Liangen Shi

Effects of extraction methods on the antioxidant activities of polysaccharides obtained from Flammulina velutipes

Four polysaccharides (CFP, UFP, MFP and EFP) were extracted from Flammulina velutipes using hot water, ultrasonic, microwave or enzymatic methods optimized by orthogonal test. Preliminary structural characterizations were conducted using physicochemical properties. Polysaccharides extracted by all four methods showed similar physicochemical characteristics and FT-IR spectra. However, SEM images of tissues of F. velutipes were significantly different. EFP demonstrated better antioxidant activities against hydroxyl radical as well as improved metal chelating activity. UFP showed higher DPPH scavenging activity, but CFP exhibited higher antioxidant activity in reducing power. Hence, these polysaccharides can be used as natural antioxidants in functional foods or medicine. Further experiments on the biological activities of these four polysaccharides are currently in progress.

Protective function of cis-mulberroside A and oxyresveratrol from Ramulus mori against ethanol-induced hepatic damage

The aim of the study was to investigate the protective effects of oxyresveratrol and cis-mulberroside A isolated from Ramulus mori on the liver of mice intoxicated with ethanol. Animals were pretreated with different doses (30 and 60mg/kg of body weight) of oxyresveratrol and cis-mulberroside A prior to the ethanol (9g/kg of body weight) orally for 7 days. Ethanol treatment induced the decrease of reduced glutathione level and antioxidant enzymes activities, the elevation of the lipid peroxidation and cytochrome P450 2E1 activity accompanied with the increase of iron concentration and mitochondrial permeability transition. Pretreatment with oxyresveratrol and cis-mulberroside A restored the changes in the above parameters up to the basal level. The protective effects of the two active compounds were further supported by attenuation of the degree of tissue damage and the regulation of the expression of TNF-α. It could be concluded that oxyresveratrol and cis-mulberroside A from R. mori could protect mice against ethanol-induced hepatic damage.

Structural elucidation and in vitro antitumor activity of a novel oligosaccharide from Bombyx batryticatus.

An oligosaccharide BBPW-2 was isolated and purified from Bombyx batryticatus, its molecular weight was 2.0 × 10(3)Da, and its structure was elucidated by compositional, methylation and NMR analysis. Our results showed that BBPW-2 consisted of β-D-(1 → 2,6)-glucopyranose and β-D-(1 → 2,6)-mannosyl units serving as the backbone, α-D-(1 → 2)-galactopyranose and α-D-(1 → 3)-mannosyl units as branches, and α-D-Manp and β-D-Glcp as terminals. The in vitro inhibitory activity of BBPW-2 was measured using MTT and and crystal violet assays, which suggested that BBPW-2 had direct cytotoxic effects on the cancer cell lines HeLa and HepG2 (particularly HeLa cells), and had a long-term antiproliferative effect on MCF-7 cells, respectively. Apoptosis and cellcycle analysis of HeLa cells showed that BBPW-2 induced cellcycle disruption in the G0/G1 and G2/M phases accompanied by an impressive increment of early apoptotic cells and late apoptotic and necrotic cells. These results suggest that BBPW-2 could be a potential chemotherapeutic drug and its antitumor effects deserve further study.

Antioxidant Properties of Ethanolic Extract from Ramulus mori (Sangzhi)

The antioxidant properties and total phenolic contents of four fractions of ethanolic extract from Ramulus mori were examined. Various experimental models including superoxide radical, hydroxyl radical, 1,1-diphenyl-2-picrylhydrazyl radicals (DPPH) scavenging activity, metal chelating activity, and reducing power were used for characterization of their antioxidant activity. The four fractions showed various degrees of efficacy in each assay in a dose-dependent manner. The third fraction with the highest amount of total phenolics was the most potent antioxidant in all assays used. In addition, the most powerful compound (oxyresveratrol) was isolated and identified followed by on-line HPLC method and characterized by different spectral analysis. Oxyresveratrol exhibited impressive antioxidant activities in scavenging the superoxide radical, hydroxide radical, and DPPH. On the basis of the results obtained, Ramulus mori may serve as a potential source of natural antioxidant due to its significant antioxidant activity and oxyresveratrol may be the most powerful antioxidant in ethanolic extracts of Ramulus mori.The antioxidant properties and total phenolic contents of four fractions of ethanolic extract from Ramulus mori were examined. Various experimental models including superoxide radical, hydroxyl radical, 1,1-diphenyl-2-picrylhydrazyl radicals (DPPH) scavenging activity, metal chelating activity, and reducing power were used for characterization of their antioxidant activity. The four fractions showed various degrees of efficacy in each assay in a dose-dependent manner. The third fraction with the highest amount of total phenolics was the most potent antioxidant in all assays used. In addition, the most powerful compound (oxyresveratrol) was isolated and identified followed by on-line HPLC method and characterized by different spectral analysis. Oxyresveratrol exhibited impressive antioxidant activities in scavenging the superoxide radical, hydroxide radical, and DPPH. On the basis of the results obtained, Ramulus mori may serve as a potential source of natural antioxidant due to its significant antioxidant...

Structural characterization and antitumor activity of a polysaccharide from ramulus mori

This study investigated the chemical characterization and antitumor effects of a polysaccharide from ramulus mori. A water-soluble polysaccharide, RMP1 with the estimated molecular weight of 137 kDa, was isolated and purified from ramulus mori through gel permeation chromatography. RMP1 is mainly composed of arabinose, xylose, glucose, galactose and rhamnose in a ratio of 0.56:0.37:0.17:1.00:0.08. Methylation and NMR analysis revealed that RMP1 had a backbone composed of 1,6-β-d-Galp, 1,3,6-β-d-Galp and 1,3-β-d-Galp residues, two main branches of 1,2-α-l-Araf, 1,3,6-β-d-Galp and 1,4-β-d-Xylp; it also had α-l-Araf and β-d-Glcp as terminals. In the MTT assay, RMP1 showed significant anticancer effects against the SGC-7901 and HeLa cells. In addition, no cytotoxicity was observed on the HEK-293 and RAW 264.7 cells. Flow cytometry showed that RMP1 exerted a stimulatory effect on the SGC-7901 cells apoptosis and induced the cell cycle arrest at the S phases. These findings suggest that RMP1 may serve as a potential novel antitumor agent.

Molecular evolutionary mechanisms driving functional diversification of α-glucosidase in Lepidoptera

The digestive tract of lepidopteran insects is unique given its highly alkaline pH. The adaptive plasticity of digestive enzymes in this environment is crucial to the highly-efficient nutritional absorption in Lepidoptera. However, little is known about the molecular adaptation of digestive enzymes to this environment. Here, we show that lepidopteran α-glucosidase, a pivotal digestive enzyme, diverged into sucrose hydrolase (SUH) and other maltase subfamilies. SUH, which is specific for sucrose, was only detected in Lepidoptera. It suggests that lepidopteran insects have evolved an enhanced ability to hydrolyse sucrose, their major energy source. Gene duplications and exon-shuffling produced multiple copies of α-glucosidase in different microsyntenic regions. Furthermore, SUH showed significant functional divergence (FD) compared with maltase, which was affected by positive selection at specific lineages and codons. Nine sites, which were involved in both FD and positive selection, were located around the ligand-binding groove of SUH. These sites could be responsible for the ligand-binding preference and hydrolytic specificity of SUH for sucrose, and contribute to its conformational stability. Overall, our study demonstrated that positive selection is an important evolutionary force for the adaptive diversification of α-glucosidase, and for the exclusive presence of membrane-associated SUHs in the unique lepidopteran digestive tract.

Agonist-mediated activation of Bombyx mori diapause hormone receptor signals to extracellular signal-regulated kinases 1 and 2 through Gq-PLC-PKC-dependent cascade

Diapause is a developmental strategy adopted by insects to survive in challenging environments such as the low temperatures of a winter. This unique process is regulated by diapause hormone (DH), which is a neuropeptide hormone that induces egg diapause in Bombyx mori and is involved in terminating pupal diapause in heliothis moths. An G protein-coupled receptor from the silkworm, B. mori, has been identified as a specific cell surface receptor for DH. However, the detailed information on the DH-DHR system and its mechanism(s) involved in the induction of embryonic diapause remains unknown. Here, we combined functional assays with various specific inhibitors to elucidate the DHR-mediated signaling pathways. Upon activation by DH, B. mori DHR is coupled to the Gq protein, leading to a significant increase of intracellular Ca(2+) and cAMP response element-driven luciferase activity in an UBO-QIC, a specific Gq inhibitor, sensitive manner. B. mori DHR elicited ERK1/2 phosphorylation in a dose- and time-dependent manner in response to DH. This effect was almost completely inhibited by co-incubation with UBO-QIC and was also significantly suppressed by PLC inhibitor U73122, PKC inhibitors Gö6983 and the Ca(2+) chelator EGTA. Moreover, DHR-induced activation of ERK1/2 was significantly attenuated by treatment with the Gβγ specific inhibitors gallein and M119K and the PI3K specific inhibitor Wortmannin, but not by the Src specific inhibitor PP2. Our data also demonstrates that the EGFR-transactivation pathway is not involved in the DHR-mediated ERK1/2 phosphorylation. Future efforts are needed to clarify the role of the ERK1/2 signaling pathway in the DH-mediated induction of B. mori embryonic diapause.

Determination of 1-deoxynojirimycin in the Larvae of the Silkworm, Bombyx mori, by High-Performance Liquid Chromatography

1-Deoxynojirimycin, known to be one of the most potent α-glycosidase inhibitors, has been used as an antihyperglycemia agent. In this study, a rapid method for the determination of 1-deoxynojirimycin in the larvae of the silkworm, Bombyx mori was established, using high-performance liquid chromatography equipped with fluorescence detection after derivatization with 9-fluorenylmethyl chloroformate. The 1-deoxynojirimycin was efficiently extracted from the larvae using 0.05 mol/L hydrochloric acid, vortexing for 15 s at room temperature, followed by sonication for 30 min. The linear dynamic range extended from 0.7 to 28 µg/mL (r = 0.9999). The average recovery of the 1-deoxynojirimycin was 104% with a relative standard deviation of 3.30%. The limit of detection was 0.05 µg/mL at a signal-to-noise ratio of 3. There were high concentrations of 1-deoxynojirimycin in the larvae, reaching 0.50% of total body mass in the gluttonous stage of the third instar and significant variation in the fifth instar, suggests that the larvae may be a valuable source of 1-deoxynojirimycin for the treatment of carbohydrate-mediated disorders.

Expression plasticity and evolutionary changes extensively shape the sugar-mimic alkaloid adaptation of nondigestive glucosidase in lepidopteran mulberry-specialist insects

During the co‐evolutionary arms race between plants and herbivores, insects evolved systematic adaptive plasticity to minimize the chemical defence effects of their host plants. Previous studies mainly focused on the expressional plasticity of enzymes in detoxification and digestion. However, the expressional response and adaptive evolution of other fundamental regulators against host phytochemicals are largely unknown. Glucosidase II (GII), which is composed of a catalytic GIIα subunit and a regulatory GIIβ subunit, is an evolutionarily conserved enzyme that regulates glycoprotein folding. In this study, we found that GIIα expression of the mulberry‐specialist insect was significantly induced by mulberry leaf extract, 1‐deoxynojirimycin (1‐DNJ), whereas GIIβ transcripts were not significantly changed. Moreover, positive selection was detected in GIIα when the mulberry‐specialist insects diverged from the lepidopteran order, whereas GIIβ was mainly subjected to purifying selection, thus indicating an asymmetrically selective pressure of GII subunits. In addition, positively selected sites were enriched in the GIIα of mulberry‐specialist insects and located around the 1‐DNJ‐binding sites and in the C‐terminal region, which could result in conformational changes that affect catalytic activity and substrate‐binding efficiency. These results show that expression plasticity and evolutionary changes extensively shape sugar‐mimic alkaloids adaptation of nondigestive glucosidase in lepidopteran mulberry‐specialist insects. Our study provides novel insights into a deep understanding of the sequestration and adaptation of phytophagous specialists to host defensive compounds.

Silkworm Pupa Protein Hydrolysate Induces Mitochondria-Dependent Apoptosis and S Phase Cell Cycle Arrest in Human Gastric Cancer SGC-7901 Cells

Silkworm pupae (Bombyx mori) are a high-protein nutrition source consumed in China since more than 2 thousand years ago. Recent studies revealed that silkworm pupae have therapeutic benefits to treat many diseases. However, the ability of the compounds of silkworm pupae to inhibit tumourigenesis remains to be elucidated. Here, we separated the protein of silkworm pupae and performed alcalase hydrolysis. Silkworm pupa protein hydrolysate (SPPH) can specifically inhibit the proliferation and provoke abnormal morphologic features of human gastric cancer cells SGC-7901 in a dose- and time-dependent manner. Moreover, flow cytometry indicated that SPPH can induce apoptosis and arrest the cell-cycle in S phase. Furthermore, SPPH was shown to provoke accumulation of reactive oxygen species (ROS) and depolarization of mitochondrial membrane potential. Western blotting analysis indicated that SPPH inhibited Bcl-2 expression and promoted Bax expression, and subsequently induced apoptosis-inducing factor and cytochrome C release, which led to the activation of initiator caspase-9 and executioner caspase-3, cleavage of poly (ADP-ribose) polymerase (PARP), eventually caused cell apoptosis. Moreover, SPPH-induced S-phase arrest was mediated by up-regulating the expression of E2F1 and down-regulating those of cyclin E, CDK2 and cyclin A2. Transcriptome sequencing and gene set enrichment analysis (GSEA) also revealed that SPPH treatment could affect gene expression and pathway regulation related to tumourigenesis, apoptosis and cell cycle. In summary, our results suggest that SPPH could specifically suppress cell growth of SGC-7901 through an intrinsic apoptotic pathway, ROS accumulation and cell cycle arrest, and silkworm pupae have a potential to become a source of anticancer agents in the future.