treatment using pH, hypoxia, glutathione (GSH), enzymes, light, magnetic fields (MF), and ultrasound (US) as stimuli. Moreover, the latest progress in emerging therapy for colorectal cancer is further summarized, including photothermal therapy (PTT), magnetothermal therapy (MTT), photodynamic therapy (PDT), sonodynamic therapy (SDT), and chemodynamic therapy (CDT). Finally, we explore the existing challenges and future directions for the better design and development of nanomedicines for clinical colorectal cancer treatment.

Full-length transcriptome sequencing and modular organization analysis of oleanolic acid- and dammarane-type saponins related gene expression patterns in Panax japonicus.

The saponins found in Panax japonicus, a traditional medicinal herb in Asia, exhibit high degrees of structural and functional similarity. In this study, metabolite analysis revealed that oleanolic acid-type and dammarane-type saponins were distributed unevenly in three tissues (rhizome_Y, rhizome_O, and secRoot) of P. japonicus. Single-molecule real-time (SMRT) sequencing and next generation sequencing (NGS) data revealed distinct and tissue-specific transcriptomic patterns relating to the production of these two types of saponins. In the co-expression network and hierarchical clustering analyses, one 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) and two 1-deoxy-D-xylulose-5-phosphate synthase (DXS) etc. transcripts were found to be key genes associated with the biosynthesis of oleanolic acid and dammarane-type saponins in P. japonicus, respectively. In addition, cytochrome p450 (CYP) and UDP-glucuronosyltransferase (UGT) family proteins that serve as regulators of saponin biosynthesis-related genes were also found to exhibit tissue-specific expression patterns. Together these results offer a comprehensive metabolomic and transcriptomic overview of P. japonicus.

Comparative transcriptome analysis of rhizome nodes and internodes in Panax. japonicus var. major reveals candidate genes involved in the biosynthesis of triterpenoid saponins.

The rhizome of P. japonicus var. major, one of the important herbs in Traditional Chinese medicine (TCM), has been used as tonic and hemostatic drugs in Tujia and Miao ethnic groups of China for thousand years. In this study, comparative metabolite and transcriptome analysis of rhizome nodes and internodes of wild P. japonicus var. major was performed to reveal their different roles in the biosynthesis of triterpene saponins. The results showed that the node was the crucial section for the synthesis of ginsenosides in the rhizome. The content of oleanane-type ginsenosides in the node was much higher than those in the internode. Most isoprenoid biosynthesis-related genes were highly expressed in the node. And, candidate UDP-glycosyltransferase (UGT) genes were also found to be differentially expressed between node and internode. Our study will provide a better understanding of the metabolism of ginsenosides in the rhizome of P. japonicus var. major.

[Study on interaction between ginsenosides Rg_1,Rb_1 and Ro and bovine serum albumin].

Small molecules with physiological or pharmacological activities need to interact with biological macromolecules in order to function in the body. As the protein with the highest proportion of plasma protein,serum albumin is the main protein binding to various endogenous or exogenous small molecules. Serum albumin interacts with small molecules in a reversible non-covalent manner and transports small molecules to target sites. Bovine serum albumin( BSA) is an ideal target protein for drug research because of its low cost and high homology with human serum albumin. The research on the interaction between drugs and BSA has become a hotspot in the fields of pharmacy,medicine,biology and chemistry. In this research,molecular docking method was used to study the interaction between three small ginsenosides with high pharmacological value( Rg_1,Rb_1,Ro) and bovine serum albumin( BSA),and the binding mode information of three ginsenosides interacting with BSA was obtained. The results of molecular docking showed that ginsenosides and amino acid residues in the active pocket of proteins could be combined by hydrophobic action,hydrogen bonding and electrostatic action. The interaction between small ginsenosides and bovine serum albumin is not the only form,and their interaction has many forms of force. The interaction between these molecules and various weak forces is the key factor for the stability of the complex. The results of this study can provide the structural information of computer simulation for the determination of the interaction patterns between active components and proteins of ginseng.

Lipid Supplement in the Cultural Condition Facilitates the Porcine iPSC Derivation through cAMP/PKA/CREB Signal Pathway.

Large numbers of lipids exist in the porcine oocytes and early embryos and have the positive effects on their development, suggesting that the lipids may play an important role in pluripotency establishment and maintenance in pigs. However, the effects of lipids and their metabolites, such as fatty acids on reprogramming and the pluripotency gene expression of porcine-induced pluripotent stem cells (iPSCs), are unclear. Here, we generated the porcine iPSCs that resemble the mouse embryonic stem cells (ESCs) under lipid and fatty-acid-enriched cultural conditions (supplement of AlbuMAX). These porcine iPSCs show positive for the ESCs pluripotency markers and have the differentiation abilities to all three germ layers, and importantly, have the capability of aggregation into the inner cell mass (ICM) of porcine blastocysts. We further confirmed that lipid and fatty acid enriched condition can promote the cell proliferation and improve reprogramming efficiency by elevating cAMP levels. Interestingly, this lipids supplement promotes mesenchymal-epithelial transition (MET) through the cAMP/PKA/CREB signal pathway and upregulates the E-cadherin expression during porcine somatic cell reprogramming. The lipids supplement also makes a contribution to lipid droplets accumulation in the porcine iPSCs that resemble porcine preimplantation embryos. These findings may facilitate understanding of the lipid metabolism in porcine iPSCs and lay the foundation of bona fide porcine embryonic stem cell derivation.

The Phosphatome of Medicinal and Edible Fungus Wolfiporia cocos.

Wolfiporia cocos is an important medicinal and edible fungus that grows in association with pine trees, and its dried sclerotium has been used as a traditional medicine in China for centuries. However, the commercial production of W. cocos sclerotia is currently limited by shortages in pine wood resources. Since protein phosphatases (PPs) play significant roles in growth, signal transduction, development, metabolism, sexual reproduction, cell cycle, and environmental stress responses in fungi, the phosphatome of W. cocos was analyzed in this study by identifying PP genes, studying transcript profiles and assigning PPs to orthologous groups. Fifty-four putative PP genes were putatively identified in W. cocos genome based on homologous sequences searching using BLASTx program against the Saccharomyces cerevisiae, Fusarium graminearum, and Sclerotinia sclerotiorum databases. Based on known and presumed functions of orthologues of these PP genes found in other fungi, the putative roles of these W. cocos PPs in colonization, hyphal growth, sclerotial formation, secondary metabolism, and stress tolerance to environment were discussed in this study. And the level of transcripts from PP genes in the mycelium and sclerotium stages was also analyzed by qRT-PCR. Our study firstly identified and functional discussed the phosphatome in the medicinal and edible fungus W. cocos. The data from our study contribute to a better understanding of PPs potential roles in various cellar processes of W. cocos, and systematically provide comprehensive and novel insights into W. cocos economically important traits that could be extended to other fungi.

De novo assembly and transcriptome analysis of sclerotial development in Wolfiporia cocos.

Wolfiporia cocos Ryvarden et Gilbertson, a well-known medicinal fungus in the Basidiomycetes, is widely distributed in East Asia. Its dried sclerotium, which is known as Fuling in China, has been used as a traditional crude drug in Chinese traditional medicine for thousand years. However, little is known about how the sclerotium is developed at the genetic level. In this study, the de novo sequencing of sclerotia of W. cocos (S1_initial stage; S2_developmental stage and S3_mature stage) was carried out by illumina HiSeq 2000 technology. 27,438 unigenes were assembled from ~30Gbp raw data, and 12,093 unigenes were significantly annotated. The analysis of expression profiles during development returned 304 differentially expressed genes (DEGs), which were clustered into four different groups according to their expression trends. Especially for the maturation stage (S3), the sclerotium exhibited a markedly different expression profile from other stages. We further showed that peroxisome, unsaturation of fatty acids and degradation pathway were respectively prevalent in S1, S2 and S3 stages as evidenced by enrichment analysis. To our knowledge, this study represents the first report of sclerotial development transcriptomics in W. cocos. The obtained results provide novel insights into the developmental biology of the sclerotia, which is helpful for future studies about cultivation and breeding of W. cocos.

De Novo Analysis of Wolfiporia cocos Transcriptome to Reveal the Differentially Expressed Carbohydrate-Active Enzymes (CAZymes) Genes During the Early Stage of Sclerotial Growth.

The sclerotium of Wolfiporia cocos has been used as an edible mushroom and/or a traditional herbal medicine for centuries. W. cocos sclerotial formation is dependent on parasitism of the wood of Pinus species. Currently, the sclerotial development mechanisms of W. cocos remain largely unknown and the lack of pine resources limit the commercial production. The CAZymes (carbohydrate-active enzymes) play important roles in degradation of the plant cell wall to provide carbohydrates for fungal growth, development, and reproduction. In this study, the transcript profiles from W. cocos mycelium and 2-months-old sclerotium, the early stage of sclerotial growth, were specially analyzed using de novo sequencing technology. A total of 142,428,180 high-quality reads of mycelium and 70,594,319 high-quality reads of 2-months-old sclerotium were obtained. Additionally, differentially expressed genes from the W. cocos mycelium and 2-months-old sclerotium stages were analyzed, resulting in identification of 69 CAZymes genes which were significantly up-regulated during the early stage of sclerotial growth compared to that of in mycelium stage, and more than half of them belonged to glycosyl hydrolases (GHs) family, indicating the importance of W. cocos GHs family for degrading the pine woods. And qRT-PCR was further used to confirm the expression pattern of these up-regulated CAZymes genes. Our results will provide comprehensive CAZymes genes expression information during W. cocos sclerotial growth at the transcriptional level and will lay a foundation for functional genes studies in this fungus. In addition, our study will also facilitate the efficient use of limited pine resources, which is significant for promoting steady development of Chinese W. cocos industry.

Polysaccharides from Rhizoma Panacis Majoris and its anti-oxidant activity.

Response surface method (RSM) was employed to optimize the extraction conditions of polysaccharides from Rhizoma Panacis Majoris (the rhizomes of Panax japonicus C. A. Mey. var. major (Burk.) C. Y. Wu et K. M. Feng) (RPMP), a well-known Chinese traditional medicine. In order to obtain the optimal processing parameters, a three-variable Box-Behnken designs (BBD) were applied for experimental designs. RSM analysis indicated the good correspondence between experimental and predicted values, the optimal conditions for the yield of polysaccharides were as follows: the ultrasound time is 31.15 min, extraction temperature is 92.50 °C, and the ratio of water to raw material is 40 mL/g. The maximum value (13.87 ± 0.16%) of the yield of polysaccharides was obtained under these optimal conditions. The molecular weight (MW) was determined to be 1.48 × 10(5)(±0.39%)Da by HPSEC-MALLS-RID chromatography system. FT-IR spectra demonstrated obvious characteristic peaks of polysaccharides. The antioxidant activities of RPMP were investigated including scavenging activity of hydrogen radicals, ABTS radicals, and free radicals of superoxide anion in vitro, and the results exhibited that RPMP had a good potential for antioxidant.

[Transcriptome profiling and analysis of Panax japonicus var. major].

The rhizome of Panax japonicus var. major have been used as the natural medicinal agent by Chinese traditional doctors for more than thousand years. Most of the therapeutic effects of P. japonicus var. major had been reported due to the presence of tetracyclic or pentacyclic triterpene saponins. In this study, Illumina pair-end RNA-sequencing and de novo splicing were done in order to understand the pathway of triterpenoid saponins in this species. The valid reads data of 15. 6 Gb were obtained. The 62 240 unigenes were finally obtained by de novo splicing. After annotation, we discovered 19 unigenes involved in ginsenoside backbone biosynthesis. Additionally, 69 unigenes and 18 unigenes were predicted to have potential function of cytochrome P450 and UDP-glycosyltransferase based on the annotation results, which may encode enzymes responsible for ginsenoside backbone modification. This study provides global expressed datas for P. japonicus var. major, which will contribute significantly to further genome-wide research and analysis for this species.

De novo characterization of Panax japonicus C. A. Mey transcriptome and genes related to triterpenoid saponin biosynthesis.

Panax japonicus C.A.Mey, the traditional medicinal herb in the Araliaceae family, has been used as a tonic, anti-inflammatory and haemostatic agent in China for more than thousand years. Its clinical effects are mainly due to the presence of triterpenoid saponins. However, little is known at the genetic level about how saponins are biosynthesized in this plant. We have therefore performed the de novo transcriptome assembly and high throughput RNA-seq analysis for P. japonicus. 66,403 unigenes were assembled from 19.6 Gbp raw data, and 34,639 unigenes were annotated. After annotation, 29 unigenes involved in putative backbone biosynthesis of triterpenoid saponin were selected. Additionally, 34 Cytochrome P450 and 18 UDP-glycosyltransferase unigenes were predicted based on the annotation, which were related to the saponin backbone modification. The expression level of related key genes were further verified by qPCR analysis. The results of this study provide the most comprehensive expressed sequence resources for P. japonicus, which will enlarge the available P. japonicus gene pool and facilitate further genome-wide research and analyses in this species.

Resource investigation of traditional medicinal plant Panax japonicus (T.Nees) C.A. Mey and its varieties in China.

ETHNOPHARMACOLOGICAL RELEVANCE: Panax japonicus, the perennial herb in the Araliaceae family, was used as the natural medicinal herb by Chinese traditional doctors for more than thousand years. Its rhizome was mainly used as a tonic, anti-inflammatory and hemostatic agent in China. Most of the therapeutic effects of P. japonicus had been reported due to the presence of tetracyclic or pentacyclic triterpene saponins. Volatile oil, polysaccharides and amino acids had also been found in P. japonicus species and reported in the pharmacological functions. AIM OF THE STUDY: A three-year survey was conducted to determine the current resource status of P. japonicus (T.Nees) C. A. Mey and its varieties (P. japonicus var. major (Burkill) C.Y.Wu & Feng and P. japonicus var. bipinnatifidus (Seem.) C.Y.Wu & Feng) in 10 provinces of southern and southwestern China. METHODS AND RESULTS: Whole plants were sampled at 64 sites. Resource distribution, habitat type, morphological variation and market trend of them were studied and discussed. The natural resource in China is rarely available due to extensive exploitation and continual environment deterioration in recent decades, Abundance of P. japonicus was much lower than previous records, mainly found in Hubei, Sichuan, Guizhou and Yunnan province. Wild resources of P.japonicus var. major and P.japonicus var. bipinnatifidus were even scarcer, only found in Guizhou and Yunan province. Despite their dramatic rise of market trend, the artificial cultivation of them was still not fully developed in China, but progressed rapidly in Hubei province. CONCLUSION: In this study, we synthesized our understandings of the current resource state of P. japonicus׳s existence, variation and cultivation in China. This study will aid further investigations and increased protection of these plants, which are very valuable to traditional herbal medicine.

Characterization and antioxidant activities of degraded polysaccharides from Poria cocos sclerotium.

Poria cocos F.A.Wolf is a Chinese traditional medicine used to treat chronic gastritis, edema, nephrosis, gastric atony, and acute gastroenteric catarrh. Polysaccharides are the main active component of P. cocos. We obtained polysaccharides PCP-1, PCP-2, and PCP-3 from the degradation of P. cocos polysaccharides (PCP) with different concentrations of H2O2 solution. Molecular weights were determined by high performance size exclusion chromatography. HPLC analysis of monosaccharide composition confirmed that PCP-1, PCP-2, and PCP-3 are heteropolysaccharides composed of glucose and arabinose. IR spectra indicated obvious characteristic peaks of polysaccharides. The antioxidant activities of these polysaccharides were evaluated by established in vitro systems, including scavenging activity of hydroxyl radicals, ABTS radicals, and ferrous ions. The degradation polysaccharides exhibited obvious and concentration-dependent antioxidant properties. In addition, DNA binding analysis showed that PCP-1 had a stronger capacity than other polysaccharides to interact with DNA. However, each polysaccharide had a certain capacity for DNA damage protection.

Polysaccharides from Panax japonicus C.A. Meyer and their antioxidant activities.

Polysaccharides named as PP1, PP2, PP3, PP4 and PP5 were extracted and isolated from the rhizomes of Panax japonicus C.A. Meyer, a well-known Chinese traditional medicine, by controlling the final concentration of solution to precipitate the polysaccharides. The molecular weight of polysaccharides was determined by HGPLC chromatography system. The monosaccharide composition was analyzed by Gas chromatography on an Agilent 6890A instrument using a DB-35MS column and flame-ionization detector. All of the polysaccharides were heteropolysaccharides and consisted of arabinose, glucose and galactose. The content of arabinose increased with the increasing of ethanol concentration and PP5 had the most arabinose content in these samples. IR spectra indicated obvious characteristic peaks of polysaccharide, the presence of uronic acids. Their antioxidant activities were evaluated by various established in vitro systems, including scavenging activity of superoxide anion, hydroxyl radical, ABTS and DPPH radicals. Both samples showed inhibitory effects on superoxide, hydroxyl, ABTS and DPPH radical. And PP5 shows more clearly and relatively stronger capacity than other polysaccharides on the protective effect of DNA damage.