Silymarin-Functionalized Selenium Nanoparticles Prevent LPS-Induced Inflammatory Response in RAW264.7 Cells through Downregulation of the PI3K/Akt/NF-κB Pathway.

Silymarin exhibits an anti-inflammatory property in various cancers and inflammatory diseases. In our previous work, silymarin-mediated selenium nanoparticles (SeNPs) (Si-SeNPs) were developed using a green synthesis technique, and its potential as an anticancer agent was confirmed. In order to further examine the extended comprehensive potential of Si-SeNPs, this investigation focuses on studying the enhanced anti-inflammatory effect of Si-SeNPs in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. Enzyme-linked immunosorbent assay and quantitative reverse transcription-polymerase chain reaction were used to evaluate the expression of pro-inflammatory mediators and cytokines. Western blotting and immunofluorescence assays were conducted to assess the protein expression of p-PI3K, p-Akt, p-NF-κB, and p-IκBα. Compared to silymarin, Si-SeNPs exhibited a significantly increased inhibitory effect on LPS-induced release of nitric oxide and the expression of pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α) and interleukin 1ß (IL-1ß) in RAW264.7 cells. A western blot assay indicated that Si-SeNPs downregulated the PI3K/Akt and NF-κB signaling pathways. The immunofluorescence assay suggested that Si-SeNPs inhibited the nuclear translocation and the activation of NF-κB. In addition, 740 Y-P (PI3K agonist) was used to demonstrate that activating the PI3K/Akt signal could partially reverse the inflammatory response, suggesting a causal role of the PI3K/Akt signaling pathway in the anti-inflammatory effect of Si-SeNPs. Consequently, these findings indicate that Si-SeNPs could be a functional agent of the attenuation of LPS-induced inflammatory responses in RAW264.7 macrophages through inhibiting the PI3K/Akt/NF-κB signaling pathway. In addition, biosynthesized Si-SeNPs could be more effective at reducing inflammation than only silymarin extracts. Thus, this study lays an experimental foundation for the clinical application of using biosynthesized SeNPs as a novel candidate in the field of inflammation-associated diseases.

Biologically synthesis of gold nanoparticles using Cirsium japonicum var. maackii extract and the study of anti-cancer properties on AGS gastric cancer cells.

Plant extract-mediated synthesis of metal nanoparticles (NPs) is an eco-friendly and cost-effective biosynthesis method that is more suitable for biological applications than chemical ones. We prepared novel gold NPs (AuNPs), Cirsium japonicum mediated-AuNPs (CJ-AuNPs), using a biosynthetic process involving Cirsium japonicum (Herba Cirsii, CJ) ethanol extract. The physicochemical properties of CJ-AuNPs were characterized using spectrometric and microscopic analyses. The in vitro stability of CJ-AuNPs was studied for 3 months. Moreover, the selective human gastric adenocarcinoma (AGS) cell killing ability of CJ-AuNPs was verified in cancer and normal cells. An in vitro study revealed that CJ-AuNPs trigger oxidative stress and iron-dependent ferroptosis in AGS cells. Mechanistically, CJ-AuNPs induced mitochondrial reactive oxygen species (ROS), Fe2+, and lipid peroxidation accumulation, and mitochondrial damage by destroying the glutathione peroxidase-4 (GPX4)-dependent antioxidant capacity. Furthermore, in a xenograft mouse model implanted with AGS cells, treatment with 2.5, 5, and 10 mg/kg CJ-AuNPs for 16 days reduced tumor xenograft growth in a dose dependent manner in vivo without systemic toxicity. These results demonstrate that CJ-AuNPs exert anticancer effects in vitro and in vivo by inducing ferroptosis-mediated cancer cell death. This study, based on green-synthesized nanodrug-induced ferroptosis, provides new insight into potential developments in cancer therapies.

Immune-enhancing effects of postbiotic produced by Bacillus velezensis Kh2-2 isolated from Korea Foods.

Postbiotics defined as soluble factors (products or metabolic byproducts) that are released after bacterial lysis or secreted by live bacteria, have attracted considerable attention because of their long shelf life, safety, and beneficial effects. In this study, we investigated the immune-enhancing activities of squid jeotgal (a traditional Korean fermented seafood)-derived Bacillus velezensis Kh2-2 (Kh2-2) postbiotics in vitro, ex vivo, and in vivo. Cell lysates of four Bacillus species were prepared by sonication. In particular, Kh2-2 lysates induced NO production by upregulating iNOS expression in RAW264.7 cells compared with the lysates of B. subtilis Kh2-1, B. vallismortis Kh8-3, and B. amyloliquefaciens Kh3-1. Furthermore, Kh2-2 lysates stimulated immune activation of macrophages by upregulating the NF-κB and MAPK signaling pathways and promoting immune-related cytokine secretion. In the ex vivo study, Kh2-2 lysates stimulated proliferation and polarized Th1 response by inducing the production of IL-2 and IFN-γ and inhibiting IL-10 expression in splenocytes. The in vivo immune-enhancing effects of Kh2-2 lysates and Kh2-2 were further evaluated using a cyclophosphamide (CTX)-induced immunosuppression mouse model. The results showed that oral administration of Kh2-2 lysates improved CTX-induced immunosuppression by enhancing innate and adaptive immunity, stimulating immune-related cytokine secretion, and modulating gut microbiota dysbiosis in mice. Thus, we concluded that Kh2-2 lysates have potential as a functional material for postbiotics with immune-enhancing effects.

A Comparative Study of Rutin and Rutin Glycoside: Antioxidant Activity, Anti-Inflammatory Effect, Effect on Platelet Aggregation and Blood Coagulation.

The effects of rutin and rutin glycoside with different solubility were compared on antioxidant activity and anti-inflammatory effects in vitro and the effects on platelet aggregation and blood coagulation in vitro and in vivo. Rutin glycoside (consisting of rutin mono-glucoside and rutin di-glucoside) was prepared via enzymatic transglycosylation from rutin. Rutin glycoside showed a higher effect than rutin on radical scavenging activity in antioxidant assays. Rutin showed a higher toxicity than rutin glycoside in murine macrophage RAW264.7 cells. They had similar effects on the levels of nitric oxide (NO), prostaglandin E (PGE) 2 and pro-inflammatory cytokines (such as tumor necrosis factor (TNF)-α, and interleukin (IL)-6) in the cells. Both rutin and rutin glycosides similarly reduced the rate of platelet aggregation compared to controls in vitro. They also similarly delayed prothrombin time (PT) and activated partial thromboplastin time (APTT) in an in vitro blood coagulation test. The effect of repeated administration of rutin and rutin glycoside was evaluated in vivo using SD rats. The platelet aggregation rate of rutin and the rutin glycoside administered group was significantly decreased compared to that of the control group. On the other hand, PT and APTT of rutin and rutin glycoside group were not significantly delayed in vivo blood coagulation test. In conclusion, rutin and rutin glycoside showed differences in antioxidant activities in vitro, while they were similar in the reduction of NO, PGE2, TNF-α and IL-6 in vitro. Rutin and rutin glycoside also showed similar platelet aggregation rates, and blood coagulation both in vitro and in vivo condition. Comparing in vitro and in vivo, rutin and rutin glycoside were effective on platelet aggregation both in vitro and in vivo, but only in vitro on blood coagulation.

Composition analysis and oral administered effects on dextran sulfate sodium-induced colitis of galactooligosaccharides bioconverted by Bacillus circulans.

Galactooligosaccharides have been known to have many health benefits as prebiotic ingredients. In this study, we examined the anti-inflammatory activity of the galactooligosaccharide, NeoGOS-P70 (Korean commercial product), in a dextran sodium sulfate-induced colitis model. Next, we performed compositional characterization of NeoGOS-P70, which confirmed that it was a 77.4% high-purity GOS products, including a large amount of 4'-galactosyllactose. Further experiments in DSS-induced colitis model showed that oral administration of NeoGOS-P70 could significantly improve DSS-induced colitis symptoms, such as weight loss, reduction in colon shortening, and suppression of inflammatory mediators, including interleukin-6, tumor necrosis factor-α, and myeloperoxidase secretion from colon of ulcerative colitis mice. Histological analysis of mucin expression in colon tissue revealed the protective effects of NeoGOS-P70. These results suggest the potential of the novel GOS, NeoGOS-P70, as an anti-ulcerative colitis agent that could regulate inflammatory responses.

Antioxidant and anti-inflammatory activities of phenolic compounds extracted from lemon myrtle (Backhousia citriodora) leaves at various extraction conditions.

Lemon myrtle leaves were extracted with ethanol at different temperatures (25, 50, and 80 °C) and times (2, 4, 6, and 10 h) to examine the effect of extraction conditions on total polyphenol contents (TPC), total flavonoid contents (TFC), their antioxidant, anti-inflammatory activities, and amount of phenolic compounds. Under optimal extraction conditions (80 °C and 6 h), the values were 23.37%, 102.72 mg gallic acid equivalents (GAE/g dry basis), 23.37 mg rutin equivalents (RE/g dry basis), 83.31%, 60.13%, and 1.10% for yield, TPC, TFC, DPPH, ABTS radical scavenging activity, and reducing power, respectively. In addition, total amount of the phenolic compounds of extract was determined as 43.9 µg/g. The anti-inflammatory effect was determined in lipopolysaccharide-stimulated RAW 264.7 cells and inhibited the production of inflammatory mediators such as nitric oxide (NO). These results indicate that extracts of lemon myrtle leaves have potential as a valuable natural product with antioxidant and anti-inflammatory.

Structural elucidation of an anti-metastatic polysaccharide from the peels of Korean citrus Hallabong.

Previously, we have reported that the polysaccharide, HBE-III, purified from the peel of the Korean citrus Hallabong, potently inhibits tumor-metastasis. In this paper, the primary structure of HBE-III was elucidated. HBE-III is a 10 kDa acidic polysaccharide, which contains 15 different monosaccharides and 23 glycosyl linkages, indicating it is a highly branched complex polysaccharide. To determine its microstructure, sequential acid hydrolysis was carried out and the resulting fragments were analyzed using composition, methylation, and MS/MS analyses. The results indicate that HBE-III is composed of a main chain with a α-(1→4)-galacturono-oligosaccharide unit and four groups of side chains including an uronic acid-rich octasaccharide (side chain A), aceric acid-containing nonasaccharide (side chain B), Rhap-(1→5)-Kdo (side chain C), and Araf-(1→5)-Dha (side chain D), which is characteristic of rhamnogalacturonan II. The results of our 11B-NMR analysis suggest that HBE-III exists as an RG-II dimer and this structure contributes to the anti-metastatic activity of HBE-III.

Signaling pathway and structural features of macrophage-activating pectic polysaccharide from Korean citrus, Cheongkyool peels.

To characterize the immuno-stimulating ingredient from the Korean citrus, Cheongkyool, a crude polysaccharide (CCE-0) was isolated from the pectinase digests of Cheongkyool peels, from which the complex polysaccharide CCE-I was purified to homogeneity by gel filtration. CCE-I highly enhanced the production of IL-6, TNF-α, and NO in RAW 264.7 cell lines. It augmented the mRNA expression of IL-6, TNF-α, and iNOS in a dose-dependent manner. Moreover, CCE-I dose-dependently induced phosphorylation of MAPKs and NF-κB related proteins and led to the nuclear translocation of p65. The effect of CCE-I on NO and IL-6 production was suppressed by treatment with specific antibodies for TLR2, TLR4, and scavenger receptors. Conversely, the primary structure of CCE-I that exhibited potent immunostimulatory activity was characterized by sugar composition, linkage analysis, and oligosaccharide analysis after ß-elimination. The results suggested that CCE-I may be a rhamnogalacturonan-I type, highly branched polysaccharide with short arabinan and galactan side chains.

Immunostimulating and Antimetastatic Effects of Polysaccharides Purified from Ginseng Berry.

Ginseng root has been used in traditional oriental medicine for the enhancement of immune system function. The immunostimulatory effects of ginseng berry polysaccharides, however, remain unclear. Effects of polysaccharides from ginseng berry on the activation of natural killer (NK) cells and inhibition of tumors are reported. A crude polysaccharide was isolated from ginseng berry as a ginseng berry polysaccharide portion (GBPP) and was further fractionated using gel filtration chromatography to obtain the three polysaccharide fractions GBPP-I, -II and -III. GBPP-I consisted of mainly galactose (46.9%) and arabinose (27.5%). GBPP-I showed a high dose-dependent anticomplementary activity. Stimulation of murine peritoneal macrophages by GBPP-I showed the greatest enhancement of interleukin (IL)-6 and IL-12 and tumor necrosis factor (TNF)- α production. In addition, an ex vivo assay of natural killer (NK) cell activity showed that oral ( p.o.) administration of GBPP-I significantly increased NK cell cytotoxicity in YAC-1 tumor cells and production of granzyme B. Prophylactic intravenous ( i.v.) and p.o. administration of GBPP-I significantly and dose-dependently inhibited lung metastatic activity in B16BL6 melanoma cells. Depletion of NK cells after injection of rabbit anti-asialo GM1 partially abolished the inhibitory effect of GBPP-I on lung metastasis, indicating that NK cells play an important role in anticancer effects. GBPP-I exerts a strong immune-enhancing activity and can prevent cancer metastasis through activation of NK cells and other immune-related cells.

Signaling pathways associated with macrophage-activating polysaccharides purified from fermented barley.

Barley is commonly used in many food and health products. We have previously demonstrated the macrophage-stimulating properties of polysaccharides derived from fermented barley. In this study, three polysaccharide fractions (BF-I-III) were purified from fermented barley and their monosaccharide composition was analyzed. Their immune-stimulatory activities and intracellular signaling pathways were also studied in RAW264.7 cells. Among the three fractions, BF-I exhibited enhanced macrophage activation properties, such as inducing the production of IL-6, IL-12, and TNF-α. However, BF-II and BF-III showed moderate effects on RAW 264.7 cells. BF-I treatment led to the phosphorylation of MAPKs, NF-κB, and c-Jun (major component of AP-1 transcription factor) and induced the nuclear translocation of p65 in RAW264.7 cells. In addition, experiments with neutralizing antibodies showed that Dectin-1, toll-like receptor (TLR) 4, scavenge receptor (SR), and CD14 were mainly involved in the stimulation of nitric oxide (NO) production by BF-I which was suppressed by the inhibition of JNK phosphorylation. These findings suggest that BF-I, isolated from fermented barley, has an immune potentiation activity on macrophages, where it activates the JNK signaling pathway via several macrophage receptors including dectin-1, TLR4, SR, and CD14.

Anti-Cancer Effects of Panax ginseng Berry Polysaccharides via Activation of Immune-Related Cells.

Panax ginseng has long been used as natural medicine and health food all over the world. Cancer is a major cause of death worldwide and its prognosis likely depends on the immune system during tumor treatment. In this study, ginseng berry polysaccharides were evaluated for their immunostimulant and anti-cancer effects. Ginseng berry polysaccharide portion (GBPP) was used to investigate its effects on anti-complementary activity, peritoneal macrophage activation, and natural killer (NK) cell cytotoxicity. Moreover, both intravenous (i.v.) and oral administration of GBPP prior to B16-BL6 melanoma implantation in mice was evaluated. GBPP significantly increased the anti-complementary activity and cytokine production including interleukin (IL)-6, IL-12, and tumor necrosis factor (TNF)-α, dose-dependently. Splenocytes obtained after i.v. administration of GBPP showed cytolytic activity in Yac-1 cells in proportion to the E/T ratio. In addition, GBPP enhanced the production of interferon (IFN)-γ and granzyme B of NK cells. For the experimental lung cancer, compared with control mice, GBPP delivered by i.v. suppressed cancer by 48% at 100 µg/mouse, while a 37% reduction was achieved by oral administration. Deficient of NK cells in animal model demonstrated that the anti-cancer effect of GBPP was through NK cell activation. Results of this study suggest that ginseng berry polysaccharides, owing to their modulation of the immune response, can be a potential curative applicant for the prevention and treatment of tumors.

Absorption rate of krill oil and fish oil in blood and brain of rats.

BACKGROUND: Krill (Euphausia superba) is a small marine crustacean with a lipid content. The mechanism of Krill oil function is not clear yet and research reports on the absorption rate of the phospholipids of krill oil in the blood and brain are very poor. METHODS: We studied the effect of oral short-term and long-term administration of Krill oils (KOs) on bioavailability in the blood and brain of rats. For short-term testing of fish and KO bioavailability, rats were divided into four groups: normal, fish oil (FO), Krill oil 1 (KO), and Krill oil 2 (CKO). The blood and brain were collected at 2, 4, 8, 12, 24, and 48 h after oral administration (1000 mg/rat). Five hundred milligrams of FO, KO, and CKO were orally administered daily for 2 weeks for long-term administration, and then the brain and blood were collected. RESULTS: Two types of KOs showed high content of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in the PL. The EPA content of CKO and KO were 41.13 and 32.49%, respectively. After short-term KO administration, KO showed a higher EPA content than CKO in the blood after 2 h. KO showed higher content of DHA than CKO even after 2 h. FO increased until 8 h, but then decreased rapidly until 12 h. Although the total unsaturated fatty acid (UFA) content of KOs was lower than the total UFS content in FO, the remaining UFS content in the brain was higher than that in FO over time. Following oral administration of FO, KO, and CKO for 1 and 2 weeks, triglycerides (TG) and PL contents in the blood for KOs were slightly higher than for FO. EPA and DHA levels in the brain were slightly higher in KOs following long-term administration, but the difference was not significant. CONCLUSIONS: Base on these findings, KOs have functional potential for the brain and vascular diseases, and can be utilized as a multi-functional material composed mainly of functional ingredients.

Antitumor and antimetastatic activities of rhamnogalacturonan-II-type polysaccharide isolated from mature leaves of green tea via activation of macrophages and natural killer cells.

To investigate the antitumor and antimetastatic polysaccharide from the mature leaves of green tea, GTE-II was purified using size exclusion chromatography. GTE-II consisted of 15 different sugars including rarely observed sugars such as 2-O-methyl-fucose, 2-O-methyl-xylose, apiose, aceric acid, 3-deoxy-d-manno-2-octulosonic acid, and 3-deoxy-d-lyxo-2-heptulosaric acid, which were characteristics of pectic polysaccharide rhamnogalacturonan-II. Treatment of peritoneal macrophages with GTE-II not only increased interleukin (IL)-6 and IL-12 production, but also had significantly increased tumoricidal activity against Yac-1 tumor cells than those obtained from untreated mice. In an assay of natural killer (NK) cell activity, intravenous administration of GTE-II significantly stimulated NK cytotoxicity against Yac-1 tumor cells. Furthermore, the depletion of NK cells by injection of rabbit anti-asialo GM1 serum eliminated the inhibitory effect of GTE-II on B16BL6 melanoma cells. These data suggest that GTE-II inhibits tumor metastasis, and its antitumor effect is associated with activation of macrophages and NK cells.

Structural elucidation of anti-metastatic rhamnogalacturonan II from the pectinase digest of citrus peels (Citrus unshiu).

The aim of this study was to characterize a polysaccharide found in citrus peels with an anti-metastatic property. CPE-II was purified by the pectinase digestion of citrus peels. During in vivo lung metastasis of Colon26-M3.1, administration of 10µg of CPE-II per mouse showed 81.3% inhibition of metastasis. CPE-II consists of 15 different monosaccharides and 22 different glycosyl linkages, characteristic of rhamnogalacturonan II (RG-II). The primary structure was elucidated based on sugar composition, methylation analysis, oligosaccharide analysis, and sequencing using GC, GC-MS, LC-MS, and ESI-MS/MS analyses. Sequential degradation using partial acid hydrolysis indicated that CPE-II contained Rhap-(1→5)-Kdo, Araf-(1→5)-Dha, an AceA-containing nonasaccharide, and an uronic acid-rich oligosaccharide in addition to an α-(1→4)-galacturono-oligosaccharide main chain. The molecular weight of CPE-II was observed to decrease from 9 to 5kDa at a pH value of <2.0, as observed by HPSEC. Thus, we propose that the anti-metastatic CPE-II is primarily present as an RG-II dimer.

Antitumor metastasis activity of pectic polysaccharide purified from the peels of Korean Citrus Hallabong.

A polysaccharide fraction, HBE-III, was successfully purified in a high yield (40.4%) from its crude polysaccharide (HBE-0) which was prepared from pectinase hydrolysates of the peels of the Korean Citrus Hallabong. In experimental lung metastasis studies of Colon 26-M3.1 carcinoma cells, prophylactic administration of HBE-III significantly inhibited lung metastasis in a dose-dependent manner. In an in vitro cytotoxicity analysis, HBE-III (up to 1000 µg/mL) did not affect the growth of Colon 26-M3.1 cells and normal cells. HBE-III enhanced production of IL-6 and IL-12 by murine peritoneal macrophages. In an assay for natural killer (NK) cell activity, HBE-III (1000 µg/mouse, i.v.) significantly augmented NK cytotoxicity against Yac-1 tumor cells. The depletion of NK cells by injection of mouse anti-asialo GM1 serum abolished the inhibitory effect of HBE-III on lung metastasis of Colon 26-M3.1 cells. These data suggest that HBE-III may inhibit tumor metastasis via activation of macrophages and NK cells.

Anti-metastatic effect of polysaccharide isolated from Colocasia esculenta is exerted through immunostimulation.

In the present study, an edible corm of the plant Colocasia esculenta, commonly known as Taro was extracted with cold water (4˚C). Finally, 10.44 g (1.04%) of the crude polysaccharide (Taro-0) was obtained from Taro. The purified active compound (Taro-4-I) was isolated using DEAE-Sepharose FF and Sephadex G-100. The anti-complementary activity of Taro-4-I (57.3±4.5%) was similar to that of polysaccharide K (used as the positive control). The molecular weight of Taro-4-I was 200 kDa and it was a polysaccharide composed of 64.4% neutral sugars and 35.6% uronic acid. Taro-4-I activated the complement system through the classical and alternative pathways. The treatment of peritoneal macrophages with Taro-4-I significantly increased the production of interleukin (IL)-6 and tumor necrosis factor-α (TNF-α) in a dose-dependent manner. However, IL-12 production showed maximal activity at 56 µg/ml and subsequently decreased. Splenocytes obtained from mice which were administered Taro-4-I intravenously showed a higher toxicity to Yac-1 cells compared to those obtained from untreated mice in a effector­to­target (E/T) ratio-dependent manner. The group treated with 50 µg/ml Taro-4-I showed a significantly increased toxicity to Yac-1 cells compared to the group treated with 500 µg/ml Taro-4-I. The administration of Taro-4-I significantly inhibited the lung metastasis of B16BL6 melanoma cells. However, the group treated with 50 µg/mouse Taro-4-I had a significantly lower number of tumors compared to the group injected with 500 µg/mouse Taro-4-I.