Longyan Zhao

Discovery of an intrinsic tenase complex inhibitor: Pure nonasaccharide from fucosylated glycosaminoglycan

Significance Selective inhibition of the intrinsic coagulation pathway is a promising strategy for developing safer anticoagulants without serious bleeding consequences. We prepared and identified a series of oligosaccharides as inhibitors of the intrinsic tenase, which is the final and rate-limiting enzyme complex in the intrinsic coagulation pathway and is an attractive but less explored target for anticoagulants due to the lack of a pure selective inhibitor. Analysis of these purified oligosaccharides reveals the precise structure of fucosylated glycosaminoglycan. Among these oligosaccharides, nonasaccharide is the minimum fragment that retains potent anticoagulant activity by selective inhibition of the intrinsic tenase while avoiding adverse effects and, thus, it may pave the way for the development of better treatments for thromboembolic diseases. Selective inhibition of the intrinsic coagulation pathway is a promising strategy for developing safer anticoagulants that do not cause serious bleeding. Intrinsic tenase, the final and rate-limiting enzyme complex in the intrinsic coagulation pathway, is an attractive but less explored target for anticoagulants due to the lack of a pure selective inhibitor. Fucosylated glycosaminoglycan (FG), which has a distinct but complicated and ill-defined structure, is a potent natural anticoagulant with nonselective and adverse activities. Herein we present a range of oligosaccharides prepared via the deacetylation–deaminative cleavage of FG. Analysis of these purified oligosaccharides reveals the precise structure of FG. Among these fragments, nonasaccharide is the minimum fragment that retains the potent selective inhibition of the intrinsic tenase while avoiding the adverse effects of native FG. In vivo, the nonasaccharide shows 97% inhibition of venous thrombus at a dose of 10 mg/kg in rats and has no obvious bleeding risk. This nonasaccharide may therefore serve as a novel promising anticoagulant.

Structural Characterization and Immunostimulatory Activity of a Homogeneous Polysaccharide from Sinonovacula constricta

Sinonovacula constricta has been widely used as a health food and medicine in China, Japan, and Korea. In the present study, a water-soluble polysaccharide fraction (SCP-1) was prepared from S. constricta by enzyme-assisted extraction and purification of chromatography with DEAE-52 cellulose anion-exchange column and Sephadex G-100 size exclusion column. On the basis of the analytical results of high-performance liquid chromatography, Fourier transform-infrared spectroscopy, methylation analysis, and NMR spectroscopy, SCP-1 was found to have an average molecular weight of 15.63 kDa and a linear backbone of (1→4)-linked α-D-Glcp residue with one branch, α-D-Glcp, attached to the main chain by a (1→6) glycosidic bond at every five α-D-Glcp units. Furthermore, it was found that SCP-1 could significantly increase the viability of macrophages, enhance the capability of macrophage phagocytosis, increase the activity of acid phosphatase, and promote the production of nitric oxide, mouse tumor necrosis factor (TNF)-α, mouse interferon (IFN)-γ, and mouse interleukin (IL)-1β. The results suggest that SCP-1 possesses potent immunomodulating effect and may be explored as a potential biological response modifier.

Structure and anticoagulant activity of fucosylated glycosaminoglycan degraded by deaminative cleavage

Fucosylated glycosaminoglycans (FGs) are complex glycosaminoglycans that exhibit potent anticoagulant activity. To study the relationship between molecular size and biological activity, oligosaccharides with (2,5)-anhydro-D-talose units at new reducing ends were prepared by hydrazine deacetylation and nitrous acid depolymerization. The product chemical structures were analyzed by one- and two-dimensional NMR methods. Additionally, anticoagulant activities were evaluated by clotting assay and chromogenic substrate cleavage. The results demonstrated that under mild deacetylation and deaminative cleavage conditions, both products were relatively homogeneous and sulfated fucose branch types and sulfate substituents remained stable. These depolymerized FGs with different molecular sizes had potent intrinsic anticoagulant activities, which were similar to those that were obtained by free-radical depolymerization with similar molecular weights. Decreasing molecular weight may weaken activity but not significantly affect factor Xase and heparin cofactor II (HCII)-mediated thrombin inhibition.

Structural Analysis and Anticoagulant Activities of the Novel Sulfated Fucan Possessing a Regular Well-Defined Repeating Unit from Sea Cucumber

Sulfated fucans, the complex polysaccharides, exhibit various biological activities. Herein, we purified two fucans from the sea cucumbers Holothuria edulis and Ludwigothurea grisea. Their structures were verified by means of HPGPC, FT-IR, GC–MS and NMR. As a result, a novel structural motif for this type of polymers is reported. The fucans have a unique structure composed of a central core of regular (1→2) and (1→3)-linked tetrasaccharide repeating units. Approximately 50% of the units from L. grisea (100% for H. edulis fucan) contain sides of oligosaccharides formed by nonsulfated fucose units linked to the O-4 position of the central core. Anticoagulant activity assays indicate that the sea cucumber fucans strongly inhibit human blood clotting through the intrinsic pathways of the coagulation cascade. Moreover, the mechanism of anticoagulant action of the fucans is selective inhibition of thrombin activity by heparin cofactor II. The distinctive tetrasaccharide repeating units contribute to the anticoagulant action. Additionally, unlike the fucans from marine alga, although the sea cucumber fucans have great molecular weights and affluent sulfates, they do not induce platelet aggregation. Overall, our results may be helpful in understanding the structure-function relationships of the well-defined polysaccharides from invertebrate as new types of safer anticoagulants.

The Mulberry (Morus alba L.) Fruit—A Review of Characteristic Components and Health Benefits

Mulberry (Morus alba L.) fruit has a high yield in one fruiting season in many countries, especially in Asia, and a long history of use as an edible fruit and traditional medicine. A great diversity of nutritive compounds such as fatty acids, amino acids, vitamins, minerals, and bioactive compounds, including anthocyanins, rutin, quercetin, chlorogenic acid, and polysaccharides have been found in mulberry fruit depending on the cultivars and maturity stages. Furthermore, the extracts and active components of mulberry fruit have demonstrated numerous biological activities, including antioxidant, neuroprotective, antiatherosclerosis, immunomodulative, antitumor, antihyperglycemic, and hypolipidemic activities in in vitro and in vivo studies, and they have received increasing interest from researchers and pharmaceutical companies. Although some mechanistic studies further substantiate these potential health benefits of mulberry fruit, a need exists to make a better understanding of the roles of these compounds in traditional medicine and the diet. This review provides recent findings regarding the chemical constituents and biological activities of mulberry fruit, which may be useful for stimulating deep research of mulberry fruit and for predicting their uses as important and safe contributors to benefit human health.

Enzyme-Assisted Extraction Optimization, Characterization and Antioxidant Activity of Polysaccharides from Sea Cucumber Phyllophorus proteus

Enzyme-assisted extraction optimization, characterization and in vitro antioxidant activity of polysaccharides from sea cucumber Phyllophorus proteus (PPP) were investigated in the present study. The optimal extraction conditions with a yield of 6.44 ± 0.06% for PPP were determined as follows: Extraction time of 2.89 h, ratio of extraction solvent to raw material of 16.26 mL/g, extraction pH of 6.83, exraction temperature of 50 °C and papain concentration of 0.15%. Three purified fractions, PPP-1a, PPP-1b and PPP-2 with molecular weights of 369.60, 41.73 and 57.76 kDa, respectively, were obtained from PPP by chromatography of FPA98Cl and Sepharose CL-6B columns. Analysis of monosaccharide compositions showed that PPP-1a consisted of N-acetyl-galactosamine (GalNAc), galactose (Gal) and fucose (Fuc), PPP-1b of Fuc as the only monosaccharide and PPP-2 of glucuronic acid, GalNAc and Fuc. Sulfate contents of PPP, PPP-1a, PPP-1b and PPP-2 were determined to be 21.9%, 20.6%, 25.2% and 28.0% (w/w), respectively. PPP and PPP-1a had higher molecular weight and intrinsic viscosity than those of the PPP-1b and PPP-2. PPP, PPP-1a, PPP-1b and PPP-2 exhibited obvious activities of scavenging 1,1-diphenyl-2-picrylhydrazyl radical, hydroxyl radical, superoxide radical and ABTS radical in different extent, which suggested that the polysaccharides from Phyllophorus proteus may be novel agents having potential value for antioxidation.

Physicochemical analysis, structural elucidation and bioactivities of a high-molecular-weight polysaccharide from Phellinus igniarius mycelia

Phellinus igniarius is a well-known edible and medicinal fungus consumed in Asia for centuries. In the present study, a polysaccharide named PIP-1 was obtained from Phellinus igniarius mycelia, its physicochemical properties were determined, its detailed structures were elucidated by analysis of its depolymerized product, and its antioxidant and antitumor activities were evaluated. Results showed that PIP-1 had a high molecular weight, a high intrinsic viscosity and a linear repeating backbone composed of glucopyranose (Glcp), galactopyranose, and mannopyranose joined by α-(1 → 4), α-(1 → 3), and α-(1 → 6) linkages, and single α‑terminal‑D‑Glcp as side chains 6-O-linked to the main chain according to the GC-MS and NMR results, and exhibited triple helical structure. Pharmacological results revealed that PIP-1 could effectively scavenge hydroxyl radicals, partly scavenge DPPH radials and chelate ferrous metal ions. Furthermore, PIP-1 showed potent inhibitory effects on growth of HT-29 and MCF-7 cells. These findings suggested that high-molecular-weight PIP-1 containing triple helical structure and only α-type glycosidic bond holds promise as novel functional foods and agents to promote human health.