Screening of natural inhibitors against peptidyl arginine deiminase 4 from herbal extracts by a high-performance liquid chromatography ultraviolet-visible based method.

Peptidyl arginine deiminase 4 (PAD4) is an important biocatalytic enzymes involved in the conversion of protein arginine to citrulline, its dysregulation has a great impact on many physiological processes. Recently, PAD4 has emerged as a potential therapeutic target for the treatment of various diseases including rheumatoid arthritis (RA). Traditional Chinese Medicines (TCMs), also known as herbal plants, have gained great attention by the scientific community due to their good therapeutic performance and far fewer side effects observed in the clinical treatment. However, limited researches have been reported to screen natural PAD4 inhibitors from herbal plants. The color developing reagent (COLDER) or fluorescence based methods have been widely used in PAD4 activity assay and inhibitor screening. However, both methods measure the overall absorbance or fluorescence in the reaction solution, which are easy to be affected by the background interference due to colorful extracts from herbal plants. In this study, a simple, and robust high-performance liquid chromatography ultraviolet-visible (HPLC-UV) based method was developed to determine PAD4 activity. The proposed strategy was established based on COLDER principle, while used hydrophilic l-arginine instead of hydrophobic N-benzoyl-l-arginine ethyl ester (BAEE) as a new substrate to determine PAD4 inhibition activity of herbal extracts. The herbal extracts and PAD4 generated hydrophobic l-citrulline were successfully separated by the HPLC, and the developed method was optimized and validated with a known PAD4 inhibitor (GSK484) in comparison with COLDER assay. The IC50 value of GSK484 measured by HPLC-UV method was 153 nM, and the detection limit of the citrulline was 0.5 nmol, respectively, with a linear range of 0.5 nmol to 20 nmol. The IC50 value of the HPLC-UV method was improved by nearly three times compared with COLDER assay (527 nM), and the results indicated the reliability of PAD4 inhibition via HPLC-UV method. The inhibitory effect against PAD4 were fast and accurately screened for the twenty-four extracts from eight herbs. Among them, Ephedra Herba extracts showed significant inhibitory activity against the PAD4 with the IC50 values of three extracts (ethanol, ethyl acetate and water) ranging from 29.11 µg/mL to 41.36 µg/mL, which may help researchers to discover novel natural compounds holding high PAD4 inhibition activity.

Optimization of Chemical Extraction Conditions of Dietary Fiber from Cistanche deserticola Residues and Its Structural Characteristics and Physicochemical and Functional Properties.

Cistanche deserticola residues are by-products of the industrial production of Cistanche deserticola, which are currently often discarded, resulting in the waste of resources. In order to achieve the efficient utilization of Cistanche deserticola, dietary fiber from Cistanche deserticola residues was extracted chemically and the optimization of the extraction conditions was performed, using the response surface methodology to study the effects of the NaOH concentration, extraction temperature, extraction time, and solid-liquid ratio on the yield of water-soluble dietary fiber (SDF). The structural, physicochemical, and functional properties of the dietary fiber were also investigated. The results showed that the optimal conditions were as follows: NaOH concentration of 3.7%, extraction temperature of 71.7 °C, extraction time of 89.5 min, and solid-liquid ratio of 1:34. The average yield of SDF was 19.56%, which was close to the predicted value of 19.66%. The two dietary fiber types had typical polysaccharide absorption peaks and typical type I cellulose crystal structures, and the surface microstructures of the two dietary fiber types were different, with the surface of SDF being looser and more porous. Both dietary fiber types had good functional properties, with SDF having the strongest water-holding capacity and the strongest adsorption capacity for nitrite, cholesterol, sodium cholate, and glucose, while IDF had a better oil-holding capacity. These results suggest that Cistanche deserticola residues are a good source of dietary fiber and have promising applications in the functional food processing industry.

Astragaloside IV ameliorates radiation-induced nerve cell damage by activating the BDNF/TrkB signaling pathway.

Radiation can induce nerve cell damage. Synapse connectivity and functionality are thought to be the essential foundation of all cognitive functions. Therefore, treating and preventing damage to synaptic structure and function is an urgent challenge. Astragaloside IV (AS-IV) is a glycoside extracted from Astragalus membranaceus (Fisch.). Bunge is a widely used traditional Chinese medicine in China with various pharmacological properties, including protective effects on the central nervous system (CNS). In this study, the effect of AS-IV on synapse damage and BDNF/TrkB signaling pathway in radiated C57BL/6 mice with X-rays was investigated. PC12 cells and primary cortical neurons were exposed to UVA in vitro. Open field test and rotarod test were used to observe the effects of AS-IV on the motor and explore the abilities of radiated mice. The pathological changes in the brain were observed by hematoxylin and eosin and Nissl staining. Immunofluorescence analysis was used to detect the synapse damage. The expressions of the BDNF/TrkB pathway and neuroprotection-related molecules were detected by Western blotting and Quantitative-RTPCR, respectively. The results showed that AS-IV could improve the motor and explore abilities of radiated mice, reduce pathological damage to the cortex, enhance neuroprotection functions, and activate BDNF/TrkB pathway. In conclusion, AS-IV could relieve radiation-induced synapse damage, at least partly through the BDNF/TrkB pathway.

Hypolipidemic mechanism of Pleurotus eryngii polysaccharides in high-fat diet-induced obese mice based on metabolomics.

Objective: In this study, the structure of Pleurotus eryngii polysaccharides (PEPs) was characterized, and the mechanism of PEP on obesity and hyperlipidemia induced by high-fat diet was evaluated by metabonomic analysis. Methods: The structure of PEPs were characterized by monosaccharide composition, Fourier transform infrared spectroscopy and thermogravimetry. In animal experiments, H&E staining was used to observe the morphological difference of epididymal adipose tissue of mice in each group. Ultrahigh performance liquid chromatography (UHPLC)-(QE) HFX -mass spectrometry (MS) was used to analyze the difference of metabolites in serum of mice in each group and the related metabolic pathways. Results: The PEPs contained nine monosaccharides: 1.05% fucose, 0.30% arabinose, 17.94% galactose, 53.49% glucose, 1.24% xylose, 23.32% mannose, 1.30% ribose, 0.21%galacturonic acid, and 1.17% glucuronic acid. The PEPs began to degrade at 251°C (T0), while the maximum thermal degradation rate temperature (Tm) appeared at 300°C. The results histopathological observation demonstrated that the PEPs had signifificant hypolipidemic activities. After PEPs intervention, the metabolic profile of mice changed significantly. A total of 29 different metabolites were selected as adjunctive therapy to PEPs, for treatment of obesity and hyperlipidemia-related complications caused by a high-fat diet. These metabolites include amino acids, unsaturated fatty acids, choline, glycerol phospholipids, and other endogenous compounds, which can prevent and treat obesity and hyperlipidemia caused by a high-fat diet by regulating amino acid metabolism, fatty acid metabolism, and changes in metabolic pathways such as that involved in the citric cycle (TCA cycle). Conclusions: The presented results indicate that PEPs treatment can alleviate the obesity and hyperlipidemia caused by a high-fat diet and, thus, may be used as a functional food adjuvant, providing a theoretical basis and technical guidance for the prevention and treatment of high-fat diet-induced obesity and hyperlipidemia.

Preliminary study on the anti-apoptotic mechanism of Astragaloside IV on radiation-induced brain cells.

With multiple targets and low cytotoxicity, natural medicines can be used as potential neuroprotective agents. The increase in oxidative stress levels and inflammatory responses in the brain caused by radiation affects cognitive function and neuronal structure, and ultimately leads to abnormal changes in neurogenesis, differentiation, and apoptosis. Astragaloside Ⅳ (AS-Ⅳ), one of the main active constituents of astragalus, is known for its antioxidant, antihypertensive, antidiabetic, anti-infarction, anti-inflammatory, anti-apoptotic and wound healing, angiogenesis, and other protective effects. In this study, the mechanism of AS-IV against radiation-induced apoptosis of brain cells in vitro and in vivo was explored by radiation modeling, which provided a theoretical basis for the development of anti-radiation Chinese herbal active molecules and brain health products. In order to study the protective mechanism of AS-IV on radiation-induced brain cell apoptosis in mice, the paper constructed a radiation-induced brain cell apoptosis model, using TUNEL staining, flow cytometry, Western blotting to analyze AS-IV resistance mechanism to radiation-induced brain cell apoptosis. The results of TUNEL staining and flow cytometry showed that the apoptosis rate of radiation group was significantly increased. The results of Western blotting indicated that the expression levels of p-JNK, p-p38, p53, Caspase-9 and Caspase-3 protein, and the ratio of Bax to Bcl-2 in radiation group were significantly increased. There was no significant difference in the expression levels of JNK and p38. After AS-IV treatment, the apoptosis was reduced and the expression of apoptosis related proteins was changed. These data suggested that AS-IV can effectively reduce radiation-induced apoptosis of brain cells, and its mechanism may be related to the phosphorylation regulation of JNK-p38.

[Identification of key enzyme genes involved in biosynthesis of steroidal saponins and analysis of biosynthesis pathway in Polygonatum cyrtonema].

Steroidal saponins, which are the characteristic and main active constituents of Polygonatum, exhibit a broad range of pharmacological functions, such as regulating blood sugar, preventing cardiovascular and cerebrovascular diseases and anti-tumor. In this study, we performed RNA sequencing(RNA-Seq) analysis for the flowers, leaves, roots, and rhizomes of Polygonatum cyrtonema using the BGISEQ-500 platform to understand the biosynthesis pathway of steroidal saponins and study their key enzyme genes. The assembly of transcripts for four tissues generated 129 989 unigenes, of which 88 958 were mapped to several public databases for functional annotation, 22 813 unigenes were assigned to 53 subcategories and 64 877 unigenes were annotated to 136 pathways in KEGG database. Furthermore, 502 unigenes involved in the biosynthesis pathway of steroidal saponins were identified, of which 97 unigenes encoding 12 key enzymes. Cycloartenol synthase, the first key enzyme in the pathway of phytosterol biosynthesis, showed conserved catalytic domain and substrate binding domain based on sequence analysis and homology modeling. Differentially expressed genes(DEGs) were identified in rhizomes as compared to other tissues(flowers, leaves or roots).The 2 437 unigenes annotated by KEGG showed rhizome-specific expression, of which 35 unigenes involved in the biosynthesis of steroidal saponins. Our results greatly extend the public transcriptome dataset of Polygonatum and provide valuable information for the identification of candidate genes involved in the biosynthesis of steroidal saponins and other important secondary metabolites.

[Transcriptome analysis of venom gland and identification of functional genes for snake venom protein in Agkistrodon acutus].

Agkistrodon acutus is a traditional Chinese herb medicine which has immunological regulation,anti-tumor,anti-inflammatory and analgesic effects,which is mainly used for the treatment of rheumatoid arthritis,ankylosing spondylitis,sjogren's syndrome and tumors. In order to excavate more important functional genes from A. acutus,the transcriptome of the venom gland was sequenced by the Illumina Hi Seq 4000,and 32 862 unigenes were assembled. Among them,26 589 unigenes were mapped to least one public database. 2 695 unigenes were annotated and assigned to 62 TF families,and 5 920 SSR loci were identified. The majority of mapped unigenes was from Protobothrops mucrosquamatus in the NR database,which revealed their closest homology. Three secretory phospholipase A_2 with different amino acid sequences showed similar spatial structures and all had well-conserved active sites. The 3 D structural models of C-type lectin showed conserved glycosylation binding sites( Asn45). This study will lay the foundation for the further study of the function of snake venom protein,and promoting the development and utilization of genome resources from A. acutus.

[Cloning and characterization of chalcone synthase and chalcone isomerase genes in Arisaema heterophyllum].

Chalcone synthase( CHS) and chalcone isomerase( CHI) are key enzymes in the biosynthesis pathway of flavonoids. In this study,unigenes for CHS and CHI were screened from the transcriptome database of Arisaema heterophyllum. The open reading frame( ORFs) of chalcone synthase( Ah CHS) and chalcone isomerase( Ah CHI) were cloned from the plant by RT-PCR. The physicochemical properties,expression and structure characteristics of the encoded proteins Ah CHS and Ah CHI were analyzed. The ORFs of Ah CHS and Ah CHI were 1 176,630 bp in length and encoded 392,209 amino acids,respectively. Ah CHS functioned as a symmetric homodimer. The N-terminal helix of one monomer entwined with the corresponding helix of another monomer. Each CHS monomer consisted of two structural domains. In particular,four conserved residues define the active site. The tertiary structure of Ah CHI revealed a novel open-faced ß-sandwich fold. A large ß-sheet( ß4-ß11) and a layer of α-helices( α1-α7) comprised the core structure. The residues spanning ß4,ß5,α4,and α6 in the three-dimensional structure were conserved among CHIs from different species. Notably,these structural elements formed the active site on the protein surface,and the topology of the active-site cleft defined the stereochemistry of the cyclization reaction. The homology comparison showed that Ah CHS had the highest similarity to the CHS of Anthurium andraeanum,while Ah CHI had the highest similarity to the CHI of Paeonia delavayi. This study provided the basis for the functional study of Ah CHS and Ah CHI and the further study on plant flavonoid biosynthesis pathway.

Transcriptome analysis of Polygonatum cyrtonema Hua: identification of genes involved in polysaccharide biosynthesis.

BACKGROUND: Polygonatum cyrtonema Hua (P. cyrtonema) is one of the most important herbs in traditional Chinese medicine. Polysaccharides in P. cyrtonema plants comprise a class of important secondary metabolites and exhibit a broad range of pharmacological functions. RESULTS: In order to identify genes involved in polysaccharide biosynthesis, we performed RNA sequencing analysis of leaf, root, and rhizome tissues of P. cyrtonema. A total of 164,573 unigenes were obtained by assembling transcripts from all three tissues and 86,063 of these were annotated in public databases. Differentially expressed genes (DEGs) were determined based on expression profile analysis, and DEG levels in rhizome tissues were then compared with their counterparts in leaf and root tissues. This analysis revealed numerous genes that were either up-regulated or uniquely expressed in the rhizome. Multiple genes encoding important enzymes, such as UDP glycosyltransferases (UGTs), or transcription factors involved in polysaccharide biosynthesis were identified and further analyzed, while a few genes encoding key enzymes were experimentally validated using quantitative real-time PCR. CONCLUSION: Our results substantially expand the public transcriptome dataset of P. cyrtonema and provide valuable clues for the identification of candidate genes involved in metabolic pathways.

Transcriptome Analysis of Clinopodium chinense (Benth.) O. Kuntze and Identification of Genes Involved in Triterpenoid Saponin Biosynthesis.

Clinopodium chinense (Benth.) O. Kuntze (C. chinense) is an important herb in traditional Chinese medicine. Triterpenoid saponins are a major class of active compounds in C. chinense with broad pharmacological activities and hemostatic, antitumor, and anti-hyperglycemic effects. To identify genes involved in triterpenoid saponin biosynthesis, transcriptomic analyses of leaves, stems, and roots from C. chinense were performed. A total of 135,968 unigenes were obtained by assembling the leaf, stem, and root transcripts, of which 102,154 were annotated in public databases. Differentially expressed genes were determined based on expression profile analysis and analyzed for differential expression of unique genes related to triterpenoid saponin biosynthesis. Multiple unigenes encoding crucial enzymes or transcription factors involved in triterpenoid saponin synthesis were identified and analyzed. The expression levels of unigenes encoding enzymes were experimentally validated using quantitative real-time PCR. This study greatly broadens the public transcriptome database for this species and provides a valuable resource for identifying candidate genes involved in the biosynthesis of triterpenoid saponins and other secondary metabolites.

Sex-specific parental care strategies via nestling age: females pay more attention to nestling demands than males do in the horned lark, Eremophila alpestris.

In many species, nestling demands vary continuously during early development and both parents have different parental care strategies at each nestling age. Sexual conflict arises when each parent expects its partner investing more in parental care. It is largely unknown how the two parents respond to the dynamics of nestling demands and resolve the sexual conflict during nestling period, especially on Qinghai-Tibetan Plateau. To address this question, we monitored parental care behaviors of horned larks (Eremophila alpestris) using video-recording systems. We found that male horned larks invested less in parental care, but had a larger body size than females, which is consistent with the parental investment hypothesis. Only the female brooded nestlings, but both parents contributed to feeding efforts. Feeding rates of males and females were negatively correlated, indicating that they used evolutionarily stable strategies. Strategies of parental care via nestling age were sex-specific. Females continuously adjusted care behaviors to follow the dynamics of nestling demands as nestling age increased, such as decreasing brood attentiveness and increasing feeding rate. By contrast, male feeding rate showed no significant correlation with nestling age, but increased with the synchrony feeding rate. We suggest the synchrony feeding behavior may act as a control measure for females to promote and assess the males' contribution. We consider low mating opportunities drive males to act as assistants for females, and correspondingly cause males to pay less attention to nestling demands than females.