Isolation, Purification, Fractionation, and Hepatoprotective Activity of Polygonatum Polysaccharides.

In this study, three homogeneous fractions, PSP-N-b-1, PSP-N-b-2, and PSP-N-c-1, were obtained from an aqueous extract of Polygonatum using DEAE cellulose column chromatography, CL-6B agarose gel chromatography, and Sephadex G100 chromatography. Their monosaccharide compositions and molecular weights were analyzed. The results revealed that PSP-N-b-1, PSP-N-b-2, and PSP-N-c-1 are primarily composed of six monosaccharides: Man (mannose), GlcA (glucuronic acid), Rha (rhamnose), GalA (galacturonic acid), Glc (glucose), and Ara (arabinose), with molecular weights of 6.3 KDa, 5.78 KDa, and 3.45 KDa, respectively. Furthermore, we observed that Polygonatum polysaccharides exhibited protective effects against CCL4-induced liver damage in HepG2 cells in vitro, operating through both anti-oxidant and anti-inflammatory mechanisms. Our research findings suggest that Polygonatum polysaccharides may emerge as a promising option in the development of hepatoprotective drugs or functional foods with anti-inflammatory and antioxidant properties.

Isolation, purification, and physicochemical characterization of Polygonatum polysaccharide and its protective effect against CCl4-induced liver injury via Nrf2 and NF-κB signaling pathways.

This study aimed to provide scientific evidence that Polygonatum polysaccharide can be developed as a dietary supplement and medication for treating liver injuries. A water-soluble polysaccharide (PSP-N-c-1), with an average molecular weight of 3.45 kDa, was isolated and purified from the water extract of Polygonatum using DEAE cellulose column chromatography, CL-6B agarose gel chromatography, and Sephadex G100 chromatography. High-performance liquid chromatography, gas chromatography-mass spectrometry, and nuclear magnetic resonance spectroscopy analyses revealed that PSP-N-c-1 might be linear α-(1 â†’ 4)-glucans with α-Glcp residues linked to the backbone at C-6. In vitro experiments revealed that PSP-N-c-1 exhibited protective effects against CCl4-induced damage in HepG2 cells. In vivo experiments demonstrated that PSP-N-c-1 exhibited a hepatoprotective effect by enhancing antioxidant enzyme activity, inhibiting lipid peroxidation, and reducing the activity of pro-inflammatory mediators. Besides, PSP-N-c-1 could attenuate oxidative stress and inflammatory responses by activating the Nrf2-mediated signaling pathways and regulating the TLR4-mediated NF-κB signaling pathways. These findings demonstrated that PSP-N-c-1 may serve as a supplement for alleviating chemical liver damage.

Multiplex-PCR method application to identify duck blood and its adulterated varieties.

This study applied and validated the Multiplex-PCR method to identify the authenticity of duck blood and four common adulterated animal blood varieties. To this end, the genomic DNAs of duck blood and its counterfeit products were extracted using an efficient high-throughput extraction method. Specific primers were designed using the cytochrome b gene. The reaction system and conditions of a multiplex (namely, Five-plex) PCR were optimized, and the proposed methodology was verified, proving its good specificity, repeatability, and sensitivity. The Five-plex PCR system detected nine duck blood samples sold in the local market, revealing the adulteration of duck blood products. The Multiplex-PCR system can accurately and quickly detect adulterated animal blood in duck blood products, effectively finding counterfeits and identifying the authenticity of genuine duck blood products.

Sika deer velvet antler protein extract modulater bone metabolism and the structure of gut microbiota in ovariectomized mice.

Osteoporosis is a systemic osteopathy characterized by bone metabolism disorders that become more serious with age increases in postmenopausal women. Recent studies have found that antler protein is the main bioactive component of cervus pantotrichum, and it has a positive regulatory effect on bone metabolism and can improve estrogen level. This study aimed to investigate the effect of velvet antler extract (VAE) on the prevention of osteoporosis and the modulation of gut microbiota in ovariectomized (OVX) mice. OVX mice treated with 12 weeks of VAE exhibited higher levels of serum BGP, Ca2+, CT, and HyP (p < .05). Micro-CT scans showed that VAE significantly elevated bone volume fraction (BV/TV), trabecular bone number (Tb.N), trabecular bone thickness (Tb.Th), trabecular bone connection density (Conn.D), decreased trabecular separation (Tb.Sp), and structural modality index (SMI) than untreated OVX mice. The right tibial retinaculum in the VAE group was clearer, with a clearer reticular structure, smaller gaps, a tighter distribution, and a more orderly arrangement. The gut microbiota of the cecal contents was analyzed by 16 s rDNA amplicon sequencing. The data indicated that VAE modulated the species, numbers, and diversity of the gut microbiota in OVX mice. Ovariectomy caused dysbiosis of the intestinal microbiota by increasing the ratio of Firmicutes to Bacteroidetes in mice, but the ratio decreased after treatment with VAE. These results suggest that VAE has a therapeutic effect on OVX mice via modulate bone-related biochemical markers in serum and structure of gut microbiota.

Effects of an Armillaria mellea Polysaccharide on Learning and Memory of D-Galactose-Induced Aging Mice.

Arm illaria mellea has been known and used in traditional medicine in East Asia for hundreds of years. It has already been reported that A. mellea extracts have various pharmacological effects, and the polysaccharides of A. mellea exhibit antioxidant and anti-apoptotic activities. In this study, a water-soluble polysaccharide (AMP-N-a-1), with an average molecular weight of 17 kD, was isolated and purified from the water extract of A. mellea using DEAE-52, Sepharose CL-4B, and Sephadex G-100 column chromatography. AMP-N-a-1 was mainly composed of Man (1.65%), Glca (1.64%), Rha (1.82%), Gala (2.49%), Glc (90.48%), Gal (0.89%), Xyl (0.42%), and Ara (0.61%). AMP-N-a-1 was used to study the effect on the learning and memory of mice and its underlying mechanisms. The results showed that AMP-N-a-1 could significantly increase the activities of catalase (CAT) and superoxide dismutase (SOD) and reduce the content of nitric oxide (NO) in mouse brain tissue. Meanwhile, AMP-N-a-1 could reduce the contents of norepinephrine (NE) and dopamine (DA) but could increase the content of 5-hydroxytryptamine (5-HT) in mouse brain tissue. In addition, the immunofluorescence experiment showed that AMP-N-a-1 could promote the proliferation of hippocampal dentate gyrus neurons. The above results indicate that AMP-N-a-1 can significantly improve the learning and memory of mice, and the mechanism may be that AMP-N-a-1 can participate in the regulation of learning and memory through a variety of ways.

Protective effect of Sika Deer bone polypeptide extract on dexamethasone-induced osteoporosis in rats

BACKGROUND: Osteoporosis attacks approximately 10% of the population worldwide. Sika Deer (Cervus nippon), one of China's precious traditional medicinal animals, has been widely recorded in ancient Chinese medical books and claimed for centuries to have numerous medical benefits including bone strengthening. This study aimed to find the use of Sika Deer bone in treating osteoporosis according to traditional records and to investigate the protective effect of Sika Deer bone polypeptide extract on glucocorticoidinduced osteoporosis (GIOP) in rats. RESULTS: Sika Deer bone polypeptide extract could increase serum Ca2+ and BGP, decrease serum P3+, ALP, PTH, and CT, but had no effect on serum NO in rats with GIOP. The immunohistochemical iNOS results of the rats' distal femur were negative in each group. Besides the model group, the eNOS color reaction in osteoblasts was strongly positive in the other three groups. CONCLUSIONS: Sika Deer bone polypeptide extract can improve pathological changes in the microstructure and stimulate the expression of eNOS in osteoblasts. The protective effect on bone might be mediated by eNOS-dependent NO generation.

Effects of compound deer bone extract on osteoporosis model mice and intestinal microflora.

The preventive and therapeutic mechanisms of CDBE on osteoporosis were studied by observing the serum bone-related biochemical indicators, bone trabecular micro-structure and intestinal flora in ovariectomized osteoporosis model mice, in order to provide a scientific theoretical basis for the further study on the effect of CDBE on osteoporosis, and the prevention and treatment of osteoporosis with clinical traditional Chinese medicines. The components in CDBE were detected by UHPLC-MS. A mouse osteoporosis model was established by the bilateral ovariectomy in female ICR mice. The biochemical indicators related to osteoporosis were detected, the right proximal tibia was scanned by Micro-CT, the intestinal microflora in the colon contents were examined, and the changes of microflora were taken as the main target to evaluate the effect of CDBE on the intestinal microflora in the model mice. A total of 16 compounds were obtained by the combined application of UHPLC-MS. CDBE could significantly increase the contents of E2, Ca2+ , CT, HyP, OCN, FOXP3, P1NP and CTX-II, in the model mice. CDBE could significantly improve the trabecular micro-structure, Tb.N, Tb.Sp, SMI and Conn.D. CDBE could make the intestinal flora of osteoporosis model mice tend to healthy mice in species and quantity. CDBE can improve the symptoms of postmenopausal osteoporosis in mice, with a positive effect on the intestinal flora of postmenopausal mice. Its mechanism of regulating the symptoms of osteoporosis may be related to the regulation of bone-related biochemical indicators in the serum of mice. PRACTICAL APPLICATIONS: This research has a positive impact on the development of functional food with anti-osteoporosis in the future.

Agaricus blazei polypeptide exerts a protective effect on D-galactose-induced aging mice via the Keap1/Nrf2/ARE and P53/Trim32 signaling pathways.

This experiment mainly optimized the extraction technology of Agaricus blazei polypeptide (ABp) and evaluated its protective effect on aging mice. In this study, a novel single component, the M is 3 kD, was isolated and purified from Agaricus blazei. An aging mouse model was established using D-galactose. After the administration of ABp, the contents of total antioxidant capacity (T-AOC), malondialdehyde (MDA), catalase (CAT), and reactive oxygen species were significantly changed. Through immunofluorescence staining, it was observed that ABp can reduce changes in brain tissue. The differential expression of genes was analyzed by RNA-seq. A total of 295 differentially expressed genes were screened out in the ABp group.RT-qPCR verified important genes and showed that the mRNA expression levels of Hsph1, Trim32, HK1, Hnrnpa1, and Grik5 were significantly increased, and those of ApoE, Atp1a3, Stxbp1, and Mapk8ip1 was significantly decreased. Western blotting showed that the protein expression levels of Keap1 and p53 were significantly lower, while the protein expression levels of Nrf2, HO-1, Hsph1, and Trim32 were significantly higher in the ABP group. ABp played an anti-aging role in an aging mouse model. The specific mechanism of action may be related to the regulation of the expression of the Keap1/Nrf2/P53 signaling pathway and related factors. PRACTICAL APPLICATIONS: The research may contribute to the development of ABp as functional foods or dietary supplements for anti-aging in the future.

Study on the effect of regulation of Cordyceps militaris polypeptide on the immune function of mice based on a transcription factor regulatory network.

BACKGROUND: The pathogenesis of the abnormality of the immune system is still not clear at present. Chemosynthetic drugs, human or animal immune products and microbiological drugs are used as the main drugs in clinics currently, but these drugs have different side effects. So researchers turned to safer natural products in order to find immunomodulatory active substances from natural products and their extracts. METHODS: Immunosuppressed mice were induced by cyclophosphamide and administered with Cordyceps militaris polypeptide (CMP) for the study on the effect of CMP on the immune function of mice and its mechanism. Based on the 1748 differential gene sets selected in our previous work, the transcription factors and their corresponding target genes were screened by integrating the TRED (Transcriptional Regulatory Element Database), a transcriptional factor-target gene regulatory network was constructed, then the role of transcription factors in the regulatory network was elucidated by statistically analyzing the key nodes, and finally, the correlation of network genes with diseases was analyzed by using the DAVID database. RESULTS: The results of animal experiments showed that CMP could increase the immune organ indexes, the number of white blood cells, the degree of delayed allergy and the content of hemolysin in the serum of mice. CMP was found to be involved in the regulation of immune function in mice through genes Kdr, Spp1, Ptgs2, Rel, and Smad3, and transcription factors Ets1, E2f2 and E2f1. E2F2 and E2F1 are members of the E2F family, so we speculated that the E2F family might play an important role, and its main regulatory pathways were the PI3K-Akt signaling pathway and TNF signaling pathway. CONCLUSION: CMP can improve the immunity of mice. CMP can regulate the immune function of mice through multiple genes and transcription factors, and may also play a role in immune-related diseases, such as cancer.

Sulfated polysaccharides from Rhodiola sachalinensis reduce d-gal-induced oxidative stress in NIH 3T3 cells.

In this study, three sulfated polysaccharides (S-RSP1-2, S-RSP1-4 and S-RSP1-8) from Rhodiola sachalinensis were produced by chlorosulfonic acid-pyridine method. d-gal was used to develop an oxidative stress model in the mouse embryonic fibroblast cell line NIH 3T3. Effects of the three sulfated polysaccharides on d-gal-induced oxidative stress were investigated. The results showed that S-RSP1-4 improved the viability of the d-gal-induced oxidative stress in NIH 3T3 cells. The sulfated polysaccharides were found to have a better protective effect against d-gal-induced oxidative stress as compared to the native polysaccharide. Scanning electronmicroscopy also showed a significant change in the surface morphology of sulfated polysaccharides. In addition, the sulfated polysaccharides had noticeable DPPH radical-scavenging activity. In summary, our results demonstrated that d-gal was able to induce oxidative stress in NIH 3T3 cells, and sulfated group might play an important role in resistance to d-gal-induced oxidative damage.

Erratum: Combined antitumor effects of P-5m octapeptide and 5- fluorouracil on a murine model of H22 hepatoma ascites.

[This corrects the article DOI: 10.3892/etm.2018.6422.].

Development of a PCR-based assay for detection of Chinese mink tissue in meat products based on the mitochondrial DNA cytochrome-b gene.

Species authentication of meat product origins has become an important subject for ensuring the health of consumers. Based on the cytochrome b gene, we developed a PCR-based assay kit for identification of Chinese mink tissues from 10 animal species in meat products, and to evaluate its quality indices including specificity, stability, sensitivity, and repeatability. Kits were made up of DNA extraction and PCR amplification systems based on species-specific, and universal primers. The reference meat mixtures and commercial samples were extracted by the kit and PCR technique was performed to identify the species of mink authenticity. The kit was effective after 20 repeated freeze-thaw cycles and it could be stored at -20 °C for 1 year. The sensitivity showed that a concentration as low as 0.1 ng/µL still can amplify the target band. The specificity test confirmed that the kit was 100% specific. The kit proved to be effective, stable, and reliable for extraction of efficient contents of the genomic DNA and routine analysis of Chinese mink source composition from meat products.

[Development on DNA detection kit of fox and its application in common meat products].

OBJECTIVE: To develop a kind of DNA extraction and detection kit for identification of fox source composition. METHODS: Using the modern DNA fingerprint technology, the DNA extraction method was improved, and DNA extraction and detection reagent was developed to obtain the fox polymerase chain reaction( PCR)detection kit. The performance parameters of the kit were evaluated. Finally, 42 samples of fox meat and its mixture with commercial meat products were detected. RESULTS: The kit was proved effective after 20 times of the repeated frozen-thaw and it could be stored at-20 ℃ for 1 year. The specificity test confirmed that fox source composition were detected from 42 samples of fox meat and its mixture with commercial meat. The specificity of the kit was 100%. The minimum detection limit of DNA was 0. 1 ng/µL. CONCLUSION: The fox DNA detection kit could be applied in rapid detection commonmeat of fox source composition, which are good specificity, high sensitivity and good stability.

Combined antitumor effects of P-5m octapeptide and 5-fluorouracil on a murine model of H22 hepatoma ascites.

The present study has demonstrated that P-5m octapeptide (P-5m) has therapeutic potential in metastatic human hepatocarcinoma, possibly through the modulation of matrix metalloproteinase-2 expression. The purpose of the present study was to evaluate the antitumor effect of P-5m combined with 5-fluorouracil (5-Fu) on the treatment of hepatoma 22 (H22) hepatocarcinoma malignant ascites in a mouse model. The inhibitory effect on the growth of mouse ascites tumors was monitored by measuring body weight gain, survival time, ascites volume, numbers of tumor cells, DNA synthesis and peritoneal capillary permeability analysis. The present data revealed a significant reduction in ascites volume and cell count in mice that were treated with P-5m plus 5-Fu. Furthermore, the median survival time in mice in the combination group was prolonged compared with the disease control group. Moreover, a significant reduction in the total H22 ascites cell count in mice from the combination group was observed when compared with the disease control group. P-5m plus 5-Fu was able to induce the cell cycle arrest and inhibit the peritoneal capillary permeability of the mice. To conclude, the present study indicated that P-5m may have therapeutic potential in ascites caused by hepatocellular carcinoma.

Data Mining Mycobacterium tuberculosis Pathogenic Gene Transcription Factors and Their Regulatory Network Nodes.

Tuberculosis (TB) is one of the deadliest infectious diseases worldwide. In Mycobacterium tuberculosis, changes in gene expression are highly variable and involve many genes, so traditional single-gene screening of M. tuberculosis targets has been unable to meet the needs of clinical diagnosis. In this study, using the National Center for Biotechnology Information (NCBI) GEO Datasets, whole blood gene expression profile data were obtained in patients with active pulmonary tuberculosis. Linear model-experience Bayesian statistics using the Limma package in R combined with t-tests were applied for nonspecific filtration of the expression profile data, and the differentially expressed human genes were determined. Using DAVID and KEGG, the functional analysis of differentially expressed genes (GO analysis) and the analysis of signaling pathways were performed. Based on the differentially expressed gene, the transcriptional regulatory element databases (TRED) were integrated to construct the M. tuberculosis pathogenic gene regulatory network, and the correlation of the network genes with disease was analyzed with the DAVID online annotation tool. It was predicted that IL-6, JUN, and TP53, along with transcription factors SRC, TNF, and MAPK14, could regulate the immune response, with their function being extracellular region activity and protein binding during infection with M. tuberculosis.

Improvement of Learning and Memory Induced by Cordyceps Polypeptide Treatment and the Underlying Mechanism.

Our previous research revealed that Cordyceps militaris can improve the learning and memory, and although the main active ingredient should be its polypeptide complexes, the underlying mechanism of its activity remains poorly understood. In this study, we explored the mechanisms by which Cordyceps militaris improves learning and memory in a mouse model. Mice were given scopolamine hydrobromide intraperitoneally to establish a mouse model of learning and memory impairment. The effects of Cordyceps polypeptide in this model were tested using the Morris water maze test; serum superoxide dismutase activity; serum malondialdehyde levels; activities of acetyl cholinesterase, Na+-k+-ATPase, and nitric oxide synthase; and gamma aminobutyric acid and glutamate contents in brain tissue. Moreover, differentially expressed genes and the related cellular signaling pathways were screened using an mRNA expression profile chip. The results showed that the genes Pik3r5, Il-1ß, and Slc18a2 were involved in the effects of Cordyceps polypeptide on the nervous system of these mice. Our findings suggest that Cordyceps polypeptide may improve learning and memory in the scopolamine-induced mouse model of learning and memory impairment by scavenging oxygen free radicals, preventing oxidative damage, and protecting the nervous system.

mRNA chip-based analysis on transcription factor regulatory network central nodes of protection targets of Deproteinized Extract of Calf Blood on acute liver injury in mice.

Our previous study found that Deproteinized Extract of Calf Blood (DECB) could protect the acute liver injury induced by carbon tetrachloride in mice, but the target-related transcription factors and their regulatory networks were not comprehensively studied. Based on the mRNA expression microarray data obtained in the previous study, the mRNA transcription factor regulatory networks were constructed by screening the transcription factors of differentially expressed genes and their corresponding target proteins, and the analysis on the functions and pathways of the regulatory network central nodes was performed. Eight genes Ltf, Tnf, Il6, Jun, Il12b, Stat3, Rel and Crem could regulate the inflammatory factors, and TNF signaling pathway and Jak-STAT signaling pathway might play an important role in the mechanism through which DECB protected the liver of mice. DECB can not only inhibit the apoptosis of hepatocytes, but also inhibit the inflammatory cytokines.

Development and evaluation of a PCR-based assay kit for authentication of Zaocys dhumnades in traditional Chinese medicine.

We developed a kind of Zaocys dhumnades DNA test kit and it's indexes including specificity, sensitivity and stability were evaluated and compared with the method recorded in Chinese Pharmacopoeia (2010 edition). The bioinformatics technology was used to design primers, sequencing and blast, in conjunction with PCR technology based on the characteristics of Z. dhumnades cytochrome b (Cyt b) gene. The efficiency of nucleic acid extraction by the kit was done in accordance with Pharmacopoeia method. The kit stability results proved effective after repeated freezing and thawing 20 times. The sensitivity results indicated that the lowest amount detected by the kit was 0. 025 g of each specimen. The specificity test of the kit was 100% specific. All repeatability tests indicated the same results when conducted three times. Compared with the method recorded in Chinese Pharmacopoeia, the PCR-based assay kit by our team developed is accurate, effective in identification of Z. dhumnades, it is simple and fast, demonstrating a broad prospect in quality inspection of Z. dhumnades in the future.

Identification and characteristics of Testudinis Carapax et Plustrum based on fingerprint profiles of mitochondrial DNA constructed by species-specific PCR and random amplified polymorphic DNA.

Traditional use of Testudinis Carapax et Plustrum (TCP) as a medicine and health food has been widely reported. We compared two DNA fingerprint profiles of mitochondrial (mtDNA) from TCP based on species-specific PCR and random amplified polymorphic DNA (RAPD) to identify their authority. A series of sequences from cytochrome b (Cyt b) of Chinemys reevesii and their counterfeits were downloaded from the Genbank, and Premier 5.0 software was used to design a set of primers. A species-specific PCR and RAPD were undertaken to obtain the different DNA fingerprints respectively. The mtDNA was successfully extracted from all samples using the modified salting-out method. A relative molecular mass of 16.6 × 103 bp was observed, and mtDNA was measured between 1.83 ± 0.02. Fragments of 78 bp were amplified from all the TCP samples tested (except adulterant animals) using species-specific PCR method. The RAPD showed different electrocardiogram between genuine and counterfeit tortoise shell goods along with stripe number and location. The salting-out method (as modified) was used to extract high-quality mtDNA from TCP. The species-specific PCR and RAPD assay proposed in this study could be used for quality control of TCP with more specificity, sensitivity, and applicability.