Epiberberine inhibits bone metastatic breast cancer-induced osteolysis.

ETHNOPHARMACOLOGICAL RELEVANCE: The anti-tumor related diseases of Coptidis Rhizoma (Huanglian) were correlated with its traditional use of removing damp-heat, clearing internal fire, and counteracting toxicity. In the recent years, Coptidis Rhizoma and its components have drawn extensive attention toward their anti-tumor related diseases. Besides, Coptidis Rhizoma is traditionally used as an anti-inflammatory herb. Epiberberine (EPI) is a significant alkaloid isolated from Coptidis Rhizoma, and exhibits multiple pharmacological activities including anti-inflammatory. However, the effect of epiberberine on breast cancer and the inflammatory factors of metastatic breast cancer-induced osteolysis has not been demonstrated clearly. AIM OF THE STUDY: Bone metastatic breast cancer can lead to osteolysis via inflammatory factors-induced osteoclast differentiation and function. In this study, we try to analyze the effect of epiberberine on breast cancer and the inflammatory factors of metastatic breast cancer-induced osteolysis. METHODS: To evaluate whether epiberberine could suppress bone metastatic breast cancer-induced osteolytic damage, healthy female Balb/c mice were intratibially injected with murine triple-negative breast cancer 4T1 cells. Then, we examined the inhibitory effect and underlying mechanism of epiberberine on breast cancer-induced osteoclastogenesis in vitro. Xenograft assay was used to study the effect of epiberberine on breast cancer cells in vivo. Moreover, we also studied the inhibitory effects and underlying mechanisms of epiberberine on RANKL-induced osteoclast differentiation and function in vitro. RESULTS: The results show that epiberberine displayed potential therapeutic effects on breast cancer-induced osteolytic damage. Besides, our results show that epiberberine inhibited breast cancer cells-induced osteoclast differentiation and function by inhibiting secreted inflammatory cytokines such as IL-8. Importantly, we found that epiberberine directly inhibited RANKL-induced differentiation and function of osteoclast without cytotoxicity. Mechanistically, epiberberine inhibited RANKL-induced osteoclastogensis via Akt/c-Fos signaling pathway. Furthermore, epiberberine combined with docetaxel effectively protected against bone loss induced by metastatic breast cancer cells. CONCLUSIONS: Our findings suggested that epiberberine may be a promising natural compound for treating bone metastatic breast cancer-induced osteolytic damage by inhibiting IL-8 and is worthy of further exploration in preclinical and clinical trials.

Rhamnan sulfate reduces atherosclerotic plaque formation and vascular inflammation.

OBJECTIVE: While lipid-lowering drugs have become a mainstay of clinical therapy these treatments only slow the progression of the disease and can have side effects. Thus, new treatment options are needed to supplement the effects of lipid lowering therapy for treating atherosclerosis. We examined the use of an inexpensive and widely available marine polysaccharide rhamnan sulfate as an oral therapeutic for limiting vascular inflammation and atherosclerosis. METHODS AND RESULTS: We found rhamnan sulfate enhanced the barrier function of endothelial cells, preventing the deposition of LDL and maintaining barrier function even in the presence of glycocalyx-degrading enzymes. Rhamnan sulfate was also found to bind directly to FGF-2, PDGF-BB and NF-κB subunits with high affinity. In addition, rhamnan sulfate was a potent inhibitor of NF-κB pathway activation in endothelial cells by TNF-α. We treated ApoE-/- mice with a high fat diet for 4 weeks and then an addition 9 weeks of high fat diet with or without rhamnan sulfate. Rhamnan sulfate reduced vascular inflammation and atherosclerosis in both sexes of ApoE-/- mice but had a stronger therapeutic effect in female mice. Oral consumption of rhamnan sulfate induced a significant decrease in cholesterol plasma levels in female mice but not in male mice. In addition, there was a marked reduction in inflammation for female mice in the liver and aortic root in comparison to male mice. CONCLUSIONS: Rhamnan sulfate has beneficial effects in reducing inflammation, binding growth factors and NF-κB, enhancing endothelial barrier function and reducing atherosclerotic plaque formation in ApoE-/- mice.

Mitochondrial toxicity evaluation of traditional Chinese medicine injections with a dual in vitro approach.

There are technical obstacles in the safety evaluation of traditional Chinese medicine (TCM) injections due to their complex chemical nature and the lack of rapid and accurate in vitro methods. Here, we established a dual in vitro mitochondrial toxicity approach combing the conventional "glucose/galactose" assay in HepG2 cells with the cytotoxic assay in mitochondrial respiration deficient cells. Using this dual in vitro approach, for the first time, we systematically assessed the mitochondrial toxicity of TCM injections. Four of the 35 TCM injections, including Xiyanping, Dengzhanhuasu, Shuanghuanglian, and Yinzhihuang, significantly reduced cellular ATP production in galactose medium in the first assay, and presented less cytotoxic in the respiration deficient cells in the second assay, indicating that they have mitochondrial toxicity. Furthermore, we identified scutellarin, rutin, phillyrin, and baicalin could be the potential mitochondrial toxic ingredients in the 4 TCM injections by combining molecular docking analysis with experimental validation. Collectively, the dual in vitro approach is worth applying to the safety evaluation of more TCM products, and mitochondrial toxic TCM injections and ingredients found in this study deserve more attention.

Homogalacturonan from squash: Characterization and tau-binding pattern of a sulfated derivative.

A pectic polysaccharide (WAP) was isolated from squash and identified as a homogalacturonan with a molecular mass of 83.2 kDa by GPC, monosaccharide composition analysis, FT-IR and NMR spectra. Sulfation modification of WAP was carried out and a sulfated derivative (SWAP) was obtained with a substitution degree of 1.81. The NMR spectrum indicated that the sulfation modification mainly occurred at the C-2 and C-3 positions of galacturonan residues. The binding pattern of SWAP to tau K18 protein was observed in 2D 1H15N HSQC spectra of tau, which resembled the tau-heparin interaction, with R2 domain as the major binding region. These results suggest that SWAP has the potential to act as a heparin mimic to inhibit the transcellular spread of tau; thus natural polysaccharide from squash may be developed into therapies for AD and related tauopathies.

Anti-SARS-CoV-2 Activity of Rhamnan Sulfate from Monostroma nitidum.

The COVID-19 pandemic is a major human health concern. The pathogen responsible for COVID-19, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), invades its host through the interaction of its spike (S) protein with a host cell receptor, angiotensin-converting enzyme 2 (ACE2). In addition to ACE2, heparan sulfate (HS) on the surface of host cells also plays a significant role as a co-receptor. Our previous studies demonstrated that sulfated glycans, such as heparin and fucoidans, show anti-COVID-19 activities. In the current study, rhamnan sulfate (RS), a polysaccharide with a rhamnose backbone from a green seaweed, Monostroma nitidum, was evaluated for binding to the S-protein from SARS-CoV-2 and inhibition of viral infectivity in vitro. The structural characteristics of RS were investigated by determining its monosaccharide composition and performing two-dimensional nuclear magnetic resonance. RS inhibition of the interaction of heparin, a highly sulfated HS, with the SARS-CoV-2 spike protein (from wild type and different mutant variants) was studied using surface plasmon resonance (SPR). In competitive binding studies, the IC50 of RS against the S-protein receptor binding domain (RBD) binding to immobilized heparin was 1.6 ng/mL, which is much lower than the IC50 for heparin (~750 ng/mL). RS showed stronger inhibition than heparin on the S-protein RBD or pseudoviral particles binding to immobilized heparin. Finally, in an in vitro cell-based assay, RS showed strong antiviral activities against wild type SARS-CoV-2 and the delta variant.

Effective Inhibition of SARS-CoV-2 Entry by Heparin and Enoxaparin Derivatives.

Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) has caused a pandemic of historic proportions and continues to spread globally, with enormous consequences to human health. Currently there is no vaccine, effective therapeutic, or prophylactic. As with other betacoronaviruses, attachment and entry of SARS-CoV-2 are mediated by the spike glycoprotein (SGP). In addition to its well-documented interaction with its receptor, human angiotensin-converting enzyme 2 (hACE2), SGP has been found to bind to glycosaminoglycans like heparan sulfate, which is found on the surface of virtually all mammalian cells. Here, we pseudotyped SARS-CoV-2 SGP on a third-generation lentiviral (pLV) vector and tested the impact of various sulfated polysaccharides on transduction efficiency in mammalian cells. The pLV vector pseudotyped SGP efficiently and produced high titers on HEK293T cells. Various sulfated polysaccharides potently neutralized pLV-S pseudotyped virus with clear structure-based differences in antiviral activity and affinity to SGP. Concentration-response curves showed that pLV-S particles were efficiently neutralized by a range of concentrations of unfractionated heparin (UFH), enoxaparin, 6-O-desulfated UFH, and 6-O-desulfated enoxaparin with 50% inhibitory concentrations (IC50s) of 5.99 µg/liter, 1.08 mg/liter, 1.77 µg/liter, and 5.86 mg/liter, respectively. In summary, several sulfated polysaccharides show potent anti-SARS-CoV-2 activity and can be developed for prophylactic as well as therapeutic purposes.IMPORTANCE The emergence of severe acute respiratory syndrome coronavirus (SARS-CoV-2) in Wuhan, China, in late 2019 and its subsequent spread to the rest of the world has created a pandemic situation unprecedented in modern history. While ACE2 has been identified as the viral receptor, cellular polysaccharides have also been implicated in virus entry. The SARS-CoV-2 spike glycoprotein (SGP) binds to glycosaminoglycans like heparan sulfate, which is found on the surface of virtually all mammalian cells. Here, we report structure-based differences in antiviral activity and affinity to SGP for several sulfated polysaccharides, including both well-characterized FDA-approved drugs and novel marine sulfated polysaccharides, which can be developed for prophylactic as well as therapeutic purposes.

Molecular mechanisms of bioactive polysaccharides from Ganoderma lucidum (Lingzhi), a review.

Ganoderma lucidum, commonly known as "Lingzhi" in Chinese, are well-known medicinal mushrooms. Lingzhi has been used in traditional Chinese herbal medicines for more than two thousand years. G. lucidum polysaccharides (GLPs) are present at high levels in G. lucidum cells and GLPs have molecular weights ranging from thousands to millions. GLPs have been widely studied for their various biological activities, such as antioxidant, antitumor, anti-inflammatory, antiviral, anti-diabetes, and immunomodulatory activities. The methods for GLPs extraction and characterization are mature, but the comprehensive research on the relationship between GLPs structure (i.e., molecular weight, tertiary structure, branching, substituents, and monosaccharide composition) and function is still quite limited. The aim of this review is to update and summarize the mechanisms of the various bioactive polysaccharides extracted from G. lucidum. The information presented on these bio-mechanisms should be valuable in the research and development of GLPs-derived therapeutics.

Structure, bioactivities and applications of the polysaccharides from Tremella fuciformis mushroom: A review.

Tremella fuciformis is an important edible mushroom that has been widely cultivated and used as food and medicinal ingredient in traditional Chinese medicine. In the past decades, many researchers have reported that T. fuciformis polysaccharides (TPS) possess various bioactivities, including anti-tumor, immunomodulatory, anti-oxidation, anti-aging, repairing brain memory impairment, anti-inflammatory, hypoglycemic and hypocholesterolemic. The structural characteristic of TPS has also been extensively investigated using advanced modern analytical technologies such as NMR, GC-MS, LC-MS and FT-IR to dissect the structure-activity relationship (SAR) of the TPS biomacromolecule. This article reviews the recent progress in the extraction, purification, structural characterization and applications of TPS.

Amphiphilic bromelain-synthesized oligo-phenylalanine grafted with methoxypolyethylene glycol possessing stabilizing thermo-responsive emulsion properties.

A thermo-responsive amphiphile was developed from oligo-phenylalanine [oligo(Phe)]. The hydrophobic moiety of the amphiphile, oligo(Phe) was synthesized via reverse hydrolysis catalyzed by bromelain in dimethyl sulfoxide and dioxane solutions. The production of oligo(Phe) increased by 80.7% by screening suitable reaction conditions. The average degree of polymerization of oligo(Phe) was determined to be four by 1H NMR. By grafting with aldehyde-ended methoxypolyethylene glycol (mPEG), oligo(Phe) was converted to amphiphilic oligo(Phe)-mPEG. The surface tension of oligo(Phe)-mPEG solution increased with decreasing chain length of the mPEG moiety. Cytotoxicity studies showed oligo(Phe)-mPEGs are biocompatible. On varying temperature, a reversible phase transition of oligo(Phe)-mPEG solutions could be observed. N-octane-in-water emulsions and 0.5% beta-carotene containing squalene-in-water emulsions stabilized by oligo(Phe)-mPEGs occurred at 25 °C but de-emulsification took place at >40 °C. Emulsification could be restored once the separated mixture cooled and re-homogenized. The emulsification/de-emulsification cycling could be repeated many times. The time required for de-emulsification decreased with elevated temperature but increased with a reduced concentration of oligo(Phe)-mPEGs and a reduction in the chain length of the mPEG moiety.

Effects of fermentation on the hemolytic activity and degradation of Camellia oleifera saponins by Lactobacillus crustorum and Bacillus subtilis.

The saponins, as components of tea seed meal, are undesirable hemolytic components and should be degraded for reducing their hemolytic activity in order to be used in animal feed. In this study, ß-glucuronidase was verified to be a potent hydrolase of tea seed saponins to reduce their hemolytic activity and a ß-glucuronidase-producing Lactobacillus crustorum strain was screened from raw bovine milk. Next, solid-state fermentation with the isolated L. crustorum and a Bacillus subtilis natto strain, which can produce cellulase and hence improve the fermentation performance of tea seed meal, was carried out for detoxification of tea seed meal. The 50% hemolytic dosage (HD50) value of tea seed saponins was increased from 6.69 to 27.43 µg mL-1. The results of LC-MS analysis showed that the percentage of saponin aglycones increased from 30.95 to 84.25% after the fermentation. According to the roles of sugar moieties in hemolytic activity, and the enzymatic hydrolysis characteristics of ß-glucuronidase, the degradation of tea seed saponins from glucosides to aglycones may contribute to the reduction of hemolytic activity. Therefore, tea seed meal may be used as animal feed after fermentation with the tested saponin-degrading microbial strains.

Isolation of a lectin binding rhamnogalacturonan-I containing pectic polysaccharide from pumpkin.

A rhamnogalacturonan-I (RG-I) containing pectic polysaccharide (PPc) was isolated from pumpkin following a low-temperature alkali treatment and a combination of gradual alcohol precipitation and ion-exchange. Monosaccharide compositional analysis of PPc revealed the presence of rhamnose, galacturonic acid, galactose, and arabinose in a molar ratio of 7.4: 25: 28: 2.6. Structural and linkage analysis by 1D NMR (1H NMR and 13C NMR), and 2D NMR (COSY, TOCSY, HSQC, and elevated temperature HMBC) suggested that PPc was a RG-I-like pectic polysaccharide, branched at the C-4 of some of the (about 29% of) rhamnosyl units, with relatively long ß-1,4-d-galactan side chains to which were attached, through the C-3 of ß-d-Gal, terminal non reducing α-Araf units. The results of surface plasmon resonance (SPR) show that PPc binds to two types of lectin, Ricinus communis agglutinin 120 (RCA120) and Galectin-3 (Gal-3). These binding studies show quick association and slow dissociation with a moderate binding affinity between PPc and Gal-3 of 1.26µM. The interaction between PPc and Gal-3 suggest the potential use of pumpkin pectic polysaccharide as a Gal-3 inhibitor in functional food or drug development applications.

Structure and bioactivity of a polysaccharide containing uronic acid from Polyporus umbellatus sclerotia.

Polyporus umbellatus is a medicinal fungus, has been used in traditional Chinese medicine for thousands years for treatment of edema, scanty urine, vaginal discharge, jaundice and diarrhea. The structure of a soluble polysaccharide (named PUP80S1), purified from the sclerotia of Polyporus umbellatus was elucidated by gas chromatography (GC), GC-mass spectrometry and nuclear magnetic resonance spectroscopy. PUP80S1 is a branched polysaccharide containing approximately 8.5% uronic acid and having an average molecular weight of 8.8kDa. Atomic force microscopy of PUP80S1 reveals a globular chain conformation in water. Antioxidant tests, Oxygen radical absorption capacity and 2,2-diphenyl-1-picrylhydrazyl radical scavenging assays indicate that PUP80S1 possesses significant antioxidant activity. But the related polysaccharide, PUP60S2, which contains more uronic acid residues and a higher level of branching, shows better antioxidant activity. These results suggest that structure features of polysaccharides play an important role in their physiological functions.

In vivo antithrombotic synergy of oral heparin and arginine: endothelial thromboresistance without changes in coagulation parameters.

On the basis of suggested clinical efficacy in an uncontrolled study in ninety-seven patients with unstable angina, an animal study was conducted to investigate antithrombotic synergy between orally administered heparin and arginine. A rat venous thrombosis model tested the difference in thrombus formation when heparin (7.5 mg/kg) and arginine (113 mg/kg) were administered, alone or in combination, by stomach tube with a minimum of 20 rats/group. Oral heparin, arginine, and heparin plus arginine reduced thrombus formation by 50%, 75%, and 90%, respectively, when compared to saline administration. Heparin was recovered from endothelium, yet there was little or no observable plasma anticoagulant activity. An orally administered low-molecular-weight anticoagulant glycosaminoglycan mixture, sulodexide (7.5 mg/kg), showed an 88% reduction in stable thrombus formation when administered alone but showed no synergy with oral arginine. A 28-day study with oral sulodexide (2.9 mg/kg) and arginine (43.9 mg/kg), 20 rats/group, showed antithrombotic activity with minimal anticoagulant activity indicating suitability for long term treatment. These findings suggest the endothelial localization of heparin and a synergistic antithrombotic effect for orally administered heparin and arginine.