Fabrication and characterization of curcumin-loaded nanoparticles using licorice protein isolate from Radix Glycyrrhizae.

Licorice was widely used in food and herbal medicine. In its extract industry, a substantial amount of licorice protein was produced and discarded as waste. Herein, we extracted Licorice Protein Isolate (LPI) and explored its potential as a curcumin nanocarrier. Using a pH-driven method, we fabricated LPI-curcumin nanoparticles with diameters ranging from 129.30 ± 3.21 nm to 75.03 ± 1.19 nm, depending on the LPI/curcumin molar ratio. The formation of LPI-curcumin nanoparticles was primarily driven by hydrophobic interactions, with curcumin entrapped in LPI being in an amorphous form. These nanoparticles significantly enhanced curcumin properties in terms of solubility, photochemical stability, and stability under varying pH, storage, and physiological conditions. Moreover, the loaded curcumin exhibited a 2.58-fold increase in cellular antioxidant activity on RAW 264.7 cells and a 1.86-fold increase in antitumor activity against HepG2 cells compared to its free form. These findings suggested that LPI could potentially serve as a promising novel delivery material.

Incidental nanoparticles in black tea alleviate DSS-induced ulcerative colitis in BALB/c mice.

As the dominant herbal drink consumed worldwide, black tea exhibits various health promoting benefits including amelioration of inflammatory bowel diseases. Despite extensive studies on the tea's components, little is known about the bioactivities of nanoparticles (NPs) which were incidentally assembled in the tea infusion and represent the major components. This study investigated the alleviative effects of black tea infusion, the isolated black tea NPs, and a mixture of caffeine, epigallocatechin-3-gallate, gallic acid and epicatechin gallate on dextran sodium sulfate (DSS)-induced ulcerative colitis. The results showed that both the black tea infusion and the NPs significantly alleviated colitis, suppressed the mRNA levels of pro-inflammatory cytokines TNF-α, IL-6, and IL-1ß, and suppressed the DSS-induced loss of cell-cell junction proteins (e.g., E-cadherin, ZO-1, and claudin-1) and increase of p-STAT3. The mixture of four tea components, which is the analogue of bioactive payloads carried by the NPs, was much less effective than the tea infusion and NPs. It shows that the NPs elevate the efficiency of polyphenols and caffeine in black tea in restoring the intercellular connection in the intestine, inhibiting mucosal inflammation, and alleviating ulcerative colitis. This work may inspire the development of tea-based therapeutics for treating inflammatory bowel diseases and have wide influences on value-added processing, quality evaluation, functionalization, and innovation of tea and other plant-based beverages.

Fish oil nano-emulsion kills macrophage: Ferroptosis triggered by catalase-catalysed superoxide eruption.

Fish oil is increasingly utilised in the form of nano-emulsion as a nutrient and function fortifier. The nano-emulsions exceptionally high content of polyunsaturated fatty acids and electron donors at the oil/water interface provide an ideal site of the redox reaction. Here we report that a vigorous superoxide production in the fish oil nano-emulsion was catalysed by mammalian catalase in acellular and cellular systems. The resulting superoxide increased cytosolic reactive oxygen species (ROS) and membrane lipid peroxidation of murine macrophage, which eventually causes fatal oxidative damages. Cell death, was significantly inhibited by a catalase-specific inhibitor 3-Amino-1,2,4-triazole (3-AT), was via ferroptosis and not apoptosis. The ferroptosis was independent of free iron or glutathione peroxidase suppression. Our findings discovered a hidden health risk of the widely acclaimed fish oil emulsion, suggesting a novel cellular damage mechanism caused by dietary unsaturated fats on the alimentary tract mucosa.

Isolation and Characterization of Bioactive Proteoglycan-Lipid Nanoparticles from Freshwater Clam (Corbicula fluminea Muller) Soup.

Nanoparticles can be prepared by several sophisticated processes but until now, it cannot be prepared by simple home cooking. Here, we report that two incidental food nanoparticles (iFNPs) consisting of proteoglycans and phytosterols were isolated from soup made from freshwater clam (Corbicula fluminea Muller), a renowned folk remedy for liver problems in China and other parts of East Asia. These two bioactive iFNPs were obtained and characterized by anionic exchange chromatography coupled with multi-angle laser light scattering measurement. Their hydrodynamic diameters and ζ-potentials were 50 ± 0.2 nm and -28.0 mV and 67 ± 0.4 nm and -9.96 mV, respectively. FT-IR revealed that the proteoglycans in the particles contained α-type heteropolysaccharides. Both iFNPs were resistant to pH changes and separation by mechanical force but responsive to temperature changes. They effectively inhibited cholesterol uptake in vitro, which resonates with the traditional belief that freshwater clam soup provides hepatoprotective benefits. This study suggests that these two proteoglycan-lipid iFNPs are the active moieties and offers a supramolecular structure-based approach to study the function of such complex matrices derived from food.

Influences of calcium and magnesium ions on cellular antioxidant activity (CAA) determination.

The cellular antioxidant activity (CAA) assay is wildly used for quantifying antioxidant activities of foods and dietary supplements in vitro. Among various incubation and handling buffers used in different laboratories, the inconsistence in concentrations of ions, particularly calcium and magnesium, has somehow been neglected. We hired the Hank's balanced salt solution with or without calcium and magnesium to perform CAA assay in Caco-2 cells and HepG2 cells, evaluating the impacts of these cations. The absence of calcium and magnesium reduced intracellular ROS level and underestimated the CAA of quercetin, Trolox and catechin. The abnormally high extracellular calcium and magnesium can also produce inaccurate results. Hank's buffer is recommended to ensure the accuracy and reproducibility. It elucidates precautions must be taken on these cations' concentrations of the buffers while conducting CAA determinations on different types of cells and when comparing foods and beverages with various calcium/magnesium contents.

Identification of protein-polysaccharide nanoparticles carrying hepatoprotective bioactives in freshwater clam (Corbicula fluminea Muller) soup.

Bioactives can impact food function either by their dosage or by their forms of dispersion, though the latter remains mostly neglected. Here we report the incidental nanoparticles (iNPs) carrying hepatoprotective bioactives identified in freshwater clam (Corbicula fluminea Muller) soup, which is a folk remedy for liver conditions in East Asia. The soup was fractionated into two iNPs containing fractions with high yield (95.8%) in 35 min by gel chromatography. With hydrodynamic diameter (Dh) range from 40 nm to 149 nm, iNPs were mainly constituted by carbohydrates and proteins. Notably, the majority of bioactives, e.g. taurine (63.2%), ornithine (68.1%) and phytosterols (60.0%), was determined to be carried by the iNPs. It suggested a possible mechanism of elevated delivery and absorption of bioactives, explaining why the clam soup can work at the bioactive concentrations way lower than the individual compound. These iNPs have great potential to be developed into a functional food with most potent nutraceutical effects.

Effects of TAT-SOD at Acupoints on Essential Hypertension by Monitoring Meridians Electrical Potential.

OBJECTIVES: To investigate the effect on essential hypertension of the topical application of TAT-Cu, Zn-superoxide dismutase (TAT-SOD) at left acupoint Zusanli (ST 36), and to observe whether the change of electrical potential difference (EPD) can be related to the change of blood pressure. METHODS: Sixteen patients with essential hypertension and 16 healthy subjects were included in the study. EPD between the left acupoints of Yanglingquan (GB 34) and Qiuxu (GB 40) was firstly screened out for the EPD detection. An intracellular superoxide quenching enzyme, TAT-SOD, was topically applied to the acupoint ST 36 within an area of 1 cm2 once a day, and the influence on EPD was investigated. The dosage applied to TAT-SOD group (n=8) was 0.2 mL of 3000 U/mL TAT-SOD cream prepared by adding purified TAT-SOD to a vehicle cream, while placebo group (n=8) used the vehicle cream instead. The left acupoints of Yanglingquan (GB 34) and Qiuxu (GB 40) were selected for EPD measurement after comparing EPD readings between 5 acupoints on each of all 12 meridians. RESULTS: EPDs between the left acupoints of GB 34 and GB 40 for 16 patients of essential hypertension and 16 healthy subjects were 44.9±6.4 and 5.6±0.9 mV, respectively. Daily application of TAT-SOD for 15 days at ST 36 of essential hypertension patients significantly decreased systolic blood pressure (SBP) and diastolic blood pressure (DBP) of 179.6 and 81.5 mm Hg to 153.1 and 74.1 mm Hg, respectively. Responding to the change in blood pressure, EPD between the left acupoints of GB 34 and GB 40 also declined from 44.4 to 22.8 mV with the same trend. No change was observed with SBP, DBP and EPD between the left acupoints of GB 34 and GB 40 with the daily application of the placebo cream. CONCLUSION: Enzymatic scavenging of the intracellular superoxide at ST 36 proved to be effective in decreasing SBP and DBP. The results reconfirm the involvement of superoxide anions and its transportation along the meridians, and demonstrate that EPD between acupoints may be an indicator to reflect its functioning status. Moreover, preliminary results suggest a close correlation between EPD and blood pressure readings, implying a possibility of using EPD as a sensitive parameter for blood pressure and to monitor the effect of antihypertensive treatment.

Boiling Licorice Produces Self-Assembled Protein Nanoparticles: A Novel Source of Bioactive Nanomaterials.

As a popular ingredient for western and traditional Chinese medicine, the root and rhizome of Chinese licorice (Glycyrrhiza uralensis Fisch.) is often administered in the form of a decoction. The protein nanoparticles (NPs) self-assembled during the process of decoction. A major constitutive protein (GLP) was purified and determined to have a molecular weight of 28 kDa with an N-terminal sequence of NPDGL IACYC GQYCW. Over 80% of the purified GLP self-assembled into spherical NPs with diameters of 74.1 ± 0.7 nm and ζ-potential of -24.3 ± 1.7 mV when boiled in Tris-HCl buffer (pH = 7.9, 20 mM) at 100 °C for 60 min. Each nanoparticle was estimated by the SEC-MALLS approach to be composed of approximately 23 protein molecules. The NPs and GLP showed low cellular toxicity upon four types of cells including MDCK, L-02, HepG2, and Caco2 cells, while the NPs promoted proliferation of normal hepatocytes by 67%. The NPs solubilized the insoluble astragaloside IV by encapsulation. The results suggest a great potential for GLP-NPs as a promising prototype of a type of drug vehicle, a novel source of bioactive nanomaterials from herbal proteins, as well as a new mode of function with herbal components.

Antidiabetic Micro-/Nanoaggregates from Ge-Gen-Qin-Lian-Tang Decoction Increase Absorption of Baicalin and Cellular Antioxidant Activity In Vitro.

The antidiabetic effects of Ge-Gen-Qin-Lian-Tang decoction (GQD) have been proven clinically. In a pharmacological study conducted on STZ-induced diabetic rats, the constitutive aggregates/sediments of Ge-Gen-Qin-Lian-Tang decoction exhibited stronger hypoglycemic and antioxidant activities compared to the soluble compositions. This study aims to demonstrate the pharmacological properties of aggregates derived from GQD by measuring permeability of the active monomer phytochemicals (e.g., baicalin) in a Caco-2 cell monolayer and determine the cellular viability, intracellular redox status (MDA and SOD), and insulin secretion of pancreatic ß-cell line, INS-1, following STZ-induced oxidative stress. The aggregates were separated into three fractions, namely, "MA (microaggregates)," "400 g supernatant," and "MNA (micro-/nanoaggregates)," by centrifugation at 400 ×g and 15000 ×g, respectively. Aggregates in the sediment increased baicalin absorption, showed little toxicity to ß-cells, elevated intracellular SOD levels, and significantly suppressed oxidative damage effects on cellular viability and functions. The "MA" fraction had a larger particle size and provided higher antioxidant cellular protection than "MNA" in vitro, implying that the sediments may be the active components in the herbal decoction. The actions of these micro-/nanoaggregates may provide a new perspective for understanding the antidiabetic effects of herbal decoctions and aid in interpretation of synergistic actions between the multiple components.

Boiling-induced nanoparticles and their constitutive proteins from Isatis indigotica Fort. root decoction: Purification and identification.

Colloidal particles are essential components of sun-dried Isatis indigotica Fort. roots (Ban-Lan-Gen in Chinese, BLG) decoction. Nanoparticles (NPs) were isolated from BLG decoction with size exclusion chromatography and characterized. Their average diameter is ∼120 nm, reversibly responding to pH and temperature changes. They promoted the growth of normal cells but suppressed that of cancerogenic cells and macrophages. Two constitutive glycated proteins were identified from the NPs, namely BLGP1 and BLGP2. Their N-terminal amino acid sequences were V-X-R-E-V-V-K-D-I and V-V-R-E-V-V-K-D-I-A-G-A-V-Q-T-N-E-Q-Y. Their full-length cDNA sequences were cloned to obtain the highly homological amino acid sequences of non-glycated proteins, whose theoretical molecular weights are 21831.64 Da and 21841.67 Da. Using pepsin hydrolysis and mass spectrometry, four possible glycation adducts were identified in BLGP1, whereas one in BLGP2. To conclude, bioactive nanoparticles isolated from the herbal decoction are intelligent nanoassemblies composed of a new boiling-stable protein. Glycation plays a critical role in heat-induced formation of these nanoassemblies. The novel, intelligent, safe and stable nano-carriers for drug delivery may be developed using BLG NPs as prototype.

Topical Application of TAT-Superoxide Dismutase in Acupoints LI 20 on Allergic Rhinitis.

Reactive oxygen species are products of cellular metabolism and assigned important roles in biomedical science as deleterious factors in pathologies. In fact, some studies have shown that the therapeutic benefits of taking antioxidants were limited and the potential for therapeutic intervention remains unclear. New evidences showed that ROS have some ability of intercellular transportation. For treating allergic rhinitis, as a novel intracellular superoxide quencher, TAT-SOD applied to acupoints LI 20 instead of directly to nasal cavity can be used to test that. TTA group apply TAT-SOD cream prepared by adding purified TAT-SOD to the vehicle cream to acupoints LI 20, while placebo group used the vehicle cream instead. TTN group applied the same TAT-SOD cream directly to nasal cavity three times daily. Symptom scores were recorded at baseline and days 8 and 15. For the overall efficacy rate, TTA group was 81.0%, while placebo group was 5.9% and TTN was 0%. Malondialdehyde levels decreased observably in TTA group, and superoxide dismutase, catalase, and glutathione peroxidase levels remained basically unaffected. Enzymatic scavenging of the intracellular superoxide at acupoints LI 20 proved to be effective in treating allergic rhinitis, while no improvement was observed with the placebo group and TTN group.

[Investigating mechanism of toxicity reduction by combination of Glycyrrhizae Radix et Rhizoma and Aconiti Lateralis Radix Preparata on terms of proteins self-assembly].

The combination of Glycyrrhizae Radix et Rhizoma and Aconiti Lateralis Radix Preparata can increase efficacy and decrease toxicity. This study started from the phenomena of protein self-assembly in the mixed decoction of Glycyrrhizae Radix et Rhizoma with Aconiti Lateralis Radix Preparata. The attenuated mechanism was explored between the combination of Glycyrrhizae Radix et Rhizoma and Aconiti Lateralis Radix Preparata by using the protein of Glycyrrhizae Radix et Rhizoma and aconitine which was the major toxic component of Aconiti Lateralis Radix Preparata. Glycyrrhizae Radix et Rhizoma protein with aconitine could form stable particles which particle mean diameter was (206.2 ± 2.02) nm and (238.20 ± 1.23) nm at pH 5.0 in normal temperature. Through the mouse acute toxicity experiment found that injection of aconitine monomer all mice were killed, and injection of Glycyrrhizae Radix et Rhizoma protein-aconitine particles with the same content of aconitine all mice survived. Survey the stability of Glycyrrhizae Radix et Rhizoma protein-aconitine shows that the colloid particles is stable at room temperature, and it has the possibility to candidate drug carrier. Glycyrrhizae Radix et Rhizoma protein can reduce the toxicity of aconitine through self-assembly.

Isolation and identification of a polygalacturonase inhibiting protein from Isatidis root.

Isatis indigotica Fort. (Chinese woad) has been widely used as a dye- and medicinal-plant in traditional Chinese medicine. Although I. indigotica has been cultivated in various regions across China in recent years, its innate immunity is little known. In this study, a protein with MW 37.5 kDa was purified from the extract of fresh Isatidis roots by SP-5PW cationic exchange and POROS HP2 hydrophobic interaction chromatography. The N-terminal amino acid sequence of the purified protein was subsequently determined as T-D-L-C-H-K-D-P-K-N-T-L-L by Edman degradation. The N-terminal sequence and PG inhibitory activity identified the purified substance as a polygalacturonase inhibiting protein. This purified Isatidis PGIP with a specific activity of 7.64x10(4) U/mg showed strong inhibitory activity against 160 U of Aspergillus niger polygalacturonase. Compared with PGIPs from guava and bean pods, Isatidis PGIP showed very poor pH and heat stabilities, which may represent the different need of plant innate immunity between plant underground and aboveground organs.

Chromatographic isolation of nanoparticles from Ma-Xing-Shi-Gan-Tang decoction and their characterization.

ETHNOPHARMACOLOGICAL RELEVANCE: The herbal decoction is a complex dispersion system containing solutes, colloid, aggregates, emulsions and precipitates. In which phase bioactive phytochemicals are dispersed determines their delivery, action and metabolism. This study took ephedrine, a well-studied and widely used phytochemical, as an example to elucidate its exact distribution in the phases of Ma-Xing-Shi-Gan-Tang decoction (MXSGT), which is an Ephedra sinica Stapf. containing traditional Chinese medicinal formula, and the biological meaning of this distribution correspondingly. It may provide an important update to the safety and efficacy assessment of the herbal decoction and its active phytochemicals. MATERIALS AND METHODS: In this study, the decoction was fractionated with size-exclusion chromatography coupled with multi-angle laser light scattering detector. The morphology of fractionated nanoparticles was observed with AFM and SEM. The bioactivities of the decoction, the ephedrine alkaloids loaded NPs (prepared by chromatography isolation) and the synthetic ephedrine were assessed by cell proliferation tests using five cell lines, namely Caco-2, L-02, Hep-G2, NR-8383, and Hela-229. RESULTS: Nanoparticles with radii of gyration ranged from 50 to 150 nm were isolated, in spherical shape. Further analysis of nanoparticles on the subsequent reversed phase chromatography revealed that the majority of ephedrine (99.7%) and pseudoephedrine (95.5%) were associated with these nanoparticles, rather than dispersed freely in the real solution. The addition of both the herbal decoction and the separated ephedrine-loaded nanoparticles reserved higher cell viability/proliferation than that of the sole synthetic ephedrine among the Caco-2, L-02, Hep-G2, and NR-8383 cells. In contrast, the nanoparticles reduced the proliferating power of ephedrine on Hela-229 cells. In general, the ephedrine-loaded NPs conducted the intermediate influences on the cell viability, in either way. CONCLUSIONS: The colloidal nanoparticles were separated from the decoction. The association of ephedrine alkaloids with nanoparticles was demonstrated and may have changed the bioactivity of the alkaloids. The naturally occurred colloidal nanoparticles may play an important role in the pharmacological properties of both the decoction and its active phytochemicals, therefore warrant further studies.

Antiviral Decoction of Isatidis Radix ( bǎn lán gen) Inhibited Influenza Virus Adsorption on MDCK Cells by Cytoprotective Activity.

The aim of this study is to elucidate how the Isatidis Radix ( bǎn lán gen) tonic, as an aqueous mixture of hundreds of compositions, interrupts the infection of influenza viruses to their host cells. The efficacy of the tonic was evaluated and expressed as cell proliferation rate and plaque reduction rate in Madin-Darby Canine Kidney (MDCK) cells, against 3 strains of influenza A and B viruses. This boiling water (at 100°C) extract of Isatidis Radix (RIE) showed antiviral activity against influenza virus A and B. The concentration for 50% inhibition of influenza virus A replication (IC50) in MDCK cell was 12.6 mg/mL with a therapeutic index >8. When cells were incubated with RIE prior to virus adsorption, the numbers of viable cell were at least doubled compared to the numbers of virus control, RIE incubation after virus adsorption and RIE incubation with virus prior to adsorption, in both influenza virus A and B. Moreover, much less virus particles were spotted by scanning electron microscope (SEM) in the RIE pre-treated cells than the cells without RIE treatment. These results indicate the antiviral activity of RIE is mainly attributed to its host cell protection effect but not actions on virus or post-virus-adsorption interruption. Cell, but not virus, is more likely to be the action target of RIE.